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1.  Arginine deiminase pathway is far more important than urease for acid resistance and intracellular survival in Laribacter hongkongensis: a possible result of arc gene cassette duplication 
BMC Microbiology  2014;14:42.
Laribacter hongkongensis is a Gram-negative, urease-positive bacillus associated with invasive bacteremic infections in liver cirrhosis patients and fish-borne community-acquired gastroenteritis and traveler’s diarrhea. Its mechanisms of adaptation to various environmental niches and host defense evasion are largely unknown. During the process of analyzing the L. hongkongensis genome, a complete urease cassette and two adjacent arc gene cassettes were found. We hypothesize that the urease cassette and/or the arc gene cassettes are important for L. hongkongensis to survive in acidic environment and macrophages. In this study, we tested this hypothesis by constructing single, double and triple non-polar deletion mutants of the urease and two arc gene cassettes of L. hongkongensis using the conjugation-mediated gene deletion system and examining their effects in acidic environment in vitro, in macrophages and in a mouse model.
HLHK9∆ureA, HLHK9∆ureC, HLHK9∆ureD and HLHK9∆ureE all exhibited no urease activity. HLHK9∆arcA1 and HLHK9∆arcA2 both exhibited arginine deiminase (ADI) activities, but HLHK9∆arcA1/arcA2 double deletion mutant exhibited no ADI activity. At pH 2 and 3, survival of HLHK9∆arcA1/arcA2 and HLHK9∆ureA/arcA1/arcA2 were markedly decreased (p < 0.001) but that of HLHK9∆ureA was slightly decreased (p < 0.05), compared to wild type L. hongkongensis HLHK9. Survival of HLHK9∆ureA/arcA1/arcA2 and HLHK9∆arcA1/arcA2 in macrophages were also markedly decreased (p < 0.001 and p < 0.01 respectively) but that of HLHK9∆ureA was slightly decreased (p < 0.05), compared to HLHK9, although expression of arcA1, arcA2 and ureA genes were all upregulated. Using a mouse model, HLHK9∆ureA exhibited similar survival compared to HLHK9 after passing through the murine stomach, but survival of HLHK9∆arcA1/arcA2 and HLHK9∆ureA/arcA1/arcA2 were markedly reduced (p < 0.01).
In contrast to other important gastrointestinal tract pathogens, ADI pathway is far more important than urease for acid resistance and intracellular survival in L. hongkongensis. The gene duplication of the arc gene cassettes could be a result of their functional importance in L. hongkongensis.
PMCID: PMC3936950  PMID: 24533585
Laribacter hongkongensis; Acid resistance; Arginine deiminase pathway; Microbe-host interaction
2.  Sequence polymorphisms of rfbT among the Vibrio cholerae O1 strains in the Ogawa and Inaba serotype shifts 
BMC Microbiology  2013;13:173.
Vibrio cholerae serogroup O1 has two major serotypes, Ogawa and Inaba, which may alternate among cholera epidemics. The rfbT gene is responsible for the conversion between the two serotypes. In this study, we surveyed the sequence variance of rfbT in the Ogawa and Inaba strains in China over a 48-year (1961-2008) period in which serotype shifts occurred among epidemic years.
Various mutation events including single nucleotide, short fragment insertions/deletions and transposases insertions, were found in the rfbT gene of the Inaba strains. Ectopically introducing an intact rfbT could overcome the mutations by converting the Inaba serotype to the Ogawa serotype, suggesting the effects of these mutations on the function of RfbT. Characteristic rfbT mutations were recognized in the Inaba strains among Inaba serotype dominant epidemic years which were separate from the Ogawa dominant epidemics. Three distinguishable mutation sites in rfbT between the classical and the El Tor biotype strains were identified and could serve as biotype-specific biomarkers.
Our results provide a comprehensive picture of the rfbT gene mutations among the V. cholerae O1 strains in different epidemic periods, which could be further used as the tracing markers in clonality analysis and dissemination surveillance of the epidemic strains.
PMCID: PMC3727987  PMID: 23889924
3.  Characters of homogentisate oxygenase gene mutation and high clonality of the natural pigment-producing Vibrio cholerae strains 
BMC Microbiology  2011;11:109.
Some microorganisms can produce pigments such as melanin, which has been associated with virulence in the host and with a survival advantage in the environment. In Vibrio cholerae, studies have shown that pigment-producing mutants are more virulent than the parental strain in terms of increased UV resistance, production of major virulence factors, and colonization. To date, almost all of the pigmented V. cholerae strains investigated have been induced by chemicals, culture stress, or transposon mutagenesis. However, during our cholera surveillance, some nontoxigenic serogroup O139 strains and one toxigenic O1 strain, which can produce pigment steadily under the commonly used experimental growth conditions, were obtained in different years and from different areas. The genes VC1344 to VC1347, which correspond to the El Tor strain N16961 genome and which comprise an operon in the tyrosine catabolic pathway, have been confirmed to be associated with a pigmented phenotype. In the present study, we investigated the mechanism of pigment production in these strains.
Sequencing of the VC1344, VC1345, VC1346, and VC1347 genes in these pigmented strains suggested that a deletion mutation in the homogentisate oxygenase gene (VC1345) may be associated with the pigmented phenotype, and gene complementation confirmed the role of this gene in pigment production. An identical 15-bp deletion was found in the VC1345 gene of all six O139 pigment-producing strains examined, and a 10-bp deletion was found in the VC1345 gene of the O1 strain. Strict sequence conservation in the VC1344 gene but higher variance in the other three genes of this operon were observed, indicating the different stress response functions of these genes in environmental adaption and selection. On the basis of pulsed-field gel electrophoresis typing, the pigment-producing O139 strains showed high clonality, even though they were isolated in different years and from different regions. Additionally all these O139 strains belong to the rb4 ribotype, which contains the O139 strains isolated from diarrheal patients, although these strains are cholera toxin negative.
Dysfunction of homogentisate oxygenase (VC1345) causes homogentisate accumulation and pigment formation in naturally pigmented strains of V. cholerae. The high clonality of these strains may correlate to an environmental survival advantage in the V. cholerae community due to their pigment production, and may imply a potential protective function of melanin in environmental survival of such strains.
PMCID: PMC3114702  PMID: 21592381
Vibrio cholerae; pigment; homogentisate oxygenase; clonality
4.  Possible use of ail and foxA polymorphisms for detecting pathogenic Yersinia enterocolitica 
BMC Microbiology  2010;10:211.
Yersinia enterocolitica is an enteric pathogen that invades the intestinal mucosa and proliferates within the lymphoid follicles (Peyer's patches). The attachment invasion locus (ail) mediates invasion by Y. enterocolitica and confers an invasive phenotype upon non-invasive E. coli; ail is the primary virulence factor of Y. enterocolitica. The ferrioxamine receptor (foxA) located on the Y. enterocolitica chromosome, together with its transport protein, transports a siderophore specific for ferric ion. Currently, ail is the primary target gene for nucleic acid detection of pathogenic Y. enterocolitica.
The genes ail and foxA in 271 pathogenic and 27 non-pathogenic Y. enterocolitica strains isolated from China and 10 reference strains were sequenced, aligned, compared to the ail and foxA sequences of Yersinia enterocolitica subsp. enterocolitica 8081 (Genbank: NC_008800), and analyzed for sequence polymorphism. The ail from the 282 strains showed 3 sequence patterns: 277 strains of serotypes O:3, O:9 and O:5, 27 with identical nucleic acid sequences formed pattern A1; 4 strains of serotype 1B/O:8 with identical nucleic acid sequences formed pattern A2; and one Chinese isolate 2/O:9 formed pattern A3. In the primary coding region of the foxA ORF (Genebank: X60447 nt 433-1866; nt 28 to 1,461 in the ORF), the sequences formed 3 groups and were further divided into 8 sequence patterns.
The ail and foxA loci of pathogenic Y. enterocolitica have been analyzed. The ail sequence was highly conserved among the same serotype strains from different sources; and foxA was highly conserved among the pathogenic strains, although there was some sequence diversity. Fewer strains were used from outside China, which is a limitation of the study.
PMCID: PMC2924855  PMID: 20691098
5.  Virulence regulator AphB enhances toxR transcription in Vibrio cholerae 
BMC Microbiology  2010;10:3.
Vibrio cholerae is the causative agent of cholera. Extensive studies reveal that complicated regulatory cascades regulate expression of virulence genes, the products of which are required for V. cholerae to colonize and cause disease. In this study, we investigated the expression of the key virulence regulator ToxR under different conditions.
We found that compared to that of wild type grown to stationary phase, the toxR expression was lower in an aphB mutant strain. AphB has been previously shown to be a key virulence regulator that is required to activate the expression of tcpP. When expressed constitutively, AphB is able to activate the toxR promoter. Furthermore, gel shift analysis indicates that AphB binds toxR promoter region directly. We also characterize the effect of AphB on the levels of the outer membrane porins OmpT and OmpU, which are known to be regulated by ToxR.
Our data indicate that V. cholerae possesses an additional regulatory loop that use AphB to activate the expression of two virulence regulators, ToxR and TcpP, which together control the expression of the master virulence regulator ToxT.
PMCID: PMC2806343  PMID: 20053280
6.  Proteins involved in difference of sorbitol fermentation rates of the toxigenic and nontoxigenic Vibrio cholerae El Tor strains revealed by comparative proteome analysis 
BMC Microbiology  2009;9:135.
The nontoxigenic V. cholerae El Tor strains ferment sorbitol faster than the toxigenic strains, hence fast-fermenting and slow-fermenting strains are defined by sorbitol fermentation test. This test has been used for more than 40 years in cholera surveillance and strain analysis in China. Understanding of the mechanisms of sorbitol metabolism of the toxigenic and nontoxigenic strains may help to explore the genome and metabolism divergence in these strains. Here we used comparative proteomic analysis to find the proteins which may be involved in such metabolic difference.
We found the production of formate and lactic acid in the sorbitol fermentation medium of the nontoxigenic strain was earlier than of the toxigenic strain. We compared the protein expression profiles of the toxigenic strain N16961 and nontoxigenic strain JS32 cultured in sorbitol fermentation medium, by using fructose fermentation medium as the control. Seventy-three differential protein spots were found and further identified by MALDI-MS. The difference of product of fructose-specific IIA/FPR component gene and mannitol-1-P dehydrogenase, may be involved in the difference of sorbitol transportation and dehydrogenation in the sorbitol fast- and slow-fermenting strains. The difference of the relative transcription levels of pyruvate formate-lyase to pyruvate dehydrogenase between the toxigenic and nontoxigenic strains may be also responsible for the time and ability difference of formate production between these strains.
Multiple factors involved in different metabolism steps may affect the sorbitol fermentation in the toxigenic and nontoxigenic strains of V. cholerae El Tor.
PMCID: PMC2714520  PMID: 19589152
7.  Pleiotropic effects of the twin-arginine translocation system on biofilm formation, colonization, and virulence in Vibrio cholerae 
BMC Microbiology  2009;9:114.
The Twin-arginine translocation (Tat) system serves to translocate folded proteins, including periplasmic enzymes that bind redox cofactors in bacteria. The Tat system is also a determinant of virulence in some pathogenic bacteria, related to pleiotropic effects including growth, motility, and the secretion of some virulent factors. The contribution of the Tat pathway to Vibrio cholerae has not been explored. Here we investigated the functionality of the Tat system in V. cholerae, the etiologic agent of cholera.
In V. cholerae, the tatABC genes function in the translocation of TMAO reductase. Deletion of the tatABC genes led to a significant decrease in biofilm formation, the ability to attach to HT-29 cells, and the ability to colonize suckling mouse intestines. In addition, we observed a reduction in the output of cholera toxin, which may be due to the decreased transcription level of the toxin gene in tatABC mutants, suggesting an indirect effect of the mutation on toxin production. No obvious differences in flagellum biosynthesis and motility were found between the tatABC mutant and the parental strain, showing a variable effect of Tat in different bacteria.
The Tat system contributes to the survival of V. cholerae in the environment and in vivo, and it may be associated with its virulence.
PMCID: PMC2698830  PMID: 19480715

Results 1-7 (7)