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Results for “Virulence profiling”

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PubMed2003

Gene expression in the tuberculous granuloma: analysis by laser capture microdissection and real-time PCR.

We have assessed the kinetics of host gene expression in granulomas of mice infected with virulent Mycobacterium tuberculosis, using an approach that incorporates the laser capture microdissection (LCM) and real-time PCR technology in conjunction with a newly derived mathematical equation. The results have provided evidence indicating that conventional use of whole infected lungs to study granuloma-specific gene expression can yield data that may not genuinely reflect intralesional events. Significantly, the expression of nine host genes known to regulate the inflammatory response to M. tuberculosis, as determined by real-time PCR analysis of microdissected granuloma-derived cDNAs, was downregulated (up to 27-fold) at around the time when the rapid growth phase of the bacilli in the lungs of infected mice ends. This downregulation was masked when whole infected lungs were used for the studies. The data suggest that the host immune system can adjust and respond to, or can be modulated by specific physiological states of the tubercle bacillus in vivo. The LCM/real-time PCR-based system described in this study can be applied to safely and accurately evaluate gene expression in any lesions that can be microscopically visualized, including those contained in biohazardous tissues.

Animals↗
PubMed2005

Differential display analysis of Porphyromonas gingivalis gene activation response to heat and oxidative stress.

BACKGROUND/AIMS: The etiologic relationship between periodontitis and Porphyromonas gingivalis is attributed to the ability of the organism to express a variety of virulence factors, many of which are cell surface components including lipopolysaccharide and arginine-specific cysteine proteases (Arg-gingipains, RgpA, and RgpB). P. gingivalis responds to the stress of rapid elevation in temperature by activating a set of genes to produce heat shock proteins that mediate the effects of sudden changes in environmental temperatures by repairing or eliminating cellular proteins denatured by that stress. METHODS: We used restriction fragment differential display (RFDD) to identify and measure the genes expressed by surrogates of environmental stresses, heat and oxidative stress. The results were then confirmed using quantitative reverse-transcription polymerase chain reaction. RESULTS: We selected 16 genes differentially induced from over 800 total expression fragments on the RFDD gels for further characterization. With primers designed from those fragments we found that a + 5 degrees C heat shock caused a statistically significant increase in expression compared 12 of 18 untreated genes tested. The exposure of P. gingivalis to atmospheric oxygen resulted in statistically significant increases in five of the target genes. These genes are likely involved in transport and synthesis of components of the lipopolysaccharide biosynthetic pathway important in anchoring the Arg-gingipains required for virulence-related activities. CONCLUSION: These results emphasize the need for studies to measure the coordinated responses of bacteria like P. gingivalis which use a multitude of interrelated metabolic activities to survive the environmental hazards of the infection process.

Adhesins, Bacterial↗
PubMed2006

Two-dimensional electrophoresis protein profile of the phytopathogenic fungus Botrytis cinerea.

Botrytis cinerea is a phytopathogenic fungi causing disease in a number of important crops. It is considered a very complex species in which different populations seem to be adapted to different hosts. In order to characterize fungal virulence factors, a proteomic research was started. A protocol for protein extraction from mycelium tissue, with protein separation by 2-DE and MS analysis, was optimised as a first approach to defining the B. cinerea proteome. Around 400 spots were detected in 2-DE CBB-stained gels, covering the 5.4-7.7 pH and 14-85 kDa ranges. The averages of analytical and biological coefficients of variance for 64 independent spots were 16.1% and 37.5%, respectively. Twenty-two protein spots were identified by MALDI-TOF or ESI IT MS/MS, with some of them corresponding to forms of malate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Two more spots matched a cyclophilin and a protein with an unknown function.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2006

Amplification generates modular diversity at an avirulence locus in the pathogen Phytophthora.

The destructive late blight pathogen Phytophthora infestans is notorious for its rapid adaptation to circumvent detection mediated by plant resistance (R) genes. We performed comparative genomic hybridization on microarrays (array-CGH) in a near genome-wide survey to identify genome rearrangements related to changes in virulence. Six loci with copy number variation were found, one of which involves an amplification colocalizing with a previously identified locus that confers avirulence in combination with either R gene R3b, R10, or R11. Besides array-CGH, we used three independent approaches to find candidate genes at the Avr3b-Avr10-Avr11 locus: positional cloning, cDNA-AFLP analysis, and Affymetrix array expression profiling. This resulted in one candidate, pi3.4, that encodes a protein of 1956 amino acids with regulatory domains characteristic for transcription factors. Amplification is restricted to the 3' end of the full-length gene but the amplified copies still contain the hallmarks of a regulatory protein. Sequence comparison showed that the amplification may generate modular diversity and assist in the assembly of novel full-length genes via unequal crossing-over. Analyses of P. infestans field isolates revealed that the pi3.4 amplification correlates with avirulence; isolates virulent on R3b, R10, and R11 plants lack the amplified gene cluster. The ancestral state of 3.4 in the Phytophthora lineage is a full-length, single-copy gene. In P. infestans, however, pi3.4 is a dynamic gene that is amplified and has moved to other locations. Modular diversity could be a novel mechanism for pathogens to quickly adapt to changes in the environment.

Chromosomes, Artificial, Bacterial↗
PubMed2005

Analysis of the growth phase-associated transcriptome of Streptococcus pyogenes.

Streptococcus pyogenes (group A streptococci, GAS) is a human pathogen which probably varies its multiplication rate and thus, growth phases in association with the type of infection caused in its host. To create a basis for future determinations of such associations, the genome-wide growth phase-related GAS transcriptome was assessed in the present study. Therefore, the published serotype M1 S. pyogenes genome sequence as well as the partially sequenced serotype M18 and M49 GAS genomes were used to produce DNA microarrays that carried 2256 oligonucleotide probes matching 3662 open reading frames (ORFs). With these microarrays, the transcriptome of the serotype M49 GAS strain 591 grown to the exponential, transition, and early stationary growth phases was assessed in seven independent experiments. The gained data were compared to real-time RT-PCR assays. Data analysis was refined by a novel approach, i.e. grouping of expressed genes to four classes according to relative transcript abundance and gene functions. At the different growth phases, 86.7%, 79.5% and 55.7% of the at least 1883 ORFs contained in the serotype M49 genome were expressed above the defined detection level. Contrary to the general trend, transcript amounts of genes in the functional groups of transport and membrane proteins as well as stress response factors peaked at the transition phase. The most prominent changes in the transcript abundances were predominantly observed for sugar compound transport and turnover-related ORFs. The majority of known virulence genes had their maximum expression during the transition phase, consistent with the proposed associated change in virulence behavior of the bacteria. With these results, it will now be feasible to assess the in situ growth phase of a given GAS strain during any type of infection by measuring the expression of selected marker genes.

DNA, Bacterial↗
PubMed1994

Lack of evidence for increased adherent growth in broth or human serum of clinically significant coagulase-negative staphylococci.

The relevance of adherent growth of coagulase-negative staphylococci (CNS) in vitro to their clinical significance is controversial. We have investigated the accumulation on polymer surfaces of clinically significant CNS strains, isolated from blood cultures and intravascular catheter tips, compared with controls. The adherent growth of significant CNS isolates in a microtitre tray assay was not greater than controls, and this finding was repeated when different combinations of target polymer surface, culture medium and incubation atmosphere type were investigated. The surface accumulation of CNS cultured in pooled human serum was poor compared with culture in broth. Whole cell and surface protein profiles of serum grown Staphylococcus epidermidis differed markedly from broth cultured cells. We recommend the use of human body fluids, such as serum, as useful culture media to assess whether potential virulence determinants are likely to be expressed in vivo. The microtitre tray assay of adherent growth is not helpful in determining the virulence of individual CNS isolates.

Bacterial Adhesion↗
PubMed1998

Phenotypic and genotypic alterations associated with the attenuation of a Theileria annulata vaccine cell line from Turkey.

Attenuated vaccines, produced by prolonged in vitro culture of the macroschizont stage of the life-cycle, are the main method of controlling Theileria annulata infections. Little is known about the mechanism(s) of attenuation. Here we present data from a Turkish cell line demonstrating that attenuation is associated with reduced ability to differentiate into microschizonts and a reduction in matrix metalloproteinase activity. We also show that attenuation results in a change in the structure of the parasite population. Using the technique of differential mRNA display, we demonstrate that gene expression profiles differ between non-attenuated and attenuated macroschizont infected leucocytes. One differentially expressed gene is of parasite origin. These data are discussed in the context of a multifactorial model for virulence.

Animals↗
PubMed1996

Iron utilization studies in Citrobacter species.

Seventy-one strains of Citrobacter were screened for iron scavenging mechanisms by biologic and chemical assays. Essentially all citrobacteria (70/71) were found to elaborate enterobactin-like siderophores by both biologic and chemical assays, however only c. koseri (C. diversus) was found to produce aerobactin. The concentration of ethylenediamine di(o-hydroxyphenylacetic acid) (EDDA) required to inhibit the growth of individual Citrobacter strains by depleting free iron ranged from 250 micrograms/ml to 100 micrograms/ml. Iron utilization studies of selected citrobacter isolates indicated that hemin and hematin could reverse the effects of iron limitation on growth under iron-stressed conditions (1000 micrograms/ml of EDDA). Two C. koseri strains grown under iron-restricted conditions showed similar changes in their whole cell protein profiles including induction of high molecular mass proteins (72-83 kDa) which may play a role in iron acquisition under iron-stressed conditions. The collective results support an additional virulence-associated mechanism for C. koseri strains which may help explain the greater pathogenic potential this group has for causing serious extraintestinal disease in humans.

Bacterial Proteins↗
PubMed1995

Molecular characterization of Pasteurella testudinis isolated from desert tortoises (Gopherus agassizii) with and without upper respiratory tract disease.

Isolates of Pasteurella testudinis recovered from clinically healthy desert tortoises (Gopherus agassizii) and tortoises with upper respiratory tract disease (URTD) were characterized in an attempt to identify strains associated with disease. Eighty-nine isolates, 52 from ill and 37 from healthy tortoises collected from Nevada (USA), June 1990 to September 1991, were genomically fingerprinted and grouped based on ribotype similarity. Twelve isolates (six from ill and six from healthy tortoises) were further characterized with regard to whole-cell protein (WCP) and outer membrane protein (OMP) composition and their ability to survive in normal tortoise plasma. The 89 isolates were initially distributed into 33 distinct ribotype groups using the restriction enzyme EcoRI; five ribotypes contained over 50% of the isolates. Only one EcoRI ribotype was comprised of multiple isolates (n = 4) exclusively recovered from tortoises with URTD. When the ten EcoRI ribotypes that contained more than one isolate per ribotype were further studied using a second restriction enzyme, EcoRV, one EcoRI/EcoRV ribotype contained five isolates recovered from URTD tortoises and none from healthy animals. The EcoRI ribotype comprised of four isolates, all from tortoises with URTD, was further separated into three distinct groups with EcoRV. All 12 isolates studied grew equally well in normal tortoise plasma, and when broth-grown WCP and OMP profiles were evaluated, no proteins were unique to isolates from URTD tortoises. Iron-regulated OMP's were produced in three isolates examined, but these OMP's apparently were not virulence-related.

Animals↗
PubMed2002

Characterization of Borrelia burgdorferi sensu lato strains isolated from human material in Slovenia.

The aim of the study was to assess genotypic and phenotypic diversity among a large number of clinical isolates of Borrelia burgdorferi sensu lato obtained from patients in Slovenia. Plasmid profiles and species identification were determined by PFGE, protein profiles by SDS-PAGE. Of 706 B. burgdorferi sensu lato human isolates 599 (85%) were found to be B. afzelii, 101 (14%) B. garinii and six (1%) B. burgdorferi sensu stricto. The vast majority of strains (605; 86%) were isolated from skin, 58 (8%) from blood, and 43 (6%) from CSF. When analysed by RFLP, B. afzelii strains were unique, while heterogeneity was found within B. garinii and B. burgdorferi sensu stricto species. An unusual plasmid content was found in 52/706 (7%) isolated strains, more often in B. garinii than in B. afzelii strains. A plasmid dimer was found in B. afzelii and B. garinii strains, whereas multiple copies of the large plasmid were associated nearly exclusively with B. garinii strains. Analysis of protein profiles revealed that OspA and OspB are expressed more often by B. afzelii strains, and OspC by B. garinii strains. Heterogeneity of Borrelia strains may play a significant role in the virulence and pathogenesis of the infection. Differences in antigenic components have an important impact on serological testing and vaccine development.

Acrodermatitis↗
PubMed2006

Extensive and genome-wide changes in the transcription profile of Staphylococcus aureus induced by modulating the transcription of the cell wall synthesis gene murF.

A murF conditional mutant was used to evaluate the effect of suboptimal transcription of this gene on the transcriptome of the methicillin-resistant Staphylococcus aureus strain COL. The mutant was grown in the presence of optimal and suboptimal concentrations of the inducer, and the relative levels of transcription of genes were evaluated genome wide with an Affymetrix DNA microarray that included all open reading frames (ORFs) as well as intergenic sequences derived from four sequenced S. aureus strains. Using a sensitivity threshold value of 1.5, suboptimal expression of murF altered the transcription of a surprisingly large number of genes, i.e., 668 out of the 2,740 ORFs (close to one-fourth of all ORFs), of the genome of S. aureus strain COL. The genes with altered transcription were distributed evenly around the S. aureus chromosome, and groups of genes involved with distinct metabolic functions responded in unique and operon-specific manners to modulation in murF transcription. For instance, all genes belonging to the isd operon and all but 2 of the 35 genes of prophage L54a were down-regulated, whereas all but one of the 21 members of the vraSR regulon and most of the 79 virulence-related genes (those for fibronectin binding proteins A and B, clumping factor A, gamma hemolysin, enterotoxin B, etc.) were up-regulated in cells with suboptimal expression of murF. Most importantly, the majority of these altered gene expression profiles were reversible by resupplying the optimal concentration of IPTG (isopropyl-beta-D-thiogalactopyranoside) to the culture. The observations suggest the coordinate regulation of a large sector of the S. aureus transcriptome in response to a disturbance in cell wall synthesis.

Bacterial Proteins↗
PubMed2005

Salmonella pathogenicity island 2 is expressed prior to penetrating the intestine.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that causes disease in mice that resembles human typhoid. Typhoid pathogenesis consists of distinct phases in the intestine and a subsequent systemic phase in which bacteria replicate in macrophages of the liver and spleen. The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI-2) is a major virulence factor contributing to the systemic phase of typhoid pathogenesis. Understanding how pathogens regulate virulence mechanisms in response to the environment, including different host tissues, is key to our understanding of pathogenesis. A recombinase-based in vivo expression technology system was developed to assess SPI-2 expression during murine typhoid. SPI-2 expression was detectable at very early times in bacteria that were resident in the lumen of the ileum and was independent of active bacterial invasion of the epithelium. We also provide direct evidence for the regulation of SPI-2 by the Salmonella transcription factors ompR and ssrB in vivo. Together these results demonstrate that SPI-2 expression precedes penetration of the intestinal epithelium. This induction of expression precedes any documented SPI-2-dependent phases of typhoid and may be involved in preparing Salmonella to successfully resist the antimicrobial environment encountered within macrophages.

Animals↗
PubMed2003

MgrA, an orthologue of Mga, Acts as a transcriptional repressor of the genes within the rlrA pathogenicity islet in Streptococcus pneumoniae.

Streptococcus pneumoniae normally resides in the human nasopharynx in a nondisease state. In response to unknown triggers this organism can descend to the lower respiratory tract and/or invade the bloodstream. Regulation and activation of virulence genes play essential roles in this process of disease development. Characterization of S. pneumoniae regulatory networks has been a recent area of interest, but despite inroads little is known about regulation of virulence genes in this pathogen. A putative transcriptional regulator in S. pneumoniae, mgrA, which exhibits homology to the virulence gene activator mga of group A streptococcus, was previously identified as a regulator that is required for development of pneumonia in a murine model. In this study we confirmed that mgrA plays a role in both nasopharyngeal carriage and pneumonia. Transcriptional profiling by microarray technology was used to show that mgrA acts as a repressor of the previously characterized rlrA pathogenicity islet. This is manifested phenotypically by a decrease in adherence to epithelial cells in tissue culture since the rlrA pathogenicity islet contains genes mediating adherence.

Animals↗
PubMedDate not supplied

Restriction fragment length polymorphism in four virulence-associated genes of Listeria monocytogenes.

We observed restriction fragment length polymorphism in 4 genes of Listeria monocytogenes associated with virulence. Using the polymerase chain reaction (PCR) and primers derived from published sequences, we amplified the following genes: hlyA coding for listeriolysin O, iap coding for a putative invasion-associated factor, mpl coding for a metalloprotease, and the prfA gene that positively regulates the hylA gene. PCR-amplified DNA were cut with several restriction endonucleases, and the restriction profiles from 29 strains, representing 6 serovars (serovars 1/2a, 1/2b, 1/2c, 3a, 3b and 4b) were compared. Based on these restriction profiles, the strains were categorized into 2 subgroups: one group contained all 10 strains of serovars 1/2a, 1/2c and 3a, the other group contained all 19 strains of serovars 1/2b, 3b and 4b. This division is in complete agreement with multilocus enzyme electrophoretic analysis data which divide the species into the same 2 subgroups. Whether the differences observed in the nucleotide sequences of the 4 virulence-associated genes for the 2 subgroups of L. monocytogenes represent salient variations in pathogenic mechanisms is not known.

DNA, Bacterial↗
PubMed1998

Classification of Listeria monocytogenes by PCR-restriction enzyme analysis in the two genes of hlyA and iap.

PCR-restriction enzyme analysis in the two virulence-associated genes was performed. The hlyA gene cording for listeriolysin O and the iap gene cording for an invasion associated factor were amplified with primers SH2 or SI3. The PCR products obtained were cleaved with 32 restriction enzymes, and restriction profiles from 12 strains, 6 each of serotypes 1/2a and 4b, were compared. We obtained two profiles for the hlyA using 4 restriction enzymes and eight profiles for the iap by using AluI, and the results of the profiles did not correlate with the serotypes. The polymorphism in the iap region was of a higher degree than that in the hlyA region, and the PCR-restriction enzyme analysis of the iap gene with primers SI3 and AluI was confirmed as one of the useful epidemiological analysis methods for listerosis outbreaks.

Bacterial Proteins↗
PubMed2006

Deletion of a Mycobacterium tuberculosis proteasomal ATPase homologue gene produces a slow-growing strain that persists in host tissues.

The in vivo rate of proliferation of Mycobacterium tuberculosis, the causative agent of tuberculosis, has been linked to the rate of progression and severity of disease. Here, we report that deletion of the gene MT2175 (Rv2115c), a putative mycobacterial proteasome-associated AAA-ATPase, leads to a reduction in the growth rate of M. tuberculosis in vitro and in vivo. Despite the reduced growth, the mutant persisted, with slow and gradual clearance in mouse lungs. The mutant elicited reduced levels of interferon-gamma production in the lungs and, when used as an immunizing agent, provided significant protection against challenge with a virulent strain of M. tuberculosis. Expression of the genes lat and MT3159 were highly up-regulated in the mutant. Thus, loss of MT2175 slows both in vitro and in vivo growth rates and compromises the lethality of M. tuberculosis in mice but has a minimal impact on the organism's ability to persist in host tissues.

Adenosine Triphosphatases↗
PubMed2001

Comparative assessment of virulence of recombinant vaccinia viruses expressing IL-2 and IL-15 in immunodeficient mice.

IL-2 and -15 belong to the four alpha-helix bundle family of cytokines and display a spectrum of overlapping immune functions because of shared signal transducing receptor components of the IL-2 receptor complex. However, recent evidence suggests a nonredundant unique role for IL-15 in the establishment and perhaps maintenance of peripheral natural killer (NK) cell populations in vivo. To explore the contribution of locally released IL-15 on peripheral NK-cell-mediated innate immune responses, we generated a recombinant vaccinia virus that expresses IL-15 and evaluated the course of vaccinial disease in athymic nude mice. Coexpression of IL-15 resulted in the attenuation of virulence of vaccinia virus, and mice inoculated with 10(5) plaque-forming units or less resolved the infection successfully. In contrast, mice inoculated with a similar dose of the control vaccinia virus failed to eliminate the virus and died of generalized vaccinial disease. Enhanced expression of IL-12 and IFN-gamma as well as induction of chemokines were evident in the mice inoculated with IL-15-expressing vaccinia virus in addition to an increase in NK cells in the spleen. However, in this model system, the degree of attenuation in viral virulence attained with coexpression of IL-15 was much less than that achieved with coexpression of IL-2, suggesting that the peripheral NK-cell-mediated events are more responsive to IL-2 than to IL-15.

Animals↗
PubMed2003

Transcriptome analysis of the Pseudomonas aeruginosa response to iron.

To successfully infect humans, Pseudomonas aeruginosa (Pa) must overcome the low iron availability in host tissues. A transcriptome comparison was carried out between iron-starved cells of Pa treated with iron and untreated controls. The present study is the first global analysis of the early transcriptional response of exponentially growing Pa to iron. Approximately 1.3% of the Pa genes displayed > or = 5.0-fold changes in mRNA levels in iron-treated cells. Treatment affected the mRNA levels of many genes required for iron acquisition as well as several genes with relevance to virulence previously known to be regulated by iron. More importantly, the analysis permitted identification of 107 Pa genes whose mRNA levels were not previously known to be affected by iron. These genes are good candidates for mutagenesis studies aimed at identifying novel functions relevant to iron metabolism in Pa. Some of these genes encode predicted siderophore receptors, iron transport systems, TonB-dependent receptors, regulatory proteins, and proteins relevant to virulence. Notably, 49 genes encode hypothetical or conserved hypothetical proteins of unknown function, suggesting that they are involved directly or indirectly in iron metabolism or metabolic adaptation to different iron-availability conditions.

Adaptation, Biological↗