Virulence Regulation and Innate Host Response in the Pathogenicity of Vibrio cholerae

Abstract

The human pathogen Vibrio cholerae is the causative agent of severe diarrheal disease known as cholera. Of the more than 200 "O" serogroups of this pathogen, O1 and O139 cause cholera outbreaks and epidemics. The rest of the serogroups, collectively known as non-O1/non-O139 cause sporadic moderate or mild diarrhea and also systemic infections. Pathogenic V. cholerae circulates between nutrient-rich human gut and nutrient-deprived aquatic environment. As an autochthonous bacterium in the environment and as a human pathogen, V. cholerae maintains its survival and proliferation in these two niches. Growth in the gastrointestinal tract involves expression of several genes that provide bacterial resistance against host factors. An intricate regulatory program involving extracellular signaling inputs is also controlling this function. On the other hand, the ability to store carbon as glycogen facilitates bacterial fitness in the aquatic environment. To initiate the infection, V. cholerae must colonize the small intestine after successfully passing through the acid barrier in the stomach and survive in the presence of bile and antimicrobial peptides in the intestinal lumen and mucus, respectively. In V. cholerae, virulence is a multilocus phenomenon with a large functionally associated network. More than 200 proteins have been identified that are functionally linked to the virulence-associated genes of the pathogen. Several of these genes have a role to play in virulence and/or in functions that have importance in the human host or the environment. A total of 524 genes are differentially expressed in classical and El Tor strains, the two biotypes of V. cholerae serogroup O1. Within the host, many immune and biological factors are able to induce genes that are responsible for survival, colonization, and virulence. The innate host immune response to V. cholerae infection includes activation of several immune protein complexes, receptor-mediated signaling pathways, and other bactericidal proteins. This article presents an overview of regulation of important virulence factors in V. cholerae and host response in the context of pathogenesis.

Keywords: V. cholerae; host response; microbiome; quorum sensing; toxins; virulence.

Figure 1

Mechanism of action of the cholera toxin. CT binds to the ganglioside receptor on the host epithelial cells, triggers endocytosis of the holotoxin. The internalized CT moves from the endosomes to the Golgi complex and endoplasmic reticulum (ER). The catalytic CT-A1 polypeptide transfers from the ER to the cytosol by retro-translocation through the action of the ER-linked degradation pathway to activate the Gsα subunit of guanine nucleotide-binding regulatory (Gα_s) protein. Activation of Gα_s-protein leads to increased adenylate cyclase (AC) activity, which cleaves ATP to cyclic adenosine monophosphate (cAMP) and subsequently activates protein kinase-A (PKA). Activation of PKA inhibits NaCl absorption through Na⁺/H⁺ exchanger (NHE transporters) and phosphorylate the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel proteins, which leads to ATP-mediated efflux of chloride ions and induce secretion of ${\text{HCO}}_3^{-}$, Na⁺, K⁺, and H₂O. Loss of chloride ions induces massive fluid secretion in the small intestine, deposing the resorptive ability of the large intestine, which results in severe watery diarrhea.

Figure 2

Schematic mechanisms of V. cholerae heat-stable eterotoxin (NAG/O1-ST). NAG/O1-ST bind to the intestinal guanylate cyclase (GC-C). Activation of intracellular catalytic domain of GC-C result in the formation of cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP). This intracellular transformation activates cGMP-cAMP-dependent PKA leads to CFTR phosphorylation. cGMP reduces Na⁺ and Cl⁻ absorption through the NHE, and also inhibits phosphodiesterase-3 (PDE3) leading to cellular accumulation of cAMP, and subsequent activation of PKA. Phosphorylation of the CFTR leads to secretion of Cl⁻ with ${\text{HCO}}_3^{-}$ and decreased NaCl absorption, which results in diarrhea.

Figure 3

Mechanisms of action of cholix toxin. Chx binds to the lipoprotein receptor-related protein (LRP-receptor) of the eukaryotic cells, followed by internalization by receptor-mediate endocytosis (RME). A furin-like enzyme is believed to be responsible for nicking cholix in its arginine-rich loop. After passing the Golgi complex, cholix follows the retrograde pathway in the endoplasmic reticulum to form the A and B-subunits. The A-subunit translocate into the cytoplasm and prevents protein synthesis by altering the diphthamide residue of elongation factor 2 (eEF2) through its ADP-ribosylation activity. Inhibition of protein synthesis by specific modification of eEF2 leads to cell death.

Figure 4

Regulation model of Tox-mediated virulence genes in V. chlerae. TcpP/TcpH and ToxR/ToxS function together in activating transcription of toxT. ToxR and TcpP need accessory proteins (ToxS and TcpH, respectively) for maximal activity. ToxT initiates transcription of the full tcp operon, ctxAB, and acf. AphA and AphB activate transcription of the tcpPH operon in response to environmental conditions. tcpPH promoter is negatively influenced by the global regulator, CRP. ToxR/ToxS, and AphA/AphB also regulate genes other than ctx and acf. ToxRS, AphAB, TcpPH, and ToxT coordinately regulate the transcription of ctx and tcp. AphA and AphB activates expression of the tcpPH. TcpPH and ToxRS regulate ctx and tcp genes through ToxT. AphB has been linked to the expression of tcpPH for anaerobiosis that enhances the production of CT. The direct activator and the second activator are differentiated by black solid and dotted arrows, respectively. Blue arrows indicate different environmental conditions sensed by the ToxRS and TcpPH, AphAB, and VarS regulatory systems. The two-component VarS-VarA system responds to environmental factors and signals toxT. ToxR, independently activates and represses transcription of ompU and ompT. ToxR modulation was shown by an arrow (ompU) and line with base (ompT) through + for the transcription of ompU and—for repressing the transcription of ompT. Red circles in ompU and ompT indicate the ToxR binding sites and the transcriptional start sites are marked with arrows for each promoter.

Figure 5

Schematic representation of V. cholerae H-NS mediated negative modulation virulence and biofilm expression. Apart from the ctxAB operon, most of the genes are silenced by H-NS is located in the tcp, rtx gene clusters, vas T6SS operon, hlyA hemolysin/cytolysin, and the vps biofilm utilization genes at low c-di-GMP. Lines with base near the intergenic regions represent H-NS repression. Red arrow indicates activation of corresponding genes.

Figure 6

Quorum sensing circuits in V. cholerae. In V. cholerae, QS outcome depends on the cell density. At a low cell density (LCD), QS will be in a switch-off mode. With low autoinducer concentration, the membrane proteins LuxPQ and CqsS act as kinases and direct phosphate (P) through LuxO to LuxU. With σ54 of RNA polymerase, LuxO initiates transcription of genes encoding regulatory Qrr ncRNAs. The Qrr ncRNAs activate translation of AphA and with Hfq, it represses translation of HapR. The above mentioned conditions promotes expression of genes encoding virulence factors and biofilm formation. At a high cell density (HCD), the QS will be in a switch-on mode. At this phase, the autoinducers are accrued, AI-2 (Δ) and CAI-1 (⋆) bind to their respective receptors LuxPQ and CqsS. Binding of AI act as phosphatases to reverse the flow of phosphate (P) across the regulatory circuit and deactivate LuxO. As a result, the Qrr ncRNAs are not formed and hence, HapR translation is not repressed and AphA translation is not activated. HapR represses the virulence-related functions. Other receptors, VarSA/CsrABCD with unknown ligands, also transduce QS information through LuxU. DNA-binding protein Fis functions together with AI-2/LuxPQ, CAI-1/CqsS, and VarS/VarA-CsrABCD systems to increase the activity of LuxO-phosphate at LCD. At the HCD, Fis and VarS/A-CsrABCD are inactive. OM and IM, indicate outer- and inner-membrane, respectively. H and D in the boxes denote histidine and aspartate sites of phosphorylation, respectively. Dotted arrows denote hypothetical interaction.