Aihua Sun

Inactivation of the fliY gene encoding a flagellar motor switch protein attenuates mobility and virulence of Leptospira interrogans strain Lai

BackgroundPathogenic Leptospira species cause leptospirosis, a zoonotic disease of global importance. The spirochete displays active rotative mobility which may contribute to invasion and diffusion of the pathogen in hosts. FliY is a flagellar motor switch protein that controls flagellar motor direction in other microbes, but its role in Leptospira, and paricularly in pathogenicity remains unknown.ResultsA suicide plasmid for the fliY gene of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai that was disrupted by inserting the ampicillin resistance gene (bla) was constructed, and the inactivation of fliY gene in a mutant (fliY-) was confirmed by PCR and Western Blot analysis. The inactivation resulted in the mRNA absence of fliP and fliQ genes which are located downstream of the fliY gene in the same operon. The mutant displayed visibly weakened rotative motion in liquid medium and its migration on semisolid medium was also markedly attenuated compared to the wild-type strain. Compared to the wild-type strain, the mutant showed much lower levels of adhesion to murine macrophages and apoptosis-inducing ability, and its lethality to guinea pigs was also significantly decreased.ConclusionInactivation of fliY, by the method used in this paper, clearly had polar effects on downstream genes. The phentotypes observed, including lower pathogenicity, could be a consequence of fliY inactivation, but also a consequence of the polar effects.

Methyl-accepting chemotaxis proteins 3 and 4 are responsible for Campylobacter jejuni chemotaxis and jejuna colonization in mice in response to sodium deoxycholate

Methyl-accepting chemotaxis proteins (MCPs), also termed transducer-like proteins (Tlps), serve as sensors in bacterial chemotactic signalling, and detect attractants and promote bacterial movement towards suitable sites for colonization. Campylobacter jejuni is a leading cause of human enteritis, but the mechanisms responsible for bacterial chemotaxis and early colonization in the jejunum of hosts are poorly understood. In the present study, we identified several types of bile and sodium deoxycholate (SDC) acting as chemotactic attractants of C. jejuni strain NCTC 11168-O in vitro, in which SDC was the most efficient chemoattractant. In mice with bile duct ligation, the wild-type strain displayed a markedly attenuated ability for colonization. Blockage of Tlp3 or Tlp4 protein with antibody or disruption of the tlp3 or tlp4 gene (Δtlp3 or Δtlp4) caused a significant inhibition of SDC-induced chemotaxis and attenuation for colonization on jejunal mucosa in mice of the bacterium. Disruption of both the genes (Δtlp3/Δtlp4) resulted in the absence of bacterial chemotaxis and colonization, while the tlp-gene-complemented mutants (CΔtlp3 and CΔtlp4) reacquired these abilities. The results indicate that SDC is an effective chemoattractant for C. jejuni, and Tlp3 and Tlp4 are the SDC-specific sensor proteins responsible for the bacterial chemoattraction.

Characterization of conserved combined T and B cell epitopes in Leptospira interrogans major outer membrane proteins OmpL1 and LipL41.

BackgroundLeptospira interrogans are bacterial pathogens of animal that cause zoonotic infections in human. Outer membrane proteins of leptospire are among the most effective antigens which can stimulate remarkable immune responses during the infection processes, and thus are currently considered leading candidate vaccine antigens. The objective of the present study is to predict and confirm major combined B and T cell epitopes of leptospiral outer membrane proteins OmpL1 and LipL41, as well as to evaluate their capacity in the induction of immune responses in BALB/c mice.ResultsIn this study, four epitopes from OmpL1 and four from LipL41 conserved regions were evaluated for their potential utilization in leptospire vaccines. Firstly, combined B and T cell epitopes were predicted by softwares and expressed using a phage display system. OmpL1 residues 87-98 and 173-191 (OmpL187-98 and OmpL1173-191) and LipL4130-48, LipL41233-256 of LipL41 were identified as immunodominant B cell epitopes by Western blot. Epitopes OmpL1173-191, OmpL1297-320 of OmpL1 and LipL41233-256, LipL41263-282 of LipL41 were identified as immunodominant CD4+ T cell epitopes through proliferation analysis of splenocytes from recombinant OmpL1 (rOmpL1) or recombinant LipL41 (rLipL41)-immunized BALB/c (H-2d) mice. These epitopes induced responses of CD4+ T cells and Th1 (T helper cells) type cytokine responses during the infection.ConclusionThis work identified combined T and B cell immunodominant epitopes in outer membrane proteins OmpL1 and LipL41 of Leptospira interrogans. OmpL1173-191 of OmpL1 and LipL41233-256 of LipL41 could be useful in a vaccine against Leptospira. The findings could also contribute to the development of effective cross-protective vaccine strategies for leptospirosis.

Human papillomavirus prevalence and type-distribution among women in Zhejiang Province, Southeast China: a cross-sectional study

BackgroundHuman papillomavirus (HPV) infection is the main etiological factor for cervical cancer and premalignant lesions of the cervix. The purposes of the present study were to determine the prevalence of type-specific HPV infections and the association of different HPV types with cervical dysplasia among women in Zhejiang province, Southeast China.MethodsA total of 15,267 women presenting to a gynaecological outpatient clinic were enrolled in this study. Women were screened for HPV in addition to routine cervical cytology testing. Microarray hybridization and liquid-based cytology tests were used to detect HPV genotypes and cervical cytology, respectively.ResultsBased on the population attending a gynaecological outpatient clinic, overall prevalence of any 23 HPV type was 22.8% and multiple HPV infection was found in 4.0% of all the outpatients. HPV prevalence showed bimodal age distribution, with a peak (55.7%) at theu2009≤20 age group and a second one (35.5%) atu2009>60 age group. In total samples, the five most frequent types were HPV 16 (4.4%), 58 (2.9%), 52 (2.7%), 33 (2.2%) and 11 (1.9%). Overall HPV prevalence increased with the severity of the cytologic result. Analysis through crude odds ratios (ORs) revealed that the cervical lesion risk of HPV-infected women increased to about 26-fold of uninfected women (OR 26.1, 95% CI 22.4 to 30.3). The five most risky HPV types associated with abnormal cytology were HPV 73, 16, 82, 45 and 51.ConclusionsThis study provided baseline data on HPV prevalence in women attending a gynecological outpatient clinic in Zhejiang province. Our data will supply guidance for the primary screening and vaccination program for cervical cancer in this area.

Eosinophils from Murine Lamina Propria Induce Differentiation of Naïve T Cells into Regulatory T Cells via TGF-β1 and Retinoic Acid

Treg cells play a crucial role in immune tolerance, but mechanisms that induce Treg cells are poorly understood. We here have described eosinophils in lamina propria (LP) that displayed high aldehyde dehydrogenase (ALDH) activity, a rate-limiting step during all-trans retinoic acid (ATRA) synthesis, and expressed TGF-β1 mRNA and high levels of ATRA. Co-incubation assay confirmed that LP eosinophils induced the differentiation of naïve T cells into Treg cells. Differentiation promoted by LP eosinophils were inhibited by blocked either TGF-β1 or ATRA. Peripheral blood (PB) eosinophils did not produce ATRA and could not induce Treg differentiation. These data identifies LP eosinophils as effective inducers of Treg cell differentiation through a mechanism dependent on TGF-β1 and ATRA.

Mononuclear-macrophages but not neutrophils act as major infiltrating anti-leptospiral phagocytes during leptospirosis

Objective To identify the major infiltrating phagocytes during leptospirosis and examine the killing mechanism used by the host to eliminate Leptospira interrogans. Methods Major infiltrating phagocytes in Leptospira-infected C3H/HeJ mice were detected by immunohistochemistry. Chemokines and vascular endothelial cell adhesion molecules (VECAMs) of Leptospira-infected mice and leptospirosis patients were detected by microarray and immunohistochemistry. Leptospira-phagocytosing and -killing abilities of human or mouse macrophages and neutrophils, and the roles of intracellular ROS, NO and [Ca2+]i in Leptospira-killing process were evaluated by confocal microscopy and spectrofluorimetry. Results Peripheral blood mononuclear-macrophages rather than neutrophils were the main infiltrating phagocytes in the lungs, liver and kidneys of infected mice. Levels of macrophage- but not neutrophil-specific chemokines and VECAMs were significantly increased in the samples from infected mice and patients. All macrophages tested had a higher ability than neutrophils to phagocytose and kill leptospires. Higher ROS and NO levels and [Ca2+]i in the macrophages were involved in killing leptospires. Human macrophages displayed more phagolysosome formation and a stronger leptospire-killing ability to than mouse macrophages. Conclusions Mononuclear-macrophages but not neutrophils represent the main infiltrating and anti-leptospiral phagocytes during leptospirosis. A lower level of phagosome-lysosome fusion may be responsible for the lower Leptospira-killing ability of human macrophages.

CagA(+) H. pylori induces Akt1 phosphorylation and inhibits transcription of p21(WAF1/CIP1) and p27(KIP1) via PI3K/Akt1 pathway.

OBJECTIVEnCytotoxin-associated protein (CagA) of H. pylori has been confirmed to be closely associated with gastric inflammation and tumorigenesis, but the mechanism behind it is little understood. In this study, we try to determine roles of CagA(+) strain in activating PI3K/Akt1 signaling pathway, and affecting expression of p21(WAF1/CIP1) and p27(KIP1), and also in releasing IL-8 in host cells.nnnMETHODSnAkt1 phosphorylation and IL-8 levels of CagA(+) and CagA⁻ strain infected AGS cells were detected by ELISAs. Two quantitative RT-PCRs were established to measure p21(WAF1/CIP1) and p27(KIP1) mRNA levels in the CagA(+) and CagA⁻ strain infected cells. LY294002, an inhibitor of PI3K/Akt pathway, was used to define effect of the pathway in IL-8 release.nnnRESULTSnCagA(+) strain could induce an obvious elevation of Akt1 phosphorylation in the infected AGS cells while CagA? strain failed to do so. The CagA(+) H. pylori strain infected AGS cells showed significant drops both in p21(WAF1/CIP1) and p27(KIP1) mRNA levels, whereas the CagA⁻ H. pylori strain caused a remarkable increase in p21(WAF1/CIP1) mRNA without affecting p27(KIP1) gene transcription in the AGS cells. Both the CagA(+) and CagA⁻ H. pylori strains enabled AGS cells to produce close elevated levels of IL-8, and the LY294002 block resulted in unexpected elevations of IL-8 levels.nnnCONCLUSIONSnCagA can activate PI3K/Akt1 pathway that plays an inhibitory role in IL-8 release in H. pylori infected AGS cells. Activation of PI3K/Akt1 pathway and subsequent negative regulation of p21(WAF1/CIP1) and p27(KIP1) expression might be involved in CagA-associated carcinogenesis.

Vibrio vulnificus cytolysin induces apoptosis in HUVEC, SGC-7901 and SMMC-7721 cells via caspase-9/3-dependent pathway

Vibrio vulnificus cytolysin (VVC) is known to be a pore-forming toxin which shows cytotoxicity for mammalian cells in culture and induces apoptosis in endothelial cells. In order to determine whether VVC induces apoptosis in vascular endothelial cells and tumor cells, the cytotoxicity induced by recombinant VVC (rVVC) and its potential mechanism in HUVEC, SGC-7901 and SMMC-7721 cells were investigated. Our study demonstrated that rVVC induced the release of intracellular K(+) from all the target cells, yet lactate dehydrogenase was not released by rVVC. It indicates that osmotic lysis might not contribute to the cytolysin-induced cytotoxicity. The study also demonstrated that rVVC induced apoptosis in HUVEC, SGC-7901 and SMMC-7721 cells in time- and dosage-dependent manners, which was associated with the activation of caspase-9 and -3, but not caspase-8. During the apoptotic process of the target cells, rVVC labeled with FITC was monitored to attach initially to the surface of the cells and entered the cytoplasma subsequently. These findings suggest that VVC may be not only a pore-forming toxin, but also a transmembrane toxin with powerful ability to induce apoptosis in human vascular endothelial cells and tumor cells.

Role of RpoS in stress resistance, quorum sensing and spoilage potential of Pseudomonas fluorescens

Microorganism activities are considered the main cause of most food spoilage, leading to great economic losses. Pseudomonas fluorescens is a Gram-negative bacterium that is widely found in food and has high spoilage activity. RpoS is considered an important global regulator involved in stress survival and virulence in many pathogens. Thus, it is very possible that RpoS plays an important role in spoilage regulation in P. fluorescens. In this study an in-frame deletion mutation of rpoS was constructed to explore its function in P. fluorescens. The results showed that RpoS positively regulated the resistance of P. fluorescens to H2O2, heat, ethanol and crystal violet, negatively regulated the resistance to acetic acid, and had no effect on the resistance to NaCl. Further studies indicated that acylated homoserine lactone (AHL) production and the transcription levels of five AHL-related genes were significantly decreased in the rpoS mutant compared with those in the wild-type strain. Interestingly, the two homologous genes coding for AHL synthases contained RpoS-dependent -10 elements, suggesting that AHL quorum sensing is directly regulated by RpoS. RpoS also contributed to the spoilage activities of P. fluorescens by regulating extracellular protease and total volatile basic nitrogen (TVB-N) production in sterilized salmon juice. Our results reveal that RpoS was a key regulatory factor involved in stress resistance, the AHL quorum sensing system, and spoilage potential of P. fluorescens. Our study may benefit food safety control and food preservation.

Transcriptomic analysis of the response of Pseudomonas fluorescens to epigallocatechin gallate by RNA-seq.

Epigallocatechin gallate (EGCG) is a main constituent of green tea polyphenols that are widely used as food preservatives and are considered to be safe for consumption. However, the underlying antimicrobial mechanism of EGCG and the bacterial response to EGCG are not clearly understood. In the present study, a genome-wide transcriptional analysis of a typical spoilage bacterium, Pseudomonas fluorescens that responded to EGCG was performed using RNA-seq technology. A total of 26,365,414 and 23,287,092 clean reads were generated from P. fluorescens treated with or without 1 mM EGCG and the clean reads were aligned to the reference genome. Differential expression analysis revealed 291 upregulated genes and 134 downregulated genes and the differentially expressed genes (DEGs) were verified using RT-qPCR. Most of the DGEs involved in iron uptake, antioxidation, DNA repair, efflux system, cell envelope and cell-surface component synthesis were significantly upregulated by EGCG treatment, while most genes associated with energy production were downregulated. These transcriptomic changes are likely to be adaptive responses of P. fluorescens to iron limitation and oxidative stress, as well as DNA and envelope damage caused by EGCG. The expression of specific genes encoding the extra-cytoplasmic function sigma factor (PvdS, RpoE and AlgU) and the two-component sensor histidine kinase (BaeS and RpfG) were markedly changed by EGCG treatment, which may play important roles in regulating the stress responses of P. fluorescens to EGCG. The present data provides important insights into the molecular action of EGCG and the possible cross-resistance mediated by EGCG on P. fluorescens, which may ultimately contribute to the optimal application of green tea polyphenols in food preservation.