A few years ago, while writing an article on the association between science and serendipity, I had the opportunity to interview Dr. Hamilton Smith, Professor of Molecular Biology and Genetics at Johns Hopkins University. Following a series of fortuitous events, Dr. Smith made a revolutionary discovery that led to his becoming the father of modern molecular biology and the recipient of the Nobel Prize in Medicine in 1978. He discovered restriction enzymes, chemicals used by bacteria to slice setments of DNA that confers upon them unparalleled genetic plasticity and adaptability.
In the early 1990s, Dr. Smith was recruited by Dr. Craig Venter, an extremely bright but controversial geneticist, to the Institute for Genomic Research (TIGR). Until then, Venter had been randomly sampling and sequencing small bits of cDNA. Soon after joining TIGR, Smith proposed a bolder approach: “shotgunning” the entire genome of an organism. The idea was dramatically simple. Using an ordinary kitchen blender, the organism's DNA was divided into millions of small fragments which were then run through automated sequeneers and reassembled into the full genome using a high-speed computer and novel software written by an in-house computer whiz.
Within a year, TIGR had published the entire genome of Hemophilus influenza, the microorganism that lead Smith to the discovery of restriction enzymes. Soon after, TIGR engaged in the race to sequence the entire human genome, a story that has recently made the news because of its political, interpersonal, and ethical implications. The great genome quest was officially called a tie between TIGR and the government-sponsored Human Genome Project, thanks to a round of “pizza diplomacy”.
Following this major milestone, one might think that any other accomplishment in genome sequencing would appear negligible. Nevertheless, the recent report by TIGR researchers on the sequence of the entire Vibrio cholerae genome (Nature 2000;406:477–483) carries major implications for the long-standing battle with a major scourge of humankind.
Cholera has been a recognized human plague for over two millennia. The study of this disease has spanned from ancient Greece, to the Roman Empire, to the famous John Snow's tracing of an outbreak to a water pump in Broad Street, London, in 1854. Thirty years later Robert Koch proposed that the agent responsible for cholera produces “a special poison” acting on the intestinal epithelium and that the symptoms of cholera could be “regarded essentially as a poisoning”. Considerable time elapsed before the existence of this hypothesized toxin was demonstrated in 1959. Ten years later, cholera toxin (CT) was purified to homogeneity. It was only recently, however, that we learned that Vibrios produce a variety of other extracellular products that enable these microorganisms to activate different intracellular signaling in the host mammalian cells, all leading to diarrhea (Am J Physiol 1999;276:C765–C776).
The CT genes (ctx AB) operon is located on a 4.5-kb region called the “core region”. Beside ctx, this region contains a series of other toxin-encoding genes, including the Zonula occludens toxin (Zot) gene, located immediately upstream of ctx, and the gene encoding the Accessory cholera toxin (Ace). Interestingly, all these toxins have secretory capabilities; however, their mechanisms of action are different, suggesting that Vibrio cholerae possesses a diversified portfolio that induces diarrhea. The clustering of pathogenic factors within a highly dynamic region of the Vibrio cholerae chromosome that can be amplified or deleted during bacterial replication was perceived as a virulence cassette of the bacterium (J Pediatr Gastroenterol Nutr 1998;26:52–532).
The complicated genetic arrangement of the Vibrio cholerae virulence cassette raises an obvious question: what does a microorganism that elaborates the most powerful enterotoxin ever described maintain in its limited genome genes that encodes other enterotoxins? The answer came a few years ago from a study by Waldor and Mekalanos, who showed that the genes upstream of ctx belongs to a filamentous phage (designed CTXφ) that replicates as a plasmid and is responsible for the horizontal transfer of pathogenic elements (ctx, zot, ace) to non-toxigenic Vibrio cholerae. The authors also proved that the toxin-coregulated pilus (TCP), surface organelles required for intestinal colonization encoded by a second phage and that are co-regulated with CT by the same expression system (the ToxR system), represents the phage receptor. These findings suggest that CTXφ gains entry in the bacterium by way of the TCP, incorporates its genes into the bacterial chromosome, and, in some manner involving the tox R gene, causes the cell to producing enterotoxins.
This series of discoveries generated great enthusiasm among scientists who felt that they now had the “keys” to win the ongoing fight against Vibrio cholerae. However, every new effort was systematically followed by disappointing failures, giving the impression that the microorganism always has a “backup” solution to continue to induce diarrhea. The general perception was that the battle against cholera was a lost cause. It is within this scenario that the importance of the report of the entire genome sequence of V. cholerae O1 (biotype El Tor) in Nature needs to be framed. This publication can properly be regarded as an historic event that will trigger a paradigm shift in the study of this organism.
Of all enteric pathogens, Vibrio cholerae is responsible for the most rapidly fatal diarrheal disease in humans, particularly in children. Although cholera is rare in developed countries, it remains a major cause of diarrheal morbidity and mortality in many parts of the developing world. However, the occurrence of both natural (i.e., earthquakes) and human-generated calamities (such as the ethnic wars in Rwanda and in the Balkans), and the spreading of cholera infection in refugee camps, in which sanitary conditions resemble those occurring in cholera endemic areas, represents a real threat worldwide.
The most intriguing, and least understood, feature of Vibrio cholerae comes from the study of its annual epidemic profile, particularly in the Bengal region of Bangladesh and India, where nearly all cases each year occur in a synchronized, massive outbreak in the months of October and November, just as the monsoon rains decline. This epidemic profile and its correlation with major transitions of climate suggest that the microorganism resides in a stable environmental reservoir. Seasonally determined changes in rainfall and sunlight then trigger its periodic and transient emergence as a human pathogen. Between peidemics, V. cholerae lives in natural aquatic habitats formed by the confluence of the Ganges and the Brahmaputra rivers. Thus, the functional repertoire of the V. cholerae O1 genome must be unusually broad as it accommodates two distinctive lifestyles such as the milieu of the human intestine and long term residence in aquatic habitats that are subject to climate-determined changes of the environment.
It is therefore interesting that the longstanding view (based on studies conducted on Escherichia coli) that bacterial cells each contain a single circular chromosome has now been subverted by the observation that the V. cholerae O1 genome consists of two circular chromosomes*. The larger chromosome, chromosome I, contains a preponderance of genes dedicated to essential cell functions and most of the genetic loci associated with virulence. The smaller chromosome II also contains essential genes, suggesting that both chromosomes have existed together for a long period and are functionally complementary, even though the authors propose disparate evolutionary origins for the two units of replication. Chromosome II also contains genes involved in the transport of sugars, metal ions and anions, in the metabolism of sugars and energy, and in two-component signal transduction, all essential functions to adapt and survive in dynamic aquatic environments. Therefore, it is tantalizing to hypothesize that the distribution of genes of known function between the large and small chromosomes confer an evolutionary advantage to Vibrio cholerae in habitats that vary with climate changes and microenvironment. Specifically, it is possible to predict that chromosomes I genes mainly adapt the organism for growth in the intestine whereas chromosome II genes are essential within environmental niches.
Can these findings help us once and for all to win the battle against cholera? Maybe so, particularly considering that we have now the tools to learn the switches used by this microorganism to adapt to different microenvironments and to change from an aquatic bacterium to an intestinal pathogen. To this end, microarray experiments will be useful to identify genes that are differentially expressed during co-cultivation with algae and zooplankton, after exposure to different salt concentrations, and within the human intestinal tract. Furthermore, microarray DNA-DNA hybridization experiments can now be performed with labeled DNA to compare the genomes of other Vibronacee (including V. cholerae non-O1) not capable of seasonal endemic outbreaks to identify genes present in the sequenced V. cholerae O1, but missing from the yet-to-be-sequenced other biotypes. The identification of these genes and the mechanisms of regulation of their expression could lead to the development of strategies to keep these bacteria where they belong; in the aquatic environment and thereby put an end to one of the most devastating plagues that continues to claim a heavy toll on the human population.