extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Medicine Chemistry

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Structures of soluble guanylate cyclase: implications for regulatory mechanisms and drug development

Activation of cGMP synthesis leads to vasodilation, and is an important mechanism in clinical treatment of angina, heart failure, and severe peripheral and pulmonary hypertension. The nitric oxide-responsive sGC (soluble guanylate cyclase) has been the target of recent drug discovery efforts. The present review surveys recent data on the structure and regulation of sGC, and the prospects of new avenues for therapeutic intervention.

Introduction
Nucleotide cyclases generate two of the most important intracellular messengers, cAMP and cGMP. cGMP causes vasodilation by relaxing vascular smooth muscle cells. It is also involved in reducing platelet aggregation, in neuronal transmission, in vision and in cancer [1][2][3]. cGMP may also have a more general role in regulating energy expenditure in cells [4]. cGMP in animal cells is generated by two classes of GCs (guanylate cyclases): sGC (soluble GC) and pGC [membrane-spanning (particulate) GC]. sGC is the receptor for nitric oxide (NO). pGCs are transmembrane receptors activated by the peptide natriuretic factors ANF (atrial natriuretic factor) and BNF (brain natriuretic factor). NO acts in a paracrine manner, secreted by endothelial cells in response to hypoxia and/or mechanical stimuli, and activating sGC in adjacent smooth muscle cells. ANF and BNF are secreted from the heart atrium and brain respectively, and act systemically on ANF receptors. Upon hormone binding, the GC of the intracellular domains is activated, leading to increased cGMP levels. cGMP then activates downstream effectors (cGMP-dependent protein kinase, Ca 2 + channels and phosphodiesterases) to eventually cause muscle relaxation and vasodilation.
sGC is the target of the earliest class of cardiovascular drugs known, nitroglycerin and other NO precursors, which have been used since the 19th Century as vasodilators to alleviate angina, myocardial infarction and other disorders [2,5]. In recent years, additional drugs with more favourable pharmacology have entered into clinical trials [6]. The structure, regulation and pharmacology of sGC are the subject of a vast literature (see, for example, [7]). In the present review, we survey recent advances in the structural biology of sGC and their possible implications on mechanistic understanding and opportunities for developing new types of modulators.

Structural organization of GCs
GCs of animal cells, as well as ACs (adenylate cyclases), derive from ancestral enzymes consisting of homodimers of catalytic domains; they have evolved into fusion proteins with regulatory or membrane-anchoring domains [8]. ACs include two catalytic half-domains within the same polypeptide, whereas GCs are either homodimers (e.g. GC-A and GC-B, the natriuretic factor receptors) or heterodimers (sGC). Human sGC is composed of two homologous subunits: α1 and β1. The domain organization of sGC is depicted schematically in Figure 1(A). The C-terminal domains of both subunits combine to form a heterodimeric catalytic domain. In addition, both sGC subunits include three distinct regulatory and structural domains: an N-terminal HNOX (haem-containing NO/oxygen-binding) domain, a PAS (Per/Arnt/Sim)-like domain, and an α-helical region capable of forming CCs (coiled coils). Only the β subunit binds haem; the corresponding region of the α subunit may have a similar structure, but lacks critical haem-binding residues. The dimer interface is complex, and involves the catalytic, CC and PAS domains. Heterodimers seem to be the preferred form in cells, but the features that determine this preference, and any possible role for homodimers, are not fully defined.

Structures of sGC domains
The catalytic core of sGC is composed of two homologous domains. The active site lies at the interface between the two domains. Ligand binding and catalysis require the precise alignment of residues from both subunits; this is very sensitive to relative motion of the subunits as well as to local changes, which underlie the modes of allosteric regulation. We have reported recently a crystal structure of the catalytic domain of human sGC [9]. Structures of GCs of algae and cyanobacteria have been reported previously [10,11], in addition to multiple structures of mammalian and microbial ACs.
The studies of the catalytic domains have revealed the following features.
(i) The isolated catalytic domains of both α and β subunits can form stable homodimers, both of which are enzymatically inactive. When mixed at high concentrations, the subunits can exchange to form heterodimers, which are active even in the absence of NO. Crystal structures were obtained for both the α-β heterodimer and homodimers of the β catalytic domain [9]. The dimer interfaces are similar between the heterodimers and the β homodimers. This promiscuity of subunit association is a feature of cyclase catalytic domains; for example, active mammalian ACs have been assembled by combining catalytic domains from AC 1 or 5 with AC 2 [12,13]. (ii) The two subunits are arranged as a wreath-like headto-tail dimer ( Figure 1B). This arrangement has an inherent pseudosymmetry, which could create a second pseudosymmetric active site (not shown). In sGC, the pseudosymmetric site lacks the critical residues for catalysis and is therefore not enzymatically active. (iii) The crystal structure of the heterodimer is in an inactive conformation: the active-site residues from the two subunits are misaligned, and the cavity that should contain the substrate is not formed. (iv) It is possible to model an active conformation by aligning the α and β subunits separately with the two subdomains of an active AC structure. This entails a 26 • rigidbody rotation of the α subunit ( Figure 1C). In this conformation, it is possible to model a molecule of GTP bound at the substrate site, with most of the substratebinding and catalytic residues in the same positions as in AC (see Figure 4 of [9]). Space-filling models of both conformations (Figures 1D and 1E) show that the conformational change generates a cavity that can contain a GTP molecule (modelled in green), as well as a pseudosymmetry-related cavity, which can engulf a small molecule (illustrated by a white atomic model).
Structures of AC with the allosteric activator forskolin [12,14] have shown precisely the same arrangement, with the active and pseudosymmetry sites occupied by ATP analogues and forskolin respectively. This immediately suggests a mechanism for direct allosteric regulation of the catalytic domain through binding of small molecules to the pseudosymmetry site, stabilizing or inducing the transition to an active conformation. (v) The question arises whether the inactive conformation of the catalytic subunits seen in the crystal ever exists in the context of the full-length protein. The catalytic domains are linked directly to an upstream CC domain, which is expected to be quite rigid. As seen in Figure 1(C), the distance between the N-termini of the catalytic α and β domains changes from 28.05 to 24.94 Å (1 Å = 0.1 nm) between the inactive and active conformations. One can imagine that the distance is determined by the arrangement of the CC region, which can favour a constitutive structure similar to that of the active conformation; more intriguingly, this could be a mechanism for transmitting regulatory influences from the upstream haem-binding domains to the catalytic domain, via changes in the intervening CC region.
Other regions of sGC have been crystallized successfully. The CC region preceding the catalytic domain of the β subunit has been crystallized as a four-helix antiparallel bundle [15]. The authors indicate that an antiparallel organization of the CC region is unlikely to occur within the full-length protein, as it places the C-termini of the two helices at a distance that is much greater than the distance between the N-termini of the catalytic domain. On the basis of the distribution of charged residues in the α-helical regions of the α and β subunits, the authors suggest a preference for an antiparallel orientation in the heterodimer. This has been corroborated by cross-linking analysis [16]. Interestingly, this suggests that linking together two domains (the catalytic and CC domains), either of which can readily form homodimers, can lead to a strong preference for heterodimerization.
The structure of a PAS domain of sGC from the moth Manduca sexta was published recently [17]. Although the structure does not immediately shed light on the association or regulatory roles of the PAS domain, comparison with other PAS domains suggests that a mobile region within this domain may generate a cavity large enough to bind small molecules. The same group studied the arrangement of the entire regulatory domain (HNOX-PAS-CC) by chemical cross-linking and SAXS [16]. This enabled them to build a rough model in which the HNOX and PAS domains are folded back along a backbone of the CC domain, poised for direct interaction with the catalytic domains (which were not present in the protein fragments used). It would be interesting to see how regulatory ligands such as NO, cinaciguat or riociguat (see below) affect the gross structure of sGC.
Finally, HNOX domains of sGC have not been crystallized, but structures of highly homologous domains from cyanobacteria have been solved [18][19][20]. These structures provide important insights on the mode of binding of NO, the discrimination between ligands, and the structural consequences of NO binding, as well as of haem-mimetic drugs [21]. Briefly, NO binding releases an iron-ligated histidine (His 105 ) on the distal face of the haem group. This leads to structural changes in helix αF that contains His 105 , which are thought to be critical in transducing the signals to the catalytic domain. The important role of this region of the HNOX domain in cyclase activation is supported by mutagenesis and by hydrogen-deuterium exchange analysis [22][23][24].

Regulation of GC activity
The simplest regulatory scheme for sGC envisions inherently active catalytic domains which are inhibited by the Nterminal domains in the absence of NO. NO binding to the haem causes a structural change in the HNOX domain which is allosterically transmitted to the catalytic domain, leading to relief of inhibition. Support for this scheme comes from the observation that the isolated catalytic domains of α1 and β1, when mixed, exhibited concentration-dependent cyclase activity; this activity could be inhibited in trans by adding large concentrations of the purified regulatory domain [25]. A protein fragment containing the HNOX, PAS and part of the CC domain (β-1-385) was considerably more effective than the isolated HNOX domain (β-1-194). However, these experiments do not fully reflect the complexities of interdomain interactions in the intact proteins. Primarily, inhibition in trans is not relieved by NO [25]. Furthermore, mutagenesis of the HNOX domain usually lead to loss of activation, but only one mutation (of Thr 110 ) reportedly led to a modest increase in the basal (i.e. NO-independent) cyclase activity of the full-length protein [22]. Another mutation in the catalytic domain (α C541S) also led to an increase in basal activity, but this may be through a direct effect on the conformation of the catalytic region [23,26]. A thorough investigation of the interactions between the different domains of rat sGC was carried out using hydrogen-deuterium exchange kinetics. Adding the HNOX and PAS domains (β-1-384) in trans resulted in localized changes in hydrogen accessibility in the catalytic domains of both the α and β subunits, primarily in the 'front' side; this footprint is consistent with the HNOX/PAS domains blocking substrate entry to the active site. Notably, when comparing the hydrogen-deuterium exchange pattern of the isolated catalytic domains with the full-length protein (in which the N-terminal region is present in cis), the footprint on the β subunit is shifted, indicating a difference in the interaction interface.
The primary activation switch of sGC is the binding of NO to a sixth co-ordination site of the haem iron in the HNOX domain of the β subunit and subsequent breaking of the bond to His 105 . However, this is not the whole mechanism, as binding of a single molecule of NO leads to only a modest activation, which is enhanced severalfold by binding of additional NO molecule(s) to lower-affinity sites [20,[27][28][29]; the location of these additional sites is not clear, and may be either at the haem iron or a protein cysteine residue. sGC can be desensitized by a number of mechanisms. Oxidation of the haem iron to Fe(III), which may be followed by the loss of the haem group, leads to inactivation and subsequent degradation. In addition, other less well-defined events such as oxidation or nitrosylation of critical cysteine residues may also lead to enzyme inactivation.
The development of drugs that allosterically activate sGC was initiated following the serendipitous discovery of YC-1, which can stimulate cyclase activity 30-40-fold in the absence of NO, but can also synergize with NO. Activation by YC-1 required the presence of Fe(II)-haem. Extensive efforts to improve the potency, selectivity and pharmacological properties led to the development of two classes of sGC modulators [6,30]. The first, which includes YC-1 and the drug candidate riociguat, are termed 'sGC stimulators' and require haem. A distinct class of molecules termed 'sGC activators', which include the drug candidate cinaciguat, are haem-independent. Crystallographic studies [21,31] showed that cinaciguat and another activator (BAY 60-2770) replace the haem in a bacterial HNOX protein, presumably generating a conformation that is comparable with that of an activated haem-NO complex. The mechanism of action of YC-1 and other stimulators is less clear, and may involve stabilization of bound NO as well as allosteric effects on the global structure of sGC. Figure 2 presents a schematic summary of some of the states and transitions of sGC. Figure 2(A) represents basal state, without NO. Binding of one ( Figure 2B) or more ( Figure 2C) NO molecules leads to full activation; this is achieved through a scission of the Fe-His 105 bond and additional covalent and conformational changes in the HNOX domain, which are transmitted to the catalytic domains through a combination of direct contacts and other long-range interactions. The enzyme may be inactivated by oxidation, nitrosylation ( Figure 2D) and haem loss ( Figure 2E). Allosteric modulators can mimic the activated state either by replacing the haem moiety (cinaciguat) or by enhancing or bypassing a haemdependent activation step (riociguat). Figure 2(F) represents a hypothetical state of the catalytic domains, which is suggested by the crystal structure of the isolated domains. In this state, the α and β catalytic fragments are misaligned, disrupting both the catalytic site and a putative pseudosymmetric binding site. At present, it is not clear whether such a conformation exists in the full-length protein, and whether it, in fact, represents the conformation of the catalytic domain in Figure 2(A) or 2(E). It is tempting to speculate that sGC activation does include a relative movement of the two half-domains, possibly transmitted through changes in the CC domain or in the multiple interdomain interactions. If this is the case, there is a distinct possibility that small molecules that bind to the pseudosymmetric site can allosterically activate cyclase activity in a manner that is distinct from that of the current stimulators and activators.

The case for allosteric regulation at the catalytic domain
We now turn to allosteric effects at the level of the isolated catalytic domain. In pGC, the dependence of the cyclase activity on GTP concentration shows a sigmoidal curve with a Hill coefficient of 1.5. This is taken as evidence of cooperative binding to two sites, probably the catalytic and the pseudosymmetry sites. ATP acts as an activator of cyclase activity; in the presence of ANF, ATP converts the GTP dependence into a Hill coefficient near 1, and decreases the K m . Interestingly, this implies a non-equivalence between the two nucleotide-binding sites [32]. There may be an additional regulatory role for ATP through a kinase homology domain in pGC [33,34].
It is instructive to compare the situation with ACs in which the catalytic core is composed of two subdomains within a single polypeptide. The plant diterpene forskolin is a signal-independent activator of AC, which binds in the pseudosymmetry site. It may work by stabilizing the association of the two subdomains; indeed, all crystal structures of mammalian ACs contain forskolin analogues in the pseudosymmetric site. As we do not know the structure in the absence of forskolin, it is possible that the drug not only stabilizes the complex, but also favours a subset of possible conformations of the dimer.
As for sGC, ATP and 2 -substituted analogues have been found to inhibit GC activity, at physiological concentrations of ATP [4,35]. Inhibition by ATP has been shown both in the NO-stimulated full-length enzyme and in the isolated catalytic domains [36]. This has been suggested as a mechanism for modulating the response to paracrine or autocrine NO signalling by the energy status of the cell, to regulate energy economy [4]. Analyses with multiple nucleotides and analogues [36,37] indicate a distinct regulatory nucleotide-binding site which is most likely to be located in the catalytic domain.

Outlook: utilization of regulatory interactions for drug development
The pharmacology of cardiovascular diseases is extremely complex, reflecting the need to modulate different components of the system in a specific temporal and spatial manner, and interact with endogenous regulators which are patientspecific. There is no doubt that sGC is an attractive target for cardiovascular disorders, ranging from angina and heart failure to peripheral and pulmonary hypertension [6,38]. Even attempts to modulate this single target face issues of delivery, desensitization and side effects [39]. Hence the drive to develop new compounds that utilize different states or regulatory mechanisms; as mentioned, haem-dependent and -independent allosteric regulators (riociguat and cinaciguat) are in clinical testing, as alternatives to the traditional NOgenerating drugs [40][41][42]. The advances in characterization of sGC suggest that new chemistry directed at allosteric sites within the catalytic domain may provide further options for modulating cGMP generation. There are vast libraries of nucleotide-mimetic compounds which could provide initial hits; the recent advances in crystallization could form the basis of fragment screening and the application of computational methods to this search.


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The Colletotrichum gigasporum species complex

In a preliminary analysis, 21 Colletotrichum strains with large conidia preserved in the CBS culture collection clustered with a recently described species, C. gigasporum, forming a clade distinct from other currently known Colletotrichum species complexes. Multi-locus phylogenetic analyses (ITS, ACT, TUB2, CHS-1, GAPDH) as well as each of the single-locus analyses resolved seven distinct species, one of them being C. gigasporum. Colletotrichum gigasporum and its close allies thus constitute a previously unknown species complex with shared morphological features. Five of the seven species accepted in the C. gigasporum species complex are described here as novel species, namely C. arxii, C. magnisporum, C. pseudomajus, C. radicis and C. vietnamense. A species represented by a single sterile strain, namely CBS 159.50, was not described as novel species, and is treated as Colletotrichum sp. CBS 159.50. Furthermore, C. thailandicum is reduced to synonymy with C. gigasporum.

The most distinctive morphological feature of C. gigasporum is the long straight conidia (up to 32 μm long, av. length 26 μm). Rakotoniriana et al. (2013) discussed the morphological differences between C. gigasporum and other species that produce large conidia, e.g. C. crassipes, C. echinatum, C. macrosporum, C. taiwanense and C. vinosum. Based on phylogenetic analyses of ITS and TUB2 sequence data, they showed C. gigasporum to belong to a distinct clade, distant from other currently accepted Colletotrichum species.
Numerous Colletotrichum isolates detected in a blastn search on GenBank have similar ITS sequences to that of the extype strain of C. gigasporum, e.g. isolates from Coffea arabica in Vietnam (Nguyen et al. 2010), Hibiscus rosa-sinensis in Thailand (Noireung et al. 2012), Magnolia liliifera in Thailand (Promputtha et al. 2007), Taxus chinensis var. mairei in China (Wu et al. 2013) and Theobroma cacao, Trichilia tuberculata and Virola surinamensis in Panama (Rojas et al. 2010). In our preliminary ITS analysis, 21 isolates retrieved from the CBS collection clustered with C. gigasporum, but showed considerable genetic variability, suggesting further species belonging to a previously unreported species complex.
The objectives of this study are to clarify the genetic and taxonomic relationships of Colletotrichum strains from various hosts and geographic areas thought to be closely related to C. gigasporum, and to describe the new species from this complex.

Isolates
Colletotrichum isolates with large conidia were obtained from the culture collection of the CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands (CBS). All descriptions are based on ex-type cultures. Features of other strains are added if deviant. Cultures of additional isolates used for morphological and phylogenetic analyses are maintained in the CBS culture collection (Table 1).

Morphological analysis
To enhance sporulation, 5-mm-diam plugs from the margin of actively growing cultures were transferred to the centre of 9-cm-diam Petri dishes containing synthetic nutrient-poor agar medium (SNA) (Nirenberg 1976) amended with autoclaved filter paper and double-autoclaved stems of Anthriscus sylvestris placed onto the agar surface. Strains were also studied after growth on oatmeal agar (OA). Cultures were incubated for 10 d at 20 °C under near UV light with a 12 h photoperiod. Measurements and photographs of characteristic structures were made according to methods described by Liu et al. (2012). Appressoria on hyphae were observed on the reverse side of colonies grown on SNA plates. Microscopic preparations were made in clear lactic acid, with 30 measurements per structure, and observed with a Nikon Eclipse 80i microscope using differential interference contrast (DIC) illumination. Colony characters and pigment production on SNA and OA incubated at 20 °C were noted after 10 d. Colony colours were scored according to Rayner (1970). Growth rates were measured after 7 and 10 d.
The DNA sequences obtained from forward and reverse primers were used to obtain consensus sequences using MEGA v. 5.1 (Tamura et al. 2011), and subsequent alignments were generated using MAFFT v. 6 (Katoh & Toh 2010), and manually edited using MEGA v. 5.1.
Sequences of the 21 Colletotrichum strains studied in this paper as well as sequences of 50 reference strains (Table 1) downloaded from GenBank (www.ncbi.nlm.nih.gov/genbank/) and NIAS GenBank (www.gene.affrc.go.jp /about_en.php) were included in individual alignments and eight single gene phylogenies were generated using a distance-based method. The ITS alignment included a further 22 sequences that were found in blastn searches in GenBank in addition to those in Table 1. Distance matrixes of the aligned sequences were calculated using the Kimura 2-parameter model (Kimura 1980), and analysed with the Neighbour-joining (NJ) algorithm (Saitou & Nei 1987) using MEGA v. 5.1, excluding positions with gaps. The reliability of the inferred trees was estimated by bootstrap analyses with 1 000 replicates.
A maximum parsimony analysis was performed on the multilocus alignment including five of the eight loci (ACT, CHS-1, GAPDH, ITS, TUB2) of a total of 71 strains (Table 1) using PAUP v. 4.0b10 (Swofford 2002). Ambiguously aligned regions were excluded from all analyses. Unweighted parsimony (UP) analysis was performed. Trees were inferred using the heuristic search option with TBR branch swapping and 1 000 random sequence additions. Maxtrees were unlimited, branches of zero length were collapsed and all multiple parsimonious trees were saved. Clade stability was assessed in a bootstrap analysis with 1 000 replicates, each with 10 replicates of random stepwise addition of taxa. A second phylogenetic analysis of the concatenated alignment using a Markov Chain Monte Carlo (MCMC) algorithm was done to generate trees with Bayesian posterior probabilities in MrBayes v. 3.2.1 (Ronquist & Huelsenbeck 2003). Nucleotide substitution models were determined using MrModeltest v. 2.3 (Nylander 2004) for each gene region and included in the analyses. Two analyses of four MCMC chains were run from random trees for 10 000 000 generations and sampled every 1 000 generations. The first 25 % of trees were discarded as the burn-in phase of each analysis and posterior probabilities determined from the remaining trees. Monilochaetes infuscans strain CBS 869.96 was used as outgroup in all analyses. Sequences derived in this study were lodged in GenBank, the multi-locus alignment and tree in TreeBASE (http://www.treebase.org/treebase-web/search/ studySearch.html) (S15175), and taxonomic novelties in Myco-Bank (www.MycoBank.org; Crous et al. 2004a).

Phylogeny
The eight NJ trees derived from the single gene sequence alignments (ACT, CAL, CHS-1, GAPDH, GS, HIS3, ITS, TUB2) confirmed that the 21 CBS isolates and the ex-type and other strains of C. gigasporum constituted a monophyletic lineage, distant from other known major clades of the genus Colletotrichum recognised by Cannon et al. (2012). The NJ trees are not shown in this study except for the phylogeny based on ITS data ( Fig. 1). Isolates studied in this paper (marked with red squares) are separated into seven subclades, which could also be confirmed with the other seven single gene phylogenies.  Fig. 2, where the 21 strains studied belong to a major clade consisting of seven subclades. More than half of the strains clustered in the largest subclade (C. gigasporum) with a high bootstrap support and Bayesian posterior probability value (100/1.00). The Bayesian tree confirmed the tree topology of the trees obtained with maximum parsimony.

Fig. 1
Neighbour-joining tree of ITS sequences from 21 isolates generated in this study and 43 isolates from other studies, retrieved from GenBank. The tree was constructed using MEGA v.5.1 software. The Kimura-2-parameter method was used. Bootstrap support values (1 000 replicates) above 50 % are shown at the nodes. Ex-type cultures are emphasised in bold, and include the taxonomic name as originally described. Our isolates are marked with a red square, and the strain number is followed by host and country of origin. Stars indicate reported pathogens, triangles indicate reported endophytes, GenBank accessions are followed by taxonomic name as originally identified, strain number, host and country of origin. The tree is rooted with Monilochaetes infuscans.
Closest match in a blastn search with the ITS sequence of strain CBS 132511 (with 99 % identity, 8 bp differences) was an endophytic isolate (DQ780412) from Magnolia liliifera probably in Thailand (Promputtha et al. 2007) and an endophytic isolate (FJ205460) from an orchid in Taiwan  Notes -Colletotrichum gigasporum is characterised by large conidia ((22-)25 -29(-32) × (6 -)7-9 μm). Phylogenetic analyses by Rakotoniriana et al. (2013) based on the ITS and TUB2 sequences placed it in a distinct clade far from the cur-rently accepted Colletotrichum species. Another species with large conidia (27-30 × 9-10 μm), C. thailandicum, was described from diseased Alocasia sp. and Hibiscus rosa-sinensis from Thailand (Noireung et al. 2012). Colletotrichum thailandicum is morphologically similar to C. gigasporum; the ITS and β-tubulin sequences of both fungi are identical or near-identical (differed in two nucleotide position in β-tubulin). In addition, phylogenetic analyses of single locus data, including ITS (Fig.  1), and multi-locus data (Fig. 2), show that the ex-type strains of the two species cluster together in one strongly supported clade. Since C. gigasporum was published online earlier (8 August 2012) than C. thailandicum (September 2012), we regard C. thailandicum as a synonym of C. gigasporum.
Strain CBS 109355, isolated from a phaeohyphomycotic cyst from a Brazilian man, was originally identified as C. crassipes, mainly based on morphology of the appressoria with crenate or deeply lobed margins and its size of conidia ). In addition, strains CBS 159.75 and IMI 302450, which were deposited as C. crassipes in the CBS and IMI culture collections, were compared morphologically with CBS 109355 by Castro et al. (2001). However, strains CBS 159.75 and CBS 109355 were reidentified as C. gigasporum in the present study (Fig. 2). Hitherto, the taxonomic status of C. crassipes as well as the genetic relationship between C. gigasporum and C. crassipes remain unclear due to the lack of an ex-type culture and DNA sequence data. Thus, an epitype is needed to stabilise the nomenclature of C. crassipes.
In addition to being a disease-causing agent of humans, C. gigasporum is also associated with Musa sp. (Fig. 1, 2), the anthracnose of which is commonly considered to be caused by C. musae that belongs to the C. gloeosporioides species complex (Weir et al. 2012). However, C. gigasporum is phylogenetically distinct from C. musae, and its conidia are significantly larger than those of C. musae. Additional Colletotrichum species associated with Musa spp. include C. cavendishii, C. liukiuensis and C. paxtonii. Colletotrichum gigasporum differs from C. liukiuensis (Sawada 1959), a species on leaves of M. liukiuensis in Taiwan, and C. cavendishii (Petrak 1925), a species on living leaves of M. cavendishii by producing larger conidia (20.5-25.5 × 6-9 μm vs 12-14 × 4.8-5.5 μm and 10-19 × 4.5-7 μm, respectively). Colletotrichum paxtonii, a species associated with banana in St. Lucia, belongs to the C. acutatum complex (Johnston & Jones 1997, Damm et al. 2012a) and is therefore not closely related to C. gigasporum.
Our 5-locus phylogram shows that several strains from diverse countries and hosts cluster with C. gigasporum (syn. C. thailandicum). Based on our blastn search in GenBank, the results of which are included in the ITS phylogeny, 22 additional ITS sequences from GenBank cluster with the ex-type strain of C. gigasporum, including sequences derived from strains isolated from plants as endophytes or pathogens and even strains that were isolated from human tissue (Fig. 1). This is in accordance with the conjecture that ecologically C. gigasporum can occur as either endophyte or pathogen (Rakotoniriana et al. 2013). The isolates from which most of these GenBank sequences were generated had been previously identified as C. crassipes, C. gloeosporioides, C. incarnatum, C. orbiculare or C. taiwanense (sexual morph Glomerella septospora) (Fig. 1).
The ascospores and conidia of C. gigasporum resemble those of C. taiwanense with respect to their size. However, C. gigasporum produces aseptate conidia and 0-1-septate ascospores (Rakotoniriana et al. 2013), while the conidia of C. taiwanense may become 1-5-septate with age and ascospores are mostly 3-septate and may become up to 6-or 8-septate when old (Sivanesan & Hsieh 1993). Colletotrichum taiwanense, originally described from Styrax formosanus in Taiwan, is currently poorly characterised using molecular methods (Hyde et al. 2009,  Cannon et al. 2012). Unfortunately, a subculture from the extype isolate of C. taiwanense (IMI 353024) is contaminated; the original strain could not be recovered. Several plant pathogenic strains from various hosts (none of them from Styrax) that were previously identified as C. taiwanense were reidentified as C. gigasporum based on the ITS-rDNA phylogram in this study (Fig. 1). Colletotrichum gigasporum differs from C. incarnatum (Zimmermann 1901), a species first described from Coffea liberica in Java, by producing larger conidia (20.5 -25.5 × 6-9 μm vs 14 -19 × 5 μm).
Some strains from Mora excelsa in Guyana had been previously identified as C. orbiculare (Lu et al. 2004) and grouped with C. gigasporum in our ITS tree. However, C. orbiculare was recently redefined and shown to belong to a different species complex together with C. lindemuthianum (Damm et al. 2013).
Although the ITS-rDNA phylogram revealed that C. gigasporum strains formed two subclades (Fig. 1), the bootstrap values are too low to support two distinct species, which could also be verified by the multi-locus phylogram (Fig. 2).
The closest matches in a blastn search in GenBank with the ITS sequence of strain CBS 398.84 were with 100 % identity EF672323 from the endophytic isolate VegaE4-36 from Coffea arabica from Hawaii, USA (Vega et al. 2010), EU686812 from an endophytic isolate from Rhipidocladum racemiflorum from Panama (Higgins et al. 2011), as well as KF436311 from the endophytic isolate TK780 from a tropical woody plant from Panama (Higginbotham et al. 2013). The closest match with the TUB2 sequence (with 96 % identity, 16 bp differences) was isolate MUCL 41702 from Orchis in Singapore (FN599826; Rakotoniriana & Munaut unpubl. data).
The name C. camelliae, although not listed by Hyde et al. (2009) and Cannon et al. (2012), is widely used for the causal agent of the brown blight disease of tea (Sosa de Castro et al. 2001, Muraleedharan & Baby 2007. However, the status of C. camelliae and its taxonomic relationship with G. cingulata f. sp. camelliae remain unresolved (Weir et al. 2012). There are 11 ITS sequences of Colletotrichum sp. from tea in GenBank (EF063686, FJ515007, EU732732, FJ216456, HQ832797, JQ809665, HQ832801, AB548281, AB218993, GQ916544, HE655519), of which sequence HQ832801 associated strain nested within the C. boninense complex in the ITS phylo genetic tree, while the others belong to several clades within the C. gloeoporioides complex (data not shown). Appropriate fresh collections associated with brown blight symptoms of tea from Sri Lanka are needed for epitypification to clarify the phylogenetic relationships of this taxon. Colletotrichum pseudomajus can be distinguished from C. camelliae by its significantly larger conidia (22-30.5 × 6.5 -9.5 μm vs 15 -17 × 4 -5 μm).
Colletotrichum pseudomajus is morphologically similar to G. major except for the presence of paraphyses and the shape of its ascospores. Paraphyses were reported to be absent in G. major, but thin-walled, hyaline and septate paraphyses are present in C. pseudomajus; ascospores of G. major are ellipsoid, not allantoid, with obtuse or subacute tips (Tunstall 1935), while those of C. pseudomajus are lunate, with more or less acute tips (Fig. 5). Currently, the phylogenetic position of G. major is unresolved due to the lack of an ex-type isolate. Thus, an epitype is needed to stabilise the nomenclature of G. major and to clarify the relationship between C. pseudomajus and G. major.
The closest matches in a blastn search with the ITS sequence of CBS 571.88 with 100 % identity were JX009424, the sequence generated from the same isolate by Weir et al. (2012), and JQ809667 from the endophytic isolate JD08-18 from Camellia sinensis in China (Fang et al. 2013), as well as JN418782 from the endophytic isolate E10202g from Otoba parvifolia in Ecuador (Barba et al. unpubl. data). Closest match with the TUB2 sequence (with 93 % identity, 32 bp differences) was isolate MUCL 41702 from Orchis in Singapore (FN599826; Rakotoniriana & Munaut unpubl. data). The blastn search with the GAPDH sequence of CBS 571.88 showed similarity with JN050231 (85 % identity, 34 bp differences) from isolate BCC 38879 from Hibiscus rosa-sinensis in Thailand (Noireung et al. 2012) which is here referred to C. gigasporum, and JX009422 (99 % identity, 1 bp difference), a sequence generated from the same isolate. The only base difference in the end of the sequence was due to sequencing error by Weir et al. (2012).
Colletotrichum radicis F. Liu,L. Cai,Crous & Damm,Fig. 6 Etymology. Referring to the host organ, a plant root, from which it was isolated.
Culture characteristics -Colonies on OA flat with entire margin, aerial mycelium lacking; colonial diam 64-71 mm in 7 d, > 90 mm in 10 d. Colonies on SNA flat with entire margin, aerial mycelium lacking, medium hyaline, buff around Anthriscus stem; colonial diam 64 -75 mm in 7 d, > 90 mm in 10 d.
Specimen examined. Costa riCa, La Selva, host plant unknown (isolated from a plant root), unknown collection date and collector (isolated by G. Weber in Mar. 1993) (holotype CBS H-21494, culture ex-type CBS 529.93).
Another species known to occur on Coffea sp. from Vietnam in this complex is C. gigasporum (CBS 125476 and CBS 125475), which can be distinguished from C. vietnamense by each of the eight genes used in this study, including ITS (Fig. 1).
The closest matches with the ITS sequence of CBS 125478 were FJ968584 (with 100 % identity), a sequence generated from the same isolate by Nguyen et al. (2010), and EF672327 (with 100 % identity) from the endophytic isolate PR61F2, also from Coffea arabica , but from coffee berries in Puerto Rico, a country in Central America (Vega et al. unpubl. data). Closest match with the TUB2 sequence was KC293665 (with 96 % identity, 20 bp differences) from isolate gnqczg15 from China (Huang et al. unpubl. data).

dISCuSSIon
Many of the strains included in the present study were deposited in the CBS culture collection as C. crassipes (Speg.) Arx. However, C. crassipes is a species with uncertain taxonomic status. There is significant confusion regarding its morphology in the literature. Spegazzini (1878) originally described this fungus as Gloeosporium crassipes from Vitis vinifera from Conegliano, Italy with conidia measuring 20 -30 × 7-8 μm. Subsequently, von Arx (1957) combined Gloeosporium crassipes in Colletotrichum as C. crassipes along with 17 synonyms. The conidial size of C. crassipes was reported as 22 -31 × 6 -8 μm, broadly matching the original description; and the appressoria as irregular, usually lobed, measuring 8 -12 μm (von Arx 1957). Sutton (1980) presented a different morphological concept of C. crassipes, which was characterised by conidia measuring 10-15 × 4.5-6.5 μm, long clavate or circular appressoria with crenate or deeply divided edges, 10.5 -14 × 7-9.5 μm, and reduced another two names to synonymy with it. However, when Sutton summarised an accepted taxa list of Colletotrichum species, C. crassipes was characterised with conidia again with a different size (14 -28 × 5 -7 μm), and he suspected that this species may consist of a number of separate taxa (Sutton 1992). Moreover, several isolates identified as C. crassipes that have sequences lodged in GenBank actually belong to C. gloeosporioides s.lat. (Weir et al. 2012). Recollecting and epitypification of this taxon is required to stabilise the phylogenetic position of C. crassipes.
Although morphological features are not stable and change under different growth conditions and with repeated subculturing, species of the C. gigasporum species complex form larger conidia than most of the other species in the genus Colletotrichum, which provides a valuable character for species complex level diagnosis. Conidia of two other species with large conidia, C. euphorbiae and C. sansevieriae, differ in shape; they are slightly clavate with a round apex tapering to a truncate to slightly acute base (Nakamura et al. 2006, Crous et al. 2013). These two species do not belong to the C. gigasporum complex.
While single gene data, especially ITS data, are usually not sufficient for species recognition in most of the Colletotrichum species complexes or groups (Cannon et al. 2012) and multilocus phylogenies are therefore now routinely used as the primary basis on which to describe new Colletotrichum species , b, Weir et al. 2012, Liu et al. 2013a, species of the C. gigasporum species complex can be easily distinguished from each other using the individual gene data included in this study (Fig. 1).
Colletotrichum gigasporum appears to have a wide host range and geographic distribution. Isolates treated in this paper and those deposited in GenBank originate mainly from Africa (East Africa, Madagascar), Central and South America (Brazil, Chile, Columbia, Ecuador, Guyana, Mexico, Panama), Asia (China, India, Japan, Korea, Thailand, Vietnam) and New Zealand (Fig. 1). Besides, this species is associated with various host plants as pathogens and endophytes, from air and stored grain, indicating that it is not host-specific and apparently has different life styles. This character is not unique to C. gigasporum, many other Colletotrichum species have been reported as both pathogens and endophytes, e.g. C. boninense, C. karstii and C. liriopes (Yang et al. 2011, Damm et al. 2012b, Tao et al. 2013. For instance, C. boninense causes diseases of Crinum asiaticum var. sinicum and Solanum lycopersicum, and is also an endophyte of Bletilla ochracea and Dacrycarpus dacrydioides (Damm et al. 2012b, Tao et al. 2013. The relationship between plant endophytic and pathogenic isolates of the same Colletotrichum species needs more research, as some endophytes may be latent pathogens (Lu et al. 2004).


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Role of gamma-secretase in human umbilical-cord derived mesenchymal stem cell mediated suppression of NK cell cytotoxicity

Background Mesenchymal stem cells (MSCs) are increasingly considered to be used as biological immunosuppressants in hematopoietic stem cell transplantation (HSCT). In the early reconstitution phase following HSCT, natural killer (NK) cells represent the major lymphocyte population in peripheral blood and display graft-vs-leukemia (GvL) effects. The functional interactions between NK cells and MSCs have the potential to influence the leukemia relapse rate after HSCT. Until date, MSC-NK cell interaction studies are largely focussed on bone marrow derived (BM)-MSCs. Umbilical cord derived (UC)-MSCs might be an alternative source of therapeutic MSCs. Thus, we studied the interaction of UC-MSCs with unstimulated allogeneic NK cells. Results UC-MSCs could potently suppress NK cell cytotoxicity in overnight cultures via soluble factors. The main soluble immunosuppressant was identified as prostaglandin (PG)-E2. Maximal PGE2 release involved IL-1β priming of MSCs after close contact between the NK cells and UC-MSCs. Interestingly, blocking gamma-secretase activation alleviated the immunosuppression by controlling PGE2 production. IL-1 receptor activation and subsequent downstream signalling events were found to require gamma-secretase activity. Conclusion Although the role of PGE2 in NK cell-MSC has been reported, the requirement of cell-cell contact for PGE2 induced immunosuppression remained unexplained. Our findings shed light on this puzzling observation and identify new players in the NK cell-MSC crosstalk. Electronic supplementary material The online version of this article (doi:10.1186/s12964-014-0063-9) contains supplementary material, which is available to authorized users.

Background
Mesenchymal stem cells (MSCs) are multipotent precursor cells that have the ability to differentiate mainly along chondrogenic, adipogenic and osteogenic lineages [1]. Although MSCs were originally isolated from bone marrow, it is now known that MSCs can be derived from various post-natal tissues like placenta, umbilical cord and cord blood, as well as adult tissues like tooth pulp, skin, adipose tissue, the nervous system and kidney [2,3]. MSCs can be defined as a heterogenous population of plastic-adherent cells with fibroblast-like morphology that express certain markers like CD90, CD73, and CD105, but are negative for surface markers of the hematopoietic lineage such as CD14, CD19, CD34, CD45, and HLA-DR [4].
MSCs have been reported to mediate profound immunosuppressive effects on T -, B -, NK cells, and dendritic cells [5]. MSCs are referred to as immunoprivileged as they possess a hypo-immunogenic profile due to lack of MHC class II molecules and classical co-stimulatory ligands as well as very low expression of MHC class I [6]. This immunoregulatory capacity together with their inherent non-immunogenicity makes them promising candidates for the use as biological immunosuppressants or tolerance inducers in alloimmune diseases like steroid resistant acute graft-versus-host-disease (GvHD) [7], organ transplantation or implantation of engineered tissues. Natural killer (NK) cells are one of the main effectors of innate immunity due to their cytolytic activity against tumour cells and virus infected cells as well as by their ability to secrete various cytokines and chemokines [8]. NK cells comprise the main lymphocyte population in the early reconstitution phase after hematopoietic stem cell transplantation (HSCT) and mediate significant graft-vs-leukemia (GvL) effect [9,10] while MSCs are being increasingly used to treat GvHD following HSCT [11]. In therapeutic settings, the interaction of NK cells with MSCs might affect NK cell functions and the transplantation outcome. Clinical intervention to prevent any such adverse outcome of NK-MSC interaction requires precise understanding of the molecular mechanisms utilised by MSCs to suppress NK cells. Some work has been done to characterise the interaction of bone marrow derived MSCs (BM-MSCs) with NK cells [12][13][14]. However, interaction of umbilical cord derived MSCs (UC-MSCs) with NK cells is largely unexplored. Although effective, BM-MSCs are inherently afflicted with the issue of low proliferative potential and only very low numbers of MSCs can be isolated from bone marrow via painful invasive procedures. There is a constant hunt for better sources of MSCs for future clinical cell-based therapies. UC-MSCs have emerged as an ideal candidate owing to their non-invasive isolation procedure, superior proliferative potential and simple accessibility in large quantities [15]. Thus we chose to study UC-MSC-NK cell functional interplay.
This project was aimed at elucidating the underlying molecular mechanisms of UC-MSC mediated immunemodulatory effects on freshly isolated, human NK cells without the presence of any NK cell activating cytokine like IL-15 or IL-2 in the co-culture system. Previous work on BM-MSC-induced suppression of NK cell functions implicated soluble factors like indoleamine 2,3-dioxygenase (IDO), transforming growth factor (TGF)-β and prostaglandin (PG)-E2. PGE2 is the soluble immunosuppressant which is common to all reports till date [12,13,16]. However, the suppression of NK activation through PGE2 was reported to be contact dependent [13]. This indicated that the inhibition via PGE2 involved a more complex pathway that is yet to be understood. We also found that the UC-MSCs suppressed NK cell cytotoxicity mainly via PGE2 but the maximal release of PGE2 occurred only in contact cultures. Small amounts of interleukin (IL)-1β present in NK cells preparations were found to act on UC-MSCs to stimulate maximal PGE2 production. Additionally, we show that efficient IL-1 receptor (IL-1R) activation in MSCs require gamma-secretase enzyme activity. These findings further corroborate the recent reports on the identification of IL-1R as a novel gammasecretase substrate [17].

Phenotypic analysis and differentiation potential of UC-MSCs
The surface antigen expression patterns on the UC-MSCs were analysed using flow cytometry ( Figure 1). CD73, CD90, and CD105 were highly expressed. MSCs lacked the expression of CD14, CD19, CD45, and HLA-DR on their cell surface. There was a very faint expression of CD34. The ability of the UC-MSCs to undergo chondrogenic, adipogenic and osteogenic differentiation was determined as described before (data not shown) [3]. Hence, the UC-MSCs fulfilled all the criteria recommended by the International Society for Cellular Therapy for identifying MSC [4].

Effect of UC-MSCs on NK cell function and phenotype
To examine the effect of MSCs on NK cells, freshly isolated, unstimulated NK cells were incubated with MSCs in direct contact at ratio 5:1, 10:1, and 20:1 for 16 hours. Following the co-culture, NK cells were harvested and exposed to K652 target cells to trigger NK cell degranulation. Surface expression of CD107a was analysed as a marker for degranulation. We found that NK cells precultured with MSCs had significantly reduced degranulation capacity compared to NK cells cultured alone ( Figure 2A). There was no significant difference in the suppression induced by different ratios of MSC during co-culture ( Figure 2A). For all subsequent experiments, the NK: MSC ratio of 10: 1 was used. To confirm that the freshly isolated NK cells did not kill the MSCs during the co-culture, we performed chromium release assay with MSCs as target cells. We observed that the NK cells could efficiently lyse K562 cells but not the MSCs (Additional file 1: Figure S1). IL-15 pre-activated NK cells were better at killing the MSCs but the lysis was still less when compared to K562 (Additional file 1: Figure S1).
Following HSCT, CD56 bright NK cell subset is the major NK cell population in peripheral blood [9,18,19]. Our study aims to understand the fate of NK cells following MSC co-transfusion in HSCT settings. Therefore, we also specifically analysed the effect of MSCs on CD56 bright NK cells. We found that co-culturing with MSCs could significantly suppress K562-induced degranulation ( Figure 2B) as well as IL-12 + IL-18 stimulated interferon-γ production by CD56 bright NK cells (Additional file 2: Figure S2). The observed suppression of NK cell cytotoxicity could have resulted from an altered expression of NK cells surface receptors which are crucial to NK cell activation. Using flow cytometry-based analysis, we looked for phenotypic changes on NK cells pre-cultured with MSCs compared to NK cells cultured alone. The panel of NK cell receptors analysed included CD16, CD27, CD62L, CD69, CD94, CD158, CD244, CD247, CD335, CD336, CD337, NKG2A, and NKG2D. Interestingly, the expression of all receptors tested was unaffected (Additional file 3: Figure S3) by MSCs, except for NKG2D. UC-MSCs significantly downregulated NKG2D receptor expression on both CD56 dim and CD56 bright NK cells ( Figure 2C,D). We also examined if perforin or granzyme contents of NK cells were altered upon co-culturing with MSCs. Intracellular staining demonstrated that perforin and granzyme contents of NK cells cultured with MSCs were comparable to NK cells cultured alone ( Figure 2E,F,G,H, Additional file 4: Figure S4).

Mechanism of MSC-mediated suppression of NK cell cytotoxic functions
To verify the role of soluble factor(s) in MSC-induced suppression of NK cell cytotoxicity, we collected conditioned media (cm) from MSC cultures (MSC cm) and MSC-NK cell co-cultures (NK-MSC cm). NK cells were cultured in normal media or MSC cm or NK-MSC cm overnight and subsequently incubated with K562 target cells. Both MSC cm and NK-MSC cm were found to inhibit NK cell degranulation. However, NK-MSC cm was able to suppress NK cell degranulation more effectively than MSC cm ( Figure 3A). These results were further corroborated by chromium release assays in which NK cells were cultured in normal media or MSC cm or NK-MSC cm overnight and subsequently incubated with radioactive chromium ( 51 Cr) labelled K562 target cells. The extent of K562 lysis was determined by the 51 Cr released into the culture supernatants. MSC cm suppressed NK cell cytotoxicity but to a significantly lower degree than NK-MSC cm ( Figure 3B). This suggested that the suppressing soluble factor was present in lower amounts in MSC cm. Only when MSCs and NK cells came into contact, the soluble factor was released in larger amounts and resulted in enhanced suppression as seen with NK-MSC cm. MSC cm reduced cytotoxicity significantly, but not maximally, while the transwell system had no significant effect (Additional file 5: Figure S5). It has been reported by other groups [12,13] that indoleamine-2,3-dioxygenase (IDO) and cyclooxygenase (COX)-2 or transforming growth factor beta (TGF)-β produced by MSCs are responsible for suppression of NK cell cytotoxicity. We investigated if soluble endproducts like prostaglandin (PG)-E2 or kynurenine (from COX-2 or IDO respectively) or TGF-β played any role in our system. Antibody mediated blocking of TGF-β and pharmacological inhibition of IDO by 1-MT did not result in increased degranulation (Additional file 6: Figure S6A,B). We found constitutive expression of COX-2 in UC-MSCs. Overnight co-culture of MSCs with NK cells resulted in significant upregulation of COX-2 ( Figure 3C) in MSCs. However, no expression of COX-2 was observed in the cocultured NK cells (Additional file 7: Figure S7). Blocking of COX-2 by a small-molecule inhibitor (NS-398) could significantly restore NK cell degranulation ( Figure 3D). This indicated that PGE2 could be the major immunosuppressive factor released by MSCs inhibiting NK cell cytotoxicity. UC-MSCs, when cultured overnight in NK cell cm (NK cm), also upregulated COX-2 (data not shown). This indicated that some secretory molecules coming from NK cells could trigger COX-2 upregulation in UC-MSCs. We found that IL-1β is present in NK cm at low levels ( Figure 3E). It is to be noted that the freshly isolated NK cells were producing IL-1β without prior exposure to MSCs. We hypothesized that this IL-1β could be responsible for the observed COX-2 upregulation in MSCs. To verify our hypothesis, we added IL-1β neutralising antibody in NK-MSC co-cultures.
This resulted in significant inhibition of COX-2 upregulation ( Figure 3F).

Role of gamma-secretase in IL-1 signalling
IL-1β is known to act via IL-1 receptor present on target cells. IL-1 receptor (IL-1R) activity has been recently Figure 2 Suppression of NK cell cytotoxicity by UC-MSCs. A: NK cells were cultured alone or with MSCs at NK cell: MSC ratio of 5:1, 10:1, and 20:1. Following overnight co-culture, CD107a degranulation assay was performed with K562 target cells. The bar graphs represent the percentage of CD107a + NK cells (n = 5) B: NK cells were cultured without MSCs or with MSCs at NK cell: MSC ratio of 10:1. CD107a degranulation assay was performed with K562 target cells (n = 5). Following overnight co-culture, CD107a expression on CD56 bright NK cells was analysed. The bar graphs represent the percentage of CD107a + cells (n = 9). C, D: NK cells were cultured overnight with or without MSCs. NKG2D expression on the NK cells was analysed by flow cytometry. The bar graphs depict the mean fluorescence intensity (MFI) of NKG2D staining on CD56 dim (C; n = 9) and CD56 bright (D; n = 8) NK cells. E, F, G, H: NK cells were cultured overnight with or without MSCs. Perforin (E; n = 7), granzyme A (F; n = 7), granzyme B (G; n = 5), and granzyme K (H; n = 7) content of the NK cells was analysed by flow cytometry.
described to be gamma-secretase proteolysis dependent in HEK293T cells as well as mouse embryonic fibroblasts (MEFS) [17]. We examined if IL-1R signaling in human UC-MSCs depended on gamma-secretase activity. We cultured UC-MSCs with varying concentrations of IL-1β overnight in presence or absence of gamma-secretase inhibitor, DAPT. IL-1β was very efficient in upregulating COX-2 levels. However, the presence of DAPT significantly reduced COX-2 upregulation ( Figure 4A). Pharmacological inhibitors can have a wide range of targets. To eliminate this possibility, we performed small interfering RNA (siRNA) mediated silencing of gammasecretase activity in MSCs. We chose to knock-down Presenilin (PSEN)-1 because it is one of the catalytic subunits of gamma-secretase complex [20,21]. siRNA targeting PSEN-1 or control scrambled (SCR) siRNAs were introduced into the MSCs by electroporation. Knock-down of PSEN-1 mRNA was analysed after 24 hours by real-time PCR. PSEN-1 siRNA caused a 75% decrease in PSEN-1 mRNA compared to SCR siRNA (Additional file 8: Figure  S8). In parallel with the assessment of mRNA knockdown, siRNA-treated MSCs were cultured for 16 hours in presence or absence of IL-1β. Similar to DAPT-mediated inhibition, PSEN-1 silencing significantly reduced COX-2 upregulation in response to IL-1β ( Figure 4B). Introduction of PSEN-1 siRNA did not affect the basal COX-2 expression in MSCs. This clearly indicates the involvement of gamma-secretase in IL-1R signalling. Next, we tested if this effect of gamma-secretase blocking stemmed from impairment of IL-1R downstream signalling. We pre-treated MSCs with DAPT for 2.5 hours followed by IL-1β stimulation for 40 minutes. The DAPT pre-treated MSCs showed significant impairment in their ability to phosphorylate c-Jun N-terminal kinases (JNK) as mirrored in reduced phosphorylation of JNK following stimulation by IL-1β ( Figure 4C). We further investigated if gammasecretase influenced IL-1R signalling in our culturesetting. Pharmacological inhibition of gamma-secretase activity in NK-MSC co-cultures resulted in a restoration of NK cell degranulative capacity ( Figure 4D). To establish that gamma-secretase blocking indeed affected the release of soluble suppressants, we performed the following experiment. Cell-free supernatants were collected from the NK-MSC co-cultures conducted in presence of DAPT or DMSO. They have been referred to as MSC-NK-DAPT conditioned medium (NK-MSC-DAPT cm) and MSC-NK-DMSO conditioned medium (NK-MSC-DMSO cm), respectively. NK cm and NK-MSC cm were also collected as controls. NK cells were cultured in these conditioned media followed by chromium release assay. NK-MSC cm Figure 4 Effect of gamma-secretase on IL-1 signalling and PGE2 content. A: UC-MSCs were cultured in presence of different doses of IL-1β stimulation with or without DAPT (gamma-secretase inhibitor). UC-MSCs were detached using accutase and stained to analyse intracellular COX-2 expression (n = 5). B: Gamma-secretase activity in UC-MSCs was silenced using siRNA targeting a catalytic subunit of gamma-secretase (PSEN-1). Scrambled siRNA (SCR) was also introduced into UC-MSCs as control. After 24 hours, PSEN-1 or SCR siRNA treated MSCs were stimulated with IL-1β overnight. UC-MSCs were detached using accutase and stained to compare intracellular COX-2 expression with or without IL-1β stimulation (n = 3). C: UC-MSCs were stimulated with IL-1β for 40 minutes, with or without 2.5 hours pre-treatment with DAPT. MSCs were detached using accutase, immediately fixed, permeabilised and stained to analyse intracellular pJNK levels (n = 4). D: NK cells were cultured with or without MSCs or co-cultured in presence of 6 μM DAPT, 10 μM DAPT or DMSO. CD107a degranulation assay was performed with K562 target cells. The bars represent the effect of gamma-secretase inhibition on degranulative capacity of NK cells (n = 5). E: NK cells were cultured in NK cell conditioned media (NK cm) or in NK-MSC conditioned media (NK-MSC cm) or NK-MSC conditioned media where they were co-cultured in presence of 10 μM DAPT or DMSO (NK-MSC-10 μM DAPT cm or NK-MSC-DMSO cm respectively). Chromium release assay was performed with K562 target cells (n = 3). F: PGE2 concentration in MSC cm, NK-MSC cm or NK-MSC-10 μM DAPT cm as determined by competitive ELISA (n = 3). caused a significant decline in lytic potential of NK cells. The cell-free co-culture supernatant generated in presence of gamma-secretase blocker, DAPT, had non-significant suppressive capacity. This indicated that NK-MSC-DAPT cm contained much lower quantity of the suppressant ( Figure 4E). To directly test if the release of PGE2 was gamma-secretase dependent, we performed ELISA to analyse the concentration of PGE2 in MSC cm, NK-MSC cm, and NK-MSC-10 μM DAPT cm. We found that the amount of PGE2 increased significantly upon NK-MSC co-culture, but there was no increase in PGE2 if gammasecretase activity was blocked ( Figure 4F).

Discussion
We observed that UC-MSCs were able to suppress NK cell effector functions in 16 hours of co-culture which is much shorter than the effective co-culture period described by other groups [12,13]. This discrepancy could be possibly explained by different culture conditions. The impairment of NK cell cytotoxicity towards K562 cells probably resulted from reduced degranulation. Interestingly, following co-culture with UC-MSCs, there was preferential downregulation of NKG2D on NK cells while no change was observed in the other activating receptors examined. NKG2D is one of the most prominent activating receptor on the NK cells [22]. The downregulation of NKG2D could be partially responsible for reduced NK cells degranulation towards K562 targets because this cell line expresses NKG2D activating ligand [23]. To understand if soluble factors have any role in the diminution of NK cell cytotoxicity, we compared cell-free supernatants from MSC cultures and NK-MSC contact cultures for their NK cells suppressive ability. It was evident from our experiments that soluble factors present in the media were responsible for the observed immunosuppression. NK-MSC cm was significantly superior to MSC cm in suppressing cytotoxic potential of NK cells indicating a need for a direct contact between the UC-MSCs and NK cells, for the maximal release of the cytotoxicity suppressant.
Soluble factors like TGF-β, kynurenine and PGE2 released by enzymatic action of COX-2 have been reported to be involved in inhibition of immune cell functions by BM-MSCs [12,13,24]. In our assays, antibody-mediated blocking of TGF-β and pharmacological inhibition of IDO via 1-methyl-tryptophan (MT) did not result in any restoration of the degranulative potential of NK cells. These differences could result from the use of unstimulated freshly isolated NK cells in our experiments. It is known that IDO is induced by IFN-γ in MSC [25], and freshly isolated NK cells did not produce considerable amount of IFN-γ (data not shown). We found that COX-2 was constitutively expressed in UC-MSCs. Blocking COX-2 resulted in significant restoration of NK cell degranulation suggesting that the soluble cytotoxicity suppressant is PGE2. IL-1β released by NK cells was measured to be in the range of 70-200 pg/ml. In a murine model of ulcerative colitis, IL-1β pre-treated MSCs were found to exhibit significantly higher therapeutic efficacy than untreated MSCs [26]. IL-1β is known to be a potent inducer of COX-2. Hence we neutralised IL-1β in our MSC-NK cell cocultures and observed that blocking IL-1β indeed resulted in a decrease in COX-2 upregulation.
The induction of COX-2 is dependent on IL-1β release from NK cells. Hence, IL-1β produced by NK cells in transwell co-cultures should also induce COX-2 and PGE2 production. However, no suppression was observed in transwell co-cultures. This can possibly be explained by the low levels of IL-1β produced by NK cells, making direct cell-cell contact a pre-requisite for attaining effective concentrations for bioactivity. In transwell co-cultures, the low IL-1β concentrations appear insufficient to upregulate COX-2 in MSCs. It is important to note that our co-culture duration is just 16 hours. In conditioned media collected from NK cultures (NK cm) after 16 hours, IL-1β has probably reached effective concentrations as implicated by COX-2 upregulation in MSCs exposed to NK cm.
Previous work has demonstrated that gamma-secretase acts on IL-1R and controls the downstream signalling following receptor activation by ligand binding [17]. We hypothesized that the IL-1R signalling in MSCs could also be gamma-secretase dependent. We inhibited gammasecretase enzyme activity using pharmacological inhibitors or siRNA-mediated knockdown. We found that the inhibition of gamma-secretase significantly reduced COX-2 upregulation in MSCs in response to IL-1R activation by IL-1β. We could also demonstrate that gamma-secretase inhibition also affected JNK phosphorylation down-stream of IL-1R activation. Therefore, we could demonstrate that IL-1R signalling in MSCs was dependent on gammasecretase activity. This dependence was also mirrored in the drastic mitigation of PGE2 release under conditions of gamma-secretase inhibition. Finally, pharmacological inhibition of gamma-secretase during NK cell-MSC co-cultures showed significant restoration of NK cell degranulation.

Conclusion
Use of MSCs for cell therapy is on the rise, and BM-MSCs have been shown to be advantageous for the treatment and prevention of GvHD as well as facilitation of engraftment following HSCT in several clinical trials [27]. The mechanistic insights obtained from in-vitro studies indicate that the anti-inflammatory effects of MSCs, that ameliorate complications following HSCT, stem from suppression of aberrant T-cell proliferation [25]. Following HSCT, the first lymphocyte population to reconstitute are NK cells, which contribute to improved engraftment, reduced rates of leukemia relapse [28] and decreased GvHD.
MSCs co-administered with HSC to suppress T-cells are also likely to suppress NK cell functions, thereby eliminating their beneficial effects.
We found that UC-MSCs indeed suppress NK cell cytotoxicity in overnight contact cultures. The suppression is mediated by the soluble suppressant PGE2. However, the maximal release of PGE2 required a cross-talk between NK cells and MSCs involving IL-1β/IL-1R signalling. The low IL-1β levels are probably more effective within the immunological synapse formed in contact cultures. This could explain the contact dependence of observed suppression. We also show that the IL-1R signalling is dependent on gamma-secretase enzyme activity. Finally, pharmacological inhibition of gamma-secretase significantly alleviated MSC-induced suppression. We need to further investigate if these immunosuppressive mechanisms also apply to MSCs derived from other sources. Identification of common molecular pathways utilised by MSCs, irrespective of the source, could have significant clinical implications, especially in case of HSCT.

Isolation and culture of UC-MSCs
This study was approved on 26th February, 2009 by the Institutional Review Board (Project No. 3037) in an extended permission # 443. After obtaining written consent, MSCs were isolated from at least four different human umbilical cords obtained from full-term infants (38-40 weeks) by explant culture, expanded and cryopreserved until the start of the experiment as described elsewhere [3,29]. αMEM supplemented with 10% human serum and 50μg/ml streptomycin, 50U/ml penicillin, 1mM glutamine and 0.5mM sodium pyruvate (Biochrom, Berlin, Germany) was used to culture UC-MSCs for all subsequent experiments.

Isolation of peripheral blood mononuclear cells (PBMC) and NK cells
Heparinised blood samples were obtained from healthy consenting donors and diluted with an equal volume of phosphate buffered saline (PBS). PBMC were separated by centrifugation over a ficoll-hypaque gradient and stained with anti-CD56 and CD3 antibodies. NK cells were sorted using MoFlo Cell Sorter (Beckman Coulter) at the MHH sorting facility. NK cells were defined as CD56 + CD3 − lymphocytes.

Cell culture
UC-MSCs were seeded in 24-or 48-well plates and allowed to adhere. After 24 hours, freshly isolated, FACS-sorted NK cells from unrelated donors were added at a MSC:NK-ratio of 1:10. Following 16 hours of co-culture, the NK cells were removed from the adherent MSCs by pipetting for phenotyping or were tested for cytokine producing ability by intracellular staining or for cytotoxic potential against K562 leukemic cells using CD107a degranulation or chromium release assay. NK cells cultured without MSCs were used as controls. Cell-free supernatants from the NK-MSC cocultures or MSC cultures were frozen for further analysis as MSC-NK-conditioned medium (NK-MSC cm) or MSC conditioned media (MSC cm), respectively. For determining the influence of conditioned media, NK cells from the unrelated donors were cultured for 16 hours in the cell-free supernatants generated as mentioned above. To determine the role of gamma-secretase activation, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) (Sigma-Aldrich, Germany), a gamma-secretase inhibitor, was added to the co-cultures at 6 μM or 10 μM concentrations. DMSO was used as vehicle control. 5 μM NS-398 was added to the co-cultures to block the action of cyclooxygenase (COX)-2. IL-1β neutralising antibody or the matched isotype control antibody was added to the cocultures at a concentration of 10 ng/ml.

Detection of intracellular proteins
For detection of perforins and granzymes, after surface staining, cells were fixed with 4% paraformaldehyde (Merck) for 10 min. Subsequently, the cells were perforated in 0.1% saponin buffer (PBS supplemented with 0.1% Saponin (Riedel-de-Haën) and 0.01M HEPES (Roth) and fluorochrome-tagged monoclonal antibodies (mAbs) were added. After 30 minutes of incubation and three washes, cells were analysed by flow cytometry.
For detection of COX-2, the MSCs were detached by Accutase (Life technologies) treatment at 37°C for 5 min and fixed with 4% paraformaldehyde followed by 30 minute incubation with anti-COX-2 mAb in saponin buffer. The cells were then washed and analysed by flow cytometry.
For detection of pJNK, MSCs were stimulated with IL-1β for 40 minutes and washed once with PBS. Detachment of MSCs using Accutase was immediately followed by fixation using 1X BD Lyse/Fix Buffer. The permeabilisation and intracellular staining for pJNK was performed according to manufacturer's protocol.
CD107a degranulation assay NK cells cultured with or without MSCs were incubated with K562 target cells in presence of anti-CD107a mAb at an Effector:Target (E:T) ratio of 10:1. Monensin (BD Biosciences) was added after 1 hour at a final concentration of 6 μg/ml and the incubation was continued for 3 hours. The cells were then analysed for the surfaceexpression of CD107a by flow cytometry.

Cytotoxicity assay against K562 target cells
Cytotoxicity of NK cells, cultured under different conditions e.g in presence/absence of MSCs or in the presence/absence of conditioned media, was determined by 4 hour chromium release assay against K562 target cells following the protocol as described elsewhere [30]. Briefly, K562 cells were labelled with radioactive 51 Cr by incubating the cells with 3 MBq Na 51 CrO 4 for 1h at 37°C. The cells were washed twice and plated in triplicates in V-bottom 96-well plates. Effector: Target ratios of 20:1, 10:1, 5:1 and 2.5:1 were used. NK cell mediated lysis of targets results in the release of 51 Cr into the supernatant. Following the 4 hour incubation, the plates were centrifuged. 25 μl cell-free supernatant was collected from each well, and the 51 Cr release was measured by a gamma counter. Lytic units (LU 20 /10 7 cells) of the assays were calculated according to the methods established by Bryant et al. [31].

Cytokine bead array
The amount of IL-1β present in the culture supernatants of NK cells was measured using the cytometric bead array kit (BD Biosciences) in combination with human IL-1β Flex set according to the manufacturer's protocol. Briefly, fluorescently labelled beads (bead position B4) were mixed with known standards or test samples followed by incubation with PE-conjugated detection antibodies. The samples were washed, measured on FACS Canto II and analysed using the BD CBA analysis software.
Prostaglandin(PG)-E2 ELISA PGE2 was measured in culture supernatants by competitive enzyme-linked immunosorbent assay (ELISA) technique using a commercially available ELISA kit (Enzo Life Sciences), according to the manufacturer's protocol. Concentrations were calculated by comparison with known PGE2 standards using a 5 parameter logistic curve fitting program.
Transfection with siRNAs was performed using the Neon transfection system (Invitrogen) at 1350 V, 10 ms,


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Triatoma maculata, the Vector of Trypanosoma cruzi, in Venezuela. Phenotypic and Genotypic Variability as Potential Indicator of Vector Displacement into the Domestic Habitat

Triatoma maculata is a wild vector of Trypanosoma cruzi, the causative agent of Chagas disease; its incursion in the domestic habitat is scant. In order to establish the possible domestic habitat of T. maculata, we evaluated wing variability and polymorphism of genotypic markers in subpopulations of T. maculata that live in different habitats in Venezuela. As markers, we used the mtCyt b gene, previously apply to evaluate population genetic structure in triatomine species, and the β-tubulin gene region, a marker employed to study genetic variability in Leishmania subgenera. Adults of T. maculata were captured in the period 2012–2013 at domestic, peridomestic (PD), and wild areas of towns in the Venezuelan states of Anzoátegui, Bolívar, Portuguesa, Monagas, Nueva Esparta, and Sucre. The phenotypic analysis was conducted through the determination of the isometric size and conformation of the left wing of each insect (492 individuals), using the MorphoJ program. Results reveal that insects of the domestic habitat showed significant reductions in wing size and variations in anatomical characteristics associated with flying, in relation to the PD and wild habitats. The largest variability was found in Anzoátegui and Monagas. The genotypic variability was assessed by in silico sequence comparison of the molecular markers and PCR-RFLP assays, demonstrating a marked polymorphism for the markers in insects of the domestic habitat in comparison with the other habitats. The highest polymorphism was found for the β-tubulin marker with enzymes BamHI and KpnI. Additionally, the infection rate by T. cruzi was higher in Monagas and Sucre (26.8 and 37.0%, respectively), while in domestic habitats the infestation rate was highest in Anzoátegui (22.3%). Results suggest domestic habitat colonization by T. maculata that in epidemiological terms, coupled with the presence in this habitat of nymphs of the vector, represents a high risk of transmission of Chagas disease.

INTRODUCTION
Triatomines (Hemiptera, Reduviidae, Triatominae) are bloodsucking insects that act as vectors of tripanosomatids such as Trypanosoma rangeli and T. cruzi (Kinetoplastida, Trypanosomatidae), the latter being the causal agent of American trypanosomiasis or Chagas disease. This is one of the parasitic diseases of great medical importance in the Neotropics. Chagas disease remains a public health problem in America, being distributed from the centralsouthern region of the United States to Southern Argentina and Chile; patients with this disease have been found in Canada and some European countries (1,2).
Transmission of Chagas disease in Venezuela and elsewhere in South America has been traditionally associated with the domestic (D) and peridomestic (PD) environments in rural areas with poor socioeconomic conditions and high presence of vectors. However, colonization by triatomines such as Triatoma maculata and Panstrongylus geniculatus of D environments has increased, whereas before these triatomines had been mostly associated with PD or wild (S) habitats (3). In Venezuela, T. maculata is found in most of the states comprising the country, with the exception of Táchira and Delta Amacuro. Distribution is established from 0 to 1,500 m of altitude, with natural habitats such as palms, dry trees, fences and bird nests, and rates of infection with T. cruzi lower than those recorded for Rhodnius prolixus. Apparently, as a result of anthropogenic changes, the characteristic habitats of T. maculata have changed the insect becoming domestic (4).

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The domiciliation of triatomines seems to be an event that can sometimes lead to the simplification of genotypic and phenotypic characteristics, which can be adaptive to macro-and microclimatic variations and reduction of wildlife mammals that serve as blood source, among others factors. These factors favor the dispersion and increase triatomine populations in anthropogenic niches (5,6). There is suggestive evidence of a recent increase in adaptive capacity of T. maculata in populated areas, hence the importance of studying this vector (7).
The taxonomic position of triatomines has been revised through phenotypic studies using various methodologies, such as analysis of biochemical markers, e.g., isoenzymes, or morphometric techniques, e.g., variability analysis of the size and shape of anatomical structures (8)(9)(10)(11)(12), and genotypic assessment methods of polymorphism of genetic markers, e.g., the mitochondrial cytochrome b (mtCyt b) sequence (13)(14)(15) and the ribosomal spacer region (ITS-2), among others. All of these methodologies have shown interspecific variability in different triatomines species and have been used to evaluate population genetic structure. The geometric morphometry analysis, which allowed differentiating domestic and wild insects of medical importance such as mosquitoes, and ontogenetic studies of triatomine populations have been useful in discriminating vectors, which cannot be identified by morphological or molecular variability studies (16)(17)(18).
The combined use of phenotypic methods such as geometric morphometry analysis and assessment methods such as genetic polymorphism of molecular markers would be useful in the evaluation of vector populations related to Chagas disease and the establishment of appropriate interventions for disease control. Analysis of changes in the wings, supported by the study of molecular markers such as mtCyt b, has been used in Colombia in the differentiation of species of Rhodnius (14). In Venezuela, comparative studies, both phenotypic and genotypic, on vectors of Chagas disease are scarce; one of these studies suggests a polymorphism related to the geographical origin of the specimens in the restriction patterns of the mtCyt b gene in T. maculata (4).
This work assesses the dispersion of this vector throughout S, PD, and D ecotopes in several Venezuelan states using both the phenotypic and genotypic approaches, in specimens of T. maculata captured in different regions of Venezuela. The phenotypic approach includes a geometric morphometry study to establish wing variability; the genotypic variability was evaluated through the polymorphism of the molecular markers mtCyt b and the β-tubulin genes region. Previously, the β-tubulin marker has been used to establish genetic variability between Leishmania subgenera.

STUDY AREA AND INSECTS
Field work was conducted in the endemic Venezuelan states Anzoátegui, Sucre, and Monagas (east of the country); Nueva Esparta (northeast, Margarita island), Bolívar (south), and Portuguesa (west). Sampling was carried out following the method proposed by Schofield (19). Specimens of T. maculata were collected through a direct search by personnel previously trained; sampling was conducted twice/year in each region and the capture effort occurred at 5 h/man by day or night for 5-day visit to each region. A total of 26 locations distributed in these states were visited to collect, in periods of high and low precipitation, specimens of T. maculata, directly from ecotopes defined following previous criteria. In D habitat, attention was put on internal walls of the houses, rooms, roofs, furniture, and in the ceiling; external domiciles or PD habitat (30 m around the exterior walls of the house), the area explored included farmyards, henhouses, and wood piles; and in S habitat (removed 30 m from the PD habitat), the area explored covered palm trees, tree holes, cave, and crops (20,21). A total of 492 adult insects, male (M) and female (F), were collected and used in this study. Three indices were calculated; the colonization index was determined as CI (%) = 100 × total numbers of houses presenting nymphs/total number of houses with adults; the dispersion as DI (%) = 100 × number of locations with adults/number of locations studied, and infection index as II (%) = 100 × number of adults infected with T. cruzi/total number of adults captured. Specimens were dissected and their intestinal contents and/or hemolymph examined under the microscope for the presence of Trypanosoma; samples of the intestinal contents were used to evaluate the presence of T. cruzi and T. rangeli by means of a PCR assay (22). After dissection, the collected specimens were preserved in 70% alcohol and stored at −20°C for further analysis. Colonies from each location were established in the laboratory for morphometry analysis.

GEOMETRIC MORPHOMETRY ANALYSIS: METRIC DATA, SHAPE, AND SIZE VARIATION
For each individual, only the left wing was examined and included in the analysis. The wings were mounted between microscopy slides and cover-slips and photographed using a digital camera Leica S6D. Nine landmarks ( Figure 1A) were identified in each wing according to previous references (18,23); the geometric coordinates of each landmark were digitized and shape variables (partial warps) were obtained using tpsDig Version 2 (24). Data were submitted to a discriminant analysis to examine differences in wing shape between male (M) and female (F) and statistical significant was evaluate by the Wilk's lambda statistics. For comparison of wing size between genders and among ecotopes within each gender, we used the isometric estimator centroid size (CS) derived from coordinate data (10,15,23). Statistical analysis was carried out using the MorphoJ software package for geometric morphometric (25). Landmark coordinate (x, y) configurations were registered and aligned using the Procrustes analysis and covariance analysis was implemented with proportions of re-classified groups and MANOVA. Then, wing shape variable and the CS were analyzed using the principal component analysis. For this analysis, M and F were processed separately due to the sexual size dimorphism of Triatominae. The relationship between shape and size was explored by a regression analysis. The significance of wing conformation due to landmark variation was established using the canonical variate analysis.

DNA EXTRACTION AND MOLECULAR MARKER AMPLIFICATION
Total genomic DNA was extracted from all six legs of each specimen using the Wizard Genomic kit (Promega, Madison, WI, USA. Cat. No. A1620). Purity and integrity of the DNA were determined by agarose gel electrophoresis. The same procedure was used to Frontiers in Public Health | Epidemiology FIGURE 1 | Differences in wing shape between genders of Triatoma maculata from Venezuela. (A) Landmark points type I identified in wing of T. maculata. Numbering of points (PAR 1-9) refer to the arrangement followed to obtain the coordinates using tps Dig 2.0. (B) Differences in wing shape architecture of T. maculata. Differences in wing shape between male (M) and female (F) of T. maculata are represented by grids deformation and variation between homologous landmark (solid circles). After superposition to the homologous consensus (X) between M (solid lane) and F (dashed lane), the differences in wing shape are represented by incongruence between homologous landmarks (arrows).
isolate the DNA from triatomine intestinal content. The extracted genomic DNA was resuspended in TE buffer (10 mM Tris, pH 7.4 and 1 mM EDTA), and stored at 4°C for further analysis.
For direct molecular identification of T. cruzi and T. rangeli, DNA isolated from the intestinal content of insects was used to amplify the variable region of the minicircles of kinetoplast DNA (kDNA) and the non-transcribed spacer region of the mini-exon (26,27).
Two markers, previously reported as fit to evaluate genetic diversity in different organisms, were used in T. maculata. First, the mtCyt b gene apply to evaluate population genetic structure in different triatomine species (28,29), and second, the β-tubulin gene region, a marker employ to study genetic variability in Leishmania subgenera (30,31).
Markers were amplified under standard polymerase chain reaction (PCR) assays as described previously; the reaction was carried out in a final volume of 25 µl containing 12.5 µl cocktail of PCR mix 2X (GoTaq Master Mix, Promega, Madison, WI, USA. Cat.# M7122), 0.4 µmol primers (stock 100 µM) and 5 ng total genomic DNA; the PCR reaction was performed in an MJ Research PTC-200 thermocycler.

PCR-RFLP OF THE β-TUBULIN GENE MARKER
The PCR fragment of the β-tubulin gene marker from specimens of different ecotopes was partially sequenced using the Sequence Navigator version 1.0.1 (Perkin Elmer Applied Biosystem) and the in silico restriction map was established (NETcutter version II). The endonucleases BamHI and KpnI (Life Technology) were selected for double digestion of the β-tubulin-PCR product following the manufacturer's instructions, and the digested DNA fragments fractionated by agarose gel electrophoresis.

ELECTROPHORESIS
The purity and integrity of the DNA were determined by electrophoresis in 0.6% agarose gel at 80 V for 1 h in TBE buffer (90 mM Tris-HCl, pH 8.0; 90 mM boric acid; 2.5 mM EDTA). The PCR products were analyzed by electrophoresis on 1.5% agarose gel in TBE buffer and the RFLP products were subjected to electrophoresis in 3% agarose gel in TBE buffer. After electrophoresis, the gel was stained with ethidium bromide, visualized with UV illumination, and recorded on a gel documentation system.

GENETIC DIVERSITY
The relationships, genetic differentiation, among the pattern fragments of the RFLP analysis of the β-tubulin marker (presence or absence of fragments) from different states and ecotopes were estimated using the NJ algorithm and the tree is based on a Kimura 2parameter distance matrix (32,33). Statistical support for branches in the NJ tree was assessed by the bootstrap method with 1,000 replicates. The analysis was conducted using the software MEGA V.4 (34).

Colonization and infection of T. maculata with T. cruzi
Of the 492 specimens of T. macula, 49.2% were M; the insects were collected from six Venezuelan states, whose distribution by location showed a majority of these specimens distributed in the PD (67.07%) and domestic (26%) ecotopes, followed to a lesser extent by the wild ecotopes (7.3%). On average, 20.73% of these www.frontiersin.org  specimens were positive for T. cruzi according to the specific kDNA and mini-exon-PCR assays ( Table 2). The higher CI found in Anzoátegui and Monagas states in relation to the other states coupled to a high II to T. cruzi in these two states suggests a higher rate of household colonization and showed the importance of T. maculata as a vector.

Size and shape variation
Average size of the membrane region of the wing was 1,984 mm for F and 1,786 mm for M. The discriminate function for gender re-classified wings, 75% for M (M 30/40) and 82% for F (F 33/40); this function in turn contained 94% of gender variance, showing significant differences in the formation of the wing according to gender (Wilk's lambda: 0.543 and 0.754 for M and F respectively; p < 0.001). When analyzing the intraspecific allometric effect (degree of deformation of the wing), using size (component 1) as the independent variable and wing conformation (component 2) as the dependent variable, the contribution of these components was 49.5% of the variation. The differences in conformation based on the deformation of grids made by discriminating analysis disclosed that the M has a lower degree of variation compared with F, and a clear sexual dimorphism (Figure 1B). The mean wing deformation (X), obtained through the overlapping of gender-related wing deformation grids, showed changes in the landmark points; the decrease in wing size was observed in at least one of the landmarks (PAR 1-9) and in some cases by the loss of anatomical landmarks ( Figure 1B). The same changes of the landmark points were found after the third filial generation in colonies from the same location established in the laboratory.
The formation of wing architecture based on the variation of CS in each ecotope (Figure 2) evidenced an association between PD and S insects with no significant difference between them (p = 0.0392 for PD and p = 0.0382 for S), whereas significant differences (p = 0.0051) were observed when comparison was carried out between PD and S specimens together with insects collected in D, regardless of gender (Figure 2).
The variation in wing size and conformation allowed the grouping of three states (Figure 3). The results showed clusters of M and F of T. maculata captured in S and PD ecotopes in Anzoátegui, Monagas, and Portuguesa. Interestingly, higher variation in the consensus tendency was found in both genders in specimens captured in D ecotopes of Anzoátegui and Monagas (Figure 3, squares a and b).

Mini-exon analysis
Twenty percent of specimens were identified as positive for T. cruzi infection and 1.2% presented a co-infection with T. rangeli as demonstrated by a PCR assay for the mini-exon. The lineage of T. cruzi circulating in all states was identified as TcI after amplification of a band of 200 bp from the non-transcribed region of the mini-exon (results not shown).

Variability of molecular markers
Genomic DNA from specimens of T. maculata representative of each state, regardless of location or ecotope, was evaluated by amplification of mtCyt b and the β-tubulin gene region (Table 1;  Figure 4). The results showed a unique PCR product of 600 bp for mtCyt b (Figure 4A), which was common in size among specimens of different locations of different states and also among the ecotopes of these locations; the low variability of this product, as determined by partial sequencing, did not allow to establish differences based on mtCyt b between specimens (results non-shown). In contrast, a unique fragment of 980 bp was generated by the amplification of the β-tubulin marker ( Figure 4B); the partial sequencing of this fragment showed differences between states and ecotopes, suggesting genetic variability in populations of T. maculata.
In order to establish the variability of the β-tubulin gene marker, the PCR product from specimens of different states and ecotopes was sequenced and in silico restriction fragment maps were established and used to identify the restriction enzymes to be used to evaluate genetic differences among specimens from different ecotopes using RFLP analysis. The results revealed a partial common pattern for double digestion with BamHI-KpnI, with quantitative and qualitative differences among specimens of the majority of states, represented for bands of 980 (a), 620 (b), 450 (c), and 300 bp (d) (Figure 4C). This pattern was independent of location www.frontiersin.org The components size and conformation make the higher contribution (70%) in wing variability. The dashed line circle represents the standard group, whereas the solid line ones represent groupings by confidence ellipses (95%) of male (black points) and female (gray points) specimens from Anzoátegui, Monagas, and Portuguese states. The boxes represent the grouping of M and F specimens that showed the greatest differences in wing size and shape when compared to the consensus configuration.
or ecotope, the exception being Portuguesa state, where differences were found between ecotopes, since in this location only band (c) was present in the S pattern. In addition, other individual fragments were observed among the different ecotopes in all states; however, differences between ecotopes of the same state were evident, e.g., Anzoátegui, Sucre, Monagas, and Portuguesa ( Figure 4C, lanes 1-9 and [16][17][18], as well as between the same ecotope when different states were compared ( Figure 4C, lanes 1, 4, 7, and 16).
The PCR-RFLP patterns obtained for the region of β-tubulin ( Figure 4C) were evaluated according to the presence or absence of bands to establish comparative marker variability among different states and particularly among ecotopes. Anzoátegui and Monagas states had the largest differences in pattern by ecotopes (genetic differentiation index, Fst 0.476, p = 0.019). Anzoátegui state showed a 60% similarity between PD and S ecotopes, and the remaining 40% was due to D ecotope. The rest of the states revealed similar groupings in their genetic profiles ( Figure 4C). This suggests that the restriction patterns obtained could be an alternative for intraspecific differentiation of T. maculata associated with different ecotopes. Comparative analysis of data from Anzoátegui and Monagas states showed that about 60% of the restriction fragments are common regardless of ecotope. Maximum Parsimony analysis with 1,000 replicates, statistically supported by bootstrapping, generating a similar clustering among specimens of S and PD ecotopes, which would indicate similarity, while the genetic pattern of D differs, causing it to clump as a synapomorphic group (Figure 5), separated according to ecotopes.

DISCUSSION
The present study demonstrated for the first time domiciliary adaptation processes of T. maculata in several Venezuelan states, using two approaches, phenotypic as the geometric morphometry of wing, and genotypic as the variability of the β-tubulin molecular marker. This, together with the presence of nymphs in houses and a high rate of infection with T. cruzi of specimens captured inside the home indicates that there is increased risk factor in the transmission of Chagas disease in Venezuela. Our results showed that there is discrimination of T. maculata according to its ecotopes, since specimens from S and PD ecotopes were more similar between them in wing architecture and variability of the β-tubulin marker in comparison with that from D ecotope. Previous studies suggest that T. maculata is contributing to increased risk of transmission of T. cruzi in the human population from various regions of Venezuela, particularly in the north-eastern region where specimens of this species showed a high percentage of infestation and a high rate of infection with T. cruzi (35).
Our results demonstrate the utility of geometric morphometry study of wing architecture to establish sexual dimorphism, phenotypic variability, and the association of these variables to different ecotopes of T. maculata. Thus, this is a robust tool to determine intraspecific differences possibly related to the geographical distribution in the macro-and microenvironment.
It has been demonstrated that Chagas disease vectors traditionally considered exclusively S have the possibility to change their behavior and colonize D habitat, with a high risk in the epidemiology of the disease (36). The association with particular habitats or ecotopes of different phenotypical and genotypical characters in triatomines has proved important in vector identification, dispersion, and colonization properties and in general in the epidemiology and control of Chagas disease in South America (14,(37)(38)(39).
Different molecular strategies have been used to study genetic diversity in populations of triatomine vectors, such as variability of the mtCyt b gene, microsatellites, and random amplified polymorphic DNA (RAPD) (14,28,37,40). Previous studies on the genetic variability of T. maculata in Venezuela using mtCyt b partially enabled to infer the occurrence of different haplotypes for populations in Anzoátegui and Portuguesa (4). In our study, mtCyt b presented the lowest level of variability, and these variations failed to discriminate T. maculata by ecotopes.
The polymorphism for β-tubulin marker in specimens of T. maculata generated evident variations between ecotopes of different locations of the Venezuelan states. This was particularly seven haplotypes for samples collected in homes and five haplotypes for copies of PD and wild ecotopes. The correspondence observed between phenotypic and genotypic grouping indicates that the joint application of both approaches is a robust tool for the study of vector domiciliation. This is the first time that the molecular marker of β-tubulin was used for evaluation of the genetic variability in T. maculata, the evaluation of this sequence being important in establishing it for use as a valuable tool in the genetic evaluation of triatomine population.

CONCLUSION
This work presents for the first time the relationship between phenotypic and genotypic approaches in the discrimination of T. maculata, a vector of Chagas disease, according to its ecotopes in Venezuela. Wing architecture and variability of the β-tubulin DNA region, the variables used to differentiate T. maculata populations, showed that specimens from S and PD ecotopes were more similar between them than those captured in the D ecotope which, together with the presence of nymphs in the houses and a high rate of infection with T. cruzi of D specimens, increases the risk factor in the transmission of Chagas disease in Venezuela.
Eradication of the vector in the domestic ecotopes followed by vigilance of re-infection will be important in reducing transmission of Chagas disease; however, integrative research is necessary to understand the vector population structure, domiciliation, and parasitic transmission.