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PubMed2004

The serine/threonine protein phosphatase SIT4 modulates yeast-to-hypha morphogenesis and virulence in Candida albicans.

SIT4 encodes the multifunctional catalytic subunit of a type 2A-related protein phosphatase of Saccharomyces cerevisiae and has been implicated in cell cycle regulation and nitrogen sensing. We have identified the Candida albicans homologue of SIT4, and we show that its disruption caused a significant reduction in general growth rate, in hyphal outgrowth and in virulence in a mouse infection model. These phenotypes were reversed by the reintroduction of the wild-type SIT4 gene. We used glass DNA microarrays to measure the transcriptional profiles of 6287 open reading frames in sit4 cells undergoing the yeast-to-hypha transition induced by serum. Although differential expression of many of the hyphae-specific genes was not affected by the SIT4 deletion, the transcription of two new hyphae-induced genes, XOG1 and YNR67, was entirely reliant upon Sit4p. Both genes represent glucanases, indicating that SIT4 may play a role in controlling cell wall biogenesis. Furthermore, sit4 cells exhibited a reduced heat shock response to treatment with serum/37 degrees C, suggesting that SIT4 acts to co-ordinate the stress response signals during morphological switching. Finally, sit4 cells displayed reduced transcript levels for the genes encoding the Hog1p MAP kinase and several modulators of protein biosynthesis. Sit4p thus plays important roles during hyphal growth in Candida albicans through the regulation of cell wall biogenesis, osmosensing and protein translation.

Animals↗
PubMed1999

Levels of tumor necrosis factor alpha, gamma interferon, and interleukins 4,6, and 10 as determined in mice infected with virulent or attenuated strains of Trypanosoma cruzi.

Inoculation of BALB/c mice with the virulent Tulahuen (TUL) strain of T. cruzi was shown to lead to progressive and eventually lethal infections, whereas infection with an attenuated strain (TCC) resulted in a hardly noticeable experimental disease producing no tissue damage. To determine whether differences in such infection outcome are associated with a particular pattern of cytokine response, a study was undertaken to investigate the serum levels of TNF-alpha, IFN-gamma, IL-4, IL-6, and IL-10 by using an enzyme-linked immunosorbent assay. Mice from both infected groups were bled at 5, 9, 15, 22, 30 and 48 days post-infection (p.i.), with the same interval being applied for obtention of serum samples in age-matched uninfected mice, a group that yielded negative results in all cases. Infection with the TUL strain of T. cruzi was accompanied by a significant increase of TNF-alpha serum concentrations at day 5 p.i., and detectable amounts of IFN-gamma by day 15 p.i., which were exclusively recorded in this group. Serum IL-4 was mostly present in TCC mice with values at day 15 pi being statistically significant in relation to TUL-infected mice. IL-10 was firstly detected at 3 weeks after infection, and showed higher levels in the TCC group, although comparisons with TUL-infected group were not significant. At our limit of detection, no samples were found to contain IL-6 serum concentrations. Infection with virulent parasites seems to be associated with presence of Th1-type cytokines, whereas challenge with the attenuated TCC strain appears as being related to a Th2-type profile.

Animals↗
PubMed2001

Phenotypic and genotypic characteristics of Escherichia coli strains of non-enteropathogenic E. coli (EPEC) serogroups that carry EAE and lack the EPEC adherence factor and Shiga toxin DNA probe sequences.

This study was conducted to characterize the virulence potential of 59 Escherichia coli strains carrying EAE and lacking the enteropathogenic E. coli adherence factor and Shiga toxin probe sequences. In hybridization studies, all strains carried the locus of enterocyte effacement (LEE)-associated DNA sequences. Of the other 15 virulence DNA sequences tested, HLY was the most frequent (44.1%); 17 combinations of these sequences were found, but strains carrying EAE only (EAE profile) were the most frequent (35.6%). Except for 1 cytodetaching strain, all others adhered to HeLa and Caco-2 cells, most of which (approximately 75.0%) showed variations of the localized adherence pattern. Actin accumulation was detected in 75.9% of the nondetaching strains. Most strains had LEE, probably inserted in pheU (49.2%), and presented a nontypeable intimin (83.1%). Translocated intimin receptor-derived DNA sequences correlated with enteropathogenic and enterohemorrhagic E. coli in 61.0% and 32.0% of the strains, respectively. Thirty-five different serotypes were found. Only strains with the EAE profile were associated with diarrhea (P=.039).

Actins↗
PubMed2003

ZmpB, a novel virulence factor of Streptococcus pneumoniae that induces tumor necrosis factor alpha production in the respiratory tract.

Inflammation is a prominent feature of Streptococcus pneumoniae infection in both humans and animal models. Indeed, an intense host immune response to infection is thought to contribute significantly to the pathology of pneumococcal pneumonia and meningitis. Previously, induction of the inflammatory response following infection with S. pneumoniae has been attributed to certain cell wall constituents and the toxin pneumolysin. Here we present data implicating a putative zinc metalloprotease, ZmpB, as having a role in inflammation. Null mutations were created in the zmpB gene of the virulent serotype 2 strain D39 and analyzed in a murine model of infection. Isogenic mutants were attenuated in pneumonia and septicemia models of infection, as determined by levels of bacteremia and murine survival. Mutants were not attenuated in colonization of murine airways or lung tissue. Examination of cytokine profiles within the lung tissue revealed significantly lower levels of the proinflammatory cytokine tumor necrosis factor alpha following challenge with the Delta zmpB mutant (Delta 739). These data identify ZmpB as a novel virulence factor capable of inducing inflammation in the lower respiratory tract. The possibility that ZmpB was involved in inhibition of complement activity was examined, but the data indicated that ZmpB does not have a significant effect on this important host defense. The regulation of ZmpB by a two-component system (TCS09) located immediately upstream of the zmpB gene was examined. TCS09 was not required for the expression of zmpB during exponential growth in vitro.

Animals↗
PubMed1995

Characterization of Streptococcus agalactiae strains by multilocus enzyme genotype and serotype: identification of multiple virulent clone families that cause invasive neonatal disease.

The chromosomal genotypes of 277 isolates of 16 serotypes of Streptococcus agalactiae were characterized by analysis of electrophoretically demonstrable allele profiles at 12 metabolic enzyme loci. The collection comprised the type strain and 276 strains recovered from French symptomatic and asymptomatic subjects. Sixty-one distinctive electrophoretic types (ETs), representing multilocus clonal genotypes, were identified. Cluster analysis of the ETs revealed two primary phylogenetic divisions separated by a genetic distance of 0.62, Division I contained 67 isolates which could be assigned to 13 ETs. Twenty-seven of these isolates were from samples of cerebrospinal fluid (CSF) from neonatal meningitis patients. Two ETs, separated by a genetic distance of 0.217, contained 26 of these 27 isolates. Division II contained 210 isolates, of which 27 were isolated from CSF. This division was more polymorphic and included 48 ETs. Spanning a genetic distance of 0.3, three clusters and one ET were identified within this group. Twenty-four of 27 strains isolated from CSF belonged to one cluster, and 19 of them belonged to two adjacent ETs with a genetic distance of 0.083. Fifty-five of the 68 serotype Ia strains and 24 of the 26 serotype Ib strains were each confined to one of the evolutionary lineages, and 85 of the 86 strains which carried protein antigen c belonged to phylogenetic division II. Most of the type III organisms were assigned to two clone families. The characteristics of this French population argue for the existence of particular groups of strains responsible for neonatal meningitis and demonstrate that serotyping can supply information about the genetic distribution of strains.

Alleles↗
PubMed2025

IBDV-SSA, a novel molecular approach for the recovery of infectious bursal disease virus whole genomes from FTA cards.

Infectious bursal disease (IBD), a highly contagious viral disease in young chickens, poses significant economic losses due to high mortality and immunosuppression. While IBD virus (IBDV) virulence is influenced by multiple genes, whole-genome sequencing (WGS) of IBDV is crucial for defining the strain pathotype and clinical profile. Flinders Technology Associates (FTA) cards are convenient for field sample collection, but their filter paper matrix can hinder nucleic acid recovery, impacting sequencing efficiency. This study evaluated two enrichment strategies, single primer amplification (SPA) and IBDV segment-specific amplification (SSA), coupled with short-read (Illumina) and long-read (Oxford Nanopore Technologies, ONT) sequencing platforms, to optimize IBDV whole-genome recovery from FTA cards. Illumina sequencing produced comparable raw read counts for both methods, yet IBDV-SSA samples achieved significantly higher genome mapping rates (76%) than IBDV-SPA (12%). Genome coverage analysis revealed that IBDV-SSA provided uniform read distribution across both genomic segments, ensuring complete coverage, while IBDV-SPA exhibited significant bias, with most reads mapping to segment B, and limited coverage of segment A. Importantly, IBDV-SSA also proved compatible with ONT long-read sequencing, providing complete genome coverage. Notably, IBDV-SSA coupled with short-read sequencing successfully characterized coinfections in two samples. This optimized approach using IBDV-SSA enables efficient and comprehensive WGS of IBDV from FTA cards, facilitating strain characterization, virulence prediction, and epidemiological investigations.IMPORTANCEThis research tackles a significant problem for poultry farmers: a virus called infectious bursal disease virus (IBDV) that harms young chickens, causing high death rates and economic losses. To fight it effectively, scientists need to analyze its complete genetic makeup. Traditionally, collecting and preserving IBDV field samples was challenging. Flinders Technology Associates (FTA) cards have simplified this process, but getting usable genetic material from them has been difficult. This study introduces a new genome enrichment method, IBDV segment-specific amplification (IBDV-SSA), which successfully allows for IBDV complete genome recovery from FTA cards. By using this improved approach, scientists can accurately identify virus strains, assess how harmful they are, and monitor their spread. This, in turn, helps to improve vaccines and protect flocks. IBDV-SSA is a powerful tool for outbreak surveillance, supporting the poultry industry and ensuring a stable food supply.

Infectious bursal disease virus↗
PubMed1996

Characterization of Aeromonas hydrophila: a comparative study of strains isolated from diarrheal feces and the environment.

Thirty-five strains of Aeromonas hydrophila isolated from feces of diarrheal patients and from the environments were collected from Thailand and Japan. The physiological, biochemical, and serological characteristics, antibiotic resistance patterns and cell surface-related properties were compared. The diarrheal and environmental isolates of A hydrophila were found to be remarkably consistent in general culture and biochemical characteristics, with the exception of the reaction to D-arabinose in which the diarrheal strains were positive and environmental strains were negative. The plasmid patterns and cell surface-related properties of the environmental and diarrheal isolates were different. All strains produced Vero cell cytotoxin, hemolysin and lecithinase at 37 degrees, 30 degrees and 15 degrees C. In contrast, 83% of the environmental strains produced these virulence factors even at 4 degrees C. All strains indicated almost uniform susceptibility to the 16 antibiotics tested. Variations were found in the plasmid profile, toxin production in relation to the differences of temperature and cell surface-related properties of the strains. These variations between the clinical and environmental isolates could have potential as epidemiological markers for the sources of strains.

Aeromonas hydrophila↗
PubMed2004

Transcription profile of Helicobacter pylori in the human stomach reflects its physiology in vivo.

BACKGROUND: Little is known about levels of expression of Helicobacter pylori genes in the human host. We therefore developed a quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) assay to measure transcript profiles of H. pylori in the human stomach. METHODS: In vivo expression of 16 genes on the cag pathogenicity island and of 18 putative virulence genes was quantitated by isolation of total RNA directly from infected human gastric mucosa. The results were compared with in vitro expression determined from H. pylori cells grown in culture. RESULTS: The highest levels of expression were found for cag1 and cag25 and for genes, such as urease and catalase, that may be important for bacterial homeostasis in the relatively hostile environment of the gastric mucosa. Transcript abundance, relative to 16S rRNA, was lower in vivo than in vitro, which suggests that H. pylori cells are in stationary phase in the gastric environment. This was particularly apparent for cagA. Since CagA is arguably of unique importance, in terms of interaction with the host, tight control of its in vivo expression might be particularly important. CONCLUSIONS: qRT-PCR is a powerful tool to measure gene expression in human or animal tissue that contains minute amounts of microbial mRNA, and the results reflect on the physiology of the pathogen in its natural host.

Adult↗
PubMed2006

Comparative and functional genomics of Listeria spp.

The genus Listeria comprises a group of non-sporulating, Gram-positive, soil bacteria belonging to the low G+C group of microorganisms. The genus consists of only six species, L. monocytogenes, L. ivanovii, L. seeligeri, L. innocua, L. welshimeri, and L. grayi.L. monocytogenes and L. ivanovii are the only known pathogens of this group. Comparative whole-genome sequencing of representative strains comprising the entire genus is currently being performed and nearing completion. In the genus Listeria, genome reduction has led to the generation of non-pathogenic species from pathogenic progenitor strains. Indeed, many of the regions absent in the non-pathogenic species represent commonly deleted genes. Speciation and diversity of strains has been achieved by horizontal gene transfer of DNA encoding novel genes probably required for niche specific survival. The sequencing of several listerial genomes has also been accompanied by studies using global strategies involving whole-genome transcriptional profiling and proteomics to examine the adaptative changes of L. monocytogenes to growth in different environments and to catalogue the genes mediating these responses. We review this data and present information on the expression profile of L. monocytogenes EGD-e inside the vacuolar and the cytosolic environments of the host cell using whole-genome microarray analysis. Of the 484 genes regulated during intracellular growth 41 genes are species-specific, being absent from the genome of the non-pathogenic L. innocua CLIP 11262 strain. There were 25 genes that are strain-specific i.e. absent from the genome of the L. monocytogenes F2365 serotype 4b strain suggesting heterogeneity in the gene pool required for intracellular survival of L. monocytogenes in host cells.

Chromosomes, Bacterial↗
PubMed2003

Low-shear modeled microgravity: a global environmental regulatory signal affecting bacterial gene expression, physiology, and pathogenesis.

Bacteria inhabit an impressive variety of ecological niches and must adapt constantly to changing environmental conditions. While numerous environmental signals have been examined for their effect on bacteria, the effects of mechanical forces such as shear stress and gravity have only been investigated to a limited extent. However, several important studies have demonstrated a key role for the environmental signals of low shear and/or microgravity in the regulation of bacterial gene expression, physiology, and pathogenesis [Chem. Rec. 1 (2001) 333; Appl. Microbiol. Biotechnol. 54 (2000) 33; Appl. Environ. Microbiol. 63 (1997) 4090; J. Ind. Microbiol. 18 (1997) 22; Curr. Microbiol. 34(4) (1997) 199; Appl. Microbiol. Biotechnol. 56(3-4) (2001) 384; Infect Immun. 68(6) (2000) 3147; Cell 109(7) (2002) 913; Appl. Environ. Microbiol. 68(11) (2002) 5408; Proc. Natl. Acad. Sci. U. S. A. 99(21) (2002) 13807]. The response of bacteria to these environmental signals, which are similar to those encountered during prokaryotic life cycles, may provide insight into bacterial adaptations to physiologically relevant conditions. This review focuses on the current and potential future research trends aimed at understanding the effect of the mechanical forces of low shear and microgravity analogues on different bacterial parameters. In addition, this review also discusses the use of microgravity technology to generate physiologically relevant human tissue models for research in bacterial pathogenesis.

Bacteria↗
PubMed2002

Response regulator important in pathogenesis of Streptococcus suis serotype 2.

In this study, we describe the first response regulator of Streptococcus suis serotype 2, designated RevS. RevS was cloned and the sequence was determined. No histidine kinase was found in the vicinity ofrevS, therefore RevS was considered to be an orphan response regulator. An isogenic knock-out mutant of RevS was shown to be attenuated in colonising the S. suis specific organs in a competitive assay in four piglets, indicating thatrevS plays a role in the pathogenesis of S. suis infections. We analysed the protein expression profiles of various fractions of the wild-type strain 10 and the mutant strain 10DeltaRevS. The expression of known virulence factors ofS. suis by wild-type and the mutant strain, was not different. However, one protein in the protoplast fraction, with unknown identity was shown to be repressed by revS in the exponential growth phase of the mutant.

Animals↗
PubMed2004

Chips with everything: DNA microarrays in infectious diseases.

Two developments are set to revolutionise research in and clinical management of infectious diseases. First, the completion of the human genome project together with the sequencing of many pathogen genomes, and second, the development of microarray technology. This review explains the principles underlying DNA microarrays and highlights the uses to which they are being put to investigate the molecular basis of infectious diseases. Pathogen studies enable identification of both known and novel organisms, understanding of genetic evolution, and investigation of determinants of pathogenicity. Host studies show the complexities of development and activation of both innate and adaptive immunity. Host-pathogen studies allow global analysis of gene expression during pathogenesis. Microarray technology will accelerate our understanding of the complex genetic processes underlying the interaction between microorganisms and the host, with consequent improvements in the diagnosis, treatment, and prevention of infectious diseases.

Animals↗
PubMed2004

Overexpression of the multidrug efflux pump SmeDEF impairs Stenotrophomonas maltophilia physiology.

OBJECTIVES: The use of antibiotics for the treatment of infectious diseases has led to important changes in the structure of pathogenic bacterial populations. However, these changes could be buffered if the expression of antibiotic resistance genes were to lead to the counter-selection of antibiotic-resistant strains in antibiotic-free environments. To test the effect of antibiotic resistance on bacterial fitness, we analysed the effect of the overproduction of the multidrug efflux pump SmeDEF on the physiology of Stenotrophomonas maltophilia. SmeDEF confers resistance to antibiotics belonging to different structural families, and its overexpression is associated with an antibiotic resistance phenotype in clinical isolates of S. maltophilia. RESULTS: Two S. maltophilia isogenic strains were analysed: the wild-type strain D457 and strain D457R, which is a SmeDEF overproducer. In co-culture experiments, under non-selective pressure the wild-type strain displaced the mutant strain D457R. Metabolic profiling showed that SmeDEF overproduction leads to several changes in S. maltophilia metabolism. Using a Dictyostelium discoideum model of bacterial virulence, we found overexpression of SmeDEF to be associated with a reduction in S. maltophilia virulence. CONCLUSIONS: Together, these data indicate that overexpression of the multidrug efflux pump SmeDEF causes a metabolic burden for S. maltophilia.

Animals↗
PubMed2003

Yeasts in apical periodontitis.

Microbiological reports of apical periodontitis have revealed that yeasts can be isolated from approximately 5-20% of infected root canals. They occur either in pure cultures or together with bacteria. Almost all isolated yeasts belong to the genus Candida, and the predominant species is C. albicans. Pheno- and genotypic profiles of C. albicans isolates show heterogeneity comparable with those of isolates from other oral sites. C. albicans expresses several virulence factors that are capable of infecting the dentin-pulp complex, including dentinal tubules. This causes, consequentially, an inflammatory response around the root apex, which suggests a pathogenic role for this organism in apical periodontitis. Yeasts are particularly associated with persistent root canal infections that do not respond favorably to conservative root canal therapy. This may be due to the resistance of all oral Candida species against a commonly used topical medicament, calcium hydroxide. However, other antimicrobial agents may offer alternative therapeutic approaches and improve the treatment of these persistent cases of apical periodontitis.

Anti-Infective Agents, Local↗
PubMedDate not supplied

[The results of the measured oral infection of white mice with strains of the causative agent of plague differing in their plasmid profile].

The oral infection of mice with Y. pestis culture with different plasmid profiles by the method of feeding them with agar granules containing a definite number of these bacteria revealed that the virulence of subcultures which had lost Pst-plasmid and the capacity for fibrinolysin and plasma-coagulase production, encoded by this plasmid, decreased 10-1000 times, the loss of Fra-plasmid produced a less pronounced effect. In contrast to subcutaneous infection, in alimentary infection Y. pestis culture grown at 28 degrees C was more virulent than that grown at 37 degrees C. During the first hours after infection Y. pestis could be detected only in the lymph nodes along the gastrointestinal tract and in the intestine walls; later generalization of the infectious process or its latent course was observed. After oral infection specific antibodies were detected in surviving animals (up to 35%).

Animals↗
PubMed2006

Contribution of the type III secretion system (TTSS) to virulence of Aeromonas salmonicida subsp. salmonicida.

The recently described type III secretion system (TTSS) of Aeromonas salmonicida subsp. salmonicida has been linked to virulence in salmonids. In this study, three TTSS effector genes, aexT, aopH or aopO, were inactivated by deletion, as was ascC, the gene encoding the outer-membrane pore of the secretion apparatus. Effects on virulence were assayed by live challenge of Atlantic salmon (Salmo salar). The DeltaascC mutant strain was avirulent by both intraperitoneal (i.p.) injection and immersion, did not appear to establish a clinically inapparent infection and did not confer protection from subsequent rechallenge with the parental strain. 1H NMR spectroscopy-based metabolite profiling of plasma from all fish showed significant differences in the metabolite profiles between the animals exposed to the parental strain or DeltaascC. The experimental infection by immersion with DeltaaopO was indistinguishable from that of the parental strain, that of DeltaaexT was delayed, whilst the virulence of DeltaaopH was reduced significantly but not abolished. By i.p. injection, DeltaaexT, DeltaaopH and DeltaaopO caused an experimental disease indistinguishable from that of the parental strain. These data demonstrate that while the TTSS is absolutely essential for virulence of A. salmonicida subsp. salmonicida in Atlantic salmon, removal of individual effectors has little influence on virulence but has a significant effect on colonization. The DeltaascC i.p. injection data also suggest that in addition to host invasion there is a second step in A. salmonicida pathogenesis that requires an active TTSS.

Aeromonas salmonicida↗
PubMedDate not supplied

Molecular typing of infectious bursal disease virus of Israeli field and vaccine strains by the reverse transcription/polymerase chain reaction/restriction fragment length polymorphism assay.

Infectious bursal disease viruses (IBDVs) were examined by testing bursa samples from 37 commercially reared chicken flocks and three vaccine strains by the reverse transcription (RT)/polymerase chain reaction (PCR)/restriction fragment length polymorphism assay (RFLP). The assay was conducted with a 717-bp fragment of the VP2 gene with the restriction enzymes BstNI and MboI. The presence of a restriction site for SspI was used to predict a very virulent phenotype. Results indicated the existence of two molecular groups within the field isolates; four samples showed one pattern of RFLPs, and the majority, 30 out of the 37 tested, showed a second RFLP pattern. Three samples tested negative for IBDV. Eight bursa samples, representing the two molecular groups, were also tested by the RT/PCR/RFLP assay as developed by Jackwood. A comparison of the RFLP profiles by the two methods indicated that four isolates belonged to molecular group 6 and 30 isolates belonged to a new molecular group. All field isolates had a very virulent phenotype. One vaccine strain, produced from a local isolate, was classified as molecular group 6. The other two vaccine strains had RFLPs that differed from those of the field isolates.

Animals↗
PubMed2006

The complete genome sequence of a chronic atrophic gastritis Helicobacter pylori strain: evolution during disease progression.

Helicobacter pylori produces acute superficial gastritis in nearly all of its human hosts. However, a subset of individuals develops chronic atrophic gastritis (ChAG), a condition characterized in part by diminished numbers of acid-producing parietal cells and increased risk for development of gastric adenocarcinoma. Previously, we used a gnotobiotic transgenic mouse model with an engineered ablation of parietal cells to show that loss of parietal cells provides an opportunity for a H. pylori isolate from a patient with ChAG (HPAG1) to bind to, enter, and persist within gastric stem cells. This finding raises the question of how ChAG influences H. pylori genome evolution, physiology, and tumorigenesis. Here we describe the 1,596,366-bp HPAG1 genome. Custom HPAG1 Affymetrix GeneChips, representing 99.6% of its predicted ORFs, were used for whole-genome genotyping of additional H. pylori ChAG isolates obtained from Swedish patients enrolled in a case-control study of gastric cancer, as well as ChAG- and cancer-associated isolates from an individual who progressed from ChAG to gastric adenocarcinoma. The results reveal a shared gene signature among ChAG strains, as well as genes that may have been lost or gained during progression to adenocarcinoma. Whole-genome transcriptional profiling of HPAG1's response to acid during in vitro growth indicates that genes encoding components of metal uptake and utilization pathways, outer membrane proteins, and virulence factors are among those associated with H. pylori's adaptation to ChAG.

Adenocarcinoma↗