The type IV pilus protein PilU functions as a PilT-dependent retraction ATPase.

Type IV pili are dynamic cell surface appendages found throughout the bacteria. The ability of these structures to undergo repetitive cycles of extension and retraction underpins their crucial roles in adhesion, motility and natural competence for transformation. In the best-studied systems a dedicated retraction ATPase PilT powers pilus retraction. Curiously, a second presumed retraction ATPase PilU is often encoded immediately downstream of pilT. However, despite the presence of two potential retraction ATPases, pilT deletions lead to a total loss of pilus function, raising the question of why PilU fails to take over. Here, using the DNA-uptake pilus and mannose-sensitive haemagglutinin (MSHA) pilus of Vibrio cholerae as model systems, we show that inactivated PilT variants, defective for either ATP-binding or hydrolysis, have unexpected intermediate phenotypes that are PilU-dependent. In addition to demonstrating that PilU can function as a bona fide retraction ATPase, we go on to make the surprising discovery that PilU functions exclusively in a PilT-dependent manner and identify a naturally occurring pandemic V. cholerae PilT variant that renders PilU essential for pilus function. Finally, we show that Pseudomonas aeruginosa PilU also functions as a PilT-dependent retraction ATPase, providing evidence that the functional coupling between PilT and PilU could be a widespread mechanism for optimal pilus retraction.

Selection of Vibrio crassostreae relies on a plasmid expressing a type 6 secretion system cytotoxic for host immune cells.

Pacific oyster mortality syndrome affects juveniles of Crassostrea gigas oysters and threatens the sustainability of commercial and natural stocks of this species. Vibrio crassostreae (V. crassostreae) has been repeatedly isolated from diseased animals, and the majority of the strains have been demonstrated to be virulent for oysters. In this study, we showed that oyster farms exhibited a high prevalence of a virulence plasmid carried by V. crassostreae, while oysters, at an adult stage, were reservoirs of this virulent population. The pathogenicity of V. crassostreae depends on a novel transcriptional regulator, which activates the bidirectional promoter of a type 6 secretion system (T6SS) genes cluster. Both the T6SS and a second chromosomal virulence factor, r5.7, are necessary for virulence but act independently to cause haemocyte (oyster immune cell) cytotoxicity. A phylogenetically closely related T6SS was identified in V. aestuarianus and V. tapetis, which infect adult oysters and clams respectively. We propose that haemocyte cytotoxicity is a lethality trait shared by a broad range of mollusc pathogens, and we speculate that T6SS was involved in parallel evolution of pathogen for molluscs.

QstR-dependent regulation of natural competence and type VI secretion in Vibrio cholerae.

During growth on chitinous surfaces in its natural aquatic environment Vibrio cholerae develops natural competence for transformation and kills neighboring non-immune bacteria using a type VI secretion system (T6SS). Activation of these two phenotypes requires the chitin-induced regulator TfoX, but also integrates signals from quorum sensing via the intermediate regulator QstR, which belongs to the LuxR-type family of regulators. Here, we define the QstR regulon using RNA sequencing. Moreover, by mapping QstR binding sites using chromatin immunoprecipitation coupled with deep sequencing we demonstrate that QstR is a transcription factor that binds upstream of the up- and down-regulated genes. Like other LuxR-type family transcriptional regulators we show that QstR function is dependent on dimerization. However, in contrast to the well-studied LuxR-type biofilm regulator VpsT of V. cholerae, which requires the second messenger c-di-GMP, we show that QstR dimerization and function is c-di-GMP independent. Surprisingly, although ComEA, which is a periplasmic DNA-binding protein essential for transformation, is produced in a QstR-dependent manner, QstR-binding was not detected upstream of comEA suggesting the existence of a further regulatory pathway. Overall, these results provide detailed insights into the function of a key regulator of natural competence and type VI secretion in V. cholerae.

Ecological implications of gene regulation by TfoX and TfoY among diverse Vibrio species.

Bacteria of the genus Vibrio are common members of aquatic environments where they compete with other prokaryotes and defend themselves against grazing predators. A macromolecular protein complex called the type VI secretion system (T6SS) is used for both purposes. Previous research showed that the sole T6SS of the human pathogen V. cholerae is induced by extracellular (chitin) or intracellular (low c-di-GMP levels) cues and that these cues lead to distinctive signalling pathways for which the proteins TfoX and TfoY serve as master regulators. In this study, we tested whether the TfoX- and TfoY-mediated regulation of T6SS, concomitantly with natural competence or motility, was conserved in non-cholera Vibrio species, and if so, how these regulators affected the production of individual T6SSs in double-armed vibrios. We show that, alongside representative competence genes, TfoX regulates at least one T6SS in all tested Vibrio species. TfoY, on the other hand, fostered motility in all vibrios but had a more versatile T6SS response in that it did not foster T6SS-mediated killing in all tested vibrios. Collectively, our data provide evidence that the TfoX- and TfoY-mediated signalling pathways are mostly conserved in diverse Vibrio species and important for signal-specific T6SS induction.

SLC2A9 (GLUT9) mediates urate reabsorption in the mouse kidney.

Uric acid (UA) is a metabolite of purine degradation and is involved in gout flairs and kidney stones formation. GLUT9 (SLC2A9) was previously shown to be a urate transporter in vitro. In vivo, humans carrying GLUT9 loss-of-function mutations have familial renal hypouricemia type 2, a condition characterized by hypouricemia, UA renal wasting associated with kidney stones, and an increased propensity to acute renal failure during strenuous exercise. Mice carrying a deletion of GLUT9 in the whole body are hyperuricemic and display a severe nephropathy due to intratubular uric acid precipitation. However, the precise role of GLUT9 in the kidney remains poorly characterized. We developed a mouse model in which GLUT9 was deleted specifically along the whole nephron in a tetracycline-inducible manner (subsequently called kidney-inducible KO or kiKO). The urate/creatinine ratio was increased as early as 4 days after induction of the KO and no GLUT9 protein was visible on kidney extracts. kiKO mice are morphologically identical to their wild-type littermates and had no spontaneous kidney stones. Twenty-four-hour urine collection revealed a major increase of urate urinary excretion rate and of the fractional excretion of urate, with no difference in urate concentration in the plasma. Polyuria was observed, but kiKO mice were still able to concentrate urine after water restriction. KiKO mice displayed lower blood pressure accompanied by an increased heart rate. Overall, these results indicate that GLUT9 is a crucial player in renal handling of urate in vivo and a putative target for uricosuric drugs.

Increased bone resorption by osteoclast-specific deletion of the sodium/calcium exchanger isoform 1 (NCX1).

Calcium is a key component of the bone mineral hydroxyapatite. During osteoclast-mediated bone resorption, hydroxyapatite is dissolved and significant quantities of calcium are released. Several calcium transport systems have previously been identified in osteoclasts, including members of the sodium/calcium exchanger (NCX) family. Expression pattern and physiological role of NCX isoforms in osteoclasts, however, remain largely unknown at the moment. Our data indicate that all three NCX isoforms (NCX1, NCX2, and NCX3) are present in murine osteoclasts. RANKL-induced differentiation of murine osteoclast precursors into mature osteoclasts significantly attenuated the expression of NCX1, while NCX2 and NCX3 expressions were largely unaffected. To study the role of NCX1 during osteoclast differentiation and bone resorption, we crossed mice with exon 11 of the NCX1 gene flanked by loxP sites with cathepsin K-Cre transgenic mice. Mature osteoclasts derived from transgenic mice exhibited an 80-90% reduction of NCX1 protein. In vitro studies indicate that NCX1 is dispensable for osteoclast differentiation, but NCX1-deficient osteoclasts exhibited increased resorptive activity. In line with these in vitro findings, mice with an osteoclast-targeted deletion of the NCX1 gene locus displayed an age-dependent loss of bone mass. Thus, in summary, our data reveal NCX1 as a regulator of osteoclast-mediated bone resorption.

Meta-analysis of genome-wide association studies identifies six new Loci for serum calcium concentrations.

Calcium is vital to the normal functioning of multiple organ systems and its serum concentration is tightly regulated. Apart from CASR, the genes associated with serum calcium are largely unknown. We conducted a genome-wide association meta-analysis of 39,400 individuals from 17 population-based cohorts and investigated the 14 most strongly associated loci in ≤ 21,679 additional individuals. Seven loci (six new regions) in association with serum calcium were identified and replicated. Rs1570669 near CYP24A1 (P = 9.1E-12), rs10491003 upstream of GATA3 (P = 4.8E-09) and rs7481584 in CARS (P = 1.2E-10) implicate regions involved in Mendelian calcemic disorders: Rs1550532 in DGKD (P = 8.2E-11), also associated with bone density, and rs7336933 near DGKH/KIAA0564 (P = 9.1E-10) are near genes that encode distinct isoforms of diacylglycerol kinase. Rs780094 is in GCKR. We characterized the expression of these genes in gut, kidney, and bone, and demonstrate modulation of gene expression in bone in response to dietary calcium in mice. Our results shed new light on the genetics of calcium homeostasis.

Furosemide stimulation of parathormone in humans: role of the calcium-sensing receptor and the renin-angiotensin system.

Interactions between sodium and calcium regulating systems are poorly characterized but clinically important. Parathyroid hormone (PTH) levels are increased shortly after furosemide treatment by an unknown mechanism, and this effect is blunted by the previous administration of a calcimimetic in animal studies. Here, we explored further the possible underlying mechanisms of this observation in a randomized crossover placebo-controlled study performed in 18 human males. Volunteers took either cinacalcet (60 mg) or placebo and received a 20 mg furosemide injection 3 h later. Plasma samples were collected at 15-min intervals and analyzed for intact PTH, calcium, sodium, potassium, magnesium, phosphate, plasma renin activity (PRA), and aldosterone up to 6 h after furosemide injection. Urinary electrolyte excretion was also monitored. Subjects under placebo presented a sharp increase in PTH levels after furosemide injection. In the presence of cinacalcet, PTH levels were suppressed and marginal increase of PTH was observed. No significant changes in electrolytes and urinary excretion were identified that could explain the furosemide-induced increase in PTH levels. PRA and aldosterone were stimulated by furosemide injection but were not affected by previous cinacalcet ingestion. Expression of NKCC1, but not NKCC2, was found in parathyroid tissue. In conclusion, our results indicate that furosemide acutely stimulates PTH secretion in the absence of any detectable electrolyte changes in healthy adults. A possible direct effect of furosemide on parathyroid gland needs further studies.

Human commensal gut Proteobacteria withstand type VI secretion attacks through immunity protein-independent mechanisms.

While the major virulence factors for Vibrio cholerae, the cause of the devastating diarrheal disease cholera, have been extensively studied, the initial intestinal colonization of the bacterium is not well understood because non-human adult animals are refractory to its colonization. Recent studies suggest the involvement of an interbacterial killing device known as the type VI secretion system (T6SS). Here, we tested the T6SS-dependent interaction of V. cholerae with a selection of human gut commensal isolates. We show that the pathogen efficiently depleted representative genera of the Proteobacteria in vitro, while members of the Enterobacter cloacae complex and several Klebsiella species remained unaffected. We demonstrate that this resistance against T6SS assaults was mediated by the production of superior T6SS machinery or a barrier exerted by group I capsules. Collectively, our data provide new insights into immunity protein-independent T6SS resistance employed by the human microbiota and colonization resistance in general.

DNA-uptake pili of Vibrio cholerae are required for chitin colonization and capable of kin recognition via sequence-specific self-interaction.

How bacteria colonize surfaces and how they distinguish the individuals around them are fundamental biological questions. Type IV pili are a widespread and multipurpose class of cell surface polymers. Here we directly visualize the DNA-uptake pilus of Vibrio cholerae, which is produced specifically during growth on its natural habitat-chitinous surfaces. As predicted, these pili are highly dynamic and retract before DNA uptake during competence for natural transformation. Interestingly, DNA-uptake pili can also self-interact to mediate auto-aggregation. This capability is conserved in disease-causing pandemic strains, which typically encode the same major pilin subunit, PilA. Unexpectedly, however, we discovered that extensive strain-to-strain variability in PilA (present in environmental isolates) creates a set of highly specific interactions, enabling cells producing pili composed of different PilA subunits to distinguish between one another. We go on to show that DNA-uptake pili bind to chitinous surfaces and are required for chitin colonization under flow, and that pili capable of self-interaction connect cells on chitin within dense pili networks. Our results suggest a model whereby DNA-uptake pili function to promote inter-bacterial interactions during surface colonization. Moreover, they provide evidence that type IV pili could offer a simple and potentially widespread mechanism for bacterial kin recognition.

Neighbor predation linked to natural competence fosters the transfer of large genomic regions in Vibrio cholerae.

Natural competence for transformation is a primary mode of horizontal gene transfer. Competent bacteria are able to absorb free DNA from their surroundings and exchange this DNA against pieces of their own genome when sufficiently homologous. However, the prevalence of non-degraded DNA with sufficient coding capacity is not well understood. In this context, we previously showed that naturally competent Vibrio cholerae use their type VI secretion system (T6SS) to actively acquire DNA from non-kin neighbors. Here, we explored the conditions of the DNA released through T6SS-mediated killing versus passive cell lysis and the extent of the transfers that occur due to these conditions. We show that competent V. cholerae acquire DNA fragments with a length exceeding 150 kbp in a T6SS-dependent manner. Collectively, our data support the notion that the environmental lifestyle of V. cholerae fosters the exchange of genetic material with sufficient coding capacity to significantly accelerate bacterial evolution.

Molecular insights into Vibrio cholerae's intra-amoebal host-pathogen interactions.

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.