Xinxiang Huang

Epidemiology of hand, foot, and mouth disease and genotype characterization of Enterovirus 71 in Jiangsu, China

BACKGROUND In the spring of 2008, an EV71-caused hand, foot, and mouth disease (HFMD) outbreak occurred in Fuyang city, Anhui Province, China. Jiangsu Province that borders Auhui to the east is presumed as a key station for the spread of EV71 to other regions of the Yangtze River Delta. OBJECTIVES To investigate the HFMD prevalence in Zhenjiang city of Jiangsu from May 2008 to October 2009, and the epidemic origin of EV71 circulating in Jiangsu. STUDY DESIGN During May 2008 and October 2009, a total of 6324 HFMD cases in Zhenjiang, Jiangsu, were investigated. Sixty throat specimens were randomly selected from different patients, and 28 nucleotide sequences of EV71 VP1 regions were successfully determined by RT-nested-PCR and sequencing. EV71 genotypes were characterized by phylogenetic analyses. RESULTS The incidence rate of HFMD was highest in the period of March-July and in the 1-4 years old age groups. Intriguingly, there was a slight predominance for boys and for children living in rural areas in HFMD infection. Phylogenetic analyses indicated that all Jiangsu EV71 strains and most China strains belonged to subgenotype C4a. CONCLUSION The C4a was the most prominent EV71 subgenotype circulating in China. Routine HFMD surveillance should be focused on the period of March-July, and more prevention efforts should be aimed at 1-4 years old children. Moreover, government efforts are urgently needed to improve public health condition and medical service quality in rural areas.

Differentiation Imbalance of Th1/ Th17 in Peripheral Blood Mononuclear Cells Might Contribute to Pathogenesis of Hashimoto’s Thyroiditis

T helper 17(Th17) cell is a new subset of CD4+ T cells that produce a proinflammatory cytokine interleukin‐17 (IL‐17). Th17 cells have recently been shown to play a critical role in many autoimmune diseases that had previously been thought to be Th1 dominant. Although Hashimoto’s thyroiditis (HT) was thought to be a Th1‐type disease, the contributions of Th17 cells to the pathogenesis remain unclear. In this study, we investigated the expression levels of Th1/Th17 cell‐associated factors in peripheral blood mononuclear cells (PBMC) and plasma from patients with HT by quantitative real‐time polymerase chain reaction (RT‐qPCR) and enzyme‐linked immunosorbent assay (ELISA). Our results showed that the expression levels of Th1 cells‐related T‐bet and interferon‐γ (IFN‐γ) mRNA in PBMC from HT significantly decreased. However, the mRNA of Th17 coherent retinoic acid‐related orphan nuclear receptor gamma t (RORγt) and IL‐17 in patients with HT increased. In addition, a negative correlation between T‐bet and RORγt mRNA expression was found in patients with HT, and the similar phenomena also appeared on the levels of mRNA and plasma concentration between IFN‐γ and IL‐17. It suggested that Th17 cells rather than Th1 cells predominated among patients suffering from HT, and Th17 cells might be involved in the pathogenesis of HT.

Enhanced HMGB1 Expression May Contribute to Th17 Cells Activation in Rheumatoid Arthritis

Rheumatoid arthritis(RA) is a common autoimmune disease associated with Th17 cells, but what about the effect of high-mobility group box chromosomal protein 1 (HMGB1) and the relationship between Th17-associated factors and HMGB1 in RA remains unknown. In the present study, we investigated the mRNA levels of HMGB1, RORγt, and IL-17 in peripheral blood mononuclear cells (PBMCs) from patients with rheumatoid arthritis by quantitative real-time PCR (RT-qPCR), and the concentrations of HMGB1, IL-17, and IL-23 in plasma were detected by ELISA. And then, the effect of HMGB1 on Th17 cells differentiation was analyzed in vitro. Our clinical studies showed that the mRNAs of HMGB1, RORγt, and IL-17 in patients were higher than that in health control (P < 0.05), especially in active RA patients (P < 0.05). The plasma HMGB1, IL-17, and IL-23 in RA patients were also higher than that in health control (P < 0.05); there was a positive correlation between the expression levels of HMGB1 and the amount of CRP, ERS, and RF in plasma. In vitro, the IL-17-produced CD4+T cells were increased with 100 ng/mL rHMGB1 for 12h, which indicated that the increased HMGB1 might contribute to Th17 cells activation in RA patients.

Identification and characterization of class 1 integrons among Pseudomonas aeruginosa isolates from patients in Zhenjiang, China.

OBJECTIVES The role of integrons in the spread of antibiotic resistance has been well established. The aim of this study was to investigate the resistance profiles of Pseudomonas aeruginosa isolated from patients in Zhenjiang to 13 antibiotics, and to identify the structure and dissemination of class 1 integrons. METHODS The Kirby-Bauer disk diffusion assay was used to determine the rate of P. aeruginosa resistance. Class 1 integrons from multidrug-resistant isolates were amplified by PCR, and their PCR products were sequenced. We also analyzed the integron structures containing the same gene cassettes by restriction fragment length polymorphism (RFLP). Isolates were genotyped by pulsed-field gel electrophoresis (PFGE). RESULTS The resistance rates were between 29.6% and 90.1%. The prevalence of class 1 integrons was 38.0%. These integrons included five gene cassettes (aadB, aac6-II, blaPSE-1, dfrA17, and aadA5). The dfrA17 and aadA5 gene cassettes were found most often. CONCLUSIONS Class 1 integrons were found to be widespread in P. aeruginosa isolated from clinical samples in the Zhenjiang area of China. The antibiotic resistance rates in class 1 integron-positive strains of P. aeruginosa were noticeably higher than those in class 1 integron-negative strains. PFGE showed that particular clones were circulating among patients.

Phenotypic and transcriptional analysis of the osmotic regulator OmpR in Yersinia pestis

BackgroundThe osmotic regulator OmpR in Escherichia coli regulates differentially the expression of major porin proteins OmpF and OmpC. In Yersinia enterocolitica and Y. pseudotuberculosis, OmpR is required for both virulence and survival within macrophages. However, the phenotypic and regulatory roles of OmpR in Y. pestis are not yet fully understood.ResultsY. pestis OmpR is involved in building resistance against phagocytosis and controls the adaptation to various stressful conditions met in macrophages. The ompR mutation likely did not affect the virulence of Y. pestis strain 201 that was a human-avirulent enzootic strain. The microarray-based comparative transcriptome analysis disclosed a set of 224 genes whose expressions were affected by the ompR mutation, indicating the global regulatory role of OmpR in Y. pestis. Real-time RT-PCR or lacZ fusion reporter assay further validated 16 OmpR-dependent genes, for which OmpR consensus-like sequences were found within their upstream DNA regions. ompC, F, X, and R were up-regulated dramatically with the increase of medium osmolarity, which was mediated by OmpR occupying the target promoter regions in a tandem manner.ConclusionOmpR contributes to the resistance against phagocytosis or survival within macrophages, which is conserved in the pathogenic yersiniae. Y. pestis OmpR regulates ompC, F, X, and R directly through OmpR-promoter DNA association. There is an inducible expressions of the pore-forming proteins OmpF, C, and × at high osmolarity in Y. pestis, in contrast to the reciprocal regulation of them in E. coli. The main difference is that ompF expression is not repressed at high osmolarity in Y. pestis, which is likely due to the absence of a promoter-distal OmpR-binding site for ompF.

Regulatory effects of cAMP receptor protein (CRP) on porin genes and its own gene in Yersinia pestis

BackgroundThe cAMP receptor protein (CRP) is a global bacterial regulator that controls many target genes. The CRP-cAMP complex regulates the ompR-envZ operon in E. coli directly, involving both positive and negative regulations of multiple target promoters; further, it controls the production of porins indirectly through its direct action on ompR-envZ. Auto-regulation of CRP has also been established in E. coli. However, the regulation of porin genes and its own gene by CRP remains unclear in Y. pestis.ResultsY. pestis employs a distinct mechanism indicating that CRP has no regulatory effect on the ompR-envZ operon; however, it stimulates ompC and ompF directly, while repressing ompX. No transcriptional regulatory association between CRP and its own gene can be detected in Y. pestis, which is also in contrast to the fact that CRP acts as both repressor and activator for its own gene in E. coli. It is likely that Y. pestis OmpR and CRP respectively sense different signals (medium osmolarity, and cellular cAMP levels) to regulate porin genes independently.ConclusionAlthough the CRP of Y. pestis shows a very high homology to that of E. coli, and the consensus DNA sequence recognized by CRP is shared by the two bacteria, the Y. pestis CRP can recognize the promoters of ompC, F, and X directly rather than that of its own gene, which is different from the relevant regulatory circuit of E. coli. Data presented here indicate a remarkable remodeling of the CRP-mediated regulation of porin genes and of its own one between these two bacteria.

Molecular Characterization of a Functional Type VI Secretion System in Salmonella enterica serovar Typhi

The type VI secretion system (T6SS) of Salmonella enterica serovar Typhi (S. typhi) is associated with Salmonella pathogenicity island 6 (SPI-6). Though the T6SS gene cluster is intact in S. typhi, the protein complex is believed to be non-functional due to the presence of a pseudogene form of SciI (VipB homolog), a key component. We detected the SciK-his6 in the supernatant of the wild type strain of S. typhi containing the plasmid over-expressing SciK (hcp homolog) with a his6 epitope at the C-terminus, which suggested that the T6SS in S. typhi is functional. We also identified four genes that were essential to T6SS function: sciC (vasA homolog), sciS (vasK homolog), sciG (clpV homolog), and vrgS (vgrG homolog). Further analysis revealed that S. typhi T6SS is cytotoxic to human epithelial cells, but does not influence bacterial growth and mobility. RcsB, PmrA, and Hfq were identified as regulators of S. typhi T6SS gene expression; however, PhoP appears to not be involved. Taken together, the data demonstrate the functionality of S. typhi T6SS and confirm the important role of T6SS for S. typhi’s ability to invade and infect epithelial cells.

Identification by cDNA cloning of abundant sRNAs in a human- avirulent Yersinia pestis strain grown under five different growth conditions

AIMS sRNA regulation is supposedly involved in the stress response of a pathogen during infection. Yersinia pestis, the etiologic agent of plague, must encounter temperature and microenvironment changes, given its lifestyle. Here, we used the cDNA cloning approach to discover full-length sRNA candidates that are highly expressed in Y. pestis under five different growth conditions. MATERIALS & METHODS The cDNA cloning approach was improved by combining the traditional cDNA library construction with the prevalent rapid amplification of cDNA ends and RNA size selection techniques. RESULTS In total, 43 RNA species, including six previously annotated sRNAs, were identified. Of these, 25 sRNAs were encoded on the antisense strand of the annotated genes. Interestingly, two of these sRNAs were found on the complementary strand of noncoding RNAs. In addition, eight novel sRNAs encoded in the intergenic regions were also revealed. Ten sRNA candidates chosen for the northern blot analysis were successfully detected. Analysis of the expression patterns of 29 candidate sRNAs showed that 24 sRNAs are highly abundant in Y. pestis upon entry into the stationary growth phase. CONCLUSION Our preliminary attempt at screening the novel sRNA candidates will lay the foundation for understanding the roles of sRNAs in Y. pestis physiology and pathogenesis.

Global transcriptional response of Salmonella enterica serovar Typhi to anti‐z66 antiserum

Recent studies have shown that flagella may modulate physiological processes by sensing environmental changes in temperature and moisture. When the z66(+) strain of Salmonella enterica serovar Typhi (S. Typhi) was exposed to an antiserum against the z66 flagellar antigen, the fljBA operon was deleted from a linear plasmid, leading to the unidirectional flagellar phase variation from FljB to FliC. We hypothesized that flagella may serve as a sensor that responded to the antiserum by altering gene expression and triggering the unidirectional flagellar phase variation. To test this hypothesis, Salmonella genomic DNA microarrays were used to determine the gene expression profile of the z66(+) wild-type strain of S. Typhi treated with the anti-z66 antiserum for 30 min. The results showed that expression levels of 187 genes were altered by more than threefold compared with the same strain treated with control serum. The microarray expression patterns of representative genes were validated by reverse transcriptase-PCR. Importantly, no significant changes in gene expression were observed in the fljB:z66 deletion mutant that was similarly treated with the anti-z66 antiserum. To the best of our knowledge, this is the first study to show the global transcriptional response of Salmonella to antiflagellin antiserum.

Identification of a fljA gene on a linear plasmid as the repressor gene of fliC in Salmonella enterica serovar Typhi

Salmonella enterica serovar Typhi z66-positive strain contains an fljBA-like operon on a linear plasmid and the fliC gene on the chromosome encoding d or j antigen. The fljB-like gene has been identified as the gene encoding z66 antigen. To investigate the function of the fljA-like gene in S. enterica serovar Typhi, a z66-positive wild-type strain GIFU10007 and its phase variation j positive strain 007j+ were used in the present study. After deletion of the fljA-like gene, the wild-type strain that only expressed FljB:z66 could express both FliC:j and FljB:z66 at the same time. A recombinant plasmid, pBADfljA, containing the fljA-like gene was prepared. SDS-PAGE and western blot analysis of secreted proteins showed that when the strain 007j+ was transformed by the recombinant plasmid, pBADfljA, the expression of FliC:j was inhibited. The fliC::lacZ fusion of the strain 007j+ was constructed. Beta-Galactosidase activity was decreased approximately 100-fold after harboring the plasmid pBADfljA in the fliC::lacZ mutant strain. However, the fliC mRNA level investigated by RT-PCR was decreased only approximately seven-fold in the strain 007j+ after harboring the plasmid pBADfljA. These results demonstrated that thefljA-like gene on a linear plasmid of S. enterica serovar Typhi is a fljA gene, the repressor of fliC gene, and the inhibition by FljA is likely at the post-transcriptional level.