Cholera, a severe disease caused by Vibrio cholerae bacteria, has had a central role in the history of infectious disease research. The cholera studies of John Snow and Robert Koch, among many others, largely gave birth to modern epidemiology and microbiology. Despite its long history as a research target, cholera continues to afflict approximately 5 million people each year and remains an important public health problem in many areas of the globe. Here we review the current knowledge of the complex regulatory network used by V. cholerae to control expression of its virulence determinants. FEATURES OF VIBRIO CHOLERAE PATHOGENICITYCholera, which is characterized by voluminous watery diarrhea, is produced when the gram-negative curved bacillus V. cholerae colonizes the upper small intestine of its human host. V. cholerae is found throughout the world in coastal areas, most often associated with aquatic fauna such as copepods and shellfish, and is transmitted to humans by contaminated food or water. Since 1817, seven cholera pandemics have been recorded, with the most recent, ongoing pandemic having begun in 1961. Although more than 200 different serogroups have been isolated from the environment, the O1 serogroup of V. cholerae was responsible for all seven pandemics. However, beginning in 1992, O139 serogroup strains were found to cause outbreaks of cholera as well; O139 V. cholerae is sometimes referred to as the eighth cholera pandemic strain.V. cholerae O1 exist in two biotypes: classical and El Tor. Classical V. cholerae was responsible for the first six cholera pandemics, whereas the seventh pandemic has been caused by El Tor V. cholerae. The two V. cholerae biotypes differ considerably. El Tor strains generally cause a milder form of cholera than that caused by classical strains and apparently evolved as better survivors in the aquatic environment; currently, El Tor strains are predominant everywhere in the world that V. cholerae O1 can be found. V. cholerae O139 likely arose by seroconversion of O1 El Tor strains (8, 72). In addition, there are subtle differences in the way that El Tor strains and classical strains regulate expression of key virulence factors, as will be discussed below.The clinical aspects of cholera are primarily induced by the activity of cholera toxin (CT), a bipartite toxin that consists of a single active A subunit and five B subunits that bind the toxin to the GM 1 ganglioside on the surface of the intestinal epithelium. Once inside epithelial cells, a proteolytically derived fragment of the CT-A subunit, CT-A 1 , ADP-ribosylates G ␣ s protein, resulting in constitutive cyclic AMP production. This leads to massive secretion of chloride and water into the lumen of the intestine. Cholera patients can lose up to 20 liters of fluid within a 24-h period, resulting in rapid dehydration, and Ͼ50% of cholera patients die without treatment. However, if patients are rehydrated orally and/or intravenously, mortality rates decrease to ϳ1%.Aside from CT, the other major V. cholerae virulence fac...
Vibrio cholerae is a gram-negative bacterium that is the causative agent of cholera, a severe diarrheal illness. The two biotypes of V. cholerae O1 capable of causing cholera, classical and El Tor, require different in vitro growth conditions for induction of virulence gene expression. Growth under the inducing conditions or infection of a host initiates a complex regulatory cascade that results in production of ToxT, a regulatory protein that directly activates transcription of the genes encoding cholera toxin (CT), toxin-coregulated pilus (TCP), and other virulence genes. Previous studies have shown that sodium bicarbonate induces CT expression in the V. cholerae El Tor biotype. However, the mechanism for bicarbonate-mediated CT induction has not been defined. In this study, we demonstrate that bicarbonate stimulates virulence gene expression by enhancing ToxT activity. Both the classical and El Tor biotypes produce inactive ToxT protein when they are cultured statically in the absence of bicarbonate. Addition of bicarbonate to the culture medium does not affect ToxT production but causes a significant increase in CT and TCP expression in both biotypes. Ethoxyzolamide, a potent carbonic anhydrase inhibitor, inhibits bicarbonate-mediated virulence induction, suggesting that conversion of CO 2 into bicarbonate by carbonic anhydrase plays a role in virulence induction. Thus, bicarbonate is the first positive effector for ToxT activity to be identified. Given that bicarbonate is present at high concentration in the upper small intestine where V. cholerae colonizes, bicarbonate is likely an important chemical stimulus that V. cholerae senses and that induces virulence during the natural course of infection.Cholera is a human disease that is characterized by massive loss of water and electrolytes, which leads to severe dehydration and hypovolemic shock if the condition is not treated. The causative agent of cholera is Vibrio cholerae, a highly motile, gram-negative, curved rod having a single polar flagellum. V. cholerae strains are classified into serogroups based on the lipopolysaccharide O antigen, and more than 200 serogroups have been identified to date. Only serogroups O1 and O139 are responsible for epidemic and pandemic cholera (46, 47). Serogroup O1 can be further divided into two biotypes, classical and El Tor, based on biochemical properties and susceptibility to bacteriophages (11,47). Classical biotype V. cholerae strains are thought to have caused the first six cholera pandemics, beginning in 1817, whereas the El Tor biotype has been responsible for the seventh pandemic, which has been ongoing since 1961 (11, 47).A major difference between the classical and El Tor biotypes is that they require different in vitro growth conditions for virulence gene induction. The classical biotype is cultured in LB medium at 30°C and pH 6.5 for maximal virulence gene expression and is cultured in LB medium at 37°C and pH 8.5 for minimal virulence gene expression (41). The El Tor biotype is cultured under biphasic condi...
SummaryThe Gram-negative, curved rod Vibrio cholerae causes the severe diarrhoeal disease cholera. The two major virulence factors produced by V. cholerae during infection are the cholera toxin (CT) and the toxincoregulated pilus (TCP). Transcription of the genes encoding both CT and the components of the TCP is directly activated by ToxT, a transcription factor in the AraC/XylS family. ToxT binds upstream of the ctxAB genes, encoding CT, and upstream of tcpA , the first gene in a large operon encoding the components of the TCP. The DNA sequences upstream of ctxAB and tcpA that contain ToxT binding sites do not have any significant similarity other than being AT-rich. Extensive site-directed mutagenesis was performed on the region upstream of tcpA previously shown to be protected by ToxT, and we identified specific base pairs important for activation of tcpA transcription by ToxT. This genetic approach was complemented by copperphenanthroline footprinting experiments that showed protection by ToxT of the base pairs identified as most important for transcription activation in the mutagenesis experiments. Based on this new information and on previous work, we propose the presence of a ToxTbinding motif -the 'toxbox' -in promoters regulated by ToxT. At tcpA , two toxbox elements are present in a direct repeat configuration and both are required for activation of transcription by ToxT. The identity of only a few of the base pairs within the toxbox is important for activation by ToxT, and we term these the core toxbox elements. Lastly, we examined ToxT binding to a mutant having 5 bp inserted between the two toxboxes at tcpA and found that occupancy of both binding sites is retained regardless of the positions of the binding sites relative to each other on the face of the DNA. This suggests that ToxT binds independently as a monomer to each toxbox in the tcpA direct repeat, in accordance with what we observed previously with the inverted repeat ToxT sites between acfA and acfD .
Transfer-messenger RNA (tmRNA, or SsrA), found in all eubacteria, has both transfer and messenger RNA activity. Relieving ribosome stalling by a process called trans-translation, tmRNAala enters the ribosome and adds its aminoacylated alanine to the nascent polypeptide. The original mRNA is released and tmRNA becomes the template for translation of a 10-amino-acid tag that signals for proteolytic degradation. Although essential in a few bacterial species, tmRNA is nonessential in Escherichia coli and many other bacteria. Proteins known to be associated with tmRNA include SmpB, ribosomal protein S1, RNase R, and phosphoribosyl pyrophosphate. SmpB, having no other known function, is essential for tmRNA activity. trans-translation operates within ribosomes stalled both at the end of truncated mRNAs and at rare codons and some natural termination sites. Both the release of stalled ribosomes and the subsequent degradation of tagged proteins are important consequences of trans-translation.
tmRNA, through its tRNA and mRNA properties, adds short peptide tags to abnormal proteins, targeting these proteins for proteolytic degradation. Although the conservation of tmRNA throughout the bacterial kingdom suggests that it must provide a strong selective advantage, it has not been shown to be essential for any bacterium. We report that tmRNA is essential in Neisseria gonorrhoeae. Although tagging per se appears to be required for gonococcal viability, tagging for proteolysis does not. This suggests that the essential roles of tmRNA in N.gonorrhoeae may include resolving stalled translation complexes and/or preventing depletion of free ribosomes. Although derivatives of N.gonorrhoeae expressing Escherichia coli tmRNA as their sole tmRNA were isolated, they appear to form colonies only after acquiring an extragenic suppressor(s).
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