Eukaryotic catecholamine hormones influence the chemotactic control of Vibrio campbellii by binding to the coupling protein CheW

In addition to their well-known role as stress-associated catecholamine hormones in animals and humans, epinephrine (EPI) and norepinephrine (NE) act as interkingdom signals between eukaryotic hosts and bacteria. However, the molecular basis of their effects on bacteria is not well understood. In initial phenotypic studies utilizing Vibrio campbellii as a model organism, we characterized the bipartite mode of action of catecholamines, which consists of promotion of growth under iron limitation, and enhanced colony expansion on soft agar. In order to identify the molecular targets of the hormones, we designed and synthesized tailored probes for chemical proteomic studies. As the catechol group in EPI and NE acts as iron chelator and is prone to form a reactive quinone moiety, we devised a photoprobe based on the adrenergic agonist phenylephrine (PE), which solely influenced colony expansion. Using this probe, we identified CheW, located at the core of the chemotaxis signaling network, as a major target. In vitro studies confirmed that EPI, NE, PE, as well as labetalol, a clinically applied antagonist, bind to purified CheW with affinity constants in the sub-micromolar range. In line with these findings, exposure of V. campbellii to these adrenergic agonists affects the chemotactic control of the bacterium. This study highlights a previously unknown effect of eukaryotic signaling molecules on bacterial motility.

Characterization and complete genome sequence of bacteriophage vB_Vc_SrVc2, a marine phage that infects Vibrio campbellii

Vibrio campbellii is widely distributed in the marine environment and is an important pathogen of aquatic organisms such as shrimp, fish, and mollusks. The emergence of multi-drug resistance among these bacteria resulted in a worldwide public health problem, which requires alternative treatment approaches such as phage therapy. In the present study, we isolated a phage vB_Vc_SrVc2 from white shrimp hepatopancreas with symptoms of AHPND. Phage vB_Vc_SrVc2 is a member of the genus Maculvirus and the family Autographiviridae, with high lytic ability against Vibrio isolates. Phage has a high resistance to a broad range of temperatures, salinity, UV radiation and chloroform. The genome size was 43,157 bp, with a GC content of 49.2% that encodes 49 putative ORFs, no tRNAs, showed three single nucleotide polymorphisms, two small deletions and one nucleotide insertion compared to SrVc9, showing slightly different infectivity profiles.. No lysogeny related genes were detected in vB_Vc_SrVc2 genome. Overall phage vB_Vc_SrVc2 has a good potential for therapeutic use in the aquaculture industry against V. campbellii infections.

First insights into a pyruvate sensing and uptake system in Vibrio campbellii and its importance for virulence

Pyruvate is a key metabolite in living cells and has been shown to play a crucial role in the virulence of several bacterial pathogens. The bioluminescent Vibrio campbellii , a severe infectious burden for marine aquaculture, excretes extraordinarily large amounts of pyruvate during growth and rapidly retrieves it by an as-yet unknown mechanism. We have now identified the responsible pyruvate transporter, here named BtsU, and our results show that it is the only pyruvate transporter in V. campbellii . Expression of btsU is tightly regulated by the membrane-integrated LytS-type histidine kinase BtsS, a sensor for extracellular pyruvate, and the LytTR-type response regulator BtsR. Cells lacking either the pyruvate transporter or sensing system show no chemotactic response towards pyruvate, indicating that intracellular pyruvate is required to activate the chemotaxis system. Moreover, pyruvate sensing and uptake were found to be important for the resuscitation of V. campbellii from the viable but nonculturable (VBNC) state and the bacterium’s virulence against brine shrimp larvae. IMPORTANCE Bacterial infections are a serious threat to marine aquaculture, one of the fastest growing food sectors on earth. Therefore, it is extremely important to learn more about the pathogens responsible, one of which is Vibrio campbellii . This study sheds light on the importance of pyruvate sensing and uptake for V. campbellii , and reveals that the bacterium possesses only one pyruvate transporter, which is activated by a pyruvate-responsive histidine kinase/response regulator system. Without the ability to sense or take up pyruvate, the virulence of V. campbellii towards gnotobiotic brine shrimp larvae is strongly reduced.

The polar flagellar transcriptional regulatory network in Vibrio campbellii deviates from canonical Vibrio species

Swimming motility is a critical virulence factor in pathogenesis for numerous Vibrio species. Vibrio campbellii DS40M4 is a wild isolate that has been recently established as a highly tractable model strain for bacterial genetics studies. We sought to exploit the tractability and relevance of this strain for characterization of flagellar gene regulation in V. campbellii . Using comparative genomics, we identified homologs of V. campbellii flagellar and chemotaxis genes conserved in other members of the Vibrionaceae and determined the transcriptional profile of these loci using differential RNA-seq. We systematically deleted all 63 predicted flagellar and chemotaxis genes in V. campbellii and examined their effects on motility and flagellum production. We specifically focused on the core regulators of the flagellar hierarchy established in other vibrios: RpoN (σ 54 ), FlrA, FlrC, and FliA. Our results show that V. campbellii transcription of flagellar and chemotaxis genes is governed by a multi-tiered regulatory hierarchy similar to other motile Vibrio species. However, there are several critical differences in V. campbellii : (i) the σ 54 -dependent regulator FlrA is dispensable for motility, (ii) the flgA , fliEFGHIJ , flrA , and flrBC operons do not require σ 54 for expression, and (iii) FlrA and FlrC co-regulate class II genes. Our model proposes that the V. campbellii flagellar transcriptional hierarchy has three classes of genes, in contrast to the four-class hierarchy in Vibrio cholerae . Our genetic and phenotypic dissection of the V. campbellii flagellar regulatory network highlights the differences that have evolved in flagellar regulation across the Vibrionaceae. Importance Vibrio campbellii is a Gram-negative bacterium that is free-living and ubiquitous in marine environments and is an important global pathogen of fish and shellfish. Disruption of the flagellar motor significantly decreases host mortality of V. campbellii , suggesting that motility is a key factor in pathogenesis. Using this model organism, we identified >60 genes that encode proteins with predicted structural, mechanical, or regulatory roles in function of the single polar flagellum in V. campbellii . We systematically tested strains containing single deletions of each gene to determine the impact on motility and flagellum production. Our studies have uncovered differences in the regulatory network and function of several genes in V. campbellii as compared to established systems in Vibrio cholerae and Vibrio parahaemolyticus .

The polar flagellar transcriptional regulatory network in Vibrio campbellii deviates from canonical Vibrio species

Vibrio campbellii is a Gram-negative bacterium that is free-living and ubiquitous in marine environments, and it is a pathogen of fish and shellfish. Swimming motility via a single polar flagellum is a critical virulence factor in V. campbellii pathogenesis, and disruption of the flagellar motor significantly decreases host mortality. To examine V. campbellii flagellar gene regulation, we identified homologs of flagellar and chemotaxis genes conserved in other members of the Vibrionaceae and determined the transcriptional profile of these loci using differential RNA-seq. We systematically deleted all 63 predicted flagellar and chemotaxis genes in V. campbellii and examined their effects on motility and flagellum production. We specifically focused on the core flagellar regulators of the flagellar regulatory hierarchy established in other Vibrios: RpoN (σ54), FlrA, FlrC, and FliA. Our results show that V. campbellii transcription of flagellar and chemotaxis genes is governed by a multi-tiered regulatory hierarchy similar to other motile Vibrio species but with two critical differences: the σ54-dependent regulator FlrA is dispensable for motility, and Class II gene expression is independent of σ54 regulation. Our genetic and phenotypic dissection of the V. campbellii flagellar regulatory network highlights the differences that have evolved in flagellar regulation across the Vibrionaceae.

Structural basis of chitin utilization by a GH20 β-N-acetylglucosaminidase from Vibrio campbellii strain ATCC BAA-1116

Vibrio species play a crucial role in maintaining the carbon and nitrogen balance between the oceans and the land through their ability to employ chitin as a sole source of energy. This study describes the structural basis for the action of the GH20 β-N-acetylglucosaminidase (VhGlcNAcase) in chitin metabolism by Vibrio campbellii (formerly V. harveyi) strain ATCC BAA-1116. Crystal structures of wild-type VhGlcNAcase in the absence and presence of the sugar ligand, and of the unliganded D437A mutant, were determined. VhGlcNAcase contains three distinct domains: an N-terminal carbohydrate-binding domain linked to a small α+β domain and a C-terminal (β/α)8 catalytic domain. The active site of VhGlcNAcase has a narrow, shallow pocket that is suitable for accommodating a small chitooligosaccharide. VhGlcNAcase is a monomeric enzyme of 74 kDa, but its crystal structures show two molecules of enzyme per asymmetric unit, in which Gln16 at the dimeric interface of the first molecule partially blocks the entrance to the active site of the neighboring molecule. The GlcNAc unit observed in subsite −1 makes exclusive hydrogen bonds to the conserved residues Arg274, Tyr530, Asp532 and Glu584, while Trp487, Trp546, Trp582 and Trp505 form a hydrophobic wall around the −1 GlcNAc. The catalytic mutants D437A/N and E438A/Q exhibited a drastic loss of GlcNAcase activity, confirming the catalytic role of the acidic pair (Asp437–Glu438).