Genomic characterization of non-O1, non-O139Vibriocholeraereveals genes for a type III secretion systemMichelle Dziejman*†, Davide Serruto*‡, Vincent C. Tam*, Derek Sturtevant*, Pornphan Diraphat*§, Shah M. Faruque¶,M. Hasibur Rahman¶, John F. Heidelberg储, Jeremy Decker**, Li Li**, Kate T. Montgomery**, George Grills**,Raju Kucherlapati††, and John J. Mekalanos*‡‡*Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115;¶Molecular Genetics Laboratory,International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka-1212, Bangladesh;储The Institute for Genomic Research, 9712 Medical Center Drive,Rockville, MD 20850; **Harvard Partners Genome Center, 65 Landsdowne Street, Cambridge, MA 02139; and††Department of Genetics, Harvard MedicalSchool, Brigham and Women’s Hospital, New Research Building, 77 Avenue Louis Pasteur, Boston, MA 02115Contributed by John J. Mekalanos, December 31, 2004Non-O1, non-O139 Vibrio cholerae can cause gastroenteritis andextraintestinal infections, but, unlike O1 and O139 strains of V.cholerae, little is known about the virulence gene content ofnon-O1, non-O139 strains and their phylogenetic relationship toother pathogenic V. cholerae. Comparative genomic microarrayanalysis of four pathogenic non-O1, non-O139 strains indicatesthat these strains are quite divergent from O1 and O139 strains.Genomic sequence analysis of a non-O1, non-O139 strain (AM-19226) that appeared particularly pathogenic in experimental an-imals suggests that this strain carries a type III secretion system(TTSS) that is related to the TTSS2 gene cluster found in a pandemicclone of Vibrio parahaemolyticus. The genes for this V. choleraeTTSS system appear to be present in many clinical and environ-mental non-O1, non-O139 strains, including at least one clone thatis globally distributed. We hypothesize that the TTSS present insome pathogenic strains of non-O1, non-O139 V. cholerae may beinvolved in the virulence and environmental fitness of thesestrains.genome 兩 virulence 兩 cholera pathogenesisPathogen ic Vibr io cholerae strains are the etiologic agents ofcholera, a severe diarrheal disease of potential high lethalit y(1). Both pathogenic and nonpathogenic V. cholerae reside in theaquatic ecosystem, but nonpathogenic strains typically dominatethis niche (2, 3). No bacterial factors have been correlated withthe prevalence of pathogenic strains in the aquatic environmentother than resistance to bacteriophages (4).V. cholerae strains are grouped into two defined biotype s (El Torand classical) and ⬎200 serogroups (5–7). The El Tor and classicalbiotype s are differentiated based on biochemical properties andphage sensitivity, whereas serogroup differentiation is based onO-antigen structure. Pathogenic clinical isolates generally expressone of two O antigens (O1 or O139). Most nonpathogenic, envi-ronmental isolates expre ss other O antigens and are referred to as‘‘non-O1, non-O139.’’ However, some non-O1, non-O139 V. chol-erae are clearly pathogenic and have caused outbreaks or sporadiccases of gastroenteritis and extraintestinal infections in humans(8–12).Pathogenic O1 and O139 isolates typically encode two criticalvirulence factors: cholera toxin (CT) and toxin-coregulated pilus(TCP) (1). CT is primarily responsible for the diarrheal purge,whereas TCP is an essential intestinal colonization factor (13).Nontoxigenic O1 strains expressing TCP can efficiently acquire theCT genes (ctxAB) through lysogenic conversion with CTX␾,afilamentous phage that encode s CT and uses TCP as its receptor(14). However, it is unclear how strains efficiently acquire thechromosomal segment that encodes TCP (15).Seven cholera pandemics have occurred since 1861, with the firstsix caused by classical biotype, O1 serogroup strains (16). Theseventh pandemic began in 1961 and is caused by a specific cloneof El Tor O1 V. cholerae (1). In 1991, the El Tor O1 seventhpandemic strain was introduced from Asia into South America andcaused a major cholera epidemic (17). In contrast to the classical O1strains that caused cholera in South America more than a centuryago but eventually vanished from the region, the El Tor O1 seventhpandemic strain has remained an endemic cause of cholera for wellover a decade in virtually all of Latin America. In 1992, the O139serogroup emerged in South Asia as the first non-O1 strain capableof causing epidemic cholera (18), and O139 strains have sinceestablished endemicity along with O1 El Tor strains in South Asia.The O139 strain clearly emerged from a CTX⫹TCP⫹seventhpandemic strain through replacement of O1 antigen genes withthose encoding the O139 antigen (19). Other than TCP and CTX,we know little about the bacterial factors that contribute to thesucce ss of the seventh pandemic and related O139 V. choleraestrains.The pathogenic mechanisms of non-O1, non-O139 strains is farless clear (3). Some of these strains carry the CTX and TCP genes,whereas others do not (2). Both pathogenic and nonpathogenicnon-O1, non-O139 strains encode a toxin referred to as RTX,whereas others encode a heat-stable enterotoxin (nag-ST) (20, 21).Although one CTX⫹TCP⫹clone of the O141 serogroup is appar-ently globally distributed (9), most pathogenic non-O1, non-O139strains are CTX⫺TCP⫺, do not cause endemic or pandemic diseaseas clones, and are highly diverse (22, 23). Thus, the geneticrelationship between pathogenic non-O1, non-O139 strains and O1and O139 strains remains unclear.The gene content of different O1 and O139 strains wasrecently examined by using DNA microarrays based on thegenomic sequence of V. cholerae strain El Tor N16961 (24, 25).These studies revealed a surprisingly high degree of genomicc onservation among TCP-positive O1 classical, O1 El Tor, andO139 strains that were previously thought to be quite diversebased on other methodologies (6, 17, 26). The microarrays alsoidentified two chromosomal islands that are uniquely present inO139 and El Tor O1 seventh pandemic strains, suggesting thatthese regions may encode factors involved in the endemic andpandemic potential of these clones (25).We performed a microarray-based analysis of four pathogenic,CTX⫺TCP⫺, non-O1, non-O139 V. cholerae strains and found thatthese strains are quite divergent from each other and from typicalAbbreviations: CT, cholera toxin; TCP, toxin-coregulated pilus; TTSS, type III secretionsystem; VSP, Vibrio