extfieldsofstudy: Biology Medicine

text:
Lactobacillus crispatus Dominant Vaginal Microbiome Is Associated with Inhibitory Activity of Female Genital Tract Secretions against Escherichia coli

Objective Female genital tract secretions inhibit E. coli ex vivo and the activity may prevent colonization and provide a biomarker of a healthy microbiome. We hypothesized that high E. coli inhibitory activity would be associated with a Lactobacillus crispatus and/or jensenii dominant microbiome and differ from that of women with low inhibitory activity. Study Design Vaginal swab cell pellets from 20 samples previously obtained in a cross-sectional study of near-term pregnant and non-pregnant healthy women were selected based on having high (>90% inhibition) or low (<20% inhibition) anti-E. coli activity. The V6 region of the 16S ribosomal RNA gene was amplified and sequenced using the Illumina HiSeq 2000 platform. Filtered culture supernatants from Lactobacillus crispatus, Lactobacillus iners, and Gardnerella vaginalis were also assayed for E. coli inhibitory activity. Results Sixteen samples (10 with high and 6 with low activity) yielded evaluable microbiome data. There was no difference in the predominant microbiome species in pregnant compared to non-pregnant women (n = 8 each). However, there were significant differences between women with high compared to low E. coli inhibitory activity. High activity was associated with a predominance of L. crispatus (p<0.007) and culture supernatants from L. crispatus exhibited greater E. coli inhibitory activity compared to supernatants obtained from L. iners or G. vaginalis. Notably, the E. coli inhibitory activity varied among different strains of L. crispatus. Conclusion Microbiome communities with abundant L. crispatus likely contribute to the E. coli inhibitory activity of vaginal secretions and efforts to promote this environment may prevent E. coli colonization and related sequelae including preterm birth.

Introduction
Preterm birth is a major health problem affecting 12.3% of live births and is a major cause of neonatal mortality [1,2]. Escherichia coli (E. coli) has been associated with preterm birth [3], is among the leading cause of neonatal sepsis in extremely low birth weight neonates [4], and is the leading cause of early-onset neonatal sepsis and meningitis [5]. Similarly, bacterial vaginosis (BV), a dysbiotic condition characterized by the replacement of a Lactobacillus crispatus (L. crispatus) predominant microbiome with anaerobic species [6,7], has also been linked to an increased risk of preterm birth, although treatment with antibiotics in late pregnancy was ineffective [8][9][10]. These findings indicate that the vaginal microbiome plays an important role in adverse pregnancy outcomes.
Recent studies suggest that the vaginal microbiome may be linked to soluble mucosal defense. Specifically, genital tract secretions have been consistently shown to possess in vitro inhibitory activity against E. coli [11][12][13][14][15]. This activity, which may reflect contributions from the host innate immune mediators such as defensins as well as from microbiota, may be critical during pregnancy and prevent dysbiotic vaginal colonization and ascending infection. Earlier studies showed that the E. coli inhibitory activity was reduced among non-pregnant women with BV and was restored following successful treatment with metronidazole [11]. In a recent cross sectional study, genital tract secretions obtained by vaginal swabs from near term healthy pregnant women were found to have significantly higher inhibitory activity against E. coli that was inversely correlated with E. coli vaginal colonization [12]. A separate study using cervicovaginal lavage (CVL) samples from healthy non-pregnant women suggested that the inhibitory activity may be mediated, at least in part, by soluble proteins secreted by lactobacilli [15]. Using biochemical techniques including mass spectrometry, four Lactobacillus proteins (three originally described as proteins of L. crispatus and one of L. jensenii) were present exclusively in CVL samples with high (.90% inhibitory activity), but not in samples with low activity (,20%). These proteins included the S-layer protein, a bacterial surface layer protein, and a cell separation protein for L. crispatus and adhesion exoprotein for L. jensenii [15].
Building on this background, the current study was designed to further evaluate the link between E. coli inhibitory activity and the vaginal microbiome and to test the hypothesis that high E. coli inhibitory activity would be associated with healthy Lactobacillus species dominant microbiome whereas low activity would be associated with a more diverse microbiome. Utilizing samples previously obtained from healthy near term pregnant and nonpregnant women, we also tested the hypothesis that pregnancy would alter the microbiome, as suggested in a prior study [16]. A subset of samples were selected from 10 pregnant and 10 nonpregnant women and within each cohort, five were selected for high inhibitory activity (defined as .90% reduction in number of E. coli colony forming units [cfu]) and five with low activity (defined as ,20% reduction in cfu).

Participants and Sample Collection
The parent study was described previously in detail [12]. Briefly, healthy pregnant women were recruited between 35 and 37 weeks of gestation and healthy non-pregnant were recruited during a routine gynecologic visit. Vaginal swabs were collected to measure soluble immune mediators and endogenous E. coli inhibitory activity. Following approval from the Montefiore Medical Center Internal Review Board, all women gave written informed consent. For this sub-study, vaginal swab pellets from 10 pregnant women between 35 and 37 weeks of gestation and 10 healthy non-pregnant women (5 with E. coli inhibitory activity . 90% and 5 with activity ,20% within each group) were selected and analyzed in a blinded manner for the vaginal microbiome. None of the participants had clinical BV using Amsel's criteria [17].
Vaginal swabs were placed in a 1.5 mL sterile eppendorf tube that was pre-filled with 0.5 mL of sterile normal saline, placed on ice, and processed within 6 hours of collection. The sample was vortexed and clarified by centrifugation at 2000 rpm for 7 minutes at 4uC and the cell pellets were re-suspended in 200 mL of phosphate buffered solution (PBS) and stored at 280uC until used for the microbiome analysis. The supernatants were divided into aliquots and used to measure E. coli inhibitory activity as well as concentrations of immune mediators [12]. The Internal Review Board of Albert Einstein College of Medicine approved the parent study; all participants signed informed consent and only subjects who agreed that samples could be used for future studies were included.

DNA Extraction and Amplification
Relatively low-cycle amplification and next generation sequencing (NGS) on the Illumina HiSeq2000 platform, in conjunction with pplacer software was used to analyze the bacterial composition of vaginal samples. DNA for sequencing was extracted from pelleted cells by incubating 150 ml of sample in 250 ml of proteinase K digestion cocktail containing 1% sodium laureth-12 sulfate at 55uC for 2 hours heated to 95u610 minutes and then the DNA was precipitated in a 0.825 M ammonium acetate/ethanol (AAE) solution, pelleted by centrifugation and re-suspended in TE (10 mM Tris, pH 8.0, 0.1 mM EDTA), as described previously [18][19][20]. The V6 region of 16S rRNA genes were amplified in an ABI 9700 Thermal Cycler (Life, Carlsbad, CA) using HotStart-IT FideliTaq DNA Polymerase (Affymetrix, Santa Clara, CA) and 50 ng of template DNA in a total volume of 50 ml. Reaction parameters included initial denaturation at 94uC for 2 min, followed with 30 cycles of denaturation at 94uC for 30 seconds, annealing at 60uC for 30 seconds, and extension at 68uC for 30 seconds; and a final extension at 68uC for 5 min. Sense target primers contained unique 8-bp Hamming DNA barcodes [21], which allowed for the identification of reads from each sample. Successfully amplified DNA from all samples was pooled, purified and isolated using gel electrophoresis and electroelution. Following quality control and library preparation, DNA was sequenced on an Illumina HiSeq 2000 using a paired-end protocol yielding 100 base pairs of sequence in each direction. Raw sequences were joined (using eautils' fastq-join), processed to remove chimeras (using uchime), and low quality reads and nucleotides, and assigned to their sample of origin by demultiplexing (using mubiomics). Demultiplexed reads were aligned to the bacterial reference library -a vaginal microbiome reference library available from http://microbiome.fhcrc.org/apps/refpkg/, containing 633 sequences, representing 138 bacterial taxa, using pyNAST. The aligned sequences were then mapped to their bacteria of origin using the classification algorithm pplacer [22] and visualized using R-scripts that were developed at Einstein [18] (see Statistical Analyses section).

E. coli Inhibitory Activity
Spent culture supernatants (SCS) from L. crispatus (ATCC 33197, M35, SJ-3C, and 60), L. iners (DSM 13335, UPII 60B-BEI HM-131, and 143D-BEI HM-126), and G. vaginalis (ATCC 49145, 14018) were prepared by centrifuging overnight cultures of bacteria at 2000 g for 15 min at room temperature and then filtering the supernatant to remove bacteria and particulate matter (0.22 mm syringe filter). L. crispatus strains were grown in MRS broth; L. iners in 1% proteose peptone, 1% beef extract, 0.5% yeast extract, 86 mM NaCl, 0.8 mM MgSO 4 , 0.3 mM MnSO 4 , 11.5 mM K 2 HPO 4 , 10% fetal bovine serum, and 2% glucose; and G. vaginalis in brain heart infusion broth supplemented with 10% FBS and 5% Fildes enrichment. The SCS from each of these cultures and their respective growth media were serially diluted in normal saline and then incubated with E. coli (ATCC strain 43827) (,10 9 cfu/mL) for 2 h at 37uC. The mixtures were then further diluted in saline (to yield 800-1000 E. coli colonies on control plates) and plated on agar enriched with trypticase soy broth [15]. Colonies were counted using ImageQuant TL v2005 after an overnight incubation at 37uC. To control for differences in cfu of the L. crispatus, L. iners or G. vaginalis cultures, the inhibitory activity was normalized to lowest bacterial yield in a post-hoc analysis or, alternatively, the SCS were diluted based on different yields prior to incubating them with E. coli. All samples were tested in duplicate and the percentage inhibition was determined relative to the colonies formed on culture media control plates.

Statistical Analyses
E. coli inhibitory activity was dichotomized as .90% (high) and ,20% (low). Categorical variables were compared between groups by Chi-square or Fisher's exact test. Continuous variables were compared by the Student t test or the Mann-Whitney U test, depending on the distribution of the data. Clinical data were analyzed using STATA (v11.0; StataCorp, Inc., College Station, TX). All plotting and statistical comparisons for NGS were performed in R v2.12.2 using a script developed in-house (available upon request). The pairwise Kantorovich-Rubinstein (KR) distances (equivalent to the weighted UniFrac distance [23] between samples were calculated with p = 1 and normalized with respect to the diameter of the reference tree. Principal component analysis was performed to determine differences between pregnancy and the microbiome and dichotomized E. coli inhibitory activity and the microbiome. Using a conservative approach, the PERMANOVA analysis was performed on the Kantorovich-Rubinstein (KR) distances between sample microbiomes, with pregnancy and E. coli inhibitory activity as the factors, respectively. Fisher's exact tests were performed with pregnancy status and E. coli inhibitory activity as binary factors against the dominant bacterial taxon. Shannon and Simpson diversity indices were calculated to determine significant differences in diversity between pregnant and non-pregnant samples and samples with high and low E. coli inhibitory activity. To ensure that sufficient sampling of the microbiome had occurred, a rarefaction analysis was performed. Bacterial taxa were grouped to reflect healthy lactobacilli (group 1), intermediate bacteria (at times associated with BV diagnosed by Nugent's score [24], group 2) and pathogenic bacteria (groups 3 and 4): Group 1: L. crispatus, L. jensenii, and L. gasseri, Group 2: L. iners, Group 3: G. vaginalis, Sneathia Sanguinegens, BVAB1, BVAB2, Megasphaera, Prevotella bivia, Prevotella melaninogenica, Prevotella genogroup 1 and genogroup 2, and Atopobium vaginae, and Group 4: Streptococcus anginosus and Staphylococcus hominis. Principal component analyses were performed using Group 1, Group 3, and Group 4 to reduce the dimensions of the data. Spearman correlation coefficients (SCC) were used to examine correlations between bacterial groups and soluble immune mediators measured in the parent study [12]. The following mediators were included in the analysis: secretory leukocyte protease inhibitor (SLPI), human neutrophil peptide 1-3 (HNP1-3), human beta defensin (HBD)-1, HBD-2, and HBD-3, and cytokines/chemokines including interleukin (IL)-1a, IL-1b, IL-1 receptor antagonist (IL-1ra), IL-6, IL-8, macrophage inhibitory protein (MIP)-1a, MIP-1b, and regulated on activation, normal T-cell expressed and secreted (RANTES). All tests were two-sided with p value of ,0.05 considered statistically significant.

Description of Participants
Sixteen of the 20 subjects had paired-end reads that were sufficient to characterize the microbiome. The characteristics of these subjects are summarized in Table 1 grouped according to E. coli inhibitory activity (10 with high activity and 6 with low activity). The two groups did not differ with respect to age, race, number of pregnant women, current smoking status, history of prior sexually transmitted infections (STI), contraceptive use,  (Table 1).

Microbiome Analysis
NGS produced 9,111,237 high quality, non-chimeric, joined, paired-end reads, of which 8,946,862 were successfully assigned a bacterial identity. Those that could not be aligned (and therefore assigned) were of non-bacterial origin. Less than 2% of the reads were determined to be chimeric and were therefore removed. The short reads were submitted to the NCBI Short-Read Archive (Submission ID: SRP034665).
Community compositions and proportional abundances of bacteria are shown in the heat map of Figure 1. All but one of the samples (JG46) was dominated by Lactobacillus species including L. crispatus and L. iners, which is consistent with the absence of clinical BV in the cohorts. Most strikingly, the majority of samples with high E. coli inhibitory activity were dominated by L. crispatus, while those with low activity were dominated by L. iners. The two exceptions were JG46 and JG50, which had a microbiome dominated by Atopobium vaginae and Lactobacillus gasseri, respectively; both samples were in the low E. coli inhibitory activity cohort. The microbiome communities found in the samples clustered into three groups: L. crispatus, L. iners, and L. gasseri and are consistent with the community types found by Ravel et al. [6] A rarefaction plot is a standard analysis used to evaluate whether sufficient reads have been tested to detect species richness. If the gradient of the rarefaction curve decreases sharply after some level of species richness, it is a good indication that increasing the read depth would discover only the very rarest of species. Due to the low gradient of the curves of the rarefaction analysis at the read depth at which the clinical samples were sequenced, only the rarest of bacteria are likely to have been missed from our analyses (data not shown).
High E. coli inhibitory activity was predictive of an L. crispatus dominated microbiome (Fisher's exact test, p value = 0.007), while low activity was not significantly associated with any dominant bacterial taxon. Furthermore, the vaginal microbiome in women with high E. coli inhibitory activity were significantly different than women with low activity by principal component analysis (p = 0.001, Figure 2A). However, there were no differences between the pregnant and non-pregnant women ( Figure 2B) and no association was found between pregnancy and any of the dominant bacterial taxa. Furthermore, we found no significant association between age, race, vaginal wall pH and L. crispatus predominance. There was also no difference in bacterial diversity between pregnant and non-pregnant women or between those with high or low E. coli inhibitory activity using Shannon and Simpson diversity indices (not shown).
In the parent study, the concentrations of protein and a subset of cytokines, chemokines, defensins and antimicrobial peptides were measured in the vaginal swab supernatants and SLPI, HBD-1, HBD-2, IL-1b, IL-6, and IL-8 correlated modestly with E. coli inhibitory activity among non-pregnant, but not pregnant women [12]. To assess whether any of these mediators were associated with the bacterial groups, Spearman correlation coefficients were measured. None of the bacterial groups correlated with the soluble immune mediators.

Spent Culture Supernatants from L. crispatus have Significant E. coli Inhibitory Activity
To gain further insight into the link between the microbiome composition and E. coli inhibitory activity, SCS from L. crispatus, L. iners, and G. vaginalis were tested for the ability to inhibit E. coli relative to the growth media for each bacteria. The bacterial yields of the cultures used to produce the SCS were as follows: L. crispatus 33197: 2.5610 9 cfu/mL; L. iners ARL1 (13335): 4.2610 8 cfu/mL; L. iners 60B: 1.0610 9 cfu/mL; L. iners 143D: 6.5610 8 cfu/mL; G. vaginalis 49145: 4.5610 8 cfu/mL; G. vaginalis 14018:1.0610 9 cfu/ mL. When the SCS were diluted 1:10 prior to mixing with E. coli, L. crispatus 33197 showed the greatest magnitude of inhibitory activity and reduced the E. coli cfu by greater than 1 log after adjusting for differences in bacterial yields, although SCS from L. iners 60B and G. vaginalis 14018 also showed significant inhibitory activity (p,0.05) (Figure 3a). To further explore the inhibitory activity of L. crispatus, we tested serial dilutions of 3 additional strains (after diluting the SCS to adjust for differences in bacterial yields as follows: L. crispatus M35: 4.1610 8 cfu/mL; L. crispatus SJ-3C: 1.3610 8 cfu/mL; and L. crispatus 60: 2610 7 cfu/mL). L. crispatus 60 displayed the most potent activity and retained significant activity even after dilution of 1:25 (Figure 3b).

Discussion
The current study demonstrates that a L. crispatus dominant vaginal microbiome community is associated with E. coli inhibitory activity and supports the contention that the microbiome contributes to host defense. This notion is further supported by the finding that culture supernatants from L. crispatus had the most potent inhibitory activity, although the activity varied between different strains. The results are consistent with prior observations that E. coli inhibitory activity is reduced in the setting of BV, a dysbiotic condition associated with loss of L. crispatus [6,7] and with a small proteomic study showing that Lactobacillus proteins (three originally described as proteins of L. crispatus and one of L. jensenii) were present exclusively in CVL samples with high but not in samples with low E. coli inhibitory activity [15]. While the microbiome from women with high E. coli inhibitory activity was dominated by L. crispatus, women with low activity (even in the absence of clinical BV) had a trend towards L. iners dominance. However, we did not observe any significant increase in bacterial diversity in samples from women with low E. coli inhibitory activity. It is possible that the other common healthy Lactobacillus species such as L. jensenii, also contribute to E. coli inhibitory activity. L. jensenii was one of the more prevalent bacteria in the women with high activity (Figure 1).
There were no differences detected in the vaginal microbiome between healthy near term pregnant and non-pregnant women. These findings are consistent with one other study in which the microbiome exhibited less diversity and richness in pregnant women sampled between 18-32 weeks gestation, but returned to the non-pregnant community structure in late gestation (.32 weeks) [16]. Both studies focused on healthy women, the majority of whom delivered at term. Larger studies in women at risk for preterm birth are needed to determine whether changes in the The spent culture supernatants obtained from three additional strains of L. crispatus (M35, SJ-3C, and 60) were normalized by diluting in culture media to that of the lowest growth (2610 7 cfu/ml) and then serial dilutions (neat, 1:5, 1:10 and 1:25) were mixed with E. coli and tested for inhibitory activity. Results are mean 6 SD from duplicate plates. The number symbol indicates that no bacterial colonies were observed on plates after incubation with undiluted L. crispatus 60 SCS. The asterisks represent a significant reduction in E. coli cfu relative to its control growth media (p,0.01). doi:10.1371/journal.pone.0096659.g003 vaginal microbiome and/or E. coli inhibitory activity will provide a biomarker of risk for adverse outcomes including E. coli colonization and associated sequelae (e.g. chorioamnionitis and neonatal sepsis).
The potential utility of E. coli inhibitory activity as a biomarker of mucosal health, however, may differ in populations where lactobacilli species are not the dominant microflora. This notion is supported by two small sub-studies of African women who were at high-risk for HIV acquisition [25]. In the latter studies, having higher E. coli inhibitory activity was associated with an increased risk of HIV acquisition and, in one study, with a higher viral set point [26]. However, the participants in these studies had relatively high Nugent scores and lower median E. coli inhibitory activity (50% inhibition) compared to that observed in the healthy U.S cohorts (.70% inhibition). Moreover, in the HIV seroconverters, E. coli inhibitory activity correlated with the concentrations of several pro-inflammatory cytokines and chemokines, suggesting that the activity may be a biomarker of inflammation in high-risk women. In contrast, in the current study, there were no significant correlations between concentrations of mucosal immune mediators and the bacterial groups further indicating that perhaps host immune factors contribute little to this antimicrobial activity in populations where the microbiome is typically dominated by protective Lactobacillus species. Thus, in populations where there is a relative paucity of protective lactobacilli (e.g. high risk African cohorts), E. coli inhibitory activity may be more influenced by inflammatory molecules and serve as a biomarker of HIV risk, whereas in populations where L. crispatus is common, high activity may be representative of a healthy vaginal microbiome.
A limitation of this exploratory study is the small sample size. Thus, as noted above, we cannot preclude a broader association with other healthy Lactobacillus species. Furthermore, conclusions about the effect of race, ethnicity, age, pH, contraceptive use and host immune mediators on the vaginal microbiome should be made with caution. Another study limitation is that we did not confirm sexual abstinence. Finally, differences in the inhibitory activity of bacterial SCS may be impacted by differences in the bacterial yields from which supernatants were obtained. In an effort to account for this difference, we adjusted the E. coli cfu by the bacterial cfu/mL used to produce the SCS in a post hoc analysis (Figure 3a) or adjusted the SCS by dilution prior to incubating the samples with E. coli (Figure 3b).
The current study highlights the possibility that E. coli inhibitory activity may be a functional feature of a Lactobacillus crispatus dominant healthy vaginal microbiome. In addition to studies with larger more diverse cohorts, future studies should also include proteomic and metabolomic analyses on the sample to further define the nature and origin (host and/or microbiome) of the molecules that contribute to the E. coli inhibitory activity. A longitudinal study in pregnancy is ongoing and will help to elucidate whether high inhibitory activity against E. coli translates to a reduction in sub-clinical or overt genital tract infection and preterm birth. These findings could promote the identification of novel strategies to enhance the antimicrobial activity of genital tract secretions. Further studies are needed to determine if introducing lactobacilli derived molecules or sustaining a L. crispatus dominant microbiome may promote a healthy vaginal environment. These approaches could lead to reduced risk of bacterial vaginosis, chorioamnionitis, preterm birth and perinatal infection and be used as a safer alternative to traditional antibiotics [27].


s2fieldsofstudy: Biology Medicine

text:
A Novel PHEX Gene Mutation in a Patient with Sporadic Hypophosphatemic Rickets

Phosphate regulating gene with homologies to endopeptidases on the X-chromosome (PHEX) is a common cause of X-linked hypophosphatemic (XLH) rickets. Diverse PHEX gene mutations have been reported; however, gene mutations in sporadic rickets are less common than in XLH rickets. Herein, we describe a 50-year-old female patient with sporadic hypophosphatemic rickets harboring a novel splicing-site mutation in the PHEX gene (c.663+1G>A) at the exon 5-intron 5 boundary. The patient had recently suffered from right thigh pain and an aggravated waddling gait. She also presented with very short stature, generalized bone pain, and muscle weakness. Despite low serum phosphate levels, her phosphate reabsorption rate was lower than normal. Additionally, her 1,25-dihydroxyvitamin D3 concentration was lower than normal, although FGF23 level was normal. After treatment with alfacalcidol and elemental phosphate, her rachitic symptoms subsided, and callus formation was observed in the fracture site on the right femur.

PHEX is located on the X chromosome at Xp22.1 [7]. PHEX has been observed in cartilage, bone, and teeth osteoblasts [8] and is thought to act on substrates that suppress phosphate excretion in the proximal renal tubule [9]. Recently, several mutational analyses of the PHEX gene in XLH rickets and sporadic hypophosphatemic rickets reported deletion, insertion, missense, nonsense, and splicing site mutations [10]. Here, we present the first report of a new splicing-site PHEX gene mutation in a Korean patient with sporadic hypophosphatemic rickets.
Copyright © 2014 Korean Endocrine Society

CASE REPORT
A 50-year-old woman visited the hospital for evaluation of right thigh pain. She had recently suffered from generalized bone pain and generalized weakness, which were aggravated after a fall 6 months prior. Additionally, her waddling gait had worsened. She was born at full term, and her body weight and height were within normal ranges at birth. She had short stature and difficulty in walking when she was a teenager but was not referred to the Department of Pediatrics for evaluation or treatment because she experienced no pain or weakness at that time. She had very short stature (129 cm, just below the 10th percentile for her age) and bowing leg deformities. She was born from healthy parents of normal heights. Her daughter and son have normal stature (her daughter is 160 cm and her son is 180 cm), and the rest of her family is healthy. DNA samples of the family were not available.
She presented with hypophosphatemia 2.0 mg/dL (normal range, 2.5 to 4.7). Her measured serum calcium level was 9.0 mg/dL (normal range, 8.7 to 10.6), alkaline phosphatase was elevated (139 IU/L; normal range, 42 to 98), parathyroid hormone was 45.68 pg/mL (normal range, 10.00 to 65.00), 25-hydroxyvitamin D3 was 11.10 ng/mL (normal range, 10.00 to 30.00), and 1,25-dihydroxyvitamin D3 was 21.6 pg/mL (normal range, 25.1 to 66.1). Nephrologic evaluation demonstrated normal diuresis (1,000 mL/24 hours) and normal renal function (estimated glomerular filtration rate of 95 mL/min/1.73 m 2 ). The tubular reabsorption rate of phosphate was 81% (normal range, 80 to 100). The maximum tubular capacity of phosphate per unit volume of glomerular filtrate was decreased to 0.51 mM (normal range, 0.88 to 1.42 for her age group), which revealed insufficient reabsorption of phosphorus in the kidneys. We also evaluated her serum fibroblast growth factor 23 (FGF23) level using sandwich enzyme-linked immunosorbent assay (ELISA) and found it was 109 RU/mL (normal range, <189 for her age group). The interindividual coefficient of variation (CV) for this ELISA assay is 3.8% to 6.4%, and the intraindividual CV is 2.5% to 6.1%.
Her plain radiograph presented diffuse bowing deformities in the upper and lower extremities and osteoporotic changes in the extremities, spine, and pelvis. There was a right subtrochanteric fracture that included the cortex of one long bone. If she had preferred surgery, it might have been possible. However, the symptoms were not severe, and she could walk by herself without orthoses. Furthermore, multiple pseudofracture lines were also observed in the right proximal femur, left iliac bone, and proximal shaft of the both fibulas with anterolateral bowing (Fig. 1). Dual energy X-ray absorptiometry measurement showed that the mean bone mineral density in the lumbar spine (L1 to L4) was 0.845 g/cm 2 , and that in the femoral neck was 0.661 g/cm 2 , resulting in T-scores of -2.7 and -2.3, respectively. After obtaining informed consent from the patient, genomic DNA was extracted from peripheral blood leukocytes using QuickGene Blood kit (Fujifilm, Tokyo, Japan). For PHEX gene analyses, all 22 exons and exonintron junctions of the PHEX gene were amplified using polymerase chain reaction (PCR) with the appropriate primers (Table 1). Subsequently sequence analyses were performed with BigDye terminator version 3.1 (Applied Biosystems, Foster City, CA, USA) and the same primers used for PCR amplification. We found a novel splicing mutation in the exon 5-intron 5 boundary of PHEX, a heterozygote of the c.663+1G>A variation, which had not been previously reported (Fig. 2).
In chromosomal analyses of 20 cells in metaphase which were performed using 72-hour culture and G banding stain of peripheral blood, mosaicism for trisomy X, and tetrasomy X were unusually expressed. We investigated serum tumor markers and whole body bone scintigraphy using Tc-99m hydroxy methylene diphosphonate (Fig. 3) to identify neoplastic lesions and found no evidence of TIO.
The patient was treated using alfacalcidol and elemental phosphate (Joulie's solution; 10 mL of 4.9 mmol phosphate in 1 mL water) four times a day. After 22 days of outpatient care, her measured serum phosphate level increased to 3.6 mg/dL (normal range, 2.5 to 4.7), alkaline phosphatase decreased to 124 IU/L (normal range, 42 to 98), and her generalized bone pain was decreased to allow daily walking. In addition, follow up X-ray findings of her right femur showed improved fracture union (Fig. 4).

DISCUSSION
XLH is the main cause of hypophosphatemic rickets. The gene that causes XLH was identified on Xp22.1, which was found to be PHEX [7]. Although a physiologically relevant PHEX function has not been established, several recent studies indicate that PHEX inactivates a phosphaturic hormone, such as FGF23, or interacts with membrane proteins that modulate phosphorus activity [5]. FGF23 is a representative phosphatonin that stimulates the renal excretion of phosphate in its fulllength state by inhibiting sodium-phosphate transport. Physiologically, FGF23 is inactivated by cleavage at the potential recognition/cleavage sites for the enzymes of the proprotein convertase family (RXXR motif) [11]. Elevated circulating levels of FGF23 were observed in autosomal dominant hypophosphatemic rickets of RXXR motif mutation and also in XLH and TIO [3]. In XLH, an elevated FGF23 level is frequently observed in association with hypophosphatemia.
Therefore, some authors have suggested that PHEX controls the inactivation of FGF23, and PHEX gene mutations, such as loss of function mutations, increase the circulating level of FGF23 [12]. In in vitro experiments, a furin-like enzyme cleaved FGF23, not directly mediated by PHEX using the Hyp mouse that is homologous to XLH. However, PHEX inactiva- Copyright © 2014 Korean Endocrine Society tion resulted in a tendency to increase FGF23 level in a mouse model. Although FGF23 is not a direct substrate of PHEX, the PHEX gene might be associated with FGF23 expression [13]. In our case, normal level of FGF23 and a lower than normal tubular reabsorption rate of phosphate might contribute to the late onset of rachitic symptoms, including generalized bone pain and muscle weakness, despite PHEX gene mutation. In the renal phosphate wasting state, an elevated FGF23 level is frequently found. Although the relationship of elevated FGF23 with PHEX mutation was not elucidated, most hypophosphatemic rickets present with elevated FGF23. However, Jonsson et al. [12] reported elevated FGF23 levels in 13 of 21 patients. Therefore, not all patients with hypophosphatemic rickets have elevated FGF23. In normal adults and children, the FGF23 levels were definitely normal [12]. Shaikh et al. [14] suggested other circulating factors controlling phosphorous metabolism such as secreted frizzled-related protein (sFRP-4), matrix extracellular phosphorglycoprotein (MEPE), and fibroblast growth factor 7 (FGF7). Especially, MEPE transcripts are increased in Hyp mice, which have poorly mineralizing bone [15]. An in vitro study reported that cathepsin B-dependent cleavage is suppressed by PHEX; therefore, MEPE and FGF have been reported as important phosphatonins affecting phosphate homeostasis and skeletal mineralization [16]. In our case, there is a possibility that another phosphatonin may have led to the resulting rickets phenotype. However, our study is limited in that we did not measure the levels of the other factors like MEPE.
Several studies have identified genotype-phenotype correlations of the PHEX gene in hypophosphatemic rickets. In genotype-phenotype analyses of Korean patients with hypophosphatemic rickets, there was no genotype-phenotype correlation, even though skeletal disease is more severe in C-terminal mutations compared to N-terminal mutations [17]. Remarkably, a novel PHEX mutation was discovered in the C terminus of exon 5 here, but the rachitic symptoms were less severe compared to other hypophosphatemic rickets diagnosed in childhood or adolescence. The patient did not suffer from bone pain or gait disturbance until middle age.
In previous cases, 124 sporadic mutations of PHEX gene have been reported (online PHEX mutation data base; http:// www.phexdb.mcgill.ca), and 22 of them are splicing mutations. Table 2 summarizes four splicing mutations in some of the previous cases [6,10,18,19]. As shown in the Table 2, mutation site does not correlate with disease severity. In one case, even though serum FGF23 was elevated to 2,430 pg/mL, the www.e-enm.org 199 patient had normal height and no bone fractures, and the maximum tubular reabsorptive rate of phosphate decreased to 0.41 mmol/L. In addition, the patient showed a mild phenotype unrelated to FGF23 level, as in our case [10]. Based on these results, we hypothesized that C-terminal splicing mutations of PHEX could represent mild clinical phenotypes. The case we report is also a sporadic gene mutation case that is expressed by a novel splicing mutation (c.663+1G>A) in the exon 5 and intron 5 junction. The radiologic and laboratory findings of the patient were reviewed to exclude TIO, and this is the first sporadic case reported with a splicing site mutation in exon 5 of PHEX and a normal FGF23 level. Although morphologic changes including short stature, leg bowing, and waddling gait were found at an early age, the rachitic symptoms were initiated by a traumatic event (falling down) in middle age. After treatment with alfacalcidol and elemental phosphate, the rachitic symptoms subsided.
In conclusion, we have identified a novel splicing site mutation in the PHEX gene in a Korean patient with sporadic hypo-POST RT ELBOW IV ANT Fig. 3. Whole body bone scan of the patient. A hot spot was found in the right femur fracture site and also in multiple pseudofractures in both lower extremities.

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After 2 months Fig. 4. After 2 months of alfacalcidol and elemental phosphate treatment, callus formation was observed in the fracture site on the right femur metaphysis. SD, standard deviation; HPT, height for piston-type imbibition in throat; DXA, dual-energy X-ray absorptionetry; FGF, fibroblast growth factor; TmP/GFR, the maximum tubular reabsorptive rate of phosphate.


New Records of Mosquitoes (Diptera: Culicidae) from Bolívar State in South Eastern Venezuela, with 27 New Species for the State and 5 of Them New in the Country

This is the first part of a series of studies related to mosquito ecological and biogeographic aspects. A total of 69 mosquito species (Diptera: Culicidae) was collected in 16 localities sampled in the Gran Sabana Municipality, Canaima National Park, and Venezuela. Twenty-seven mosquito species are recorded for the first time from Bolívar State, Venezuela. Five of them species are reported for the first time in Venezuela: Anopheles malefactor Dyar and Knab (1907); Chagasia bonneae Root (1927); Chagasia ablusa Harbach (2009); Culex anduzei Lane (1944), and Uranotaenia leucoptera Theobald (1907). Their medical importance is commented, and ecological and epidemiological aspects are discussed. A checklist of the mosquito species reported in the Gran Sabana County is given.

INTRODUCTION
The occupation of Amazonia in Brazil and Venezuela, has formed part of the integration processes that both governments are implementing in their common border, which makes evident that both governments are aware of the importance that the Amazonia has in our present world (1). Diseases such as malaria, dengue, Chikungunya, Yellow Fever, Mayaro virus, West Nile virus, and several emerging and reemerging arboviruses, which are responsible for millions of cases of sickness and death among people living in the tropical regions, continue to be of great concern to the World Health Organization authorities in our present world (2). Dengue is an important arbovirus that affects humans; it is transmitted by Aedes aegypti (Linnaeus) and Aedes albopictus Skuse. A. aegypti and A. albopictus species have been involved in Dengue transmission in the Manaus rural areas, Brazilian Amazon (3). A. albopictus a secondary dengue vector in Asia, has spread to America and Europe largely, due to the international trade of used tires (a typical larval habitat), timber, and other goods such as "lucky bamboo" (a decorative house plant that is marketed worldwide). This species has a wide geographical distribution; it is particularly resistant, and can survive in both rural and urban environments. Mosquito's eggs are highly resistant and can remain viable throughout dry season, and can survive in cold temperature regions of Europe (3). A. albopictus and A. terrens (Walker) species are two potential Yellow Fever vectors in jungle. These species utilize a wide variety of natural larval microhabitats, such as tree-holes, bamboo internodes, and artificial containers and may be found in the same natural environments as Haemagogus species (1). Haemagogus species have been involved in Sylvain yellow fever transmission in Venezuela (4). Human malaria, is one of the most serious parasitic diseases in tropical ecosystems, it is caused by parasites of the genus Plasmodium (Apicomplexa: Plasmodidae) and transmitted among human hosts by bites of the infected Anopheles female (1). Five parasite species cause malaria in humans. Plasmodium falciparum and P. vivax are the two most common. P. falciparum is the most dangerous, with the highest rates of mortality (1).
In 2012, Venezuela reported the highest recorded incidence of malaria in its history with 51,264 cases. In Bolívar State, incidence cases increased to 44,180 (86.2% of the country); with three counties (Sifontes, Gran Sabana, and Cedeño) in "epidemic" and two counties (Piar and Sucre) in "alarm" status (5). Anopheles darlingi Root has been considered as human malaria's principal vector in South America. In Amazonas and Bolívar states, it is responsible for 90% of malaria cases reported in Venezuela (1,6).
Mosquito borne diseases such as malaria, dengue, Venezuelan Equine Encephalitis (VEE), West Nile virus and others equine encephalitis, Mayaro, or Chikungunya are zoonoses with increasing incidence in the current decade in tropical and temperate countries. These diseases emerge as a consequence of changes made to terrains that often increase the natural and artificial mosquito larval habitats. Many of these mosquito species are of public health importance. Mosquito's population increases result in a risk increased of tropical diseases transmission (1). There are many factors that can accelerate the emergence of zoonoses, such as environmental changes, habitat modifications, variations of human and animal demography, deterioration of strategies of vector control, or changes in pathogen genetics (1). Efforts to control such species that transmit emerging diseases have primarily been concentrated on the use of synthetic insecticides. Unfortunately, this has resulted in the appearance of physiological mosquito resistance, toxicity problems to human, environmental contamination, ecological imbalance, and economic burden (1). Such problems have created the need to look for alternative, environmentally friendly control mechanisms, based on those found in nature.
These include the essential oils from plants, some of which have been used by people for medicinal purposes; also biological control (e.g., Bacillus sphaericus against Anopheles) and biochemical control with synthetics juvenile hormones (7,8).
It is well known that the rate of species lost worldwide surpasses that by which taxonomic knowledge is increased (9). Thus, it is necessary to intensify studies focusing on diversity and from areas with an acceptable level of conservation (e.g., Canaima National Park, Venezuela). The Gran Sabana Region (Canaima National Park) is an undulating plain grass-dominated upland savanna covering close to 18,000 km 2 , with altitudes ranging from 750 to 1,450 m (10). Most of the Gran Sabana uplands have a humid submontane climate, with average annual temperatures ranging between 18 and 24°C, average annual rainfall between 2,000 and 3,000 mm, and a short dry season occurring from December to March. This area is drained by tributaries of the Orinoco River (Venezuelan part of the igneous metamorphic Guyana Shield), most of them black-water Rivers, with very acidic and low mineral waters (10). The ecological studies in the Gran Sabana are relatively scarce, especially for short and long-term evaluations on possible changes induced by human activities (e.g., climatic change). Additionally, ecological studies on richness and distribution at regional levels are few. It is well known that Culicidae larvae are dependent on habitat characteristics and that they are sensitive to biotic factors, as predators and also to several abiotic factors (pH, temperature, dissolved oxygen, salinity, and conductivity).
The present article, refers to the finding of 69 species and 17 genera of mosquito (Diptera: Culicidae), collected in the Gran Sabana county, Bolívar State, Venezuela. Twenty-seven mosquito species are recorded for the first time from Bolívar State, and five species are reported for the first time in the country.

STUDY AREA
The study area is located in the southern of Gran Sabana Municipality, Canaima National Park, a natural protected area in southeastern Venezuela, Amazonian Region that borders Brazil and Guyana (Figures 1-3). This study is based on material collected in indigenous territory (Pemón ethnic group) of Gran Sabana, Canaima National Park, located in Bolívar State, and roughly occupies the same area as the Gran Sabana region (11). The park was established on 12 June 1962. It is the second largest park in the country, after Parima-Tapirapecó in Amazonas State, and sixth biggest national park in the world (1). About 65% of the park is occupied by plateaus of rock called tepuis, which are a kind of plateau of 1.7-1.8 billion years old. The oldest Roraima sandstone is estimated to be 1.6 billion years old. These ancient mountains of the Guiana Shield (Figure 1) in the Amazonian Region (12) constitute a unique biological environment and are of great geological interest, which makes them one of the oldest formations in the world (11). The park is home to indigenous Pemón Indians (Pemón ethnic group), part of the Carib linguistic group (11). Pemón Indians have an intimate relationship with the tepuis, and believe they are the home of the "Mawari" spirits. Most transport within the park is done by light plane, from airstrips built by various Capuchin missions, or by foot and canoe. Pemón indigenous have developed some basic and luxurious camps, which are mainly visited by tourists from around the world and Venezuelan tourists. In 1994, Canaima National Park was named a World Heritage Site by UNESCO, as a natural reserve that has abrupt relief special and unique around the world, "The Tepuis," The park includes the entire watershed of the right bank of the Caroní River, and two of the highest waterfalls in the world, the Angel Falls and the Kukenán Falls and plenty of waterfalls of lower altitude (11).
Field surveys of mosquito adults (human bait) and mosquito larvae sampling, were carried out in the Gran Sabana County. The annual mean temperature is 22°C (18-24°C); the total annual rainfall is 1,500-5,700 mm, with annual average rainfall between 2,000 and 3,000 mm, with altitudes ranging from 750 to 1,450 m and annual mean of 205 days with rain per year, and a very short dry season occurring from December to March (10). The estimated population is 39,000, mainly concentrated in the capital, Santa Elena of Uairén (Figures 2 and 3).
Sixteen localities and four rivers were sampled for mosquitoes in the Gran Sabana County near the common border Brazil-Venezuela, between August 2011 and November 2013. These sites were selected as representative areas of the Gran Sabana, Venezuelan Canaima National Park. All localities are at altitudes >600 m. These altitudes are variables and can range from 600 to 1,450 m. These localities and rivers are: Santa Elena de Uairén, Waramasén, Manak-Krú, Maurak, Colinas de la Laguna, Altamira, San Antonio, Kinok-Pon-Parú, La Primavera, Chiricayén, Chiririka, Uaiparú, Betania, Kamaiwa, El Paraíso, El Paují, Chiririka, Uairén, Uaiparú, and Kukenán rivers.

COLLECTION AND IDENTIFICATION OF MOSQUITOES
A sampling program of mosquito larvae was carried out between August 2011 and November 2013 by visiting the study area periodically (dry and rainy season every year). Mosquito samples were taken with a standard dipper. The natural aquatic habitats (not-Phytotelmata) were a priori classified into four categories: lagoons, streams, rivers, and freshwater herbaceous swamps. At each breeding site, 30 dips for mosquito larvae samples were made. Most of the mosquito collections were made in tropical humid forests and edges of streams, rivers, and herbaceous swamps (morichales) of savanna areas in Gran Sabana County. Additionally, mosquito larvae were collected in artificial and natural containers. In Phytotelmata, larvae were collected by extracting water with a plastic pipettes from tree-holes, cut bamboo internodes, leaf axils of bromeliads, foliar axils of Araceae, fallen leaves of Musaceae, fallen palm spates, especially the "Moriches" palms (Mauritia flexuosa), and from floral bracts of Heliconiaceae. Immature mosquitoes were collected from 40 samples (water-holding structures) per plant species per locality. Collected immature specimens (IV instars larvae) from half the samples (20 samples) were preserved in ethanol (90%) for identification purposes. Specimens (larvae and pupae) from the rest of samples (50%) were pooled, transported, and reared in the field laboratory to obtain the associated specimens. The species presents, were recognized on the basis of correlated anatomical features in associated life stages. Sampling to adult collections on human landing catches (Figures 4A,B) were carried out in the same localities and rivers. The taxonomic determination of the specimens was based on direct observation of morphological characters, through a stereoscopic microscope (adults) and transmitted light microscope (larvae), using several taxonomic keys and descriptions and re-descriptions of species. The abbreviations employed for mosquito genera and subgenera are those proposed by Reinert (13).

RESULTS
A list of mosquito taxa of the Gran Sabana Municipality is presented. A total of 69 mosquito species (Diptera: Culicidae) and 17 mosquito genera from the indigenous territory (Pemón ethnic group) are reported ( Table 1). A total of 19 species of anophelines were collected; 17 of them belonging to genus Anopheles www.frontiersin.org Meigen and 2 species belonging to genus Chagasia Cruz. Additionally, 50 species of culicines were collected, belonging to 14 genera of Culicinae and one genus (Toxorhynchites) of Toxorhynchitinae ( Table 1).
Special attention should be placed in the new species records for Bolívar State and for Venezuela. A total of 27 mosquito species are recorded for the first time in Bolívar State. Five of them are new for the country (Tables 1 and 2) and they namely: Anopheles (Anopheles) malefactor Dyar and Knab (1907); Chagasia bonneae  (Tables 1 and 2). These species are not potential malaria vectors. Adults and immature specimens were identified, according to keys proposed by Wilkerson (26) www.frontiersin.org and Harbach and Howard (20). Larval specimens of the three species were collected in Waramasén ( Figure 5) and reared to obtain associated specimens; larval habitats of the three species are streams edges, especially sites with algae and partial shade ( Figure 5). The same larval habitats for A. malefactor and Anopheles punctimacula Dyar and Knab (1906) were found by Wilkerson (26). The geographical distribution of A. malefactor was restricted to Panamá and northwestern Colombia (26). With this new record (A. malefactor) their geographical distribution in Central and South America now includes Panamá, Colombia, and Venezuela. Literature records indicate that A. punctimacula was found naturally infected with malaria parasites in Panamá and Colombia (26). However, A. malefactor is not a potential malaria vector in South America (26). The species Anopheles (Kertezsia) cruzii Dyar and Knab (1908) and Anopheles (Stethomyia) nimbus Theobald (1902) are reported for the first time in Bolívar State ( Table 1). Larvae of A. nimbus were collected in river edges of Chiririka River (Figure 6) especially in habitats with algae and partial shade. This species does not transmit diseases of medical importance to man. Adults were not collected. Larvae were reared to obtain the associated specimens. A. nimbus was identified, according to key proposed by Navarro (22). Females of A. cruzii are very aggressive crepuscular biters and they are potential malaria vectors (31); their larvae live in water of leaf axils of bromeliads (31). Adult females were captured on human landing catches, in the forest in El Paraíso and El Paují, between 16:30 and 18:45 h. Immature specimens were not collected. Diagnostic and differential characters of A. cruzii were confirmed in adults specimens, using descriptions of Wilkerson and Peyton in basis to 11 females collected in Iguape, Brazil (28). Anopheles darlingi Root (Major malaria vector in the southern Bolívar State) is reported for the first time in the Gran Sabana Municipality; also reports for the first time in South America, the two highest mosquito records for A. darlingi. These altitude records are: Colinas de la Laguna, with 870 MSL and Santa Elena, with 893 MSL. Larvae of A. darlingi were only collected in shaded pools in the forest. When the water level dropped during the dry season, pools formed in or near the river bed (Uaiparú and Uairén rivers). These localities with A. darlingi larval habitats are at altitudes >600 m. These altitudes can range from 600 to 1,200 m. The lower altitude record for A. darlingi in the Gran Sabana was found in Uaiparú River, with 628 m, and the highest altitude record was found in Santa Elena, near of the Uairén River (Figures 6 and 7) with 893 m. The same larval habitats of A. darlingi were found in Suriname by Rozendaal (32).
In this article, we also report for the first time in Venezuela, the presence of Culex (Carrollia) anduzei. With this new record, their geographical distribution in South America now includes Brazil and Venezuela. Immature specimens were identified, according to keys proposed by Valencia (25). C. anduzei females do not transmit diseases of medical importance to man (25). Larvae were collected in plastic and metallic artificial containers in Chiririka (Figure 6), El Paraíso, El Paují, and Santa Elena (Figure 7). Adult specimens of C. anduzei were not captured. Additionally, we report for the  first time the presence of Culex (Lutzia) bigoti Bellardi in Bolívar State. This species does not transmit diseases of medical importance to man. Larval specimens were collected in Waramasén and were found in artificial containers (tires, and plastic and metallic containers). It is well known that Cx. bigoti larvae are predators in several artificial containers (tires, tin cans, old paint cans, plastic and metallic containers). In Paraná State, Brazil, larval specimens of C. bigoti were found in tires, cans, and cut bamboo (33). In Venezuela, this species was collected in artificial containers (tires and plastic containers) but, was not collected in epiphytic and terrestrial bromeliads (12,34). Toxorhynchites (Lynchiella) theobaldi Dyar and Knab (1906) and Toxorhynchites (Lynchiella) haemorroidalis Fabricius (1787) were collected in tires and tree-holes; they are a potential predators used for mosquito control. A longterm investigation in Florida (USA), demonstrated a reduction in tree-holes, mosquitoes, attributable to predation by Toxorhynchites rutilus (35).
Additionally The genus Haemagogus Williston includes mosquitoes with diurnal activity and immature habitats on Phytotelmata (treeholes and cut bamboo internodes). Adult females bite in forests during the day (40). Haemagogus species have been involved in yellow fever transmission, a virus circulating in forest areas in Latin America among arboreal primates and marsupials by means of mosquito bite (40). The genus comprises 28 species; 9 of them are present in Venezuela (4). The presence of Haemagogus anastasionis Dyar (1921), Haemagogus janthinomys Dyar (1921), and Haemagogus celeste Núñez-Tovar (1927) was detected in forest areas of Chiririka and Santa Elena (Figures 6 and 7). Adult specimens were identified, according to photographical key proposed by Liria and Navarro (4). These three species were captured on human landing catches in the forest, between 16:30 and 18:45 h, near of the Uairén and Chiririka rivers edges (Figures 6 and  7). Immature specimens were not collected.  (Figure 7). This species is also a potential vector of sylvatic yellow fever and jungle cycle of Mayaro virus. The Mayaro alpha virus produces non-specific, sub-lethal disease symptoms, often confused with dengue, but with symptoms of arthralgias (arthrosis) that can cause incapacitating disability (40). The Mayaro virus shows a great plasticity in vertebrate host infection, whereas high specificity in the mosquitoes of Culicidae family, vectors in the jungle cycle. Risk factors of infection are associated with forest areas of northern South America and the rainy season (40). The enzootic cycle is similar to the jungle cycle of yellow fever, which involves Haemagogus mosquitoes and monkeys as reservoirs (40). However, the involvement of others secondary vectors (e.g., Sabethes) and others hosts may be important in spread of the virus. Humans may have high levels of viremia and efficient experimental transmission has been demonstrated in Aedes aegypti, Aedes albopictus, and Aedes scapularis, suggesting a significant risk to public health in urban, rural, and domestic locations close to enzootic foci of Mayaro virus (40). In addition, we collected adults of A. aegypti and A. scapularis Rondani (1848) in urban and periurban areas of Santa Elena, Waramasén, Chiririka and Maurak, representing a significant risk to the inhabitant's populations of theses localities. A. albopictus specimens were not collected in the Gran Sabana.
Aedes aegypti and A. albopictus species have been involved in dengue transmission in the Manaus rural areas, Brazilian Amazon (3). The presence of A. albopictus in the Brazilian Amazon represents a potential risk of transmission of Chikungunya and Mayaro virus in urban, rural, domestic, and wild environments (1). The occurrence of its larvae and pupae, breeding in the same containers with other domestic species, associated with several sources of blood meal available in urban, rural, and wild environments, reveal its gradual establishment in the indoor of households and its potential involvement in the transmission of zoonotic pathogens to humans. The ability that their eggs may remain viable in nature for long periods of diapauses and the demonstrated transovarial transmission occurrence of several arboviruses has raised the need to expand the strategies directed toward combating A. albopictus in Vector Control Programs in South American (1). In Venezuela, A. albopictus was detected for the first time in 2009 (41), suggesting a significant risk to public health in urban, rural, domestic, and wild environments (1). Dormancy of the egg stage (and drought resistance) is considered to be a reproductive strategy for the long-term survival of multivoltine mosquitoes that develop in temporary habitats, such as tree-holes and other natural water containers that are subject to water fluctuations (42). Egg diapause involves a long stable arrest of hatching, even when environmental conditions are favorable for hatching (42).
Additionally, in this article we also report for the first time three new records of Sabethini Tribe mosquitoes for the Bolívar State ( Table 1); and they are: Wyeomyia celaenocephala Dyar and Knab (1906), Limatus asulleptus Theobald (1903), and Johnbelkinia ulopus Dyar and Knab (1906). Adult females were collected on human landing catch during the day in Waramasén and Chiririka (Figures 5 and 6). These three species are potential arboviruses vectors in Venezuela (43) and J. ulopus larvae are facultative predators (43).
Species of the genus Runchomyia ( Table 1) does not transmit diseases of medical importance to man; their larvae live in leaf axils of epiphytic and terrestrial bromeliads and in floral bracts (19). In 1986, the species Runchomyia frontosa Theobald was reported in carnivorous bromeliads from the Gran Sabana, Venezuela. The immature mosquitoes were collected living in the fluids held by the carnivorous bromeliad Brocchinia reducta Baker (44). The larvae of Runchomyia frontosa is a facultative predator, filter feeding, or consuming large prey that it captures with its enlarged maxillae in carnivorous bromeliads (44). Adult females of Runchomyia frontosa were collected on human landing catch during the day in El Paraíso and Waramasén ( Table 1). This species does not transmit diseases of medical importance to man (19).
In addition we report, five new records of the genus Uranotaenia in Bolívar State, they are: Uranotaenia typhlosomata Dyar and Knab (1907); Uranotaenia calosomata Dyar and Knab (1907); Uranotaenia nataliae Arribalzaga (1891); Uranotaenia leucoptera Theobald (1907); and Uranotaenia lowii Theobald (1901). Species of genus Uranotaenia were found mainly in ground-water habitats, including springs, stream margins, herbaceous swamps, and temporary pools with vegetation (19). Some species have been found in tree-holes, plant axils, and artificial containers. Females of some species are known to feed on frogs, birds, and mammals, but are normally not attracted to humans (19). They are avian malaria vectors in Venezuela. In the Llanos of Venezuela, was found a high endemicity of avian malaria (45,46). Immature specimens were collected in lagoons and herbaceous swamps (Morichales) in Santa Elena de Uairén, Waramasén, Maurak, Colinas de la Laguna, Altamira, San Antonio, Kinok-Pon-Parú, La Primavera, Chiricayén, Manak-Krú, Maurak, and Uaiparú. Uranotaenia leucoptera was collected only in tree-holes in Waramasén and Manak-Krú. This species is recorded for the first time from Venezuela. This study also extends the geographical distributions of Uranotaenia leucoptera in South America to Venezuela.
These findings shows the importance of further studies related to mosquito's geographical distribution, ecological aspects, arbovirus detection, epidemiological surveillance, and possible epidemiological link with emerging and reemerging arboviruses in the common border of Brazil and Venezuela. The entomological surveillance has an important role among the preventive measures against emerging diseases transmitted by insects, particularly by mosquitoes.


extfieldsofstudy: Medicine Biology

text:
RfaH suppresses small RNA MicA inhibition of fimB expression in Escherichia coli K-12.

The phase variation (reversible on-off switching) of the type 1 fimbrial adhesin of Escherichia coli involves a DNA inversion catalyzed by FimB (switching in either direction) or FimE (on-to-off switching). Here, we demonstrate that RfaH activates expression of a FimB-LacZ protein fusion while having a modest inhibitory effect on a comparable fimB-lacZ operon construct and on a FimE-LacZ protein fusion, indicating that RfaH selectively controls fimB expression at the posttranscriptional level. Further work demonstrates that loss of RfaH enables small RNA (sRNA) MicA inhibition of fimB expression even in the absence of exogenous inducing stress. This effect is explained by induction of σ(E), and hence MicA, in the absence of RfaH. Additional work confirms that the procaine-dependent induction of micA requires OmpR, as reported previously (A. Coornaert et al., Mol. Microbiol. 76:467-479, 2010, doi:10.1111/j.1365-2958.2010.07115.x), but also demonstrates that RfaH inhibition of fimB transcription is enhanced by procaine independently of OmpR. While the effect of procaine on fimB transcription is shown to be independent of RcsB, it was found to require SlyA, another known regulator of fimB transcription. These results demonstrate a complex role for RfaH as a regulator of fimB expression.

L ike many adhesins, type 1 fimbriation is controlled by phase variation (the reversible on-to-off switching in gene expression that produces a mixed population). fim phase variation involves the site-specific inversion of an ϳ300-bp promoter element (fimS) that contains a promoter for the fimbrial structural genes (1). Inversion is catalyzed by recombinases FimE (on-to-off switching) and FimB (low-frequency switching in either direction), encoded by genes situated adjacent to fimS (2), as well as by alternative recombinases situated elsewhere in the chromosome in some pathogenic strains (3,4).
Regulation of the fim inversion is complex and involves changes in both recombinase activity and expression. For example, the availability of the branched-chain amino acids and alanine exerts a direct effect on the fim inversion by altering the interaction of the leucine-responsive regulatory protein (Lrp) with the invertible element (5). In contrast, sialic acid and N-acetylglucosamine inhibit fimB expression, and hence FimB recombination, selectively (6,7). Moreover, the orientation of fimS controls fimE expression, ensuring that fimE expression is lower in afimbriate than fimbriate cells (8,9). Type 1 fimbriae are a virulence factor in urinary tract and other infections, and attachment of fimbriate cells is proinflammatory and facilitates intracellular invasion (10)(11)(12). We have proposed that, by repressing type 1 fimbrial expression in response to signals like sialic acid, Escherichia coli is better able to avoid host defenses (6).
NusG and its homolog RfaH regulate transcriptional pausing and termination. While NusG has a generalized effect on gene expression and is essential for viability, RfaH is dispensable (13). RfaH controls the expression of a specific subset of genes in E. coli, including those involved in lipopolysaccharide (LPS) core biosynthesis (14)(15)(16), as well as virulence factors (17)(18)(19)(20). Uropathogenic E. coli (UPEC) mutants lacking rfaH are attenuated for virulence in an ascending mouse model of urinary tract infection (21). Remarkably, RfaH has also been shown to stimulate translation by binding to protein S10 of the 30S ribosomal subunit (22). Unlike NusG, both activities of RfaH require the presence of cis-acting ops (operon polarity suppressor) sites in the DNA that serve to recruit RfaH to a paused RNA polymerase (RNAP) transcription elongation complex (13,23). The ability of RfaH to switch between transcriptional regulator and translational activator involves an unprecedented refolding of the RfaH carboxy-terminal domain (RfaH-CTD) from an all-␣ to all-␤ confirmation, enabling RfaH to bind to ribosomal protein S10 (13).
fimB, which has a sigma 70 promoter, has a large (271-bp) 5= untranslated region (5= UTR), suggesting that the recombinase gene may be subject to extensive control following transcription initiation (24,25). While this possibility has yet to be investigated in detail, fimB expression has been shown recently to be inhibited by the E -dependent small RNA (sRNA) MicA (26). Here, we report that fimB expression is also enhanced by RfaH and that this effect requires MicA.

MATERIALS AND METHODS
Bacterial strains and plasmids. The bacterial strains used in this work are listed in Table 1. All bacterial strains are derivatives of E. coli K-12. To combine mutant fimB alleles with a FimB-LacZ protein fusion, PCR-generated mutant DNAs were first cloned into plasmid pIB347, a derivative of the temperature-sensitive vector pMAK705 (27), to replace the wild-type EcoO109I-SphI (⌬1 mutation) or SphI-ClaI (⌬2 and ⌬3, OLE mutations) regions. Likewise, to combine the OLE mutation with the fimB-lacZ transcriptional fusion, the mutant DNA was cloned into plasmid pIB342 to replace the SphI-ClaI region. Allelic exchange was then used to transfer the mutations from the plasmids into the chromosomal fim region of strain BGEC043 or KCEC840 using sacB and sucrose counterselection as described previously (28). A micA-lacZYA fusion replacing the wild-type micA gene was constructed by inserting an XbaI fragment containing the promoterless lacZYA genes isolated from plasmid pIB341 into a micA vector (pAM011) to generate pAM012. The micA-lacZYA construct was then transferred into the genome by allelic exchange between pAM012 and strain AAEC100 (MG1655 ⌬lacZYA) (28). Strain AAEC189 (⌬fim) was used as the host strain for recombinant plasmids to ensure that the DNA was suitably methylated to allow subsequent transformation of the strain MG1655 (23). P1 transduction was carried out using P1 vir as described previously (29).
Media and growth conditions. The media included L broth (5 g of sodium chloride, 5 g of yeast extract, and 10 g of tryptone per liter [Difco]) and L agar (L broth with 1.5% agar [Difco]). Sucrose agar, used to select recombinant bacteria (13), is L agar supplemented with 6% sucrose in the absence of sodium chloride. The antibiotics chloramphenicol (25 g/ml) and kanamycin (25 g/ml) were included in selective media as required (Sigma). Lactose MacConkey plates (BD) were used as an indicator medium to determine the proportion of switch-on to switch-off bacteria. For rich defined (RD) medium, minimal MOPS [3-(N-morpholino)propanesulfonic acid] medium was prepared as described by Neidhardt et al. (30). The media were supplemented with 10 mM thiamine, 0.4% glycerol, bases, vitamin B supplement, and amino acids as reported originally by Neidhardt et al. (30). In experiments that included an rseA mutant control, the medium was supplemented with 1 mM nicotinic acid. All re- agents were obtained from Sigma unless otherwise indicated. Liquid cultures were grown aerobically at 37°C, and culture densities were monitored spectrophotometrically at 420 or 600 nm. Analysis of fimB and fimE expression. fimB and fimE expression was measured using a FimB-LacZ or FimE-LacZ translational fusion or fimB-lacZ transcriptional fusion situated in the chromosome at fim as described previously (6,7,31). ␤-Galactosidase assays were conducted as described by us previously (31), following growth in RD medium at 37°C with rapid aeration to an optical density at 600 nm (OD 600 ) of approximately 0.2. Experiments were repeated at least twice, and the values shown in Miller units represent the mean of at least four samples with 95% confidence intervals included for each value.
Determination of inversion frequencies. Inversion of the fim switch was measured following growth in RD medium as described previously (32). Inversion frequencies were measured by inoculating 25 cultures with approximately 0.3 cells per tube. The ratio of on to off cells was estimated by plating at least five replicates onto lactose-MacConkey indicator medium after approximately 22 generations of growth at 37°C with rapid aeration.
DNA manipulations. DNA manipulations were carried out using standard protocols (33). Plasmid DNA was isolated using the miniprep or midiprep kit (Qiagen). Restriction enzymes and DNA ligase were pur-chased from either Promega or New England BioLabs. DNA sequencing was performed by Source BioScience, Nottingham, United Kingdom. Oligonucleotide synthesis was performed by MWG-Biotech AG or by Qiagen Operon, Germany. DNA was amplified by PCR using Pwo polymerase (Boehringer Mannheim) as described previously (25) or Q5 master mix (New England BioLabs). Oligonucleotides used in this study are listed in Table S1 in the supplemental material.

RfaH is a positive regulator of fimB expression.
To determine if RfaH controls fimB expression, a ⌬rfaH mutant of strain BGEC905 (MG1655 ⌬lacZYA FimB-LacZ) was constructed by P1 vir transduction. The level of ␤-galactosidase produced by the mutant was diminished by almost 3-fold relative to the wild type in this FimB-LacZ protein fusion background (Fig. 1). FimB recombination parallels fimB expression closely, and FimB recombination was also diminished around 9-fold in the rfaH mutant as anticipated (Fig. 2) (6,7,34).
In contrast to the FimB-LacZ fusion, expression of a comparable fimB-lacZYA operon fusion increased to a modest extent (ϳ1.2-fold). Likewise, expression of a FimE-LacZ protein fusion also increased slightly (ϳ1.2-fold; data not shown). As a control, the ⌬rfaH-fimB mutant phenotype was complemented by an ectopic copy of the rfaH gene inserted into the chromosome at lac (data not shown). As an additional control, the effect of SlyA on the ␤-galactosidase produced by the two fusions was also measured (Fig. 1). SlyA activates fimB transcription by inhibiting H-NS repression, and its loss decreased expression of both fusions as expected (34). Thus, RfaH affects fimB expression mainly at the posttranscriptional level, to produce a net stimulatory effect on fimB expression.
Identification of a region of the fimB 5= UTR required for RfaH control. In addition to its effects on transcription termination, RfaH stimulates translation initiation by binding to protein S10 of the 30S ribosomal subunit (22). This suggested to us that RfaH might activate fimB translation directly. Alternatively, we supposed that RfaH could activate fimB expression indirectly by  Direct control by RfaH requires a cis-acting ops (operon polarity suppressor; consensus of 5= R GGCGGTAGYNT) site downstream of the transcriptional start site, typically positioned far upstream of the translational start site (35,36). fimB has a large (271-bp) 5= UTR, and to screen for cis-active sequences required for RfaH control, three deletions were constructed in this region and transferred into the chromosome at fim to replace the wildtype regulatory region of the FimB-LacZ fusion (Fig. 3). The first deletion (⌬1) extends from immediately adjacent to the Ϫ10 region of the fimB promoter (Ϫ277 bp to Ϫ203 bp relative to the fimB open reading frame [ORF]) to an SphI restriction endonuclease site. The second (⌬2) and third (⌬3) deletions extend from the SphI site to Ϫ58 bp and Ϫ20 bp relative to the fimB ORF, respectively. The best match (5=AAGGGA) to the consensus Shine-Dalgarno sequence (5= AGGAGG) extends from Ϫ12 bp to Ϫ7 bp relative to the fimB ORF.
All of the deletion mutations increased the expression of the FimB-LacZ fusion to a greater or lesser extent, suggesting that the long intergenic region of fimB has a detrimental effect on fimB expression overall (Fig. 4). While the ⌬1 mutation increased the response to RfaH considerably (3-fold in the wild type to 7-fold in the mutant), the ⌬2 mutation decreased the response to RfaH to 2-fold and the ⌬3 mutation eliminated it almost entirely.
Effect of RfaH on MicA inhibition of fimB expression. The E -controlled regulatory sRNA MicA inhibits fimB expression (26). The mRNA binding target for MicA lies immediately upstream of the fimB Shine-Dalgarno sequence (extending from Ϫ9 to Ϫ46) and is thus conserved in its entirety in the ⌬2 mutation but absent from the ⌬3 mutation (Fig. 3). These observations suggested to us that RfaH might activate fimB expression by somehow preventing MicA inhibition. Although MicA surprisingly had a net stimulatory effect on fimB expression in the wild-type background, mutation of micA nevertheless suppressed the stimulatory effect of RfaH on fimB expression entirely (Fig. 5). As a control, the effect of RseA on fimB expression was also tested. RseA prevents induction of the E regulon by sequestering the sigma factor to the inner membrane (37). As expected, fimB expression was inhibited strongly in the rseA mutant background and this effect was also suppressed in an rseA micA double mutant. The results of these experiments thus support the conclusion that RfaH somehow prevents MicA inhibition of fimB expression.
Loss of RfaH induces micA and rpoHP3 expression. The results described above suggested that RfaH might exert an indirect effect on fimB expression by controlling micA expression. To test this hypothesis, the effect of both RfaH and RseA on expression of a micA-lacZ transcriptional fusion was determined. The results of this experiment (Fig. 6) demonstrate that loss of RfaH results in the induction of micA transcription, albeit to a lesser extent than that observed in the rseA mutant background (Ͼ5-fold versus Ͼ13-fold).
We supposed either that RfaH could activate micA expression directly or that, more likely, its loss leads to induction of the E regulon in general. To distinguish between these possibilities, the effect of RfaH on expression of the E -specific rpoHP3 promoter (38) was also tested (Fig. 7). As expected, expression of the rpoHP3-lacZ construct was induced significantly by mutation of rseA (27-fold). Mutation of rfaH had a smaller (10-fold) effect, consistent with the more modest effect of RfaH on both fimB and micA expression.
Notwithstanding the results described above, it still seemed possible that RfaH might also exert a direct effect on fimB expression by binding to an ops-like element in the fimB 5= UTR. This seemed plausible because the ⌬2 mutation, which removes a significant part of the 5= UTR, diminished the effect of RfaH on fimB expression (Fig. 4). Moreover, a search of the 5= UTR of fimB highlighted a potential ops-like site (5= TGGCGTTTGTAT; nonops-matching bases underlined) positioned around 180 bp upstream of the fimB translational start (Fig. 3). This ops-like sequence (here termed OLE for ops-like element) lies 8 bp downstream of the SphI site present in the fimB 5= UTR and hence is deleted in both the ⌬2 and ⌬3 mutants. However, the effect of rseA on fimB expression was also decreased from Ͼ10-fold in the wild-type background to Ͻ4-fold in the ⌬2 mutant background, suggesting that the ⌬2 mutation diminishes the effect of MicA on fimB expression (data not shown). Moreover, while mutation of OLE from 5= TGGCGTTTGTAT to TCCGCTATCAAT did decrease fimB expression Ͼ8-fold, this effect did not require RfaH (data not shown). Furthermore, the OLE mutation also decreased the expression of the fimB-lacZ transcription fusion (data not shown), which mutation of rfaH does not (Fig. 1). We thus conclude that loss of RfaH leads to induction of E , and hence micA, and that this effect accounts for most, if not all, of the stimulatory effect of RfaH on fimB expression.
The effect of procaine on fimB expression and FimB recombination. E , and hence MicA, is induced by procaine and by  ethanol (39,40). As noted above, although MicA had a net stimulatory effect on fimB expression under noninducing conditions (Fig. 5), inclusion of increasing amounts of procaine (Fig. 8) or of ethanol (data not shown) led to a dose-dependent decrease in fimB expression as expected. FimB recombination was also inhibited by procaine as anticipated (Fig. 9). Procaine induces E by activating the EnvZ-OmpR two-component regulatory system (39). In agreement with this assertion, the effect of procaine on fimB expression was also diminished in an ompR mutant background. Moreover, the level of fimB expression in a micA ompR double mutant was indistinguishable from that in the ompR single mutant background (data not shown).
Surprisingly, inhibition of fimB expression by both procaine (Fig. 8) and ethanol (data not shown) was still apparent, albeit to a decreased extent, in the micA mutant background. Unexpectedly, RfaH actually inhibited fimB expression in the absence of MicA under E -inducing conditions. Further work demonstrated that procaine also inhibits expression of the fimB-lacZ transcrip-tional fusion and that this effect requires RfaH (Fig. 10) but not OmpR (data not shown).
The results described above suggest that procaine might trigger an alternative envelope stress response pathway that is also controlled by RfaH. According to this scenario, we supposed that both procaine and RfaH activate the pathway and that the cognate regulator of the system inhibits fimB transcription. It has been shown previously that fimB transcription is controlled by the response regulator RcsB that forms part of the Rcs phosphorelay system, a regulatory pathway that is also responsive to envelope stress (41). However, it was found that fimB transcription was unaffected by RcsB under the growth conditions used in this study and that the effect of procaine on the expression of the fimB-lacZ transcriptional fusion remained intact in this mutant background (data not shown). In contrast, the response of fimB transcription to procaine was found to be dependent upon SlyA (Fig. 10). Furthermore, the inhibitory effect of RfaH on fimB transcription was enhanced in the slyA mutant background.

FIG 5
The effects of micA, rfaH, and micA rfaH double mutations on the ␤-galactosidase produced by a FimB-LacZ fusion. The wild-type (wt) and mutant strains indicated were grown and processed as described in Materials and Methods, except that the growth medium used contained 1 mM nicotinic acid to allow growth of the rseA mutants which contain a linked nadB::Tn10 mutation.

FIG 6
The effects of rfaH and rseA on the ␤-galactosidase produced by a micA-lacZ fusion. The wild-type (wt) and mutant strains indicated were grown and processed as described in Materials and Methods, except that the growth medium used contained 1 mM nicotinic acid to allow growth of the rseA mutants which contain a linked nadB::Tn10 mutation.

FIG 7
The effect of rfaH and rseA on the ␤-galactosidase produced by a rpoHP3::lacZ fusion. The strains indicated were grown and processed as described in Materials and Methods, except that the growth medium used contained 1 mM nicotinic acid to allow growth of the rseA mutants which contain a linked nadB::Tn10 mutation.
The results presented above are consistent with our model that RfaH activates fimB expression by preventing MicA inhibition. However, they also suggest that RfaH can, at least in the presence of procaine, somehow inhibit fimB transcription by a mechanism that involves neither OmpR nor RcsB but which does require SlyA. These results highlight the complexity of the RfaH regulatory circuit that controls fimB expression.

DISCUSSION
RfaH-binding ops elements are characteristically found in long 5= UTRs, far upstream of ORFs. Moreover, UPEC mutants lacking rfaH are attenuated for virulence in an ascending mouse model of urinary tract infection (21). Since fimB has a relatively large (271bp) 5= UTR and type 1 fimbriation is a virulence factor in the mouse model, we considered it possible that RfaH is an activator of fimB expression. Here, we demonstrate that RfaH does indeed enhance fimB expression, but further analysis reveals that this effect is indirect.
In support of the hypothesis that RfaH enhances fimB expression, it was found in an initial experiment that expression of a FimB-LacZ protein fusion was diminished around 3-fold in an rfaH deletion mutant. Surprisingly, however, expression of a comparable fimB-lacZ transcriptional fusion was elevated slightly in the absence of RfaH, indicating that RfaH activates fimB expression selectively at the posttranscriptional level. Moreover, deletion analysis of the fimB 5= UTR indicated that, rather than requiring sequences far upstream of the fimB ORF, RfaH control is dependent upon sequences close to the ribosome binding site. Further work demonstrates that RfaH activates fimB expression indirectly by controlling induction of E and hence the sRNA MicA (Fig. 11).
In addition to controlling the expression of a number of virulence factors, RfaH enhances expression of the waaQ operon required for LPS core biosynthesis (15,16). Alterations to LPS core biosynthesis, apparently by inducing misfolding of outer membrane proteins, can also induce E (42). While not proven here, we postulate that induction of E in the rfaH mutant reflects the involvement of RfaH in LPS biosynthesis (Fig. 11). We note that the increased autoaggregation factor antigen 43 (Ag43)-enhanced biofilm formation observed in an rfaH mutant background was also attributed in part to changes in LPS biosynthesis (17).
The effect of procaine on E induction, and hence micA expression, was reported previously to be dependent upon OmpR (39). Our results agree with this since the effect of procaine on fimB expression was diminished in an ompR mutant and the level of fimB expression in the micA ompR double mutant was indistinguishable from that of the ompR single mutant across the range of procaine levels (0 to 20 mM) tested (data not shown). Surprisingly, the residual effect of procaine on fimB expression in a micA mutant is largely dependent on RfaH (Fig. 8). Further work shows that procaine inhibits expression of the fimB-lacZ transcriptional fusion as well and that this effect requires RfaH (Fig. 10) but not OmpR (data not shown). We suppose that this additional effect of procaine involves an alternative stress-response pathway and an unidentified transcriptional repressor (Rx) (Fig. 11). RfaH has also been shown somehow to inhibit transcription of flu, which encodes Ag43 (17). This effect, which is due neither to changes in flu phase variation per se nor to altered control by known regulator OxyR or Dam, suggests that fimB and flu transcription may both be repressed by Rx.
The effect of RfaH and procaine on fimB transcription is not dependent upon OLE (data not shown). Moreover, we have also  ruled out involvement of the Rcs phosphorelay system, which has been reported to control fimB expression and which is also responsive to envelope stress (data not shown) (41). On the other hand, the effect of procaine on fimB transcription requires SlyA (Fig. 10). Although these results are open to interpretation, we favor a model in which RfaH is necessary for expression of the alternative stress-response pathway while loss of SlyA leads to its constitutive activation (Fig. 11). We suppose that SlyA activates the expression of an additional unidentified factor (Fx) that somehow alters the signaling pathway to make it responsive to procaine (Fig. 11).
Type 1 fimbriae, which are anchored in the bacterial outer membrane, facilitate the delivery of LPS to the TLR4 signaling pathway in CD14-negative epithelial cells (10). Furthermore, in contrast to phase-locked-off bacteria, MicA inhibits fimB expres-sion in phase-locked-on fimbriate cells even in the absence of exogenous inducers of E (our unpublished data). This suggests to us that fimbrial biosynthesis itself imposes significant stress on the outer membrane, as has been reported for other outer membrane proteins (38). We thus propose that suppressing fimB expression, and hence type 1 fimbriation, in response to the integrity of the bacterial outer membrane is an adaptation that enhances bacterial survival both by diminishing envelope stress and by limiting the host inflammatory response. The fact that RybB, a second Edependent sRNA, inhibits fimA expression would provide an additional mechanism to limit fimbrial expression in phase-on bacteria (26). Indeed, we suppose that this explains why nonfimbriate cells are produced even when fimS is locked in the on phase, or when fimS is replaced with the isopropyl-␤-D-galactopyranoside (IPTG)-inducible tac promoter (43). Mutation of surA also induces E (44), and as expected, we have found that fimB expression was also decreased in a surA mutant (data not shown). While SurA enhances the correct localization of the fim usher (FimD) to the outer membrane, our results indicate that decreased fimB expression, and hence off-to-on inversion of fimS, as well as induction of rybB, also contributed to diminished type 1 fimbriation observed in a surA mutant (45).
A BLAST search of the nucleotides of the fimB mRNA predicted to bind to MicA demonstrates that these sequences are perfectly conserved in all of the E. coli strains for which DNA sequence data are available. Thus, the regulation of fimB expression by MicA, and its control by factors such as RfaH and SurA, is likely to have general significance for the control of type 1 fimbriation in most, if not all, E. coli strains. Further work will determine how RfaH controls E , as well as the response of fimB transcription to procaine, and the full extent of these control pathways on type 1 fimbriation in commensal and pathogenic strains alike.

ACKNOWLEDGMENTS
We thank Carol Gross, Monica Gogol, Simon Andrews, and Ian Stansfield for providing some of the strains used in this study. We also thank Hanna Esser and Elise Green for technical assistance.
We thank the Nuffield Foundation for their financial support. I.C.B., L.S., and K.G. were supported by grant 085931/Z/08/Z from the Wellcome Trust, and A.M. and L.W. were supported by postgraduate studentships from the University of Kent. Procaine was included at the concentrations (0 to 20 mM) specified. The wild-type (wt) and mutant strains indicated were grown and processed as described in Materials and Methods.  (37,46). (b) Procaine (Pc) activates EnvZ/OmpR to also induce E and hence micA expression (39). (c) Mutation of rfaH also leads to changes in LPS core biosynthesis that cause misfolding of outer membrane proteins and hence induction of E and hence micA expression. (d) Procaine activates an alternative envelope stress pathway (Sx-Rx). (e) RfaH is postulated to inhibit fimB transcription indirectly by activation expression of Sx and/or Rx. (f and g) SlyA enhances fimB expression directly (34) (f) but is also postulated (g) to enhance expression of an unknown factor (Fx) that prevents Sx-Rx signaling in the absence of procaine. (h) FimB catalyzes off-to-on inversion of fimS to enhance expression of type 1 fimbriae (T1F). (i) Type 1 fimbriae stimulate inflammation by enhancing LPS-activated host signaling pathways (10)(11)(12). Stimulatory and inhibitory interactions are indicated by arrows and diamonds, respectively. Dotted lines represent speculative pathways. OM, outer membrane; IM, inner membrane.