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Infection and Immunity, June 2003, p. 2981-2982, Vol. 71, No. 6
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.6.2981-2982.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Bacteriophage and the Evolution of Epidemic Cholera

Jeff F. Miller^*

Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, California 90095

Top Introduction References

 
In January 1991, the seventh pandemic of cholera reached the west coast of Peru. It spread rapidly throughout South and Central America, and within a period of 11 months, over 300,000 cases of diarrhea were reported to the Pan American Health Organization (11). The Vibrio cholerae strain responsible was an O1 serogroup El Tor biotype, a close relative of the isolate that initiated the same pandemic 30 years earlier on the Indonesian island of Sulawesi (4). As South America was contending with an explosive outbreak of secretory diarrhea, a new epidemic began in 1992 in Madras, India, and Southern Bangladesh. The cause was a non-O1 V. cholerae strain representing an entirely new serogroup, later designated O139 (5). This came as a major surprise; it had previously been assumed that only O1 serogroup strains had the capacity to cause epidemic or endemic disease. In retrospect this assumption was naïve, considering the capacity of bacterial pathogens to undergo horizontal gene exchange. O139 strains continue to cause outbreaks with expanding geographic distribution, potentially heralding the eighth pandemic of cholera.

The evolutionary events responsible for the emergence of epidemic cholera are as fascinating and important as they are enigmatic. It is likely that the ability to cause new waves of epidemic disease in immune populations requires significant antigenic change. It is also likely that multiple horizontal gene transfer events lie at the core of this evolutionary potential. A wealth of evidence suggests that the O139 strain that emerged in 1992 was derived from a seventh pandemic El Tor clone by a series of events, which include deletion of the O1-antigen-specific gene cluster and the insertion of O139-specific genes (1, 5). Neither the donor of the newly acquired locus nor the mechanism of genetic exchange has so far been identified. More recent studies indicate that O139 isolates do not belong to a single clone, but represent disparate lineages that share the same serogroup (5).

A precise accounting of the determinants that differentiate nonpathogenic V. cholerae strains from those causing disease in humans is not yet available, although progress is certainly being made (3). It is clear, however, that the ToxRS regulatory system and the gene clusters encoding cholera toxin and the toxin-coregulated pilus (TCP) are nearly always present in pathogenic strains (4). The capacity for horizontal transfer is by no means limited to loci encoding O-antigen. The discovery by Waldor and Mekalanos that the genes encoding cholera toxin are carried on the genome of a filamentous phage (CTX) represents the clearest example of horizontal transfer of virulence genes between V. cholerae strains (13). Several observations in this seminal report are relevant to the controversy described below. First, the core region of the cholera toxin prophage contains four loci that are conserved in sequence and/or genomic organization with morphogenesis genes of other filamentous phage. Second, although CTX transduction could be readily demonstrated in vitro, by far the most efficient transfer occurred during gastrointestinal infection. This undoubtedly reflects a relationship between regulated gene expression and the efficiency of horizontal exchange. Finally, the receptor for CTX was shown to be the type IV TCP, which is itself a colonization factor in human and animal models (7, 12). This observation implied a temporal pathway for the evolution of pathogenesis. Acquisition of the TCP cluster followed by infection with CTX could represent sequential steps in the conversion of a nonpathogenic environmental isolate into a highly virulent strain. Although filamentous phage had not previously been recognized as being responsible for the lysogenic conversion of bacterial pathogens, they seem quite well suited for this purpose (13). The flexibility of their capsid structure accommodates packaging of heterologous DNA, phage production occurs without host cell lysis and in the presence of bacterial multiplication, and the pili they use as receptors are ubiquitous colonization factors for gram-negative bacteria.

The purported discovery in 1999 of yet a second filamentous phage, encoding TCP, was met with widespread interest (9) along with some healthy skepticism (C. A. Lee, Letter, Trends Microbiol. 7:391-392, 1999; D. K. R. Karaolis and J. B. Kaper, Authors' Reply, Trends Microbiol. 7:393). It was previously recognized that TCP are encoded on a 39.5-kb pathogenicity island, which includes putative integrase and transposase genes and is flanked by att-like sequences (8, 10). This V. cholerae pathogenicity island (VPI) is associated with epidemic and pandemic strains, and it clearly bears the markings of a horizontally acquired element. The suggestion that the VPI is in fact a filamentous phage (VPI) (9) was somewhat surprising, given the lack of convincing sequence similarity to morphogenesis genes of canonical filamentous phage. Nonetheless, substantiating evidence included the ability to amplify VPI sequences (but not chromosomal genes) in VPI preparations, the ability to transduce recipients with cell-free phage preparations from a VPI-tagged strain, and the apparent identification of a double-stranded VPI replicative form. It was further suggested that the TCP pilin subunit, TcpA, serves as the VPI coat protein as well as the CTX receptor. Given the importance of the publication that appeared in May 1999 (9), the lack of follow-up reports characterizing VPI has been a source of consternation.

In this issue of Infection and Immunity, Faruque et al. present results that appear to contradict the existence of VPI (6). A collection of clinical and surface water isolates containing VPI genes were cultivated under conditions identical to those described in the report by Karaolis et al. (9). Phage preparations were made and tested for VPI DNA (tcpA) and CTX DNA (ctxA) by a PCR assay capable of detecting an estimated 1 phage particle produced by 10⁸ cells. While many preparations were positive for ctxA, they were uniformly negative for tcpA. Neither mitomycin C nor UV irradiation induced VPI production, and attempts to transduce multiple Km^r-marked versions of the VPI cluster were negative. Transduction failed to occur even during infection of infant mice. The authors conclude that the TCP pathogenicity island is unable to support the production of VPI. There are several possible explanations for the inability of Faruque and colleagues to replicate the results of Karaolis et al. For example, minor differences in growth conditions could have a profound effect on the production of VPI.

Is the absence of evidence evidence of absence? Although the question is open for debate, a challenge has clearly been issued. It is reasonable to expect that an initial observation of importance will be followed up by the original authors and verified by other groups. Neither of these expectations has been met. Although the validity of the report by Karaolis et al. is in considerable doubt (2), the case for horizontal transfer of the TCP pathogenicity island remains as strong as ever (8). It may comprise an autonomous genetic element, but a more likely scenario is that it represents a "satellite" element that requires helper phage for transmission (6). The goal now is to discover the true mechanism or mechanisms that function in nature to promote VPI transfer.

The ecology of V. cholerae is complex. Defining the genetic exchange mechanisms that give rise to virulent strains will undoubtedly represent a quantum leap in our understanding of the evolution of a deadly disease. It may also improve the value of environmental surveillance efforts as a means of predicting impending epidemics. In light of this hope based on the power of molecular epidemiology, it is interesting to recall that in 1854, John Snow, a British anesthesiologist, abruptly stopped an epidemic of cholera that was sweeping through the Soho district of west London. This was accomplished not with the benefit of advanced technology or an understanding of microbial genetics, but by removing the handle from a pump that was dispensing contaminated water. Although our understanding of cholera has increased dramatically, the ultimate solution to the problem remains the same.

 
J.F.M. is supported by grants from the the National Institute of Allergy and Infectious Diseases and by the National Cancer Institute.

Many thanks go to Peggy Cotter, Catherine Miller, and members of my laboratory for thoughtful discussions and comments on the manuscript.

 
* Mailing address: Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine at UCLA, 10833 Le Conte Ave., Los Angeles, CA 90095-1747. Phone: (310) 206-7926. Fax: (310) 267-2774. E-mail: jfmiller{at}ucla.edu.

The views expressed in this Commentary do not necessarily reflect the views of the journal or of ASM. Editor: V. J. DiRita

Top Introduction References

 

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1. Bik, E. M., A. E. Bunschoten, R. D. Gouw, and F. Mooi. 1995. Genesis of the novel epidemic Vibrio cholerae O139 strain: evidence for horizontal transfer of genes involved in polysaccharide synthesis. EMBO J. 14:209-216.[Medline]
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2. Davis, B. M., and M. K. Waldor. 2003. Filamentous phages linked to virulence of Vibrio cholerae. Curr. Opin. Microbiol. 6:35-42.[CrossRef][Medline]
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3. Dziejman, M., E. Balon, D. Boyd, C. M. Fraser, J. F. Heidelberg, and J. J. Mekalanos. 2002. Comparative genomic analysis of Vibrio cholerae: genes that correlate with cholera endemic and pandemic disease. Proc. Natl. Acad. Sci. USA 99:1556-1561.[Abstract/Free Full Text]
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4. Faruque, S. M., M. J. Albert, and J. J. Mekalanos. 1998. Epidemiology, genetics, and ecology of toxigenic Vibrio cholerae. Microbiol. Mol. Biol. Rev. 62:1301-1314.[Abstract/Free Full Text]
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5. Faruque, S. M., D. A. Sack, R. B. Sack, R. R. Colwell, Y. Takeda, and G. B. Nair. 2003. Emergence and evolution of Vibrio cholerae O139. Proc. Natl. Acad. Sci. USA 100:1301-1309.
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6. Faruque, S. M., J. Zhu, Asadulghani, M. Kamruzzaman, and J. J. Mekalanos. 2003. Examination of diverse toxin-coregulated pilus-positive Vibrio cholerae strains fails to demonstrate evidence for vibrio pathogenicity island phage. Infect. Immun.71:2993-2999.
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7. Herrington, D. A., R. H. Hall, G. A. Losonsky, J. J. Mekalanos, R. K. Taylor, and M. M. Levine. 1988. Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. J. Exp. Med. 168:1487-1492.[Abstract/Free Full Text]
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8. Karaolis, D. K. R., J. A. Johnson, C. C. Bailey, E. C. Boedeker, J. B. Kaper, and P. R. Reeves. 1998. A Vibrio cholerae pathogenicity island associated with epidemic and pandemic strains. Proc. Natl. Acad. Sci. USA 95:3134-3139.[Abstract/Free Full Text]
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9. Karaolis, D. K. R., S. Somara, D. R. Maneval, Jr., J. A. Johnson, and J. B. Kaper. 1999. A bacteriophage encoding a pathogenicity island, a type-IV pilus and a phage receptor in cholera bacteria. Nature 399:375-379.[CrossRef][Medline]
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10. Kovach, M. E., M. D. Shaffer, and K. M. Peterson. 1996. A putative integrase gene defines the distal end of a large cluster of ToxR-regulated colonization genes in Vibrio cholerae. Microbiology 142:2165-2174.[Abstract/Free Full Text]
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13. Waldor, M. K., and J. J. Mekalanos. 1996. Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 272:1910-1914.[Abstract]

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Infection and Immunity, June 2003, p. 2981-2982, Vol. 71, No. 6
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.6.2981-2982.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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