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Results for “host:pathogen interactions”

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PubMed2002

NF-kappaB family of transcription factors: central regulators of innate and adaptive immune functions.

Transcription factors of the Rel/NF-kappaB family are activated in response to signals that lead to cell growth, differentiation, and apoptosis, and these proteins are critical elements involved in the regulation of immune responses. The conservation of this family of transcription factors in many phyla and their association with antimicrobial responses indicate their central role in the regulation of innate immunity. This is illustrated by the association of homologues of NF-kappaB, and their regulatory proteins, with resistance to infection in insects and plants (M. S. Dushay, B. Asling, and D. Hultmark, Proc. Natl. Acad. Sci. USA 93:10343-10347, 1996; D. Hultmark, Trends Genet. 9:178-183, 1993; J. Ryals et al., Plant Cell 9:425-439, 1997). The aim of this review is to provide a background on the biology of NF-kappaB and to highlight areas of the innate and adaptive immune response in which these transcription factors have a key regulatory function and to review what is currently known about their roles in resistance to infection, the host-pathogen interaction, and development of human disease.

Animals↗

PubMed2004

Proteomic analysis of Candida albicans cell walls reveals covalently bound carbohydrate-active enzymes and adhesins.

Covalently linked cell wall proteins (CWPs) of the dimorphic fungus Candida albicans are implicated in virulence. We have carried out a comprehensive proteomic analysis of the covalently linked CWPs in exponential-phase yeast cells. Proteins were liberated from sodium dodecyl sulfate (SDS)-extracted cell walls and analyzed using immunological and advanced protein sequencing (liquid chromatography-tandem mass spectrometry [LC/MS/MS]) methods. HF-pyridine and NaOH were used to chemically release glycosylphosphatidylinositol-dependent proteins (GPI proteins) and mild alkali-sensitive proteins, respectively. In addition, to release both classes of CWPs simultaneously, cell walls were digested enzymatically with a recombinant beta-1,3-glucanase. Using LC/MS/MS, we identified 14 proteins, of which only 1 protein, Cht2p, has been previously identified in cell wall extracts by using protein sequencing methods. The 14 identified CWPs include 12 GPI proteins and 2 mild alkali-sensitive proteins. Nonsecretory proteins were absent in our cell wall preparations. The proteins identified included several functional categories: (i) five CWPs are predicted carbohydrate-active enzymes (Cht2p, Crh11p, Pga4p, Phr1p, and Scw1p); (ii) Als1p and Als4p are believed to be adhesion proteins. In addition, Pga24p shows similarity to the flocculins of baker's yeast. (iii) Sod4p/Pga2p is a putative superoxide dismutase and is possibly involved in counteracting host defense reactions. The precise roles of the other CWPs (Ecm33.3p, Pir1p, Pga29p, Rbt5p, and Ssr1p) are unknown. These results indicate that a substantial number of the covalently linked CWPs of C. albicans are actively involved in cell wall remodeling and expansion and in host-pathogen interactions.

Amino Acid Sequence↗

PubMed2006

Apoptosis-inducing factor participation in bovine macrophage Mycobacterium bovis-induced caspase-independent cell death.

Mycobacterium tuberculosis complex species survive and replicate in phagosomes of the host cell. Cell death (CD) has been highlighted as one of the probable outcomes in this host-pathogen interaction. Previously, our group demonstrated macrophage apoptosis as a consequence of Mycobacterium bovis infection. In this study, we aimed to identify the contribution of apoptotic effector elements in M. bovis-induced CD. Bovine macrophages were either infected with M. bovis (multiplicity of infection, 10:1) or treated with an M. bovis cell extract (CFE). Structural changes compatible with CD were evaluated. Chromatin condensation was increased three times by the CFE. On the other hand, a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay demonstrated that levels of DNA fragmentation induced by M. bovis and CFE were 53.7% +/- 24% and 38.9% +/- 14%, respectively, whereas control cells had a basal proportion of 8.9% +/- 4.1%. Rates of DNA fragmentation were unaffected by the presence of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (z-VAD). Cells treated with 100 mug of CFE for 12 h had a fivefold decrease in the level of mitochondrial outer membrane permeabilization compared to that of untreated cells. Neither M. bovis infection nor CFE treatment induced activation of caspase 3, 8, or 9. Translocation of apoptosis-inducing factor (AIF) to the nucleus was identified in 32% +/- 3.5% and 26.3% +/- 4.9% of M. bovis-infected and CFE-treated cells, respectively. Incubation of macrophages with z-VAD prior to infection did not alter the percentage of cells showing AIF translocation. Our data suggest that M. bovis-induced CD in bovine macrophages is caspase independent with AIF participation.

Animals↗

PubMed1990

The Borrelia burgdorferi flagellum-associated 41-kilodalton antigen (flagellin): molecular cloning, expression, and amplification of the gene.

Monoclonal antibodies directed against the major Borrelia burgdorferi flagellar protein, the 41-kilodalton (kDa) protein flagellin, were used to monitor cloning and expression of the flagellin gene from a Borrelia burgdorferi genomic library. The structure of the gene was analyzed, and recombinant nonfusion flagellin was produced in Escherichia coli. A DNA sequence analysis of the 41-kDa flagellin gene revealed the presence of an open reading frame that encoded a protein having 336 amino acid residues and a calculated molecular mass of 35.8 kDa, indicating that there was posttranslational modification of the natural 41-kDa flagellin protein. Upstream from the AUG start codon sequence we identified motifs corresponding to consensus procaryotic promoter elements which could be utilized by the cloned flagellin gene when it was expressed in E. coli MC1061. The deduced flagellin protein sequence exhibited high levels of homology to sequences of flagellin proteins from Bacillus subtilis and Salmonella typhimurium. The levels of sequence similarity for the amino- and carboxy-terminal portions were about 65 and 56%, respectively. DNA sequence information on the flagellin gene was used to design oligonucleotides for gene amplification by the polymerase chain reaction method, and by using this method 0.01 pg of Borrelia burgdorferi DNA could be detected. Our results provide a basis for further biochemical analysis of the 41-kDa flagellin protein, investigation of the role of this protein in host-pathogen interactions, and development of a standardized reagent for diagnostic systems for Borrelia burgdorferi infections.

Amino Acid Sequence↗

PubMed1996

Construction and characterization of a Yersinia enterocolitica O:8 high-temperature requirement (htrA) isogenic mutant.

The high-temperature requirement (HtrA) family of stress response proteins are induced by different environmental stress conditions in a variety of bacteria and have been shown to contribute to the pathogenicity of some of these species. In this study, the htrA gene from Yersinia enterocolitica O:8 was amplified, cloned, and sequenced. Analysis of the deduced amino acid sequence predicted that the putative HtrA homolog contains a serine protease active site and a catalytic triad characteristic of trypsin-like serine proteases, structural features characteristic of previously described HtrA proteins. In order to evaluate the biological functions of Y. enterocolitica HtrA, an isogenic mutant was constructed by a reverse-genetics PCR-based approach. Characterization of the mutant provided evidence supporting a stress response function for the Y. enterocolitica htrA gene product. In contrast to the parent strain, the mutant showed increased sensitivity to killing by H2O2, O2- and temperature stress (50 degrees C). The mutant was avirulent in the murine yersiniosis injection model and offered partial protection to mice challenged with the parent strain. Further studies with the Y. enterocolitica htrA mutant should increase our knowledge of the host-pathogen interactions which occur during Yersinia infections.

Amino Acid Sequence↗

PubMed1997

Lipopeptides of Borrelia burgdorferi outer surface proteins induce Th1 phenotype development in alphabeta T-cell receptor transgenic mice.

Induction of the appropriate T helper cell (Th) subset is crucial for the resolution of infectious diseases and the prevention of immunopathology. Some pathogens preferentially induce Th1 or Th2 responses. How microorganisms influence Th phenotype development is unknown. We asked if Borrelia burgdorferi, the spirochete which causes Lyme arthritis, can promote a cytokine milieu in which T cells which are not specific for B. burgdorferi are induced to produce proinflammatory cytokines. Using alphabeta T-cell receptor transgenic mice as a source of T cells with a defined specificity other than for B. burgdorferi, we found that B. burgdorferi induced Th1 phenotype development in ovalbumin-specific transgenic T cells. Small synthetic lipopeptides corresponding to the N-terminal sequences of B. burgdorferi outer surface lipoproteins had similar effects. B. burgdorferi and its lipopeptides induced host cells to produce interleukin-12. When the peptides were used in delipidated form, they did not induce Th1 development. These findings may be of pathogenic importance, since it is currently assumed that a Th2-mediated antibody response is protective against B. burgdorferi. Bacteria associated with reactive arthritis, namely, Yersinia enterocolitica, Shigella flexneri, and Salmonella enteritidis, had different effects. The molecular definition of pathogen-host interactions determining cytokine production should facilitate rational therapeutic interventions directing the host response towards the desired cytokine response. Here, we describe small synthetic molecules capable of inducing Th1 phenotype development.

Animals↗

PubMed1998

Expression and characterization of group A Streptococcus extracellular cysteine protease recombinant mutant proteins and documentation of seroconversion during human invasive disease episodes.

A recent study with isogenic strains constructed by recombinant DNA strategies unambiguously documented that a highly conserved extracellular cysteine protease expressed by Streptococcus pyogenes (group A Streptococcus [GAS]) is a critical virulence factor in a mouse model of invasive disease (S. Lukomski, S. Sreevatsan, C. Amberg, W. Reichardt, M. Woischnik, A. Podbielski, and J. M. Musser, J. Clin. Invest. 99:2574-2580, 1997). To facilitate further investigations of the streptococcal cysteine protease, recombinant proteins composed of a 40-kDa zymogen containing a C192S amino acid substitution that ablates enzymatic activity, a 28-kDa mature protein with the C192S replacement, and a 12-kDa propeptide were purified from Escherichia coli containing His tag expression vectors. The recombinant C192S zymogen retained apparently normal structural integrity, as assessed by the ability of purified wild-type streptococcal cysteine protease to process the 40-kDa molecule to the 28-kDa mature form. All three recombinant purified proteins retained immunologic reactivity with polyclonal and monoclonal antibodies. Humans with a diverse range of invasive disease episodes (erysipelas, cellulitis, pneumonia, bacteremia, septic arthritis, streptococcal toxic shock syndrome, and necrotizing fasciitis) caused by six distinct M types of GAS seroconverted to the streptococcal cysteine protease. These results demonstrate that this GAS protein is expressed in vivo during the course of human infections and thereby provide additional evidence that the cysteine protease participates in host-pathogen interactions in some patients.

Animals↗

PubMed1998

Characterization of major surface glycoprotein genes of human Pneumocystis carinii and high-level expression of a conserved region.

To facilitate studies of Pneumocystis carinii infection in humans, we undertook to better characterize and to express the major surface glycoprotein (MSG) of human P. carinii, an important protein in host-pathogen interactions. Seven MSG genes were cloned from a single isolate by PCR or genomic library screening and were sequenced. The predicted proteins, like rat MSGs, were closely related but unique variants, with a high level of conservation among cysteine residues. A conserved immunodominant region (of approximately 100 amino acids) near the carboxy terminus was expressed at high levels in Escherichia coli and used in Western blot studies. All 49 of the serum samples, which were taken from healthy controls as well as from patients with and without P. carinii pneumonia, were reactive with this peptide by Western blotting, supporting the hypothesis that most adult humans have been infected with P. carinii at some point. This recombinant MSG fragment, which is the first human P. carinii antigen available in large quantities, may be a useful reagent for investigating the epidemiology of P. carinii infection in humans.

AIDS-Related Opportunistic Infections↗

PubMed1999

Non-serum-dependent chemotactic factors produced by Candida albicans stimulate chemotaxis by binding to the formyl peptide receptor on neutrophils and to an unknown receptor on macrophages.

Serum-free culture filtrates of six Candida species and Saccharomyces cerevisiae were found to contain chemoattractants for human polymorphonuclear leukocytes (PMNs) and a mouse macrophage-like cell line, J774. The chemotactic factors differed for the PMN and J774 cells, however, in terms of heat stability, kinetics of liberation by the yeast cells, and divalent cation requirements for production. The chemoattractant in Candida albicans culture filtrates appeared to act through the formyl peptide receptor (FPR) of PMNs, since it was found to induce chemotaxis of Chinese hamster ovary (CHO) cells that were expressing the human FPR but did not induce chemotaxis of wild-type CHO cells. The C. albicans culture filtrates also induced migration of PMNs across confluent monolayers of a human gastrointestinal epithelial cell line, T84; migration occurred in the basolateral-to-apical direction but not the reverse direction, unless the epithelial tight junctions were disrupted. J774 cells did not migrate toward the formylated peptide (fMet-Leu-Phe; fMLF), and chemotaxis toward the C. albicans culture filtrate was not inhibited by an FPR antagonist (t-butoxycarbonyl-Met-Leu-Phe), suggesting that a different receptor mediated J774 cell chemotaxis. In conclusion, we have identified a receptor by which a non-serum-dependent chemotactic factor (NSCF) produced by C. albicans induced chemotaxis of PMNs. Additionally, we have shown that NSCF was active across epithelial monolayers. These findings suggest that NSCFs produced by C. albicans and other yeast species may influence host-pathogen interactions at the gastrointestinal tract mucosal surface by inducing phagocytic-cell infiltration.

Animals↗

PubMed1999

Detection of Als proteins on the cell wall of Candida albicans in murine tissues.

A murine model of disseminated candidiasis was utilized to determine whether Candida albicans Als proteins are produced in vivo. The kidneys, spleen, heart, liver, and lungs were collected from mice inoculated with one of three C. albicans strains (SC5314, B311, or WO-1). Immunohistochemical analysis of murine tissues by using a rabbit polyclonal anti-Als serum indicated that Als proteins were produced by each C. albicans cell in the tissues examined. Patterns of staining with the anti-Als serum were similar among the C. albicans strains tested. These data indicated that Als protein production was widespread in disseminated candidiasis and that, despite strain differences in ALS gene expression previously noted in vitro, Als protein production in vivo was similar among C. albicans strains. The extensive production of Als proteins in vivo and their presence on the C. albicans cell wall position these proteins well for a role in host-pathogen interaction.

Animals↗

PubMed1999

Fibrinogen cleavage by the Streptococcus pyogenes extracellular cysteine protease and generation of antibodies that inhibit enzyme proteolytic activity.

The extracellular cysteine protease from Streptococcus pyogenes is a virulence factor that plays a significant role in host-pathogen interaction. Streptococcal protease is expressed as an inactive 40-kDa precursor that is autocatalytically converted into a 28-kDa mature (active) enzyme. Replacement of the single cysteine residue involved in formation of the enzyme active site with serine (C192S mutation) abolished detectable proteolytic activity and eliminated autocatalytic processing of zymogen to the mature form. In the present study, we investigated activity of the wild-type (wt) streptococcal protease toward human fibrinogen and bovine casein. The former is involved in blood coagulation, wound healing, and other aspects of hemostasis. Treatment with streptococcal protease resulted in degradation of the COOH-terminal region of fibrinogen alpha chain, indicating that fibrinogen may serve as an important substrate for this enzyme during the course of human infection. Polyclonal antibodies generated against recombinant 40- and 28-kDa (r40- and r28-kDa) forms of the C192S streptococcal protease mutant exhibited high enzyme-linked immunosorbent assay titers but demonstrated different inhibition activities toward proteolytic action of the wt enzyme. Activity of the wt protease was readily inhibited when the reaction was carried out in the presence of antibodies generated against r28-kDa C192S mutant. Antibodies produced against r40-kDa C192S mutant had no significant effect on proteolysis. These data suggest that the presence of the NH(2)-terminal prosegment prevents generation of functionally active antibodies and indicate that inhibition activity of antibodies most likely depends on their ability to bind the active-site region epitope(s) of the protein.

Animals↗

PubMed1999

Phage-displayed T-cell epitope grafted into immunoglobulin heavy-chain complementarity-determining regions: an effective vaccine design tested in murine cysticercosis.

A new type of immunogenic molecule was engineered by replacing all three complementarity-determining-region (CDR) loops of the human immunoglobulin (Ig) heavy-chain variable (V(H)) domain with the Taenia crassiceps epitope PT1 (PPPVDYLYQT) and by displaying this construct on the surfaces of M13 bacteriophage. When BALB/c mice were immunized with such phage particles (PIgphage), a strong protection against challenge infection in very susceptible female hosts was obtained. When specifically stimulated, the in vivo-primed CD4(+) and CD8(+) T cells isolated from mice immunized with PT1, both as a free peptide and as the PIgphage construct, proliferated in vitro, indicating efficient epitope presentation by both major histocompatibility complex class II and class I molecules in the specifically antigen-pulsed macrophages used as antigen-presenting cells. These data demonstrate the immunogenic potential of recombinant phage particles displaying CDR epitope-grafted Ig V(H) domains and establish an alternative approach to the design of an effective subunit vaccine for prevention of cysticercosis. The key advantage of this type of immunogen is that no adjuvant is required for its application. The proposed strategy for immunogen construction is potentially suitable for use in any host-pathogen interaction.

Animals↗

PubMed2000

Inverse relation between disease severity and expression of the streptococcal cysteine protease, SpeB, among clonal M1T1 isolates recovered from invasive group A streptococcal infection cases.

The streptococcal cysteine protease (SpeB) is one of the major virulence factors produced by group A streptococci (GAS). In this study we investigated if differences exist in SpeB production by clonally related M1T1 clinical isolates derived from patients with invasive infections. Twenty-nine of these isolates were from nonsevere cases and 48 were from severe cases, including streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF) cases. The expression and amount of the 28-kDa SpeB protein produced were determined by quantitative Western blotting, and protease activity was measured by a fluorescent enzymatic assay. A high degree of variation in SpeB expression was seen among the isolates, and this variation seemed to correlate with the severity and/or clinical manifestation of the invasive infection. The mean amount of 28-kDa SpeB protein and cysteine protease activity produced by isolates from nonsevere cases was significantly higher than that from STSS cases (P = 0.001). This difference was partly due to the fact that 41% of STSS isolates produced little or no SpeB compared to only 14% of isolates recovered in nonsevere cases. Moreover, the cysteine protease activity among those isolates that expressed SpeB was significantly lower for STSS isolates than for isolates from nonsevere cases (P = 0.001). Increased SpeB production was also inversely correlated with intact M protein expression, and inhibition of cysteine protease activity blocked the cleavage of the surface M protein. Together, the data support the existence of both an "on-off" and a posttranslational regulatory mechanism(s) controlling SpeB production, and they suggest that isolates with the speB gene in the "off" state are more likely to spare the surface M protein and to be isolated from cases of severe rather than nonsevere invasive infection. These findings may have important implications for the role of SpeB in host-pathogen interactions via regulation of the expression of GAS virulence genes and the severity of invasive disease.

Antigens, Bacterial↗

PubMed2000

Nonpolar inactivation of the hypervariable streptococcal inhibitor of complement gene (sic) in serotype M1 Streptococcus pyogenes significantly decreases mouse mucosal colonization.

Group A Streptococcus (GAS) is a human pathogen that commonly infects the upper respiratory tract. GAS serotype M1 strains are frequently isolated from human infections and contain the gene encoding the hypervariable streptococcal inhibitor of complement protein (Sic). It was recently shown that Sic variants were rapidly selected on mucosal surfaces in epidemic waves caused by M1 strains, an observation suggesting that Sic participates in host-pathogen interactions on the mucosal surface (N. P. Hoe, K. Nakashima, S. Lukomski, D. Grigsby, M. Liu, P. Kordari, S.-J. Dou, X. Pan, J. Vuopio-Varkila, S. Salmelinna, A. McGeer, D. E. Low, B. Schwartz, A. Schuchat, S. Naidich, D. De Lorenzo, Y.-X. Fu, and J. M. Musser, Nat. Med. 5:924-929, 1999). To test this idea, a new nonpolar mutagenesis method employing a spectinomycin resistance cassette was used to inactivate the sic gene in an M1 GAS strain. The isogenic Sic-negative mutant strain was significantly (P < 0.019) impaired in ability to colonize the mouse mucosal surface after intranasal infection. These results support the hypothesis that the predominance of M1 strains in human infections is related, in part, to a Sic-mediated enhanced colonization ability.

Animals↗

PubMed2000

Streptococcal erythrogenic toxin B abrogates fibronectin-dependent internalization of Streptococcus pyogenes by cultured mammalian cells.

Streptococcus pyogenes secretes several proteins that influence host-pathogen interactions. A tissue-culture model was used to study the influence of the secreted cysteine protease streptococcal erythrogenic toxin B (SPE B) on the interaction between S. pyogenes strain NZ131 (serotype M49) and mammalian cells. Inactivation of the speB gene enhanced fibronectin-dependent uptake of the pathogen by Chinese hamster ovary (CHO-K1) cells compared to that in the isogenic wild-type strain. Preincubation of the NZ131 speB mutant with purified SPE B protease significantly inhibited fibronectin-dependent uptake by both CHO-K1 and CHO-pgs745 cells. The effect was attributed to an abrogation of fibronectin binding to the surface of the bacteria that did not involve either the M49 protein or the streptococcal fibronectin-binding protein SfbI. In contrast, pretreatment of the NZ131 speB mutant with SPE B did not influence sulfated polysaccharide-mediated uptake by CHO-pgs745 cells. The results indicate that the SPE B protease specifically alters bacterial cell surface proteins and thereby influences pathogen uptake.

Adhesins, Bacterial↗

PubMed2001

Temperature-regulated protein synthesis by Leptospira interrogans.

Leptospira interrogans is an important mammalian pathogen. Transmission from an environmental source requires adaptations to a range of new environmental conditions in the organs and tissues of the infected host. Since many pathogenic bacteria utilize temperature to discern their environment and regulate the synthesis of appropriate proteins, we investigated the effects of temperature on protein synthesis in L. interrogans. Bacteria were grown for several days after culture temperatures were shifted from 30 to 37 degrees C. Triton X-114 cellular fractionation identified several proteins of the cytoplasm, periplasm, and outer membrane for which synthesis was dependent on the culture temperature. Synthesis of a cytoplasmic protein of 20 kDa was switched off at 37 degrees C, whereas synthesis of a 66-kDa periplasmic protein was increased at the higher temperature. Increased synthesis of a 25-kDa outer membrane protein was observed when the organisms were shifted from 30 to 37 degrees C. A 36-kDa protein synthesized at 30 but not at 37 degrees C was identified as LipL36, an outer membrane lipoprotein. In contrast, expression of another lipoprotein, LipL41, was the same at either temperature. Immunoblotting with convalescent equine sera revealed that some proteins exhibiting thermoregulation of synthesis elicited antibody responses during infection. Our results show that sera from horses which aborted as a result of naturally acquired infection with L. interrogans serovar pomona type kennewicki recognize periplasmic and outer membrane proteins which are differentially synthesized in response to temperature and which therefore may be important in the host-pathogen interaction during infection.

Antigens, Bacterial↗

PubMed2001

Three-dimensional tissue assemblies: novel models for the study of Salmonella enterica serovar Typhimurium pathogenesis.

The lack of readily available experimental systems has limited knowledge pertaining to the development of Salmonella-induced gastroenteritis and diarrheal disease in humans. We used a novel low-shear stress cell culture system developed at the National Aeronautics and Space Administration in conjunction with cultivation of three-dimensional (3-D) aggregates of human intestinal tissue to study the infectivity of Salmonella enterica serovar Typhimurium for human intestinal epithelium. Immunohistochemical characterization and microscopic analysis of 3-D aggregates of the human intestinal epithelial cell line Int-407 revealed that the 3-D cells more accurately modeled human in vivo differentiated tissues than did conventional monolayer cultures of the same cells. Results from infectivity studies showed that Salmonella established infection of the 3-D cells in a much different manner than that observed for monolayers. Following the same time course of infection with Salmonella, 3-D Int-407 cells displayed minimal loss of structural integrity compared to that of Int-407 monolayers. Furthermore, Salmonella exhibited significantly lower abilities to adhere to, invade, and induce apoptosis of 3-D Int-407 cells than it did for infected Int-407 monolayers. Analysis of cytokine expression profiles of 3-D Int-407 cells and monolayers following infection with Salmonella revealed significant differences in expression of interleukin 1alpha (IL-1alpha), IL-1beta, IL-6, IL-1Ra, and tumor necrosis factor alpha mRNAs between the two cultures. In addition, uninfected 3-D Int-407 cells constitutively expressed higher levels of transforming growth factor beta1 mRNA and prostaglandin E2 than did uninfected Int-407 monolayers. By more accurately modeling many aspects of human in vivo tissues, the 3-D intestinal cell model generated in this study offers a novel approach for studying microbial infectivity from the perspective of the host-pathogen interaction.

Apoptosis↗

PubMed2001

Identification of Rgg-regulated exoproteins of Streptococcus pyogenes.

Streptococcus pyogenes secretes many proteins that influence host-pathogen interactions. Despite their importance, relatively little is known about the regulation of these proteins. The rgg gene (also known as ropB) is required for the expression of streptococcal erythrogenic toxin B (SPE B), an extracellular cysteine protease that contributes to virulence. Proteomics was used to determine if rgg regulates the expression of additional exoproteins. Exponential- and stationary-phase culture supernatant proteins made by S. pyogenes NZ131 rgg and NZ131 speB were separated by two-dimensional electrophoresis. Differences were identified in supernatant proteins from both exponential- and stationary-phase cultures, although considerably more differences were detected among stationary-phase supernatant proteins. Forty-two proteins were identified by peptide fingerprinting with matrix-assisted laser desorption mass spectrometry. Mitogenic factor, DNA entry nuclease (open reading frame [ORF 226]), and ORF 953, which has no known function, were more abundant in the culture supernatants of the rgg mutant compared to the speB mutant. ClpB, lysozyme, and autolysin were detected in the culture supernatant of the speB mutant but not the rgg mutant. To determine if Rgg affected protein expression at the transcriptional level, real-time (TaqMan) reverse transcription (RT)-PCR was used to quantitate Rgg-regulated transcripts from NZ131 wild-type and speB and rgg mutant strains. The results obtained with RT-PCR correlated with the proteomic data. We conclude that Rgg regulates the transcription of several genes expressed primarily during the stationary phase of growth.

Bacterial Outer Membrane Proteins↗