Cholera is caused by toxigenic strains of Vibrio cholerae, a curved, Gram-negative bacterium that can live in aquatic environments and infect the human small intestine. Its ability to cause severe watery diarrhoea depends on several bacterial traits working together. Two are especially important: the toxin-coregulated pilus (TCP), which supports intestinal colonization, and cholera toxin (CT), which alters ion and water transport in intestinal epithelial cells. Remarkably, the genes responsible for these traits were acquired through mobile genetic elements rather than arising together as a single permanent bacterial trait.

Three genetic elements are particularly important for understanding the evolution of toxigenic Vibrio cholerae: the cholera toxin phage CTXφ, the Vibrio pathogenicity island VPI-1, and the CTXφ-associated element RS1. They have different identities and functions. CTXφ carries the cholera toxin genes; VPI-1 encodes the toxin-coregulated pilus (TCP) and associated colonization factors; and RS1 can influence CTXφ regulation and transfer. Their relationship illustrates how horizontal gene transfer and bacterial gene regulation contribute to cholera pathogenicity (Waldor & Mekalanos, 1996; Davis & Waldor, 2003; Kumar et al., 2020).

How does Vibrio cholerae cause cholera?

Although many environmental Vibrio cholerae strains lack the cholera toxin genes, epidemic cholera is primarily associated with toxigenic strains of serogroups O1 and O139. Infection usually follows ingestion of contaminated water or food. In the small intestine, toxin-coregulated pili support colonization and microcolony formation. The bacteria secrete cholera toxin, an AB₅ toxin composed of an enzymatically active A component and five receptor-binding B subunits.

The B subunits bind the GM1 ganglioside receptor on intestinal epithelial cells, allowing the toxin to enter the cells. The A1 enzymatic domain modifies the host Gsα signalling protein by ADP-ribosylation, maintaining activation of adenylate cyclase. Intracellular cyclic AMP increases, disrupting normal electrolyte handling, including chloride secretion. Water follows the resulting osmotic gradient into the intestinal lumen. This produces the characteristic profuse watery diarrhoea and can lead to rapid dehydration and electrolyte loss. Disease severity varies among infected individuals (WHO, 2026; Ramamurthy et al., 2020).

Clinical management is therefore centred on rapid rehydration, using oral rehydration solution (ORS) or intravenous fluids when indicated; antibiotics are useful in selected severe cases. Studying the phage genetics explains the emergence of toxigenic strains, not a separate infectious disease caused by phages in the patient (WHO, 2026).

1. CTXφ: the bacteriophage that carries cholera toxin genes

The pivotal discovery came in 1996, when Waldor and Mekalanos showed that the cholera toxin genes, ctxA and ctxB, belong to the genome of an infectious filamentous phage called CTXφ. This provided direct evidence for lysogenic conversion: a previously non-toxigenic, appropriately susceptible bacterium can acquire toxin-encoding DNA through phage infection. Acquisition of these genes is an essential part of the classic genetic route to toxin production, but it does not by itself guarantee epidemic potential.

What does CTXφ carry?

CTXφ has a single-stranded DNA genome in the infectious particle. Its genetic organization includes a core region containing ctxAB and genes involved in filamentous phage particle formation, and a regulatory/replication region often called RS2. The RS2 region includes rstR (a repressor), rstA (replication-associated) and rstB (integration-associated). The presence, arrangement and sequence of these regions can vary among strains and CTXφ lineages (Davis & Waldor, 2003).

Why does CTXφ recognize TCP?

A central feature of this system is the dependence of CTXφ entry on a surface structure produced by the bacterium: the toxin-coregulated pilus, or TCP. TCP is a type IV pilus assembled largely from the bacterial pilin TcpA. This makes TCP unusual because it acts both as a host-colonization factor and as a receptor used by CTXφ. In the well-characterized canonical pathway, bacteria that cannot express a suitable TCP are poor recipients for CTXφ. Phage susceptibility also depends on strain background, regulatory conditions and additional host factors. CTXφ therefore cannot convert every V. cholerae strain into a toxin producer (Waldor & Mekalanos, 1996; Kumar et al., 2020).

Integration and expression are separate questions

After infection, CTXφ DNA can be maintained in integrated prophage forms in the bacterial chromosomes. The architecture of CTXφ and adjacent repeated sequences varies across V. cholerae lineages. Integrating the genes is not the same event as producing large amounts of toxin. In a toxigenic bacterium, toxin expression is controlled by environmental and cellular regulatory networks, particularly the ToxR/ToxS, TcpP/TcpH and ToxT system. Thus, the level of toxin production depends on transcriptional regulation and bacterial physiology as well as on the presence of ctxAB (Kumar et al., 2020; Ramamurthy et al., 2020).

2. VPI-1: the pathogenicity island that produces the toxin-coregulated pilus

The Vibrio pathogenicity island 1 (VPI-1) is a horizontally acquired region of the bacterial chromosome of roughly 40 kb, although its precise size and composition can vary. It contains genes for TCP biogenesis and the transcriptional regulator ToxT, together with other factors relevant to intestinal colonization. Its importance is twofold: it supports the bacterium’s interaction with the intestinal environment, and its TCP apparatus provides the receptor for CTXφ. This helps explain why horizontal acquisition of the colonization island and the toxin phage are biologically linked (Kumar et al., 2020).

The VPIφ hypothesis and the identity of VPI-1

A 1999 study by Karaolis and colleagues proposed that VPI-1 could be associated with an infectious filamentous phage designated VPIφ. Later investigations did not establish an independently reproducible VPIφ infection cycle, and VPI-1 is currently described as a chromosomal pathogenicity island. This distinction matters because horizontal acquisition of DNA does not by itself establish that the DNA is an infectious phage genome (Karaolis et al., 1999; Davis & Waldor, 2003; Kumar et al., 2020).

An important distinction for students is that a pathogenicity island may be horizontally acquired without itself being an independently demonstrated phage. Evolutionary mobility and viral identity are different claims. VPI-1 is evidence for the role of mobile DNA in virulence, but its mode of historical acquisition is not fully reduced to a single verified viral life cycle.

3. RS1: a satellite element that influences CTXφ biology

RS1 is a small CTXφ-related genetic element frequently found adjacent to CTX prophage sequences in toxigenic V. cholerae. It resembles the RS2 regulatory region of CTXφ but contains an additional gene, rstC. Unlike CTXφ, RS1 generally lacks its own full complement of genes required for filamentous phage morphogenesis. Under experimentally established conditions, RS1 DNA can be packaged and transferred using structural and assembly functions supplied by CTXφ; it is therefore described as a satellite phage-related element (Faruque et al., 2002; Davis et al., 2002).

RstC acts as an antirepressor of RstR, thereby affecting repression of CTXφ-associated genes and influencing phage production or transfer in suitable genetic backgrounds. RS1 does not encode cholera toxin and is not required in every toxigenic V. cholerae strain (Davis et al., 2002; Davis & Waldor, 2003).

Does RS1 require an existing CTXφ infection to enter?

Laboratory studies found that RS1-containing particles could infect suitable TCP-expressing recipient cells, including nontoxigenic recipients. The stronger dependence was for productive generation of new RS1 phage-like particles in the characterized helper-dependent system, which required CTXφ morphogenesis functions. Other reported mechanisms underline the need to avoid treating the satellite relationship as one invariable rule for every isolate (Faruque et al., 2002).

How CTXφ, VPI-1 and RS1 work together

The elements contribute different functions to the same virulence system. VPI-1 encodes the pilus needed for intestinal colonization and for CTXφ entry in the canonical infection pathway. CTXφ carries the cholera toxin genes, whereas RS1 can modify CTXφ regulation and facilitate the movement of phage-related DNA. Cholera develops through the action of toxin produced by the bacterium on intestinal epithelial cells; the bacteriophages do not directly infect human cells.

Horizontal gene transfer and the emergence of epidemic lineages

The acquisition of mobile DNA can change bacterial phenotypes over evolutionary time. In V. cholerae, toxin phages, genomic islands, and other elements have contributed to combinations of colonization, toxin production, and environmental fitness. However, the acquisition of CTXφ and VPI-1 is not a complete explanation of pandemic success. Regulatory circuits, ecological persistence, strain background, interactions with other phages, and factors influencing transmission also matter (Kumar et al., 2020; Ramamurthy et al., 2020).

Some CTXφ and RS1 variants differ among classical and El Tor biotypes, and their chromosomal arrangements influence phage production and transfer. Consequently, results from one reference strain cannot automatically be extended to every O1, O139 or non-O1/non-O139 isolate. The species name alone is insufficient to establish toxigenic potential; toxin-gene detection and direct demonstration of toxin production are distinct kinds of evidence (Davis & Waldor, 2003).

What genomic studies can–and cannot–show

CTXφ offers a textbook example of phage-mediated acquisition of a virulence determinant. VPI-1 illustrates a pathogenicity island that supplies a colonization factor and an entry receptor for a toxin phage. RS1 illustrates satellite-element dependence and regulatory interplay. Together they show why interpreting a bacterial genome requires attention to both gene content and mechanism: a prophage sequence may be intact or defective, a gene may be present but poorly expressed, and a predicted mobile element may lack evidence for infectious particles.

In research, whole-genome sequencing can reveal the presence and organization of ctxAB, TCP genes and nearby mobile-element sequences. Comparative genomics can trace associations among strains, while transcriptional and phenotypic experiments are required to establish gene expression, pilus function, phage production or toxin activity. The presence of a sequence alone does not establish that a strain is making a given virulence factor under the conditions being studied.

Conclusion

The molecular basis of cholera virulence involves distinct but interacting genetic elements. Current evidence identifies CTXφ as the cholera-toxin-encoding phage, VPI-1 as the TCP-encoding pathogenicity island and RS1 as a CTXφ-related satellite element. Their interactions help explain how bacterial virulence traits can be assembled and regulated through horizontal gene transfer.

This interpretation also helps avoid two common misunderstandings: not all V. cholerae strains cause epidemic cholera, and the presence of a virulence-associated gene does not automatically prove active toxin production or severe disease. The relationship between phages and bacterial pathogenicity is important precisely because it links microbial evolution, gene regulation and host disease without reducing them to one mechanism.

References

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