Vibrio cholerae, which causes the diarrheal disease cholera, is a species of bacteria commonly found in aquatic habitats. Within such environments, the bacterium must defend itself against predatory protozoan grazers. Amoebae are prominent grazers, with Acanthamoeba castellanii being one of the best-studied aquatic amoebae. We previously showed that V. cholerae resists digestion by A. castellanii and establishes a replication niche within the host's osmoregulatory organelle. In this study, we decipher the molecular mechanisms involved in the maintenance of V. cholerae's intra-amoebal replication niche and its ultimate escape from the succumbed host. We demonstrate that minor virulence features important for disease in mammals, such as extracellular enzymes and flagellum-based motility, have a key role in the replication and transmission of V. cholerae in its aqueous environment. This work, therefore, describes new mechanisms that provide the pathogen with a fitness advantage in its primary habitat, which may have contributed to the emergence of these minor virulence factors in the species V. cholerae.
The diarrheal disease cholera is not extinct. Seven cholera pandemics have been recorded in modern history and the latest is still ongoing1,2. Cholera is caused by ingestion of the bacterium Vibrio cholerae. Toxigenic strains of this species are capable of damaging the host due to the presence of so-called virulence factors, which refers "to the elements (i.e., gene products) that enable a microorganism to colonize a host niche where the organism proliferates and causes tissue damage or systemic inflammation"3. Bacterial strains without these factors are usually attenuated with respect to the infection process.
For V. cholerae, the two major virulence factors, cholera toxin and the toxin-coregulated pilus, have a pivotal role in the infection, but additional minor virulence factors have also been identified. These include factors such as outer membrane porins, a pore-forming hemolysin, diverse proteases, N-acetyl-glucosamine binding protein, flagellum-based motility, to name a few4,5,6,7. The hemagglutinin/protease (HapA), for example, is a zinc-metalloprotease8, which was first identified due to its mucinase activity9. This enzyme was later demonstrated to not only cause hemagglutination but to also hydrolyze fibronectin, mucin, and lactoferrin, all of which were thought to contribute to the host defense against V. cholerae10. HapA also causes cell rounding, loss of the barrier function of the epithelial layer, and, ultimately, detachment of cells under tissue culture conditions11,12. Consistent with these in vitro activities, a tenfold increase in the 50% lethal dose in the absence compared to the presence of the HapA protease for V. cholerae strains that otherwise lack cholera toxin was reported13.
A second well-characterized minor virulence factor that is widespread among Vibrio species14 is hemolysin (HlyA), which is also known as V. cholerae cytolysin or vacuolating toxin. Hemolysin is a secreted protein belonging to the family of pore-forming toxins (PFTs), as it forms aqueous channels in host cells in vitro15. In vivo data by Ichinose et al.16 showed that purified hemolysin induced intestinal fluid accumulation in rabbits and in orally inoculated suckling mice, two commonly used animal models of cholera. The authors therefore concluded that hemolysin was an enterotoxic factor that contributed to gastroenteritis caused by non-pandemic V. cholerae strains16. The role of HlyA as enterotoxic or diarrheagenic factor, especially in pandemic isolate-derived vaccine strains lacking cholera toxin, was later confirmed by Alm et al.17 who also demonstrated that mice infected with hemolysin-deficient V. cholerae survived longer than those inoculated with their hemolysin-positive parental strains. More recent studies using streptomycin-treated adult mice and pandemic isolates of V. cholerae showed that hemolysin was the major cause of lethality in this animal model of cholera18. Moreover, it has been suggested that HlyA and other secreted accessory toxins modify the host environment, thereby contributing to prolonged disease-free colonization that could contribute to disease transmission via asymptomatic carriers19.
It was proposed that hlyA might be located on a pathogenicity island due to the close proximity of additional genes that likewise encode extracellular enzymes20. This region encodes apart from hemolysin (hlyA) a lipase (lipA), a metalloprotease (prtV), and a lecithinase/phospholipase (lec; also known as thermolabile hemolysin). The lecithinase activity of the latter enzyme was characterized21,22, but molecular details and its concrete function are still unknown.
To date, a limited number of studies have shown that minor virulence factors, such as those mentioned above, are also advantageous in an environmental context23,24. Indeed, despite V. cholerae being an aquatic bacterium that is well adapted for survival in this environment, the molecular details about V. cholerae's environmental lifestyle are still lacking. To address this knowledge gap, we aimed to elucidate the molecular mechanisms that V. cholerae would use as part of its environmental lifestyle, such as its interactions with grazing amoebal predators. In this context, we recently demonstrated that V. cholerae survives predation by Acanthamoeba castellanii, a free-living amoebal species25. This amoeba is frequently encountered in aquatic reservoirs and follows a bi-phasic lifecycle: the motile trophozoite form, which grazes on bacterial prey, and the double-walled resistant cyst stage, which forms under harsh conditions26. We previously showed that V. cholerae interacts with A. castellanii through the evolution of two distinct phenotypes. Firstly, upon phagocytosis by the feeding amoebal trophozoite, a fraction of the ingested V. cholerae population can resist amoebal digestion, then exit the phagosomal pathway by exocytosis. Notably and in contrast to a previous study27, we never observed free V. cholerae cells in the cytosol of such trophozoites25, indicating that the bacteria are not released from food vacuoles intracellularly. Secondly, the pathogen colonizes the amoeba's contractile vacuole (CV), which is its osmoregulatory organelle. This organelle expands and contracts in a rhythmic manner based on the accumulation and expulsion of excess water from the cytosol of the cell28. While there is still a significant knowledge gap with respect to the detailed mechanisms underlying its osmoregulatory function, it is currently thought that water enters the CV due to a ~1.5 times higher osmolarity of the CV fluid compared to that of the cytosol29. The water discharge process, on the other hand, is based on the fusion of the CV and the plasma membrane, a process that is actively driven by myosin-I30. This discharge process is essential, as interference with the water expulsion leads to an overfilling of the CV and, ultimately, amoebal cell lysis30. With respect to V. cholerae, we recently showed that the bacterium enters this organelle through vacuolar fusion of V. cholerae-containing food vacuoles with the CV. This creates a replication niche for the pathogen in which V. cholerae is maintained even upon amoebal encystement, before it ultimately lyses the host25. However, the underlying molecular mechanisms of this host–pathogen interaction remained elusive and were therefore addressed in the current study. We show that V. cholerae fine-tunes extracellular enzymes to avoid premature intoxication of its host, allowing the bacterium to take full advantage of the intra-amoebal replication niche for undisturbed and non-competitive proliferation before finally lysing its amoebal host. We also demonstrate that flagellum-based motility is of importance for V. cholerae to ultimately escape the ruptured host niche to return to the aquatic environment.
Using confocal laser scanning microscopy, we recently demonstrated that V. cholerae could enter the CV of A. castellanii and replicate within this niche25, though underlying molecular mechanisms of this host–pathogen interaction remained largely unknown. This is partially due to the low throughput of transmission electron microscopy (TEM), which only allowed for the imaging of hyper-colonizing V. cholerae mutant strains, due to their lack of quorum-sensing, while spatial information on wild-type (WT) bacteria within the amoebal vacuoles remained missing25. To circumvent this problem, we developed a correlative light and electron microscopy (CLEM) imaging strategy, which allowed us to study this specific host–pathogen interaction for WT V. cholerae for the first time at high resolution. We identified V. cholerae-infected A. castellanii cells by pathogen-produced green fluorescent protein GFP and then targeted these cells for TEM. Using this method allowed us get information on the morphology of the colonizing bacteria and their spatial distribution. As shown in Fig. 1, the morphologies of such intra-vacuolar WT bacteria possessed the typical Vibrio shape. Moreover, the membrane surrounding the CV was largely intact, which was consistent with the non-encysted phenotype of the amoeba (Fig. 1a).
We estimated the number of objects—in this case, bacteria—in the CV using an unbiased stereological approach31. To do this, a representative WT V. cholerae-colonized amoeba, at 20 h post-primary contact (p.p.c.), was identified in the fluorescent channel. After sample preparation and imaging, the number of bacteria in the vacuole was estimated to be 194 (±60). This number, and the apparent density of bacteria in the 3D reconstruction (Fig. 1b and Supplementary Movie 1), correlates well with the active growth of V. cholerae in this organelle, as the vacuole's initial colonization only involved one or a few bacteria25.
As the 3D reconstruction suggested that V. cholerae cells were densely packed within the vacuole at this later point of the infection, we wondered how the bacteria eventually escaped the vacuole after amoebal encystation and how the timing was properly regulated. To answer these questions, we first took an educated guess approach, which has its limitation but turned out as useful in this study. We therefore tested diverse knockout mutants of V. cholerae for their intra-amoebal behavior. We were especially interested in strains that lacked certain extracellular enzymes, as some of those were known to be important in the pathogen's intra-human lifestyle and its transmission to new hosts.
We challenged A. castellanii with a HapA-deficient V. cholerae strain (Supplementary Table 1) and compared the amoebal response to the WT-challenged condition. By doing this, we observed that a vast majority (79.8%) of infected amoebal cells showed an aberrant morphology upon co-incubation with the hapA-minus strain for 20 h, which occurred significantly less often for WT-infected amoebae (11.6%, Fig. 2a, b). These morphological abnormalities ranged from a shrinking or compacting phenotype towards pseudopodia retraction and detachment, all of which ultimately abolished amoebal grazing (Fig. 2 and Supplementary Fig. 1). Complementation assays, involving a genetically engineered derivative of the hapA-minus strain with a new copy of hapA on the large chromosome (Supplementary Fig. 2), were performed using the amoebal infection protocol and fully restored the WT-infected amoebal morphologies (Fig. 2a, b).
Seeing these striking differences between WT-infected and hapA-minus-infected amoebae, we wondered whether the latter might cause "amoebal constipation", meaning the accumulation of undigested phagosomal V. cholerae without efficient exocytosis. Indeed, based on the confocal microscopy images, we could localize the accumulated bacteria due to their green fluorescence. However, recognition of the CV in the transmitted light channel was often difficult for hapA-minus mutant-infected amoebae due to their compaction and malformation (Fig. 2a). Therefore, it seemed possible that the bacteria were contained in digestive food vacuoles of the endosomal pathway, blocking amoebal digestion as opposed to being localized to the CV, as is the case for WT V. cholerae. To distinguish between both scenarios (localization within a digestive vacuole or within the CV), we labeled the amoebal endosomal pathway with fluorescently labeled dextran. As the dextran- and bacteria-derived fluorescent signals did not overlap, these experiments confirmed the intra-CV localization of the hapA-minus strain (Fig. 2c).
Next, we wondered whether, despite these unusual amoebal morphologies, the lifecycle between hapA-minus V. cholerae and A. castellanii would proceed as previously reported for the WT25. Such a progression included amoebal encystation followed by the escape of V. cholerae from the CV into the cyst's cytosol, leading, ultimately, to lysis of the amoebal host. To determine this, we imaged infected amoebae at a later time point (30 h p.p.c.) to allow the lifecycle progression to develop. To better distinguish aberrantly rounded cells from cysts, we used calcofluor staining, as this fluorescence stain is known to bind to the deposited cellulose cell wall layer in Acanthamoeba cysts32. As shown in Fig. 2d (and quantified in Fig. 2e), the ability to form cellulose-positive cysts was severely impaired in amoebae infected with the hapA-minus mutant strain. This impaired encystation contrasted greatly to that of both the WT-infected A. castellanii and the complemented hapA-minus strain (Fig. 2d, e). We therefore concluded that the absence of the HapA protease leads to premature amoebal intoxication and, consequently, a defect in the encystation process.
To better understand the defect in the encystation process, we again used CLEM to obtain high-resolution images of colonized amoebae. The WT-infected CVs seemed to maintain their internal pressure, leading to a clear separation between the content of the CV and the amoebal cytosol (Figs. 1a and 2f), though this evident distinction was lacking in host cells that were infected by the mutant V. cholerae strain, which showed signs of impaired membrane integrity (Fig. 2f).
We speculated that the seemingly collapsed CV membranes might have been due to amoebal membrane rupture caused by the hapA-minus mutant strain. Given that bacterial pathogens often cause membrane rupture through the secretion of PFTs33, we wondered whether the secreted hemolysin of V. cholerae (HlyA) might be involved in the observed amoebal intoxication. Consistent with this idea was a study by Tsou and Zhu34 that showed that the HapA protease degrades HlyA in an in vitro assay. This led us to hypothesize that HapA protease-deficient strains of V. cholerae would display enhanced hemolysis, which, ultimately, could cause the observed amoebal intoxication. To test this hypothesis, we generated several V. cholerae strains that lacked hapA, hlyA, or both genes simultaneously (Supplementary Table 1) and tested them for hemolysis and proteolysis on blood and milk agar plates, respectively. As shown in Supplementary Fig. 2, there was a strong increase in hemolysis in strains lacking hapA, while the absence of hlyA fully abolished this activity. Complementation assays, in which the mutant strain contained a new copy of the missing gene elsewhere on the chromosome (Supplementary Table 1), restored the system to that of the WT (Supplementary Fig. 2a and b). Interestingly and consistent with the extracellular localization of both enzymes, we showed that the secreted HapA protease from a WT strain was also able to inactivate HlyA that was released by a co-cultured hapA-minus mutant strain. However, when we engineered an hlyA-overexpression strain (Supplementary Table 1), the protease activity exerted by the strain itself or from the co-cultured WT bacteria was insufficient to abolish hemolysis (Supplementary Fig. 2).
With these newly constructed strains in hand, we then tested their effect on A. castellanii. Consistent with our hypothesis that a hapA mutant of V. cholerae would possess enhanced hemolysin activity, which, ultimately, would result in amoebal intoxication, we found that the absence of hlyA in the protease-minus background restored normal host morphology and cyst formation (Fig. 3a, b and Supplementary Fig. 3a–d). Infection of A. castellanii by the V. cholerae double-mutant (ΔhapAΔhlyA) also resulted in an intact integrity of the CV membrane (Fig. 3c and Supplementary Fig. 3e). Overproduction of HlyA in a WT background strain, however, fully phenocopied or even aggravated the protease-minus phenotypes (Fig. 3a, b and Supplementary Fig. 3a–d). These data, therefore, support the notion that HlyA is causing amoebal intoxication and that the HapA protease counteracts this effect.
To determine whether HlyA impacts the amoebae through activity inside of the CV instead of in the surrounding medium, we co-infected A. castellanii with WT V. cholerae (labeled with dsRed) and either a second WT strain as a control or, alternatively, the hapA-minus strain or the HlyA-overexpression strain at a 1:1 ratio (all labeled with GFP). We then imaged the amoebae and quantified the number of colonized ones that showed either a normal or aberrant morphology (Supplementary Fig. 4). This experiment suggested that the secreted protease in the WT strain rescues the amoebae from intoxication by protease-minus and, therefore, HlyA-producing V. cholerae if both strains occurred within the same CV (Supplementary Fig. 4b and Supplementary Table 2). This rescuing phenotype was not observed when the second strain was the HlyA-overproducing strain (Supplementary Fig. 4c and Supplementary Table 2). Amoebae containing mono-colonized CVs showed the same phenotype as we had observed in the single strain infection experiments (Supplementary Fig. 4 compared to Figs. 2 and 3). These in vivo observations are, therefore, fully consistent with the in vitro hemolysin activity described above and suggest that both enzymes are present and active inside the amoebal CV.
To test whether these HapA-related and HlyA-related phenotypes are conserved in V. cholerae strains, we repeated the above-described experiments with diverse pandemic and non-pandemic V. cholerae isolates. First, we compared the well-studied pandemic O1 El Tor isolate A155235, which we primarily use in our laboratory, with two other O1 El Tor strains, namely C6706 and N16961 (Supplementary Table 1). Strain A1552 was isolated in California from a traveller returning from South America36, which links this strain to the Peruvian cholera outbreak in 1990s. V. cholerae strain C6706 was isolated in Peru in 1991. Notably, as we recently showed that a laboratory-domesticated version of this strain with a severe quorum-sensing defect has spread amongst V. cholerae laboratories around the globe37, we decided to work with an old stock of strain C6706 (named C6706-original in this study; gift from J. Mekalanos, Harvard). We also included the first sequenced V. cholerae strain N1696138, which was isolated in Bangladesh in 1975. This strain carries a frameshift mutation in the gene encoding the major regulator of quorum sensing HapR39, which we genetically repaired (named N16961-hapRRep in this study; Supplementary Table 1). As shown in Supplementary Fig. 5, all three O1 El Tor pandemic isolates behaved similarly in our amoebal infection assay and confirmed the hemolysin-mediated toxicity of the hapA-minus variants. We also extended our analysis to the non-pandemic V. cholerae strain Sa5Y, an isolate from the California coast40. Interestingly, this strain exerted enhanced hemolysis in vitro compared to the pandemic isolates (Supplementary Fig. 5a) and therefore phenocopied the hapA-minus mutants of the pandemic isolates with respect to its toxicity towards A. castellanii (Supplementary Fig. 5c). Consistent with what we described above for the pandemic isolates, this toxicity towards the amoeba was almost completely abrogated in hemolysin-deficient mutant derivatives (Supplementary Fig. 5c).
As the experiments above suggested that the PFT hemolysin intoxicates amoebal trophozoites, we wondered if two other PFTs of V. cholerae, RTX and VasX, would result in similar outcomes. RTX is a >4000 amino acid protein, which belongs the family of multifunctional-autoprocessing repeats-in-toxin (MARTX) toxins. Pore formation by RTX serves to translocate effector domains of this large protein across the plasma membrane of eukaryotic cells41. Notably, an earlier study suggested that the RTX protein of V. cholerae does not impair the membrane integrity of eukaryotic cells (e.g., HEp-2 cells)42, while a more recent study showed that the RTX toxin of Vibrio vulnificus caused pores in human erythrocytes43. To test whether RTX contributes to V. cholerae's lifecycle within A. castellanii, we engineered an rtxA-minus strain of V. cholerae (Supplementary Table 1) and compared its behavior to the WT control. As shown in Fig. 4a, no difference in bacterial localization was observed when these two strains were compared, indicating that RTX is not required for V. cholerae to establish its replication niche within the amoeba's CV.
The third PFT of V. cholerae, VasX, is transported into eukaryotic and prokaryotic cells by the type VI secretion system (T6SS)44,45,46. The T6SS is a multi-protein complex that delivers effector proteins into adjacent cells in a contact-dependent manner47,48. Interestingly, the T6SS was first discovered in a non-pandemic strain of V. cholerae, strain V52, due to its toxic effect towards the soil amoeba Dictyostelium discoideum49. To test whether VasX or any other T6SS effector protein (such as the lipase TseL or the actin-cross-linking domain of VgrG144,45,46) was involved in V. cholerae's interaction with A. castellanii, we tested a T6SS-defective bacterial mutant that lacked a core structural component of the secretion system (A1552ΔvipA; Supplementary Table 1) in the amoebal infection assay. No change in the bacterial lifecycle was observed for this mutant when compared to its parental WT strain (Fig. 4b).
Notably, pandemic isolates of V. cholerae are known to be T6SS-silent under laboratory conditions49 and instead require specific environmental cues for T6SS induction50,51. We therefore wondered whether the absence of induction would mask potential effects that T6SS-delivered effector proteins could have on A. castellanii. To address this question, we took advantage of V. cholerae strain ATCC25872 (Supplementary Table 1), a toxigenic O37 serogroup strain that is closely related to strain V52 but in contrast to the latter52 quorum sensing proficient53. First, we confirmed its constitutive T6SS production by imaging a derivative of this strain that carries a translational fusion between the T6SS sheath protein VipA and super-folder GFP50,54 (Fig. 4c). Constitutive T6SS activity was further demonstrated in an interbacterial predation assay in which strain ATCC25872, but not its vipA-negative derivative, efficiently killed co-cultured Escherichia coli cells (Fig. 4d). Next, we aimed at challenging amoebae with this T6SS-active strain. However, strain ATCC25872 produced high levels of hemolysin (Fig. 4e), consistent with what was previously shown for strain V5249. To avoid that these high levels of HlyA would mask any T6SS-mediated effects due to premature intoxication of the amoebae, we engineered T6SS-proficient or T6SS-deficient mutants in an hlyA-minus background. As shown in Fig. 4f, no difference was observed between T6SS-active and T6SS inactive strains in the amoebal infection model or when strain ATCC25872 was compared to the same genetically engineered mutants of pandemic strain A1552. We therefore concluded that neither the RTX toxin nor the T6SS contribute to V. cholerae's interaction with A. castellanii under the tested conditions.
While the experiments described above highlighted the essential nature of the HapA protease for reducing the cytotoxicity of V. cholerae towards its amoebal host at early time points, it remained to be discovered how the pathogen eventually escaped from its amoebal host to return to the environment. We reasoned that the pathogen would need to disrupt the host's plasma membrane. The plasma membrane of A. castellanii contains lecithin, which is a mixture of glycerophospholipids that includes a high percentage of phosphatidylethanolamine and phosphatidylcholine55. We therefore speculated that V. cholerae might use its lecithinase22 to disrupt this membrane. We generated a lec-minus strain and confirmed its impaired lecithinase activity on egg yolk plates (Supplementary Fig. 6). We also complemented the lec-minus strain by placing a new copy of lec that was preceded by its native promoter onto the chromosome (Δlec::lec). The same genetic constructs were also added into the WT background to generate a lec-merodiploid strain of V. cholerae (WT::lec). All of the strains were tested for their lecithinase activity in vitro and behaved as expected (Supplementary Fig. 6).
Next, we infected A. castellanii with this set of genetically engineered strains. We observed that while the lec-deficient strains were able to colonize the CV and escape from it in the cyst stage, the bacteria were unable to ultimately lyse the amoebal host, as visualized through the exclusion of extracellular dextran (Fig. 5). The complemented strain had its cyst lysis capability restored, a phenotype that was enhanced for the lec-merodiploid bacteria (Fig. 5). We therefore concluded that the lecithinase enzyme indeed targets and permeabilizes the plasma membrane of the host, thereby triggering the death of the cyst, a phenotype that was conserved in other pandemic and non-pandemic V. cholerae strains (Supplementary Fig. 6c and d).
As we observed that V. cholerae is highly motile within the amoebal host (Supplementary Movie 2), we wondered whether this motility contributed to the pathogen's intra-amoebal lifestyle and its escape from the succumbed host. We therefore generated non-motile mutants of V. cholerae by deleting either the gene that encodes the major flagellin subunit FlaA or the flagellar motor protein PomB (Supplementary Table 1). While the first approach resulted in non-flagellated bacteria, the latter approach led to rotation-deficient but fully flagellated bacteria (Fig. 6a). The motility deficiency of the mutants was further confirmed in in vitro motility assays (Supplementary Fig. 7). Next, we infected A. castellanii with these mutant strains. While both mutant strains still infected the amoebal CV, the strains were more static within this niche (Fig. 6b and Supplementary Movies 3 and 4). We therefore wondered whether motility would have a role in the escape of V. cholerae from the lysed CV and lysed cysts. To test this idea, we infected amoebae with the WT and the flaA-minus strains, which we labeled with different fluorescent proteins, namely dsRed and GFP, respectively. We then took time-lapse movies over several hours to observe the colonizing bacteria of those amoebae that contained both strains within the same CV (Fig. 6c and Supplementary Movie 5). These experiments showed that, compared to the WT strain, the non-motile mutant was retained in the lysed CV (Supplementary Movie 6) and its escape back to the environment was severely impaired after the final lysis of the cyst (Supplementary Movie 7). We concluded, therefore, that motility has a major role in V. cholerae's interaction with A. castellanii.
V. cholerae, the bacterial agent responsible for cholera, still poses a global threat to human health. However, apart from its chitin-induced phenotypes, which include chitin catabolism, interbacterial competition, and horizontal gene transfer50 (reviewed by refs.56,57,58,59), we know very little about its environmental lifestyle and its potential interactions with non-human hosts. Such ancient host–pathogen interactions are, however, often considered as evolutionary precursors to modern interactions that occur between bacteria and their human hosts60. Moreover, aquatic predators are recognized for their contribution to pathogen emergence due to the selection pressure they exert on their bacterial prey61. Here, we examined the molecular mechanisms that V. cholerae uses to interact with the aquatic amoeba A. castellanii and to maintain a favorable replication niche within the amoebal osmoregulatory organelle. Based on the molecular checkpoints that were deciphered in the current study, we expanded our model of the pathogen's intra-amoebal lifecycle (Fig. 7). Specifically, we showed the importance of several extracellular enzymes in this host–pathogen interaction. The production of the HapA protease, which cleaves the pore-forming hemolysin toxin, proved essential for avoiding premature intoxication of the amoebal host (Fig. 7, label A). In contrast to intracellular pathogens, such as Listeria monocytogenes or Shigella flexneri, that use PFTs to escape from acidic vacuolar compartments to reach the host cell's cytosol, the situation described herein is very different. In this case, the host–pathogen interaction relies on V. cholerae residing in a non-digestive vacuole in which it can readily replicate25. This non-digestive CV is an essential osmoregulatory organelle of the amoeba meaning that HlyA-mediated rupture of the vacuolar membrane would, therefore, release V. cholerae into the cytosol, though, at the expense of rapid host cell death. We speculated, therefore, that this HapA-mediated disintegration of HlyA might have evolved to allow V. cholerae to maximize its growth output within this intra-amoebal replication niche by avoiding the premature death of its host. Notably, the HapA protease is considered a minor virulence factor that contributes to disease outcomes in animal models of cholera13,62, along with HlyA itself, as described above. In this study, we did not observe any obvious phenotype for V. cholerae strains lacking HlyA with respect to their ability to first colonize and then escape the amoebal CV. However, at this point we cannot exclude the contribution of HlyA as well as other, potentially redundant, enzymes to the other discussed phenotype in which the V. cholerae pathogen escapes from the A. castellanii phagosomal pathway25. Alternatively, HlyA might have a role in another context in the pathogen's environmental lifestyle. It was shown that HlyA causes developmental delays and lethality in the nematode Caenorhabditis elegans through intestinal cytopathic changes63, and similar effect might occur with other hosts in the pathogen's aquatic reservoirs. Notably, both enzymes (HapA and HlyA) are widely distributed among pathogenic Vibrios14, which reinforces the notion that they have important roles in environmental settings.
While the production of the HapA protease protects the CV from premature lysis, such lysis still occurs when the amoeba has undergone encystation and the CV is fully occupied by the bacteria. We have previously shown that the rupture of the vacuolar membrane is dependent on the production of the Vibrio extracellular polysaccharide (VPS; Fig. 7, label B), and we hypothesized that this might be due to a VPS-dependent agglutination effect25. Consistent with this idea is a recent study on the importance of the osmotic pressure generated by the hydrogel-like VPS matrix of V. cholerae64. These authors suggested that the capacity of V. cholerae to escape the amoebal CV, which we had previously reported25, could indeed be related to the osmotic-pressure response of the VPS biofilm matrix64. Alternatively, the accumulation of the VPS might hinder the CV from contracting and therefore from expelling the accumulated water. Given the continued influx of water from the cytosol such a malfunctioning would likewise lead to the lysis of the vacuole, as observed (ref.25 and this study) and consistent with a previous report in which the authors blocked the vacuole's contractility using antibodies against myosin-I30.
After V. cholerae escaped from the vacuole and upon further growth within the cytosol of the amoebal cyst, another extracellular enzyme, the lecithinase/phospholipase, was required to ultimately kill the host and return the bacteria to the environment (Fig. 7, label E). This lecithinase is encoded in close proximity to the hemolysin gene, a region that was previously speculated to represent a pathogenicity island20. Unfortunately, apart from the absence of a phenotype for the lec-minus mutant in rabbit-ligated ileal loops22, a model of cholera that primarily aims at judging cholera toxin-mediated fluid accumulation, this enzyme had not been extensively studied in vivo (for current 7th pandemic O1 El Tor strains). However, a recent study based on an activity-based protein profiling method showed that the lecithinase/phospholipase is active in cecal fluids of infected infant rabbits65. Further studies are therefore required to conclusively determine whether this enzyme should also be considered as a minor virulence factor for animals, as we demonstrated here for amoebae.
Lastly, we showed that bacterial motility is required to efficiently escape from the lysed CV and the succumbed host (Fig. 7, labels C and D), which, ultimately, allows the bacteria to return to the aquatic environment. One could speculate that the lysed cyst might generate a nutrient gradient that attracts novel predators, a disadvantageous scenario for any prey. Indeed, we frequently observed that dead cysts were readily ingested by feeding trophozoites, which could counter-select for non-motile mutants that cannot escape from the cellular debris. Likewise, motility has also been demonstrated to be of importance for virulence in animal models. Indeed, non-motile mutants exerted less fluid accumulation in rabbit ileal loops62 and were severely attenuated for the colonization of infant mouse small intestines4. These correlations between the contribution to virulence in animal models and in the environmental host–pathogen interaction described herein therefore support the "coincidental evolution hypothesis". This hypothesis suggests that "virulence factors result from adaptation to other ecological niches" and, in particular, from "selective pressure exerted by protozoan predator"66.
All bacterial strains used in this study are listed in Supplementary Table 1. V. cholerae strains are derivatives of the 7th pandemic O1 El Tor strain A1552 if not indicated otherwise. Bacteria were grown on LB plates (1.5% agar) or under shaking conditions in liquid LB medium. Antibiotics were supplemented when needed for genetic engineering and selection at the following concentrations: 100 µg/ml of ampicillin; 50 µg/ml of gentamicin, and 75 µg/ml of kanamycin. Amoebal infection media were free of antibiotics.
For counter-selection of site-directly modified phenylalanyl-tRNA synthetase (pheS*)-carrying strains (Trans2 method; see below) 4-chloro-phenylalanine (cPhe; C6506, Sigma-Aldrich, Buchs, Switzerland) was added to the medium before autoclaving. Optimization experiments showed that a concentration of 20 mM of cPhe was best for V. cholerae counter-selection.
All oligonucleotides used for genetic engineering are listed in Supplementary Table 3. Genetic engineering of strains to delete/insert genes or to add gene fusions onto the V. cholerae chromosomes was performed using the previously described TransFLP method67,68,69,70. An alternative protocol, based on two rounds of transformation, was established in this study (named Trans2) to generate complemented strains or overexpression constructs. This method was adapted from Gurung et al.71 to work in V. cholerae and is based on the counter-selectablility of bacteria carrying a mutated version of the α subunit of phenylalanyl-tRNA synthetase in cPhe-containing medium. The gene encoding the phenylalanyl-tRNA synthetase of V. cholerae (α subunit; pheS or VC1219) was integrated into plasmid pBR-FRT-Kan-FRT251 to generate the plasmid pBR-FKpheSF (carrying the aminoglycoside 3′-phosphotransferase-encoding gene [aph(3′)] and pheS flanked by FRT sites). This and subsequent plasmids served as templates for inverse PCR to generate the site-directly mutated pheS in the pBR-FKpheSF[A294G] and pBR-FKpheSF[A294G/T251A] plasmids (renamed for simplicity as pFRT-aph-pheS*). This double-mutant template was generated based on a study in E. coli that showed improved counter-selectablility of this version of pheS (pheS*) on cPhe-containing agar plates72. In contrast to a previous study71, the pheS* allele used in this study (pheS[A294G/T251A]) was placed downstream of the constitutively expressed aph(3′) to ensure expression independent of the subsequent insertion site on the chromosome. The aph(3′)-pheS* construct was PCR amplified using the pFRT-aph-pheS* plasmid as a template and fused by overlapping PCR with fragments carrying parts of the 5′- and 3′-region of the lacZ gene. The resulting PCR fragment (lacZ'-aph(3′)-pheS*-'lacZ) was used as a transforming material for chitin-induced naturally competent V. cholerae cells68. Transformants were selected on kanamycin-containing LB agar plates. The growth impairment of the transformants due to the presence of pheS* was verified on cPhe-containing agar plates followed by verification through Sanger sequencing. These strains underwent a second round of transformation on chitin (therefore the name Trans2). The second-round transforming material was devoid of any resistance gene and carried the desired genetic construct (generated by PCR amplification) flanked by the 5′- and 3′-parts of the lacZ gene. Screening for the integration of this construct was done using cPhe-containing agar plates. Transformants that grew were tested by colony PCR for the replacement of the aph(3′)-pheS* genes and, ultimately, confirmed for marker- and scar-less integration using Sanger sequencing.
Variants of the mini-Tn7 transposon carrying constitutively expressed gfp, an optimized version of dsRed (dsRed.T3[DNT]50,73), or mCherry were stably integrated into the V. cholerae chromosome through triparental mating74.
Semi-quantitative assays in specific media were used to determine enzymatic activities. To perform these assays, 5 µl of the respective overnight bacterial cultures were spotted onto the following three types of agar plates. (i) Milk agar was used to determine protease activity due to the hydrolysis of casein. The modified recipe used in this study contained 0.5% tryptone, 0.25% yeast extract, 0.1% dextrose, 1% skim milk powder, and 1.25% agar. The bacteria were incubated on these plates for 24 h at 30 °C. (ii) Trypticase Soy Agar II with 5% sheep blood (BD, Heidelberg, Germany) was used to test for hemolysis (24 h at 30 °C). (iii) Egg yolk agar plates (Hardy Diagnostic, Santa Maria, CA, USA) were used to evaluate lecithinase activity (72 h at 30 °C). Lecithinase activity was quantified after taking pictures with a tabletop scanner and analyzing these images using ImageJ75. Values are given according to the formula (area of bacteria + area of precipitate) − (area of the bacteria) = area of precipitate [cm2].
The relative gene expression compared to the gene gyrA was determined using quantitative reverse transcription PCR (qRT-PCR)-based transcript scoring in V. cholerae76. Data are based on three biologically independent experiments (±standard deviation [s.d.]). Primers used for qPCR are indicated in Supplementary Table 3.
The amoeba Acanthamoeba castellanii (ATCC strain 30010) served as the host in all of the infection experiments. Supplemented peptone-yeast-glucose (PYG) medium (ATCC medium 712) was used to propagate uninfected amoebae and half-concentrated defined artificial seawater (0.5× DASW; buffered with 50 mM HEPES)77 was used as the infection medium.
For infection experiments, amoebae were diluted with fresh PYG at a concentration of 1 × 105 amoebae/ml and seeded into a µ-Dish (low wall 35 mm ibidiTreat devices; 80136-IBI, Vitaris, Baar, Switzerland). After three hours of static incubation at 24 °C, adherent amoebae were washed three times with the infection medium. Exponentially growing bacteria were likewise extensively washed in infection medium and added to the amoebae at a multiplicity of infection (MOI) of 1000. Co-cultures were incubated statically at 24 °C for the indicated time post-primary contact (p.p.c.)25.
Confocal lasers scanning microscope (CLSM) imaging was done using a Zeiss LSM 700 inverted microscope (Zeiss, Feldbach, Switzerland). To label the endosomal pathway of the amoebae, Alexa Fluor 647-labeled dextran (MW 10,000; D22914, Thermo Fisher Scientific, Waltham, Massachusetts, USA) was added to the infection medium at a final concentration of 100 µg/ml. The fluorescent stain calcofluor white was supplemented wherever indicated at a final concentration of 0.2%, as this stain is known to bind to cellulose (18909-100ML-F, Sigma-Aldrich, Buchs, Switzerland). Fluorescent signals were quantified using the open-source imaging software ImageJ75 (NIH, Bethesda, MD, USA). Bacterial motility inside the amoebae (see Supplementary Movies 2, 3, and 4) was assessed using short frame intervals and/or a reduced scanning speed.
For scanning electron microscopy (SEM) imaging, a 10 mm in diameter silicon wafer was coated with 100 µg/ml of poly-d-lysine hydrobromide (P6407, Sigma-Aldrich, Buchs, Switzerland) for 2 h at room temperature, then washed three times with bi-distillated water. Exponentially grown bacteria were washed with PBS and allowed to settle for 20 min at room temperature onto the silicon wafer. The supernatant was removed, and a 0.1 M phosphate buffer solution of 1.25% glutaraldehyde and 1% tannic acid was gently added. After 1 h, the fixative was removed and replaced by cacodylate buffer. This was transferred to a 1% solution of osmium tetroxide in cacodylate buffer for 30 min, washed in water, and then dehydrated through a series of increasing concentrations of alcohol. The sample was finally dried at the critical point of carbon dioxide and coated with a 2-nm-thick layer of osmium metal using an osmium plasma coater. Images were collected in an SEM (Merlin, Zeiss NTS) at 1.8 kV electron beam tension.
For the correlative light and electron microscopy (CLEM), 18 mm glass coverslips were used. These were coated with a thin (10 nm) layer of carbon onto which an alphanumeric grid had been outlined using a thicker, 20 nm, carbon layer. The coverslips were incubated with poly-d-lysine hydrobromide (P6407, Sigma-Aldrich, Buchs, Switzerland) at a concentration of 100 µg/ml for 30 min at room temperature and then washed three times with PBS. At 20 h p.p.c., colonized amoebae were detached from the tissue culture flask using a cell scraper, plated onto coated coverslips, and allowed to adhere for 30 min at room temperature. The supernatant was removed, and a buffered aldehyde solution of 1% glutaraldehyde and 2% paraformaldehyde was gently added. After one hour at room temperature, these were washed in cacodylate buffer. The corresponding confocal images were acquired. The samples were then post-fixed and stained with 1% osmium tetroxide followed by 1% uranyl acetate. After dehydration through increasing concentrations of alcohol, the coverslips were embedded in EPON resin and polymerized at 65 °C for 48 h. Cells of interest were located using the alphanumeric grid that remained on the resin surface after the grid had been removed. Blocks were trimmed around the cell and between 200 and 300 serial thin sections, at a thickness of 50 nm, were cut from the entire structure using a diamond knife and ultra-microtome. Sections were collected onto copper slot grids, carrying a formvar support film, and then further stained with uranyl acetate and lead citrate. Serial images of the cell were taken with a transmission electron microscopy (TEM; Tecnai Spirit, Fischer Scientific) at a voltage of 80 kV.
The number of bacteria in the vacuole was estimated using an unbiased stereological approach31 by counting bacteria in five probes across a series of 55 TEM sections cut through the middle of a single vacuole. Each section was 50 nm thick and each probe consisted of a reference section and a look up section that were five sections apart. The volume of each probe was 8.3 cubic micrometers. From these counts we calculated a mean density of 1.35 ± 0.42 bacteria per cubic micron. Given the approximate total volume of the CV of 143.8 µm3, based on the assumption of a spherical shape, the total number of V. cholerae cells was estimated to be 194 (±60). While this experimental approach was elaborate and, therefore, not applicable to large numbers of individual amoeba, an average number of 150–200 V. cholerae cells per organelle is also theoretically supported given an average volume of ~1 µm3 (www.BioNumber.org) and 150 µm3 (based on a maximum diameter of up to 6.6 µm78) for bacteria and the CV of A. castellanii, respectively.
To generate a 3D model of the colonized CV, serial TEM images were first aligned manually using image software (Photoshop, Adobe). Each structure was then segmented using TrakEM279 operating in the FIJI software80 (www.fiji.sc). The 3D models were then exported into the Blender software (www.blender.org) for smoothing and rendering into a final image or movie.
To visualize T6SS production and assembly in V. cholerae, epifluorescence microscopy images were acquired using a Zeiss Axio Imager M2 microscope using standard settings50,76. Images in Fig. 4c were taken with the same exposure times for the green (GFP) channel.
T6SS-mediated killing of E. coli prey by diverse V. cholerae strains was performed by co-incubation on solid LB agar surfaces. Recovered prey was enumerated on selective medium50,51.
One-way ANOVA was performed using GraphPad Prism version 7 for Mac (GraphPad Software, San Diego, CA, USA) followed by Tukey's post hoc test for multiple comparisons. Statistical analyses were performed on three biologically independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001; ns (not significant), p > 0.05. The amoebal population counted manually was each time 6000 (derived from three biologically independent experiments each with n = 2000).
All data supporting the findings of this study are available from the corresponding author upon request.
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We thank members of the Blokesch group at EPFL and the Geneva/Lausanne amoeba club for fruitful discussions. We also acknowledge J. Mekalanos (Harvard) for strain C6706 (original stock) and former members of the Blokesch lab for provision of genetically engineered V. cholerae strains. This work was supported by EPFL intramural funding, a Starting Grant from the European Research Council (ERC; 309064-VIR4ENV), and a Consolidator Grant from the European Research Council (ERC; 724630-CholeraIndex) to MB. M.B. is a Howard Hughes Medical Institute (HHMI) International Research Scholar (grant 55008726).
C.V.d.H. and M.B. designed the study; C.V.d.H., A.S.V., N.C.D.D., G.K., and M.B. planned the experiments; C.V.d.H., A.S.V., N.C.D.D., S.S., C.S., T.S., S.C., C.M., and M.B. performed experiments; C.V.d.H., A.S.V., N.C.D.D., G.K., and M.B. analyzed the data; C.V.d.H. and M.B. wrote the manuscript; M.B. revised the manuscript. All authors approved the final version of the manuscript.


What Does "Back to School" Mean in South Africa?
After a devastating fire at the Church where school was previously held occurred, Principal Elizabeth moved the 'school' into her own home for the sake of the children. The dedication of Elizabeth and others to making sure this community had a school after theirs was lost was inspiring. The Community as a whole is committed to the education of their children and has been participating in rebuilding this school. The community understands that education will help their children by providing more opportunities in their lives. In many cases, the meal the children receive at school is their only meal that day.
KET believes that "every child deserves at least one good meal a day and the right to a reasonable pre-school care and a competitive preschool education. All children from the disadvantaged communities will be able to compete equally against the more privileged counterparts once they start attending primary school." This initiative is something that Collette Cares has supported since 2007 with over $170,000 in funding used to directly impact approximately 2,250 students through the KET Adopt a Child's Education, renovating existing schools, adding Blackboards to over 30 schools, Holiday party support, backpack program and building new schools. Our Spectacular South Africa Tours goes into the townships that KET supports and meets with the students we have helped. Our guests have also been tremendous in supporting this endeavor while on tour as well as doing fundraisers after the tour. We are very proud of all that we have been able to accomplish these past eight years and are thrilled to see so many Happy Faces.
South Africa Hippo Rollers: Recent Delivery!
A group of Collette Foundation volunteers recently did a Hippo Roller drop off in the village of Kgautswane, South Africa. To date, more than 800 hippo rollers have been delivered to this community, truly enhancing the daily lives of those who live there.
From the recipients: The Collette Foundation once again personally delivered another 100 Hippo water Rollers to community members living at Kgautswane in South Africa. Clara Masinga facilitated and coordinated the handover which left an indelible mark on the donors. The beneficiaries were so grateful to receive their own Hippo roller – at last!
About this project: In South Africa, water is life. Millions of people worldwide are forced to walk long distances on a daily basis to collect the water needed for their families. The Collette Foundation is supporting rural communities in South Africa through a partnership with Hippo Water Rollers. In most communities the water is collected from afar and carried by women in small buckets on their heads. The hard, exhausting task not only leads to injury but simply does not provide enough clean water to support their families. Hippo Water Rollers were designed to alleviate the problem. These barrel-shaped containers hold, transport and store 90 liters of water. The rollers are so easy to use, children and the elderly are able to operate a full roller over any terrain. The use of Hippo Rollers in these remote villages allows for approximately five times the amount of water to be collected in far less time.
The Collette Foundation enthusiastically announces the Little Rascals School, located in South Africa, is officially open! Having worked closely with the Knysna Education Trust, we were able to revamp their initial building and develop a safe, stable and modern establishment through funding. Focusing on Early Childhood Development Centers (ECD), the Knysna Education Trust assists 26 facilities in hopes of providing disadvantaged children with the opportunity to obtain a solid education. Through their support, children receive an integrated education giving them the chance to rightfully benefit and build from their ever-progressing surroundings. Now the children of Little Rascals will be able to participate in preschool activities with a more focused approach due to their motivational atmosphere. We look forward to hearing more about the benefits of the new school!
Good progress is being made to date at the Little Rascals Preschool in South Africa — largely in part to the funding of the Collette Foundation — and the school principal and children are very happy and cannot wait to move into the new premises. We are so excited to see the school coming along so quickly.
The focus for this week will be to get the new septic (sewage) tanks ready and finalized. The foundations for the new building, additional classrooms, will also be completed this week. Internal work also continuing as all of the ceiling boards need to be replaced and the floor needs to be redone.
They have a wonderfully committed team of builders who are excited as well to see the children happy in their new school. The principal is currently accommodating the children in her home, and coping well under the circumstances. She has been very involved in the upgrade and has been selecting pain colors, choosing wall murals and we are assisting her with Curtaining and other such materials.
The South African team is working closely with Knysna Educational Trust (KET) on the Little Rascals School. Currently, the project is underway revamping parts of the current structure; roof cleaning, paint stripping, fixing the floor and a multitude of other task to upgrade the current structure. The Municipal approvals for the new building are in the final stages. Once these are granted it will be full steam ahead with construction of the new school. During the construction Katie, the principal, has taken all of the children out of the existing building and is running the school from her home. Both Katie and the students are very excited about the new school.
The Collette Foundation has always had a soft-spot for our Hippo Roller project in South Africa. These cylindrical containers keep villages more hydrated and allow for a much more efficient and safe transport system to get the water to the villages. In the years we have been delivering hippo rollers to the village of Kgautswane we have witnessed their positive effect on the village's people.
Word about these marvelous containers is spreading fast… all the way to France! The Hippo Roller is one of 70 selected solutions (from over 1100 solutions) that will be exhibited at the Village of Solutions during the 6th World Water Forum in a few days in Marseille, France – the world's largest water event!
Knysna, South Africa Student Graduates!
Iyema from Noluthando preschool has left and has started Grade 1 at 'big school'. Thank you so very much for allowing him the opportunity to be well prepared for Grade.
The Collette Foundation is excited to deliver 75 new Hippo Rollers in April to the village of Kgaustwane which we have been supporting consistently for five years. We have ordered them to be manufactured and a group visiting the site in April will personally deliver them!
What are Hippo Rollers? They are barrel-shaped containers that hold, transport and store 90 liters of water! The rollers are easy to use; in fact, children and the elderly are able to operate a full roller over any terrain. They are needed in many South African villages where water is collected from afar and carried by women in small buckets on their heads — a transport system that has led to injuries and did not provide enough clean water for full families. Hippo Rollers collect five times the amount of water in far less time. The Collette Foundation has delivered over 600 Hippo Rollers to the village of Kgaustwane so far.
I receive updates quite often from our friends at the Knysna Educational Trust on children at preschools in the Knysna Township area, including Isiseko Educare and Noluthando. Through the trust, the Collette Foundation sponsors the education for at-risk children, has funded preschool renovations and provided education to the teachers to create a positive environment for the young children.
The latest news was very uplifting. The children are progressing well, and all meet the developmental requirements for their respective age groups. The preschool at Isiseko was recently gifted with a great set of educational resources including art equipment, musical equipment, math sets, language sets, fantasy clothing, books and toys. The children are happy with the new resources — and the teachers benefit from it as well!
I always enjoy receiving artwork the children make and progress reports that showcase how eager they are to learn and how far they've come. These messages are especially warming during the holidays.
Artwork from one of the students benefitting from the Knysna Educational Trust and Collette Foundation.


text:
Q: How to retain this object when inserting to mutable array? update: I have found the bug in my copyWithZone method in A. Thanks everyone.
update: sorry, I do have the @properties declared, I thought it was obvious so I skipped them in my OP. Sorry about that.
The crash message  is: objA was released (zombie) memory when trying to access the str.

My data structure looks like this:
@class A
{
   NSString *str;
}
@property (retain) NSString *str;  // str synthesized in .m

@class B
{
   A *objA;
}
@property (copy) A *objA;  // objA synthesized in .m

What I am trying to do is:
B *newB = [[B alloc] init];
[someMutableArray addObject: newB];

However, I will crash some times when I try to access like this:
B *myB = [someMutableArray objectAtIndex: index];

someLabel.text = myB.objA.str;

I guess the objA & objA.str were not retained when inserting B into the array. But I don't know how to make sure they are retrain.
Any help is appreciated
-Leo

A: You should be using properties for Class A and B:
@interface A : NSObject {
    NSString *str;
}

@property (nonatomic, retain) NSString *str;
@end

The use @synthesize str; in .m file, this will retain the str, don't forget to release the str in the dealloc method:
@implementation A

@synthesize str;

- (void) dealloc {
   [str release], str= nil;
   [super dealloc];
}

@end;

A: You should look in to
http://developer.apple.com/library/mac/#documentation/Cocoa/Conceptual/ObjectiveC/Chapters/ocProperties.html%23//apple_ref/doc/uid/TP30001163-CH17
to learn about properties and how they manage memory.

A: Have you tried:
[someMutableArray addObject: [newB copy] ];

A: At this line
someLabel.text = myB.A.str;

it should be...
someLabel.text = myB.objA.str;

Yes, also you should be using properties for the members of your class. That will retain them. Just don't forget to release in dealloc

redpajama_set_name: RedPajamaStackExchange


However, just because one voices some concerns as to how the FCPA is enforced (as a committee that Clayton chaired did in a 2011 report titled "The FCPA and its Impact on International Business Transactions – Should Anything Be Done to Minimize the Consequences of the U.S.'s Unique Position on Combating Offshore Corruption?") does not make one a "public critic of the FCPA" (the law itself).
In recent years there was a high-profile and vocal critic of not only how the FCPA was enforced, but the FCPA itself. And that person was Andrew Weissmann, who in January 2015 joined the Obama Department of Justice to become Chief of the DOJ's Fraud Section.
Did Podesta ever write about Weissmann being a "public critic of the FCPA" when he joined the Obama administration?
By the way, Weissmann recently relinquished his Fraud Section position to join Special Counsel Robert Mueller's staff.
ISO 37001 is hardly a "new" standard if you are familiar with the numerous sources of best practices in the anti-bribery space. Indeed IS0 37001 is a complete yawner, as well as a disappointment as several best practices are not even captured in the purported best practices document.
As to the "much-needed level of anti-bribery guidance" assertion, the notion that IS0 37001 is somehow the magical potion that has eluded the compliance community up until now is laughable. There have been numerous sources of anti-bribery guidance that have long been in the public domain prior to ISO 37001 (which by the way you have to pay to access).
Regarding the "gaining traction" assertion, ISO 37001 has been public now for about 7 months and all that has happened from a corporate perspective it seems is that two companies under FCPA scrutiny are thinking about being ISO 37001 certified (for reasons likely due to optics, not substance). This is a pretty generous meaning of gaining traction.
As evidence that ISO 37001 is the next greatest thing, the article cites a person who helped create ISO 37001 and another individual who is selling compliance services related to it (indeed the individual has an ISO 37001 compliance website and an e-mail address ending in iso37001compliance.com). That hardly establishes the truth of the matter asserted.


Tag Archives for: "SSN 2"
For the end of season 2 of the Mo' Money Podcast, I do a special solo episode to reflect on the 30 biggest life lessons I've learned so far.
TV host and best-selling author Gail Vaz-Oxlade shares her journey to becoming Canada's #1 money guru as well as her top money tips everyone can learn from.
I interview my grandma Audrey about her immigration from Scotland to Canada and how life is now that she's a retired teacher in Terrace.
Licensed insolvency trustee Doug Hoyes and I chat about the key things everyone should know about debt and bankruptcy.
Money coach Jen Hemphill shares her tips on how to balance family, money and career and uplift other women to take control of their personal finances.
Brad Baldridge from Taming the High Cost of College shares his tips on how to get a degree without plunging into major student debt in the process.
Todd Tresidder from Financial Mentor went from hedge fund manager to retiree to money coach. In this episode, he shares his journey plus some helpful tips on how you can find financial freedom too.
Kristina Wilson from the Real Estate Council of Ontario (RECO) shares her expertise when it comes to house hunting dos and don'ts.
I interview UFile tax specialist Gerry Vittoratos on what tips we should all know so we can do our taxes like a pro and get the biggest tax refund.
I chat with Retirement Answer Man Roger Whitney about how you can plan for your tomorrow without sacrificing your today.


text:
'use strict';

const RSVP = require('rsvp');
const EventEmitter = require('events').EventEmitter;
const path = require('path');

function MockExpressServer() {
  EventEmitter.apply(this, arguments);
  this.tracks = [];
  this.trackTimings = [];
  this.trackErrors = [];
}

module.exports = MockExpressServer;

MockExpressServer.prototype = Object.create(EventEmitter.prototype);

MockExpressServer.prototype.then = function() {
  let promise = RSVP.resolve({
    directory: path.resolve(__dirname, '../fixtures/express-server'),
  });
  return promise.then.apply(promise, arguments);
};

redpajama_set_name: RedPajamaGithub


It's Nicki Minaj's world and we all just live in it. If you don't believe it, check out the femcee's latest promo for her hosting gig at the MTV European Music Awards, taking place in Glasgow on November 9th.
There will be Nicki impersonators, Nicki burgers, a Nicki television station, and even Nicki Minaj laundry detergent. Now, that's being a bawse.
In addition to hosting, the rapper, along with Charli XCX, Calvin Harris, Ariana Grande and Ed Sheeran, will perform.
Angelina Jolie and Brad Pitt continue to give us so many reasons to love them. The couple is reportedly on a mission to co-produce a film based on the life of James Foley, an American journalist who was beheaded by ISIS in Syria on August 9.
Brad has reportedly reached out to the journalist's family and they are open to the idea. Since nothing has been confirmed however, all other details are being kept on the DL.
So Aaron Paul is not one to mess with. The actor, who typically flies under the radar, lashed out at Toys 'R Us after the retail store pulled a Breaking Bad toy stores off its shelves.
Aaron, on the other hand, was having none of that. The actor took to his Twitter and questioned the store's decision to keep Barbie and violent video games.
He also shared a link to an opposing petition, which urged the retailer to bring the toys back.
Ish is about to get real this holiday season when Into the Woods premieres in theaters. The new film combines all of your favorite fairy tales with all of your favorite actors. Featuring Meryl Streep, Johnny Depp, Ana Kendrick, Chris Pine, and Emily Blunt, just to name a few, this one is set to be a blockbuster.
In a newly released featurette, we see a singing Meryl, a cunning Johnny, and a charming Chris.
Into the Woods is a modern twist on the beloved Brothers Grimm fairy tales, intertwining the plots of a few choice stories and exploring the consequences of the characters' wishes and quests. This humorous and heartfelt musical follows the classic tales of Cinderella (Anna Kendrick), Little Red Riding Hood (Lilla Crawford), Jack and the Beanstalk (Daniel Huttlestone), and Rapunzel (MacKenzie Mauzy) — all tied together by an original story involving a baker and his wife (James Corden & Emily Blunt), their wish to begin a family and their interaction with the witch (Meryl Streep) who has put a curse on them.


Unless a Republican wins the the Presidential election in 2017, the 7-year debate over whether to build the Keystone pipeline is probably over. Gone will be the endless TV and newspaper ads describing the dangers of either building or not building it. Waiting in the wings, however, is another energy/climate debate that will make the Keystone controversy look like a flyspeck on the pages of history.
I am talking about the coming debate over what now is variously known as "cold fusion," "Low Energy Nuclear Reactions," or "Condensed Matter Nuclear Reactions", all of which describe same the heat producing reaction which has been observed for the last 25 years. This new debate, which seems likely to start sometime next year, may be one of the most acrimonious in US history for these new energy-producing technologies have the potential to, over time, seriously disrupt or even destroy much of the oil, natural gas, coal, nuclear, hydro, and wind industries. In short this and/or similar developments could easily be the mother of all disruptive technologies leaving the world a far different place from the one we know.
For the last 25 years, the industries and scientific establishments that might be threatened by "cold fusion" and similar technologies have been able to keep the news of the technologies' development under wraps through a variety of stratagems and keep government funding from speeding its development.
This situation is about to change radically. In recent years, principal developers of this new technology, tired of all the skepticism and accusations of fraud, have adopted the position that they will continue to work until a commercially viable product is ready and then put it on the market.
To those of us who have been following the issue, the artificial wall of secrecy that has existed for over 25 years is about to fall. A few tentative stories have begun to appear in the press and last week a California company, Brillouin Energy, that has been making progress on its LENR technology in recent years, was invited to make a presentation on Capital Hill. The key event which could turn all this around will be the completion of the one-year trial that a North Carolina company, Industrial Heat, has been conducting. This trial is due to be finished this winter and if successful, the company says it will disclose the results and if all goes well announce that they have a product for sale.
Thus, the results of Industrial Heat's one-year test will be critical to what happens next. If the reactor has indeed been able to produce a megawatt of pollution-free heat continuously for a year and the verified costs of operating the device are sufficiently below any other source of energy to justify taking a gamble on a new technology, then we are clearly in a new age. Industrial Heat says they already have over $3 billion in tentative orders awaiting the results of the trial.
Assuming that no technical showstoppers emerge from the test and that energy producing reactors become available for sale in the next year or so, there would seem to only one possibly insurmountable problem remaining. That will be the disruption this will eventually cause to existing energy producing industries. If they have not already been made aware of what could happen, then a few minutes after the news hits the cable networks they will. High-level meetings will gather across the US and around the world to figure out how to stop or at least delay this technology for as long as possible.
We have seen this scenario before. Remember cigarettes and cancer? Exxon and global warming? It is clear that a well financed campaign to spread doubt and confusion can be highly effective. In the case of cigarettes, it was decades before tobacco consumption began to drop. In the case of global warming, enough money and PR has about half of the American people convinced that global warming is all an environmentalist hoax, even as sea levels rise, California fries, and unprecedented storms tear the land apart.
In the case of low energy nuclear reactions, it will be easy to conjure up fears. In a matter of days after it becomes apparent that the technology is valid and could shake the economy to its core, television ads will start claiming that the technology is bad for your health, and that it should be tested for another 30 or 40 years before the public is endangered. The TV ads naturally will be accompanied by a rush of lobbyists to Capital Hill seeking to outlaw or at least mandate years of testing before it can be released to the public. It will sound a lot like the campaign against AC electricity 120 years ago.
It should be noted that in 25 years of testing, no experimenter has detected harmful emissions or residue resulting from the technology.
Fortunately, however, the leadership of Industrial Heat, which at least for the minute seems to be the company that could be the first to offer this technology for sale, has a stratagem to circumvent to what will surely become widespread opposition to the technology. Industrial Heat simply took the technology to China where PR firms, lobbyists, congressmen and TV commercials have zero impact on decision makers. Moreover, China, where people are dying from their own coal smoke, is desperately in need of a clean source of energy ASAP.
Last month some sort of an agreement to set up an R&D establishment in China was signed between Industrial Heat and the Chinese government. It seems likely that the technology being tested by Industrial Heat will be transferred to the Chinese government, who are in sore need of a fix for their environmental and economic problems, will evaluate the technology on its own merits and with little consideration for the disruption it could cause to existing industries. The world just might be saved from global warming after all.


A common short term used for osteopathic treatment is 'osteo'. This is similar to chiropractors being called 'chiros'. However, the term 'osteo' has been associated with osteopaths. In the province of Ontario, 'osteopath' is a restricted title to members that belong to the College of Physicians and Surgeons of Ontario (CPSO).


text:
NABJ Announces Melanie Eversley as Executive Editor of Its Soon-to-Launch News Platform
Posted by nabjpress
NABJ Addresses Diversity Concerns with CNN Plus 'War Room' Photo
NABJ Congratulates Long-time Member Elise Durham on New Role Leading Business Diversity Efforts at Atlanta's Hartsfield-Jackson
The National Association of Black Journalists (NABJ) has announced longtime member and veteran reporter Melanie Eversley as the new Executive Managing Editor for the soon-to-launch NABJ Black News and Views (BNV) platform.
Eversley will oversee content, personnel and other resources earmarked for BNV. Once launched later this year, BNV will make history as NABJ's first-ever digital news platform focused on issues and perspectives relevant to the Black community.
"It has been a vision of our organization for several years to launch our own news platform," said NABJ President Dorothy Tucker. "Melanie's experience and passion for storytelling will help enhance the work BNV staff and the NABJ Board have already been doing behind the scenes to realize that vision."
"We are excited to welcome Melanie to the team we are building at BNV," added Drew Berry, NABJ Executive Director.
Eversley is an award-winning multimedia journalist with expertise in national politics, race, criminal justice and breaking news. Her career has spanned stellar coverage of all news formats, from developments spurred by the death of George Floyd to the Boston Marathon bombing.
She previously contributed to the BNV team as a writer preparing content for the platform's launch. Currently an independent writer based in New York City, her past roles include Washington correspondent (Atlanta Journal-Constitution); breaking news and national reporter (USA Today); race relations, statehouse and municipal reporter (Detroit Free Press); news writer (WPIX); and interim editor (Urban Faith Magazine).
Eversley's work has also appeared in/on The Undefeated, National Geographic, BET.com, Fortune.com, NBC News/NBCBLK, The Grio, Diverse: Issues in Higher Education, Gannett News Service, ESSENCE, and Edible New Jersey. She has volunteered for multiple years with NABJ as a program co-chair for several of its past conventions and as immediate past chair of its Board of Directors elections committee.
She received a bachelor's degree from Oberlin College and a master's from Columbia University Graduate School of Journalism.
Media Contact: press@nabj.org

redpajama_set_name: RedPajamaCommonCrawl


Join the diagonal which will divide the length 4 into two parts. Extend the "5" line upwards [in your diagram] a further length "3". Join that point to the top left corner of the square. You now have a right triangle with two sides 8 and 4 and the diagonal of the square. Find the length of the diagonal and the side of the square [and area] from that.
I'm sorry, what does "join the diagonal" mean?
Edit: okay so you mean to join the lines "5" and "3" together?
I should have been more clear. The diagonal of the square, top left to bottom right.


Published by Lottie Wakefield, at Tuesday, November 20th , 2018 - 02:04:44 AM, in House & Home Design category.
Published by Emerson Sackville, at Friday, November 02nd , 2018 - 22:03:28 PM, in Inspiration House category.
Published by Emerson Sackville, at Friday, November 02nd , 2018 - 23:26:00 PM, in Interior & Exterior category.
Published by Lottie Wakefield, at Friday, November 02nd , 2018 - 22:21:44 PM, in House & Home Design category.
Published by Emerson Sackville, at Friday, November 02nd , 2018 - 23:48:33 PM, in Architecture & Landscape category.


text:
Q: Inherently discrete concepts Are there any concepts which are naturally defined only for the integers and so far has resisted any attempts at extension to other fields such as rationals or reals?
Does not meet criteria:
Cardinalities of sets
n! / Gamma function
Differentiation / Fractional differentation

A: I've not yet seen, that the "rank of a matrix" has been interpolated to fractional ranks (but I'm surely not very profound with literature...)
(side remark: also your question focuses on whether it has "resisted to attempts" to interpolate ... such attempts may or may not exist, but to know this needed even a bigger radius of insight into literature and non-literature manuscripts...)

A: Turing machine is a discrete model.

A: The Arithmetic derivative.

A: I don't know if this works, because someone may actually have done something along these lines.  I'm by no means an expert on mathematics in general.  That said, some of the concepts of number theory may well work.  For example, what would the concept of a prime number, composite number, "versatile" number (highly composite number), etc. mean in terms of real numbers (or rational numbers for that matter)?  5 has only 2 positive integer factors (other than 1), but it has an infinity of factors in just the rational numbers.  So, if such an extension of the concept of prime number or composite number exists, I don't know how it works at least, and it would take some explaining.
The Fundamental Theorem(s) of Arithmetic don't seem extendable to the reals, since we don't seem to have the notion of a prime number in the reals, in the sense that some numbers exist in the real numbers which have exactly two factors.
I'll add that I know of at least two concepts which can get defined for the integers, but simply can't get extended to the rationals or reals (and never will legitimately).
For any given number n, there exists a least number o, such that o>n in the integers, where ">" indicates the usual ordering relation of "greater than".  There does not exist any such number in the rationals or reals.  One might say that in the integers, every integer has a distinct-least-upper-bound or distinct-supremum.
For any given number m, there exists a greatest number l, such that m>l in the integers.  There does not exist any such number in the rationals or reals.   One might say that in the integers, every integer has a distinct-greatest-lower-bound or distinct-infimum.
In other words, given a "direction" which either takes towards larger numbers, or smaller numbers, for any number x, there exists a "next" number "y" and a previous number "v".  This does not hold true for the reals or rationals.

A: The principle of mathematical induction.
Reduction modulo $n$, as a map of rings.  It exists only as a map of additive groups for $\Bbb{Q}$ and $\Bbb{R}$.


text:
Foodgrain production during the year 2016-17 has broken all the previous records: Radha Mohan Singh
The Union Minister for Agriculture and Farmers Welfare, Shri Radha Mohan Singh addressing a press conference on the achievements of the Ministry during 3 years of NDA Government, in New Delhi on May 22, 2017. The Minister of State for Agriculture & Farmers Welfare and Parliamentary Affairs, Shri S.S. Ahluwalia and the Principal Director General (M&C), Press Information Bureau, Shri A.P. Frank Noronha are also seen.
New Delhi: Union Minister for Agriculture and Farmers Welfare, Shri Radha Mohan Singh said the initiatives taken under the leadership of Prime Minister, Shri Narendra Modi for the welfare of farmers and the agriculture sector have started showing positive and encouraging results. The commitment of the Modi government towards the welfare of the farmers is resulting in qualitative improvement in the lives of farmers. Shri Radha Mohan Singh said that the Ministry of Agriculture is working to double farmers' income by 2022, a goal set by the Prime Minister. Shri Singh was speaking at a press conference in National Media Centre on the completion of three years of the Modi government.
Shri Radha Mohan Singh said in the last three years, the Modi government has created new and transparent working style for the growth of the nation. Under the guidance of the Prime Minister, the implementation of the farmers' welfare scheme was turned into a mission by the government. From the perspective of good governance, innovative and reformist approach, our government laid the foundation of future-oriented India.
Shri Singh said the Modi Government has succeeded in creating awareness among the farmers about initiatives taken for the development of the agriculture sector. In three years, continuous and robust efforts have been made to bring qualitative changes in lives of farmers and rural areas.
Union Agriculture Minister said during UPA government, the expenditure made by the Ministry used to be less than the budgetary allocation. For instance, during the year 2011-12 budgetary allocation was Rs.24, 526 crores and the amount spent was just Rs.23, 290 crores. Similarly, in the year 2012-13 budgetary allocation was Rs.28, 284 crores and the amount spent was Rs.24, 630 crores. In the year 2013-14, the budgetary allocation was Rs.30, 224 crore and the amount spent was Rs.25, 896.
On the other hand, under Modi government, the Ministry is spending more than the budgetary allocation for the welfare of the farmers. For instance, in the year 2016-17, the budgetary allocation was Rs. 45,035 crores, but it was increased to Rs. 57,503 crores in the revised budget.
Keeping the welfare of the agriculture sector and farmers in mind, Modi government is increasing the budget every year For example, the total budget for the welfare of the agriculture sector during the UPA government's four years – 2010-11 to 2013-14 – was Rs.1,04,337 crores, while the present government has allocated Rs.1,64,415 crore during the period of 2014-15 to 2017-18, which is 57.58 percent more.
Shri Singh said that in the initial two years of the last three years, the government provided security and confidence to farmers during two consecutive years of monsoon shortfall. Apart from focusing on the distribution of Soil Health Card, extension of irrigation facilities, low-cost organic farming, national agricultural market, horticulture development, Agroforestry, bee keeping, dairy, fish and egg production, it also focused on agricultural education, research and extension. More investment has been made to strengthen cooperatives. In the last three year, several initiatives were taken to make the country self-reliant in pulses and oilseeds. The farmers were provided unprecedented security through Prime Minister's Fasal Bima Yojana, which has the lowest premium and covers various risks. The government has given priority to the interests of farmers by providing the District-wise contingency action plans and increasing the relief funds for the farmers affected by drought and hailstorm.
In the three years of the Modi government, the initiatives taken for the welfare of farmers and the agriculture sector through the successful implementation of the schemes have started showing positive results. The achievement of the Modi Government in the agriculture sector is as follows:
Ø Foodgrain production of the year 2016-17 has broken all previous records. This year, the growth rate in agriculture and allied sector has been approximately 4.4 %.
Ø According to 3rd advance estimate for 2016-17, the total foodgrain production is estimated to be 273.38 million tonnes which is 8.67 % higher than that of the year 2015-16.
Ø It is also worth mentioning that this production is also 6.37% higher than the average production of last 5 years.
Ø For pulses, the production during 2016-17 is expected to be 22.40 million tonnes which will be a record production till date and is higher than that of the year 2015-16 by 37%.
Ø As on 16.05.2017, 725 lakh soil health cards have been distributed to the farmers. Testing of samples and printing of cards are under process. Remaining farmers would be provided cards in the next three months.
Ø During the year 2011-14, cumulative area under organic farming has increased from 7.23 lakh hectare to 20 lakh hectares during the year 2014-17.
Ø So far, 417 regulated market from 13 states have joined the scheme.
Ø Till 15.05.2017, trading of 84 lakh tones of agri produce worth Rs 20,000 crores has been done. Till 15.05.2017, 46 lakh farmers, 90,000 traders and 46,411 Commission Agents has been registered on the portal.
Ø Milk production was 398.01 million tonnes during the years 2011-14 which increased to 465.5 million tonnes during the year 2014-17. The three year growth rate has been 16.9%.
Ø Egg production was 210.93 billion during the years 2011-14 which increased to 248.73 billion during the year 2014-17. The three year growth rate has been 17.92%.
Ø Fish production was 272.88 lakh tonnes during the years 2011-14 which increased to 327.74 lakh tonnes during the year 2014-17. The three year growth rate has been 20.1%.
Ø 223 Farmer Producer Organizations (FPOs) were formed during 2011-14, whereas 383 FPOs were formed during 2014-17.
Ø During 2007-14, 6.7 lakh Joint Liability Groups (JLGs) were constituted with an assistance of Rs 6,630 crore whereas during just three years of 2014-17, 15.85 lakh JLGs have been constituted with an assistance of Rs. 16,268 crore.
Ø Rs. 5.94 crore were spent on bee keeping during 2011-14. Whereas, Rs. 18.14 crore were spent on bee keeping during 2014-17. This is an increase of 205%.
Ø States were sanctioned Rs. 33,580 crore from State Disaster Relief Fund during 5 years of UPA government. Whereas, Modi Government has allocated Rs. 61,620 crore for 5 years.
Ø During 2011-12 to 2013-14 i.e. 3 years of UPA Government, states were granted Rs. 9,099 crore from National Disaster Relief Fund (NDRF). Whereas, during 2014-15 to 2016-17 i.e. 3 years of Modi Government, States have been granted Rs. 29,194 crore from NDRF. Assistance for Karnataka, Kerala and Puducherry is under consideration.
Ø Under Pradhan Mantri Krishi Sinchai Yojana, a record coverage of 8 lakh 40 thousand hectares has been brought under micro irrigation during the year 2016-17.
Ø Under the National Agriculture Insurance Scheme, six crore farmers have got insurance for the Kharif season during three years of 2011-14. Among them, there were only 77 lakh non-lonee farmers. While in the 3 years between 2014-17, 9 crore 47 lakh farmers were insured for the Kharif season, of which 2 crore 61 lakh are non-lonee farmers. The Pradhan Mantri Fasal Bima Yojana has increased the number of non-lonee farmers by 238.96%. And in the last three years, the number of non-lonee farmers increased by 128.50% in Rabi Crops compared to the preceding three years.
With the able-guidance and leadership of Prime Minister Shri Narendra Modi ji, proactive officers of the ministry and agricultural scientists, active cooperation of state governments and hard work of farmers, we have not only faced different challenges of the agriculture sector but have also won over them and brought positive and revolutionary changes.
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