[Show abstract][Hide abstract] ABSTRACT: BackgroundO-antigen (O-polysaccharide) of the lipopolysaccharide is a highly variable cell component of the outer membrane in Shigella flexneri. It defines the serospecificity and plays an important role in the pathogenesis of shigellosis. There are two distinct O-antigen forms for the 19 serotypes of S. flexneri: one for serotypes 1¿5, X, Y, 7 (and their subtypes), and the other for serotype 6. Although having different basal O-polysaccharide structures, the two forms share a common disaccharide fragment [¿2)-¿-l-Rhap III-(1¿¿¿2)-¿-l-Rhap II]. In serotype 6 and some non-6 serotypes, RhaIII is O-acetylated at position either 3 or 4 (3/4-O-acetylation), conferring to the hosts a novel antigenic determinant named O-factor 9. An acyltransferase gene (oacB) responsible for this modification has been identified in serotypes 1a, 1b, 2a, 5a, and Y, but not in serotype 6.ResultsUsing genetic, serological, and chemical approaches, another acyltransferase gene named oacC was demonstrated to be responsible for the 3/4-O-acetylation on RhaIII in the O-antigen of S. flexneri serotype 6. Inactivation of the oacC gene resulted in the loss of the 3/4-O-acetyltion, and the cloned oacC gene restored this modification upon transformation. In accordance with the similarity in the acceptor substrate structure and high sequence homology (72% identity) between oacC and oacB, oacC has the interchangeable function with the oacB gene in mediation of the 3/4-O-acetylation. The oacC gene is located in a prophage on the chromosome and presented in all 77 serotype 6 strains tested.Conclusions
Identification and functional characterization of the O-acetyltransferase encoding gene, oacC, shows that it is involved in O-antigen modification by 3/4-O-acetylation on RhaIII specific to serotype 6.
[Show abstract][Hide abstract] ABSTRACT: Shigella flexneri O-antigen is an important and highly variable cell component presented on the outer leaflet of the outer membrane. Most Shigella flexneri share an O-antigen backbone composed of →2)-α-l-Rhap(III)-(1→2)-α-l-Rhap(II)-(1→3)-α-l-Rhap(I)-(1→3)-β-d-GlcpNAc-(1→ repeats, which can be modified by adding various chemical groups to different sugars giving rise to diverse O-antigen structures and, correspondingly, to various serotypes. The known modifications include glucosylation on various sugar residues, O-acetylation on Rha(I) or/and Rha(III), and phosphorylation with phosphoethanolamine on Rha(II) or/and Rha(III). Recently, a new O-antigen modification, namely O-acetylation at position 6 of N-acetylglucosamine (GlcNAc), has been identified in S. flexneri serotypes 2a, 3a, Y, and Yv. In this study, the genetic basis of the 6-O-acetylation of GlcNAc in S. flexneri was elucidated. An O-acyltransferase gene designated oacD was identified to be responsible for this modification. The oacD gene is carried on serotype-converting bacteriophage SfII, which is integrated into the host chromosome by lysogeny to form a prophage responsible for the evolvement of serotype 2 of S. flexneri. The OacD-mediated 6-O-acetylation also occurs in some other S. flexneri serotypes that carry a cryptic SfII prophage with a dysfunctional gtr locus for type II glucosylation. The 6-O-acetylation on GlcNAc confers the host with a novel O-antigen epitope, provisionally named O-factor 10. These findings enhance our understanding of the mechanisms of the O-antigen variation and enable further studies to understand the contribution of the O-acetylation to the antigenicity and pathogenicity of S. flexneri.
Journal of Bacteriology 08/2014; · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Shigella flexneri is the major cause of shigellosis in developing countries. Except for serotype 6, all serotypes share a common O-antigen backbone composed of →2)-α-l-Rhap(III)-(1→2)-α-l-Rhap(II)-(1→3)-α-l-Rhap(I)-(1→3)-β-d-GlcpNAc-(1→ tetrasaccharide repeat. It can be modified by additions of glucosyl group to one or more sugar residues, and/or O-acetyl group to Rha(I), and/or phosphoethanolamine to Rha(II) or/and Rha(III). These modifications give rise to type I, IC, II, IV and V as well as group 6, 7,8 and MASF IV-1 specific antigenic determinants, which comprise the current serotyping scheme of S. flexneri. Recently, another O-antigen modification by adding an O-acetyl group to Rha(III) at position either 3 or 4 (3/4-O-acetylation) has been found in S. flexneri serotypes 1a, 1b, 2a, 5a, Y and 6. A new O-acyltransferase gene named oacB has been shown to mediate the 3/4-O-acetylation in serotypes 1a, 1b, 2a, 5a and Y but not 6. In this work, we studied the distribution of the 3/4-O-acetylation in S. flexneri and the antigenicity that resulted from this modification. PCR screening of the oacB gene in clinical isolates of S. flexneri demonstrated that the oacB-mediated 3/4-O-acetylation is widespread in serotypes 1a, 1b, 2a, 5a and Y. Serological analysis indicated that this modification confers the host with a novel antigenic determinant provisionally named group O-factor 9. These findings enhance our understanding on the varieties of O-antigenic determinants related to O-antigen modification in S. flexneri, and will assist epidemiological studies and vaccine development.
Journal of clinical microbiology 03/2014; · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: O-antigen (O-polysaccharide) is an important and highly variable cell component presented on the surface which defines the serospecificity of Gram-negative bacteria. Most O-antigens of Shigella flexneri, a cause of shigellosis, share a backbone composed of →2)-α-l-Rhap(III)-(1→2)-α-l-Rhap(II)-(1→3)-α-l-Rhap(I)-(1→3)-β-d-GlcpNAc-(1→ repeats, which can be modified by adding various substituents giving rise to 19 serotypes. The known modifications include glucosylation on various sugar residues, O-acetylation on Rha(I) and phosphorylation with phosphoethanolamine on Rha(II) or/and Rha(III). Recently, two new O-antigen modifications, namely O-acetylation at position either 3 or 4 of Rha(III) and position 6 of GlcNAc, have been identified in several S. flexneri serotypes. In this work, the genetic basis for the 3/4-O-acetylation on Rha(III) was elucidated. Bioinformatic analysis of the genome of S. flexneri serotype 2a strain Sf301 that carries 3/4-O-acetylation on Rha(III) revealed an O-acyltransferase gene designated oacB. Genetic studies combined with O-antigen structure analysis demonstrated that this gene is responsible for the 3/4-O-acetylation in serotypes 1a, 1b, 2a, 5a and Y but not serotype 6, which has a different O-antigen backbone structure. The oacB gene is carried by a transposon-like structure located in the proA-adrA region on the chromosome, which represents a novel mechanism of mobilization of O-antigen modification factors in S. flexneri. These findings enhance our knowledge of S. flexneri O-antigen modifications and shed light on the origin of new O-antigen variants.
Journal of bacteriology 02/2014; · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The O-antigens of all S. flexneri serotypes, except serotype 6, share a linear tetrasaccharide repeat composed of one N-acetylglucosamine and three L-rhamnose residues, and differences between the serotypes are due to modification of various monosaccharide residues with glucosyl and/or O-acetyl and/or phosphoethanolamine (PEtN) groups. Plasmid-borne opt (formerly lpt-O) gene encoding a PEtN transferase which modifies the O-antigens of S. flexneri serotype X, 4a and Y strains and converts the hosts into MASF IV-1 (E1037) positive 'variant' (v) Xv, 4av and Yv serotypes, respectively. In this study we showed that the opt-carrying plasmid pSFxv_2 can transform strains of all S. flexneri serotypes (1 to 6) to confer them with the MASF IV-1 epitope recognized by monoclonal antibody MASF IV-1 and typing antiserum IV. The transformants possessed modified O-antigens with a PEtN group(s) at position 3 of one or two rhamnose residues. In some serotypes, the PEtN modification competed or/and interfered with glucosylation and O-acetylation at the same or its neighboring sugar residue. We also showed that the plasmid pSFxv_2 is mobilizable to other S. flexneri strains by conjugation. Although pSFxv_2-harboring S. flexneri strains found in clinical infections are restricted to serotypes Xv, 4av, Yv and, possibly, 6v, our results demonstrate a high potential of dissemination of this plasmid in S. flexneri and emergence of new S. flexneri serotypes.
[Show abstract][Hide abstract] ABSTRACT: We report on the isolation of 5 Shigella flexneri strains displaying a novel serotype, 1d, that shares serologic features from both S. flexneri serotypes 1a and X. The 1d strains contained serotype-converting bacteriophages SfI and SfX in tandem on the chromosome. These strains were likely originated from serotype X strains through SfI infection.
[Show abstract][Hide abstract] ABSTRACT: Yersinia enterocolitica is a Gram-negative enteric pathogen responsible for a number of gastrointestinal disorders; the most pathogenic bio-serotype is 1B/O: 8. In this study, we compared the antigenicity of the outer membrane proteins and proteomics of the whole-cell proteins of a pathogenic bio-serotype 2/O: 9 isolated in China and a bio-serotype 1B/O: 8 strain isolated in Japan. Using two-dimensional gel electrophoresis, we showed that the outer membrane proteins A (OmpA), C (OmpC) and F (OmpF) were the major antigens for both strains, although proteins located on the bacterial cell membrane and enzymes involved in energy metabolism were also identified as antigenic. We compared the whole-cell proteins of the two strains cultured at 25°C and 37°C and found portions of the outer membrane proteins (OmpX, OmpF and OmpA) were downregulated when the bacteria were cultured at 37°C, whereas urease subunit gamma (UreA), urease subunit alpha (UreC) and urease accessory protein (UreE), which are involved in urease synthesis, were upregulated when the bacteria were grown at 37°C. These observations will lay a foundation to selection of diagnostic markers for pathogenic Yersinia enterocolitica, and maybe contribute to choose the vaccine targets.
Microbiology and Immunology 06/2012; 56(9):583-94. · 1.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Yersinia enterocolitica is an extracellular pathogen and its related antigens interact with the host immune system. We investigated the difference in immunological characteristics between a highly pathogenic and poorly pathogenic strain of Y. enterocolitica.
We used SDS-PAGE and western blotting to characterize lipopolysaccharide (LPS), Yersinia outer membrane proteins (Yops), membrane proteins, and whole-cell proteins from poorly pathogenic Y. enterocolitica bio-serotype 2/O:9, isolated from China, and highly pathogenic bio-serotype 1B/O:8, isolated from Japan.
These two strains of Y. enterocolitica had different LPS immune response patterns. Comparison of their Yops also showed differences that could have accounted for their differences in pathogenicity. The membrane and whole-cell proteins of both strains were similar; immunoblottting showed that the 35 kD and perhaps the 10 kD proteins were immunogens in both strains.
The major antigens of the two strains eliciting the host immune response were the LPS and membrane proteins, as shown by comparing protein samples with reference and purified preparations.
Biomedical and Environmental Sciences 06/2012; 25(3):282-90. · 1.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An Escherichia coli O157:H7 outbreak in China in 1999 caused 177 deaths due to hemolytic uremic syndrome. Sixteen outbreak associated isolates were found to belong to a new clone, sequence type 96 (ST96), based on multilocus sequence typing of 15 housekeeping genes. Whole genome sequencing of an outbreak isolate, Xuzhou21, showed that the isolate is phylogenetically closely related to the Japan 1996 outbreak isolate Sakai, both of which share the most recent common ancestor with the US outbreak isolate EDL933. The levels of IL-6 and IL-8 of peripheral blood mononuclear cells induced by Xuzhou21 and Sakai were significantly higher than that induced by EDL933. Xuzhou21 also induced a significantly higher level of IL-8 than Sakai while both induced similar levels of IL-6. The expression level of Shiga toxin 2 in Xuzhou21 induced by mitomycin C was 68.6 times of that under non-inducing conditions, twice of that induced in Sakai (32.7 times) and 15 times higher than that induced in EDL933 (4.5 times). Our study shows that ST96 is a novel clone and provided significant new insights into the evolution of virulence of E. coli O157:H7.
PLoS ONE 04/2012; 7(4):e36144. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Shigella flexneri is the major pathogen causing bacillary dysentery. Fifteen serotypes have been recognized up to now. The genesis of new S. flexneri serotypes is commonly mediated by serotype-converting bacteriophages. Untypeable or novel serotypes from natural infections had been reported worldwide but have not been generated in laboratory.
A new S. flexneri serotype-serotype 1 d was generated when a S. flexneri serotype Y strain (native LPS) was sequentially infected with 2 serotype-converting bacteriophages, SfX first and then SfI. The new serotype 1 d strain agglutinated with both serotype X-specific anti-7;8 grouping serum and serotype 1a-specific anti- I typing serum, and differed from subserotypes 1a, 1b and 1c. Twenty four S. flexneri clinical isolates of serotype X were all converted to serotype 1 d by infection with phage SfI. PCR and sequencing revealed that SfI and SfX were integrated in tandem into the proA-yaiC region of the host chromosome.
These findings suggest a new S. flexneri serotype could be created in nature. Such a conversion may be constrained by susceptibility of a strain to infection by a given serotype-converting bacteriophage. This finding has significant implications in the emergence of new S. flexneri serotypes in nature.
[Show abstract][Hide abstract] ABSTRACT: Streptococcus suis is an important agent of swine and human meningitis. Sequence type (ST) 7 emerged in China and was responsible for the human epidemic caused by S. suis in 2005. The virulence of S. suis ST7 is greater than the wild type pathogenic S. suis, ST1; however, the mechanisms for this increased pathogenicity are unknown. The aim of this study was to determine the role of different toll-like receptors (TLRs) involved in regulating the host response to the S. suis infection and to speculate on differing mechanisms used by ST7 strains to induce disease. Here we compared two ST7 strains isolated in the 2005 Sichuan outbreak to two ST1 strains. Our data show TLR2, 6 and 9 are involved in the recognition of heat-killed S. suis independent of the ST type. We found the TLR-dependent cytokine production differed between the two types of strains using whole cell lysate proteins. TLR6 played a greater role in cytokine production induced by the whole cell lysate proteins from the ST7 strain than in that induced by the ST1 strain lysates. The data suggest that mechanisms of inflammation induced by S. suis strains differ where this will be useful in designing efficient strategies in combating streptococcal toxic shock-like syndrome caused by the S. suis ST7 strains.
[Show abstract][Hide abstract] ABSTRACT: Shigella spp. are the causative agent of shigellosis with Shigella flexneri serotype 2a being the most prevalent in developing countries. Epidemiological surveillance in China found that a new serotype of S. flexneri appeared in 2001 and replaced serotype 2a in 2003 as the most prevalent serotype in Henan Province. The new serotype also became the dominant serotype in 7 of the 10 other provinces under surveillance in China by 2007. The serotype was identified as a variant of serotype X. It differs from serotype X by agglutination to the monovalent anti-IV type antiserum and the group antigen-specific monoclonal antibody MASF IV-I. Genome sequencing of a serotype X variant isolate, 2002017, showed that it acquired a Shigella serotype conversion island, also as an SfX bacteriophage, containing gtr genes for type X-specific glucosylation. Multilocus sequence typing of 15 genes from 37 serotype X variant isolates and 69 isolates of eight other serotypes, 1a, 2a, 2b, 3a, 4a, 5b, X, and Y, found that all belong to a new sequence type (ST), ST91. Pulsed-field gel electrophoresis revealed 154 pulse types with 655 S. flexneri isolates analyzed and identified 57 serotype switching events. The data suggest that S. flexneri epidemics in China have been caused by a single epidemic clone, ST91, with frequent serotype switching to evade infection-induced immunity to serotypes to which the population was exposed previously. The clone has also acquired resistance to multiple antibiotics. These findings underscore the challenges to the current vaccine development and control strategies for shigellosis.
Journal of clinical microbiology 12/2009; 48(2):419-26. · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Streptococcus suis emerged to cause an unusual outbreak of streptococcal toxic-shock-like syndrome (STSLS) in 2005. The mechanisms involved are unknown.
Clinical, laboratory, and epidemiologic data on patients infected with culture-confirmed S. suis were analyzed. The strain involved in the outbreak, "epidemic" strain ST7, was compared with both a classical highly pathogenic strain, ST1, and an intermediately pathogenic strain, ST25, to determine both its capacity to induce cytokines in experimentally infected mice and its genomic difference.
Of 38 patients infected with culture-confirmed S. suis, 14 presented with STSLS. During the early phase of the disease, serum levels of interleukin (IL)-1beta, IL-6, IL-8, IL-12p70, interferon-gamma, and tumor necrosis factor-alpha were more elevated in patients with STSLS than in those with meningitis only. Serum levels of proinflammatory cytokines were significantly higher in mice infected with ST7 than in those infected with either ST1 or ST25. Genomic comparisons with ST25 showed that ST1 had acquired 132 genomic islands, including 5 pathogenicity islands, and that ST7, the epidemic strain, had acquired an additional 5 genomic islands.
Intermediately pathogenic strain ST25 has evolved to become highly pathogenic strain ST1, which, in turn, has more recently evolved to become epidemic strain ST7. ST7 has the ability to stimulate the production of massive amounts of proinflammatory cytokines, leading to STSLS.
The Journal of Infectious Diseases 12/2008; 199(1):97-107. · 5.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Streptococcus suis sequence type (ST) 7 has been spreading throughout China. To determine events associated with its emergence, we tested 114 isolates. In all 106 ST7 strains responsible for human outbreaks and sporadic infections, the tetracycline-resistance gene, tetM, was detected on the conjugative transposon Tn916. Horizontal transmission of tetM is suspected.
[Show abstract][Hide abstract] ABSTRACT: To identify antigenic proteins secreted by Streptococcus suis (S. suis) type 2 strain SC84.
Two-dimensional electrophoresis (2-DE), western-blot assay and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis were performed to search and identify antigenic proteins secreted by S. suis strain SC84, which triggered an outbreak of the disease in Sichuan province,China, in 2005.
A total number of 14 western blot spots were found on PVDF membrane. 11 spots which could be found the existence of matching protein on coomassie G-250-stained 2-DE gel were identified by MALDI-TOF MS. The 11 proteins, all located at extra-cellular or cell wall, were classified into 8 kinds of proteins. Among of them, muramidase-released protein (MRP), suilysin (Sly) and extra-cellular factor (EF) were the known antigenic proteins, but several proteins such as putative 5'-nucleotidase, ribo-nucleases G and E, and predicted metal-loendo-peptidase were newly found antigenic proteins. All the identified protein were found to have had the coding gene in genomic of S. suis strain 05ZYH33, isolated from patients in Sichuan province, China in 2005.
The newly found proteins could be used as voluntary antigens for detection and vaccination of S. suis.
Zhonghua liu xing bing xue za zhi = Zhonghua liuxingbingxue zazhi 04/2008; 29(3):267-71.
[Show abstract][Hide abstract] ABSTRACT: To understand the variation of Shiga toxin (stx) genes of Escherichia coli O157:H7 strains isolated in China.
Polymerase chain reaction (PCR) was used to identity the types of stx genes and the nucleotide sequences of the amplified stex variants genes were determined. Compare to the cytotoxicity of Stx,variants were tested by HeLa cell assay.
We found novel stx2 genes in 3 of 289 strains of Shiga toxin-producing E. coli O157:H7 isolated from 1999 to 2002 in China. The novel stx2 genes were inserted by a 1.3-kb insertion sequence (IS) and the nucleotide sequences of IS showed 100% homology with that of IS1203 variant (IS1203v). The IS1203v inserted in the stx2 genes of three E. coli O157:H7 strains at different sites and the direction of the open reading frames (ORFs) of IS1203v of each strain was different. In addition to the above mentioned findings, the nucleotide sequences of three stx2 genes were completely identical and the type of the three Stx2 was Stx2 prototype. Compare to the cytotoxicity of Stx2 prototype, the novel Stx2 was found to be obviously lower.
E. coli O157:H7 strains harboring stx2::IS1203v genes were isolated in China. Consequently, the results of HeLa cell assay showed that the insertion of IS1203v could lead to low cytotoxicity of Stx2.
Zhonghua liu xing bing xue za zhi = Zhonghua liuxingbingxue zazhi 12/2007; 28(11):1119-22.
[Show abstract][Hide abstract] ABSTRACT: An outbreak of Streptococcus suis serotype 2 emerged in the summer of 2005 in Sichuan Province, and sporadic infections occurred in 4 additional provinces of China. In total, 99 S. suis strains were isolated and analyzed in this study: 88 isolates from human patients and 11 from diseased pigs. We defined 98 of 99 isolates as pulse type I by using pulsed-field gel electrophoresis analysis of SmaI-digested chromosomal DNA. Furthermore, multilocus sequence typing classified 97 of 98 members of the pulse type I in the same sequence type (ST), ST-7. Isolates of ST-7 were more toxic to peripheral blood mononuclear cells than ST-1 strains. S. suis ST-7, the causative agent, was a single-locus variant of ST-1 with increased virulence. These findings strongly suggest that ST-7 is an emerging, highly virulent S. suis clone that caused the largest S. suis outbreak ever described.