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PubMed1993

Molecular characterization of Yersinia strains isolated from human and environmental sources.

Thirty-seven strains of Yersinia isolated from human and environmental sources were investigated for virulence and molecular characteristics. Clinical isolates of Y. enterocolitica serotypes 0:3 and 0:9, harbouring the 45 MDa virulence plasmid were Ca++ dependent and responded positively to the CV-binding assay. The environmental Y. kristensenii strain, although harbouring a 45 MDa plasmid proved to be negative for both assays. All the environmental strains showed pyrazinamidase activity, while isolates from human sources proved negative. Two different plasmid profiles were disclosed: one Y. enterocolitica strain (serotype 0:7, 8) showed one plasmid of 67 MDa and one plasmid of 1.5 MDa. Eleven human isolates of Y. enterocolitica and one environmental strain of Y. krinstensenii showed only one plasmid of 45 MDa. Restriction endonuclease analysis of the 45 MDa plasmids supported the hypothesis that the environmental Y. krinstensenii plasmid is unrelated to the family of homologous virulence plasmids of Y. enterocolitica.

Amidohydrolases↗
PubMedDate not supplied

Characterization of non-virulence plasmids with homology to the virulence plasmid of Salmonella dublin.

Six wild-type (wt) strains of Salmonella typhimurium, one wt strain of S. heidelberg and 12 wt strains of Escherichia coli were isolated based on both hybridization to a 6-kb HindIII fragment of the non-virulence coding part of the S. dublin serovar-specific virulence plasmid and the absence of hybridization to the virulence genes (spv genes) of the same plasmid. Such hybridization was shown to be caused by resident plasmids in all strains and to involve the same region of 30 to 37 kb of consecutive HindIII fragments on the S. dublin virulence plasmid, suggesting a common origin of this plasmid DNA. Nine of the plasmids were selected for detailed characterization and were shown not to be of the same plasmid species. They varied in size between 44 and 88 kb, they showed incompatibility with the plasmid K-MP10, or belonged to incompatibility group X, and with the exception of five plasmids from E. coli, they showed different HindIII restriction profile patterns.

DNA, Bacterial↗
PubMed2004

DD1.5k, the gene preferentially expressed in bloodstream isolates of vancomycin-resistant Enterococcus faecium.

Vancomycin-resistant Enterococcus faecium (VREFM) is becoming a threatening pathogen. We identified a gene called DD1.5K by differential display-PCR, which was preferentially expressed in the bloodstream isolates of VREFM. Due to its amino acid similarity to transfer complex protein, trsE, and tissue-specific expression, this gene may be involved in virulence of VREFM.

Adaptation, Physiological↗
PubMed2001

Silencing of oxidative stress response in Mycobacterium tuberculosis: expression patterns of ahpC in virulent and avirulent strains and effect of ahpC inactivation.

Intracellular pathogens such as Mycobacterium tuberculosis are able to survive in the face of antimicrobial products generated by the host cell in response to infection. The product of the alkyl hydroperoxide reductase gene (ahpC) of M. tuberculosis is thought to be involved in protecting the organism against both oxidative and nitrosative stress encountered within the infected macrophage. Here we report that, contrary to expectations, ahpC expression in virulent strains of M. tuberculosis and Mycobacterium bovis grown in vitro is repressed, often below the level of detection, whereas expression in the avirulent vaccine strain M. bovis BCG is constitutively high. The repression of the ahpC gene of the virulent strains is independent of the naturally occurring lesions of central regulator oxyR. Using a green fluorescence protein vector (gfp)-ahpC reporter construct we present data showing that repression of ahpC of virulent M. tuberculosis also occurred during growth inside macrophages, whereas derepression in BCG was again seen under identical conditions. Inactivation of ahpC on the chromosome of M. tuberculosis by homologous recombination had no effect on its growth during acute infection in mice and did not affect in vitro sensitivity to H2O2. However, consistent with AhpC function in detoxifying organic peroxides, sensitivity to cumene hydroperoxide exposure was increased in the ahpC::Km(r) mutant strain. The preservation of a functional ahpC gene in M. tuberculosis in spite of its repression under normal growth conditions suggests that, while AhpC does not play a significant role in establishing infection, it is likely to be important under certain, as yet undefined conditions. This is supported by the observation that repression of ahpC expression in vitro was lifted under conditions of static growth.

Animals↗
PubMed2006

Vibrio cholerae virulence regulator-coordinated evasion of host immunity.

To successfully propagate and cause disease, pathogenic bacteria must modulate their transcriptional activities in response to pressures exerted by the host immune system, including secreted immunoglobulins such as secretory IgA (S-IgA), which can bind and agglutinate bacteria. Here, we present a previously undescribed flow cytometry-based screening method to identify bacterial genes expressed in vitro and repressed during infections of Vibrio cholerae, an aquatic Gram-negative bacterium responsible for the severe diarrheal disease cholera. We identified a type IV mannose-sensitive hemagglutinin (MSHA) pilus that is repressed specifically in vivo. We showed that bacteria that failed to turn off MSHA biosynthesis were unable to colonize the intestines of infant mice in the presence of S-IgA. We also found that V. cholerae bound S-IgA in an MSHA-dependent and mannose-sensitive fashion and that binding of S-IgA prevented bacteria from penetrating mucus barriers and attaching to the surface of epithelial cells. The ability of V. cholerae to evade the non-antigen-specific binding of S-IgA by down-regulating a surface adhesin represents a previously undescribed mechanism of immune evasion in pathogenic bacteria. In addition, we found that repression of MSHA was mediated by the key virulence transcription factor ToxT, indicating that V. cholerae is able to coordinate both virulence gene activation and repression to evade host defenses and successfully colonize intestines.

Animals↗
PubMed1999

Experimental approaches to understanding virulence in toxoplasmosis.

Toxoplasma gondii is a widespread protozoan parasite that causes severe disease only in immunocompromised individuals. Equipped with excellent animal models and relatively advanced systems for genetics, T. gondii provides an excellent system for understanding pathogenesis. Resistance to toxoplasmosis is governed by rapid innate and adaptive immunity that is characterized by a Th1 type profile of cytokines. Despite this effective response, acute infections can cause considerable damage and the parasite effectively establishes a long-term chronic infection that predisposes the host to reactivation and provides a means of eventual transmission. This complex interaction is brought about by the differentiation of the parasite from a rapidly replicating, lytic form (known as the tachyzoite) to a slow-growing form (known as the bradyzoite) that gives rise to chronic infection. The population structure of T. gondii is remarkably clonal, consisting of just three predominant lineages that are geographically widespread and found in a variety of hosts including humans. Acute virulence is strongly associated with the type I genotype which exhibits an enhanced replication rate in vitro and higher tissue burdens in vivo relative to non-virulent lineages. The pathology associated with acute infection appears to be due to excessive production of acute inflammatory mediators, suggesting that disease is partly due to over-response of the host immune system. A combination of refined animal models and newly developed genetic tools for establishing the relative contribution of genes to pathogenesis will enable a comprehensive analysis of the molecular basis of virulence in toxoplasmosis.

Acute Disease↗
PubMed2005

Cell cycle arrest during S or M phase generates polarized growth via distinct signals in Candida albicans.

Treatments that perturb DNA synthesis or mitosis will activate checkpoints that prevent cell cycle progression and cell proliferation. In yeast-form cells of the fungal pathogen Candida albicans, exposure to hydroxyurea (HU) or shutting off expression of the polo-like kinase CaCDC5 blocked nuclear division and spindle elongation, but activated a highly polarized growth mode. We have used transcription profiling both to characterize the initiation and progression of this polar growth pattern and to determine how cell elongation may be linked to the cell cycle in C. albicans. Different gene expression patterns during early stages of cell elongation support the concept that CaCdc5p-depleted and HU-exposed cells were blocked at different stages of the cell cycle, and suggest that different signals may generate the common polarized growth phenotype. Consistent with this, BUB2 expression was modulated in CaCdc5p-depleted cells, and absence of BUB2 prevented the maintenance of cell polarization, resulting in multibudded, pseudohyphal cells with constrictions. In contrast, HU-induced filaments did not modulate or require BUB2, but were dependent on the GTPase Ras1p. However, at later stages of cell elongation, transcription profiles were more similar, and comparisons with serum-induced hyphae revealed that the cell cycle-arrested filaments expressed several targets of the hyphal signalling pathways. Thus, arresting the yeast cell cycle in S or M phase generates a polarized growth pattern through different mechanisms in C. albicans, and maintenance of the polar growth mode can ultimately lead to the expression of hyphal-associated cell wall and virulence-related factors, in the absence of any external stimuli.

Candida albicans↗
PubMed2006

Global transcriptome analysis of Staphylococcus aureus response to hydrogen peroxide.

Staphylococcus aureus responds with protective strategies against phagocyte-derived reactive oxidants to infect humans. Herein, we report the transcriptome analysis of the cellular response of S. aureus to hydrogen peroxide-induced oxidative stress. The data indicate that the oxidative response includes the induction of genes involved in virulence, DNA repair, and notably, anaerobic metabolism.

Gene Expression Profiling↗
PubMed2001

Comparison of the hydrophobic properties of Candida albicans and Candida dubliniensis.

Although Candida dubliniensis is a close genetic relative of Candida albicans, it colonizes and infects fewer sites. Nearly all instances of candidiasis caused by C. dubliniensis are restricted to the oral cavity. As cell surface hydrophobicity (CSH) influences virulence of C. albicans, CSH properties of C. dubliniensis were investigated and compared to C. albicans. Growth temperature is one factor which affects the CSH status of stationary-phase C. albicans. However, C. dubliniensis, similar to other pathogenic non-albicans species of Candida, was hydrophobic regardless of growth temperature. For all Candida species tested in this study (C. albicans, C. dubliniensis, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis), CSH status correlated with coaggregation with the anaerobic oral bacterium Fusobacterium nucleatum. Previous studies have shown that CSH status of C. albicans involves multiple surface proteins and surface protein N-glycans. The hydrophobic surface glycoprotein CAgp38 appears to be expressed by C. albicans constitutively regardless of growth temperature and medium. C. dubliniensis expresses a 38-kDa protein that cross-reacts with the anti-CAgp38 monoclonal antibody; however, expression of the protein was growth medium and growth temperature dependent. The anti-CAgp38 monoclonal antibody has been shown to inhibit adhesion of C. albicans to extracellular matrix proteins and to vascular endothelial cells. Since protein glycosylation influences the CSH status of C. albicans, we compared the cell wall mannoprotein content and composition between C. albicans and C. dubliniensis. Similar bulk compositional levels of hexose, phosphate, and protein in their N-glycans were determined. However, a component of the C. albicans N-glycan, acid-labile phosphooligomannoside, is expressed much less or negligibly by C. dubliniensis, and when present, the oligomannosides are predominantly less than five mannose residues in length. In addition, the acid-labile phosphooligomannoside profiles varied among the three strains of C. dubliniensis we tested, indicating the N-glycan of C. dubliniensis differs from C. albicans. For C. albicans, the acid-labile phosphooligomannoside influences virulence and surface fibrillar conformation, which affects exposure of hydrophobic surface proteins. Given the combined role in C. albicans of expression of specific surface hydrophobic proteins in pathogenesis and of surface protein glycosylation on exposure of the proteins, the lack of these virulence-associated CSH entities in C. dubliniensis could contribute to its limited ability to cause disseminated infections.

Blotting, Western↗
PubMed1994

Reduced virulence of rifampicin-resistant mutants of Francisella tularensis.

Rifampicin-resistant mutants of a live vaccine strain (LVS) of Francisella tularensis were produced and screened for virulence in mice; 4 avirulent clones with intraperitoneal (ip) LD50s > 10(6) cfu, compared with 10(2) cfu for LVS, were characterized. Growth characteristics at 37 degrees C, surface envelope proteins, and lipopolysaccharide profiles of resistant mutants were identical to those of LVS. Polymerase activity of the mutants was more resistant than the enzyme from LVS to the inhibitory action of rifampicin. Growth rates for mutants and LVS were similar during the first 5 h at 42 degrees C, but viability of the mutants decreased to < 0.01% at 24 h. LVS and mutants differed in their ability to grow in vitro in host macrophages: LVS increased 580-fold over 72 h; mutants increased 33-fold. After ip inoculation of the organisms into mice, increasing numbers of LVS from peritoneal cells were isolated; mutants decreased over 4 days.

Animals↗
PubMed1994

Effect of sub-MIC antibiotics on the cell surface and extracellular virulence determinants of Pseudomonas cepacia.

The effects of sub-MICs of ciprofloxacin and tobramycin on the cell surface characteristics and extracellular virulence factors of Pseudomonas cepacia were evaluated. Cells were grown in batch culture under iron-deficient and iron-replete conditions. At sub-MIC levels that did not affect bacterial growth cell surface hydrophobicity decreased under both iron-replete and iron-depleted conditions with ciprofloxacin, but increased with tobramycin under iron-sufficient conditions. Exopolysaccharide synthesis, lipase production and siderophore production were all significantly increased by the presence of ciprofloxacin under both growth conditions. Outer membrane protein and lipopolysaccharide profiles were not affected by exposure to the two antibiotics.

Burkholderia cepacia↗
PubMed2002

Differential expression of virulence-related genes in Enterococcus faecalis in response to biological cues in serum and urine.

Enterococci rank among leading causes of nosocomial bacteremia and urinary tract infection and are also a leading cause of community acquired subacute endocarditis. Limited evidence suggests that biological cues in serum and urine may play an important role in modulating enterococcal virulence at sites of infection. To determine the extent to which biological cues affect enterococcal virulence-associated gene expression, we used quantitative real-time PCR to compare mRNA levels in Enterococcus faecalis cultures grown in serum or urine to that achieved in laboratory medium. Both environment- and growth phase-specific variations were observed, demonstrating the occurrence of as-yet-uncharacterized mechanisms for control of gene expression in E. faecalis that may play an important role in vivo.

Enterococcus faecalis↗
PubMed2006

The Brucella abortus xthA-1 gene product participates in base excision repair and resistance to oxidative killing but is not required for wild-type virulence in the mouse model.

Exonuclease III, encoded by the xthA gene, plays a central role in the base excision pathway of DNA repair in bacteria. Studies with Escherichia coli xthA mutants have also shown that exonuclease III participates in the repair of oxidative damage to DNA. An isogenic xthA-1 mutant (designated CAM220) derived from virulent Brucella abortus 2308 exhibited increased sensitivity to the alkylating agent methyl methanesulfonate (MMS) compared to the parent strain. In contrast, 2308 and the isogenic xthA-1 mutant displayed similar levels of resistance to the DNA cross-linker mitomycin C. These phenotypic properties are those that would be predicted for a strain defective in base excision repair. The B. abortus xthA-1 mutant also displayed reduced resistance to killing by H2O2 and the ONOO(-)-generating compound 3-morpholinosydnonimine (SIN-1) compared to strain 2308, indicating that the xthA-1 gene product participates in protecting B. abortus 2308 from oxidative damage. Introducing a plasmid-borne copy of the parental xthA-1 gene into CAM220 restored wild-type resistance of this mutant to MMS, H2O2, and SIN-1. Although the B. abortus xthA-1 mutant exhibited increased sensitivity to oxidative killing compared to the parental strain in laboratory assays, CAM220 and 2308 displayed equivalent spleen colonization profiles in C57BL/6 [corrected] mice through 8 weeks postinfection and equivalent intracellular survival and replication profiles in cultured murine macrophages. Thus, although the xthA-1 gene product participates in base excision repair and resistance to oxidative killing in B. abortus 2308, XthA-1 is not required for wild-type virulence of this strain in the mouse model.

Animals↗
PubMed2006

Genome scale analysis of diffusible signal factor regulon in Xanthomonas campestris pv. campestris: identification of novel cell-cell communication-dependent genes and functions.

The bacterial pathogen Xanthomonas campestris pv. campestris (Xcc) recruits a diffusible signal factor (DSF), which has recently been structurally characterized as cis-11-methyl-2-dodecenoic acid, as a cell-cell communication signal to synchronize virulence gene expression and biofilm dispersal. In this study, we showed that despite the existance of phenotype variations in different Xcc isolates, the DSF-mediated functions were in general conserved. To investigate the genomic profiles of DSF regulation, we designed and conducted oligomicroarray analysis by comparison of the gene expression patterns of wild-type strain XC1 and its DSF-deficient mutant XC1dF, as well as those of XC1dF in the presence or absence of DSF signals. The analyses led to identification of 165 genes, whose expression was significantly influenced by DSF signals. These genes encode proteins and enzymes belonging to at least 12 functional groups. In addition to those previously known DSF-dependent activities such as production of extracellular enzymes and extracellular polysaccharides, microarray analyses also revealed new functions mediated by DSF, such as flagellum synthesis, resistance to toxins and oxidative stress, and aerobic respiration. Phenotype analyses confirmed that DSF signalling contributed to resistance to toxin acriflavin and hydrogen peroxide, and to the survival of bacterial cells at different temperatures. We conclude that DSF cell-cell signalling is not only essential for co-ordinating the expression of virulence genes but also plays a vital role in keeping up the general competence of the pathogen in ecosystems.

Base Sequence↗
PubMed2005

In vivo assessment of human vaginal oxygen and carbon dioxide levels during and post menses.

Previous in vitro and in vivo animal studies showed that O(2) and CO(2) concentrations can affect virulence of pathogenic bacteria such as Staphylococcus aureus. The objective of this work was to measure O(2) and CO(2) levels in the vaginal environment during tampon wear using newly available sensor technology. Measurements by two vaginal sensors showed a decrease in vaginal O(2) levels after tampon insertion. These decreases were independent of the type of tampons used and the time of measurement (mid-cycle or during menstruation). These results are not in agreement with a previous study that concluded that oxygenation of the vaginal environment during tampon use occurred via delivery of a bolus of O(2) during the insertion process. Our measurements of gas levels in menses showed the presence of both O(2) and CO(2) in menses. The tampons inserted into the vagina contained O(2) and CO(2) levels consistent with atmospheric conditions. Over time during tampon use, levels of O(2) in the tampon decreased and levels of CO(2) increased. Tampon absorbent capacity, menses loading, and wear time influenced the kinetics of these changes. Colonization with S. aureus had no effect on the gas profiles during menstruation. Taken collectively, these findings have important implications on the current understanding of gaseous changes in the vaginal environment during menstruation and the potential role(s) they may play in affecting bacterial virulence factor production.

Adult↗
PubMed1995

Exclusion of temperature bacteriophage P22 by virulent bacteriophage MB78 of Salmonella typhimurium: competition for association with the replication complex.

MB78, a virulent bacteriophage of S. typhimurium does not allow other bacteriophages like P22 and 9NA to multiply in its presence. The exclusion of P22 by MB78 is found to be due to competition for common binding site(s) in the host cell membrane. As a result, P22 DNA fails to replicate in presence of MB78 DNA. Further, the sedimentation profile of P22 DNA in cells infected simultaneously with P22 and MB78 suggested fragmentation of P22 DNA. This may also contribute to the exclusion phenomenon.

Bacteriophage P22↗
PubMed2026

wbp-encoded LPS O-antigen architecture as a prognostic and therapeutic target in Pseudomonas aeruginosa keratitis.

BACKGROUND: Pseudomonas aeruginosa (P. aeruginosa) keratitis can progress rapidly to vision-threatening disease, even with intensive therapy. Virulence-associated genes are key determinants of ocular-surface pathogenesis. We therefore sought to develop a composite wbp-exo genotyping framework for risk stratification and to guide wbp-dependent, LPS-directed, levofloxacin-polymyxin B (LVX-POL) combination therapy for high-risk corneal infections. METHODS: A well-characterised clinical P. aeruginosa keratitis cohort was integrated with whole-genome sequencing. Based on comprehensive virulence-gene identification and annotation, the relationship between strain-level genetic features and clinical prognosis was analysed. The differences between WBP1 strains and WBP2 strains in adhesion, invasion, and biofilm formation in corneal epithelial cells were further evaluated. To establish biological plausibility, wbp genotypes were correlated with LPS O-antigen electrophoretic profiles and in vivo corneal inflammatory phenotypes in murine infection, including the observation of leucocyte recruitment and cytokine responses. To further confirm the key role of wbp gene status and LPS O-antigen in pathogenicity, wbpL knockout and reconstitution strains were constructed. Their appearances in vitro and in vivo were evaluated. Finally, a mechanistic rationale for an LPS-directed LVX-POL regimen was tested in a high-risk WBP1 P. aeruginosa murine keratitis. FINDINGS: Whole-genome sequencing was performed on 46 clinical P. aeruginosa isolates and identified an average of 332 virulence- and fitness-associated genes per strain. The exo and wbp gene families were significantly associated with patient prognosis. A fusion model (AUC = 0.86) outperformed single-gene-family models (EXO: 0.66; WBP: 0.72) for predicting clinical outcomes. Intact wbp cassettes were enriched in poor-outcome isolates, and electrophoretic LPS profiles indicated that WBP1 strains produce highly polymerised O-antigen associated with sustained neutrophil recruitment and cytokine production. Murine experiments further implicated wbp genes in clinical pathogenesis, showing stronger immune responses and higher expression of TLR4, MyD88, TRAF6, p65, p-p65, IL-6, TNF-&#x3b1;, and IL-1&#x3b2; throughout the inflammatory course. After knocking out wbpL gene, the WBP1 strain got stronger in biofilm formation and adhesion but weaker in inflammation and ocular surface survival. In the high-risk WBP1 P. aeruginosa keratitis model, LVX-POL combinations achieved complete ulcer resolution and markedly improved stromal infiltration and hypopyon, outperforming LVX monotherapy. INTERPRETATION: The wbp gene family was identified as a key genetic factor that contributes to the LPS O-antigen structure, inflammatory intensity, bacterial ocular surface survival and poor prognosis in P. aeruginosa keratitis. A WBP1-targeted, LPS-directed LVX-POL regimen was proposed as a mechanistically informed option for high-risk strains. FUNDING: This research was supported by Beijing Public Health High-level Talent Training Program (Phase III-03-14), Prevention and Control of Emerging and Major Infectious Diseases-National Science and Technology Major Project (2026ZD01909300) and Beijing Natural Science Foundation "QiYan" Undergraduate Research Fund (QY26496).

Pseudomonas aeruginosa↗
PubMed2006

Regulation of type IV fimbrial biogenesis in Dichelobacter nodosus.

Type IV fimbriae are expressed by several bacterial pathogens and are essential for virulence in Dichelobacter nodosus, which causes ovine footrot. We have identified a two-component signal transduction system (PilR/S) and an alternative sigma factor (sigma 54) that were shown by insertional inactivation to be required for the regulation of fimbrial biogenesis in D. nodosus. Western blots showed that in both pilR and rpoN mutants, fimbrial subunit production was significantly reduced by a process that was shown to occur at a PilR- and sigma 54-dependent promoter. The mutants lacked surface fimbriae, which were shown to be required for the adherence of D. nodosus cells to tissue culture monolayers. The reduction in fimbrial subunit production in these mutants also resulted in a concomitant loss of the ability to secrete extracellular proteases. A maltose binding protein-PilR fusion protein was purified and was shown to bind specifically to a region located 234 to 594 bp upstream of the fimA transcriptional start point. To determine additional targets of PilR and sigma 54, genome-wide transcriptional profiling was performed using a whole-genome oligonucleotide microarray. The results indicated that PilR and sigma 54 regulated genes other than fimA; these genes appear to encode surface-exposed proteins whose role in virulence is unknown. In conclusion, this study represents a significant advancement in our understanding of how the ability of D. nodosus to cause ovine footrot is regulated, as we have shown that the biogenesis of type IV fimbriae in D. nodosus is regulated by a sigma 54-dependent PilR/S system that also indirectly controls protease secretion.

Bacterial Adhesion↗