Genes for the majority of group A streptococcal virulence factors and extracellular surface proteins do not confer an increased propensity to cause invasive disease

Background. The factors behind the reemergence of severe, invasive group A streptococcal (GAS) diseases are unclear, but it could be caused by altered genetic endowment in these organisms. However, data from previous studies assessing the association between single genetic factors and invasive disease are often conflicting, suggesting that other, as-yet unidentified factors are necessary for the development of this class of disease. Methods. In this study, we used a targeted GAS virulence microarray containing 226 GAS genes to determine the virulence gene repertoires of 68 GAS isolates (42 associated with invasive disease and 28 associated with noninvasive disease) collected in a defined geographic location during a contiguous time period. We then employed 3 advanced machine learning methods (genetic algorithm neural network, support vector machines, and classification trees) to identify genes with an increased association with invasive disease. Results. Virulence gene profiles of individual GAS isolates varied extensively among these geographically and temporally related strains. Using genetic algorithm neural network analysis, we identified 3 genes with a marginal overrepresentation in invasive disease isolates. Significantly, 2 of these genes, ssa and mf4, encoded superantigens but were only present in a restricted set of GAS M-types. The third gene, spa, was found in variable distributions in all M-types in the study. Conclusions. Our comprehensive analysis of GAS virulence profiles provides strong evidence for the incongruent relationships among any of the 226 genes represented on the array and the overall propensity of GAS to cause invasive disease, underscoring the pathogenic complexity of these diseases, as well as the importance of multiple bacteria and/ or host factors.

Identification and characterization of pptA: a gene involved in the phase-variable expression of phosphorylcholine on pili of Neisseria meningitidis

Pili of pathogenic Neisseria are major virulence factors associated with adhesion, cytotoxicity, twitching motility, autoaggregation, and DNA transformation. Pili are modified posttranslationally by the addition of phosphorylcholine. However, no genes involved in either the biosynthesis or the transfer of phosphorylcholine in Neisseria meningitidis have been identified. In this study, we identified five candidate open reading frames (ORFs) potentially involved in the biosynthesis or transfer of phosphorylcholine to pilin in N. meningitidis. Insertional mutants were constructed for each ORF in N. meningitidis strain C311#3 to determine their effect on phosphorylcholine expression. The effect of the mutant ORFs on the modification by phosphorylcholine was analyzed by Western analysis with phosphorylcholine-specific monoclonal antibody TEPC-15. Analysis of the mutants showed that ORF NMB0415, now defined as pptA (pilin phosphorylcholine transferase A), is involved in the addition of phosphorylcholine to pilin in N. meningitidis. Additionally, the phase variation (high frequency on-off switching of expression) of phosphorylcholine on pilin is due to changes in a homopolymeric guanosine tract in pptA.

Community-acquired methicillin-resistant Staphylococcus aureus carrying Panton-Valentine leukocidin genes: Worldwide emergence

Infections caused by community-acquired (CA)-methicillin-resistant Staphylococcus aureus (MRSA) have been reported worldwide. We assessed whether any common genetic markers existed among 117 CA-MRSA isolates from the United States, France, Switzerland, Australia, New Zealand, and Western Samoa by performing polymerase chain reaction for 24 virulence factors and the methicillin-resistance determinant. The genetic background of the strain was analyzed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). The CA-MRSA strains shared a type IV SCCmec cassette and the Panton-Valentine leukocidin locus, whereas the distribution of the other toxin genes was quite specific to the strains from each continent. PFGE and MLST analysis indicated distinct genetic backgrounds associated with each geographic origin, although predominantly restricted to the agr3 background. Within each continent, the genetic background of CA-MRSA strains did not correspond to that of the hospital-acquired MRSA.

Effector ExoU from the type III secretion system is an important modulator of gene expression in lung epithelial cells in response to Pseudomonas aeruginosa infection

Pseudomonas aeruginosa is an important pathogen in immunocompromised patients and secretes a diverse set of virulence factors that aid colonization and influence host cell defenses. An important early step in the establishment of infection is the production of type III-secreted effectors translocated into host cells by the bacteria. We used cDNA microarrays to compare the transcriptomic response of lung epithelial cells to P. aeruginosa mutants defective in type IV pili, the type III secretion apparatus, or in the production of specific type III-secreted effectors. Of the 18,000 cDNA clones analyzed, 55 were induced or repressed after 4 It of infection and could be classified into four different expression patterns. These include (i) host genes that are induced or repressed in a type III secretion-independent manner (32 clones), (ii) host genes induced specifically by ExoU (20 clones), and (iii) host genes induced in an ExoU-independent but type III secretion dependent manner (3 clones). In particular, ExoU was essential for the expression of immediate-early response genes, including the transcription factor c-Fos. ExoU-dependent gene expression was mediated in part by early and transient activation of the AN transcription factor complex. In conclusion, the present study provides a detailed insight into the response of epithelial cells to infection and indicates the significant role played by the type III virulence mechanism in the initial host response.

CandiVF - Candida albicans virulence factor database

Candida albicans is a pathogen commonly infecting patients who receive immunosuppressive drug therapy, long-term catheterization, or those who suffer from acquired immune deficiency syndrome (AIDS). The major factor accountable for pathogenicity of C. albicans is host immune status. Various virulence molecules, or factors, of are also responsible for the disease progression. Virulence proteins are published in public databases but they normally lack detailed functional annotations. We have developed CandiVF, a specialized database of C. albicans virulence factors (http://antigen.i2r.a-star.edu.sg/Templar/DB/CandiVF/) to facilitate efficient extraction and analysis of data aimed to assist research on immune responses, pathogenesis, prevention, and control of candidiasis. CandiVF contains a large number of annotated virulence proteins, including secretory, cell wall-associated, membrane, cytoplasmic, and nuclear proteins. This database has in-built bioinformatics tools including keyword and BLAST search, visualization of 3D-structures, HLA-DR epitope prediction, virulence descriptors, and virulence factors ontology.

Deficiency of iNOS contributes to Porphyromonas gingivalis-induced tissue damage

Periodontitis is a chronic inflammatory disease that results in extensive soft and hard tissue destruction of the periodontium. Porphyromonas gingivalis possesses an array of virulence factors and has been shown to induce expression of inducible nitric oxide synthase (iNOS) in inflammatory cells. The aim of this study was to investigate the effect of eliminating iNOS in a murine model of P. gingivalis infection. This was achieved by utilizing a P. gingivalis-induced skin abscess model, and an alveolar bone loss model employing an oral infection of P. gingivalis in iNOS knockout mice. The results indicated that iNOS knockout mice exhibit more extensive soft tissue damage and alveolar bone loss in response to P. gingivalis infection compared to wild-type mice. The local immune response to P. gingivalis in iNOS knockout mice was characterized by increased numbers of polymorphonuclear monocytes, while the systemic immune response was characterized by high levels of interleukin-12. The iNOS is required for an appropriate response to P. gingivalis infection.

The asymptomatic bacteriuria Escherichia coli strain 83972 outcompetes uropathogenic E-coli strains in human urine

Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU). In contrast to uropathogenic E. coli (UPEC), which causes symptomatic urinary tract infections (UTI), very little is known about the mechanisms by which these strains colonize the human urinary tract. The prototype ABU E. coli strain 83972 was originally isolated from a girl who had carried it asymptomatically for 3 years. Deliberate colonization of UTI-susceptible individuals with E. coli 83972 has been used successfully as an alternative approach for the treatment of patients who are refractory to conventional therapy. Colonization with strain 83972 appears to prevent infection with UPEC strains in such patients despite the fact that this strain is unable to express the primary adhesins involved in UTI, viz. P and type 1 fimbriae. Here we investigated the growth characteristics of E. coli 83972 in human urine and show that it can outcompete a representative spectrum of UPEC strains for growth in urine. The unique ability of ABU E. coli 83972 to outcompete UPEC in urine was also demonstrated in a murine model of human UTI, confirming the selective advantage over UPEC in vivo. Comparison of global gene expression profiles of E. coli 83972 grown in lab medium and human urine revealed significant differences in expression levels in the two media; significant down-regulation of genes encoding virulence factors such as hemolysin, lipid A, and capsular pollysaccharides was observed in cells grown in urine. Clearly, divergent abilities of ABU E. coli and UPEC to exploit human urine as a niche for persistence and survival suggest that these key differences may be exploited for preventative and/or therapeutic approaches.

Structure of TcpG, the DsbA protein folding catalyst from Vibrio cholerae

The efficient and correct folding of bacterial disulfide bonded proteins in vivo is dependent upon a class of periplasmic oxidoreductase proteins called DsbA, after the Escherichia coli enzyme. In the pathogenic bacterium Vibrio cholerae, the DsbA homolog (TcpG) is responsible for the folding, maturation and secretion of virulence factors. Mutants in which the tcpg gene has been inactivated are avirulent; they no longer produce functional colonisation pill and they no longer secrete cholera toxin. TcpG is thus a suitable target for inhibitors that could counteract the virulence of this organism, thereby preventing the symptoms of cholera. The crystal structure of oxidized TcpG (refined at a resolution of 2.1 Angstrom) serves as a starting point for the rational design of such inhibitors. As expected, TcpG has the same fold as E. coli DsbA, with which it shares similar to 40% sequence identity. Ln addition, the characteristic surface features of DsbA are present in TcpG, supporting the notion that these features play a functional role. While the overall architecture of TcpG and DsbA is similar and the surface features are retained in TcpG, there are significant differences. For example, the kinked active site helix results from a three-residue loop in DsbA, but is caused by a proline in TcpG (making TcpG more similar to thioredoxin in this respect). Furthermore, the proposed peptide binding groove of TcpG is substantially shortened compared with that of DsbA due to a six-residue deletion. Also, the hydrophobic pocket of TcpG is more shallow and the acidic patch is much less extensive than that of E. coli DsbA. The identification of the structural and surface features that are retained or are divergent in TcpG provides a useful assessment of their functional importance in these protein folding catalysts and is an important prerequisite for the design of TcpG inhibitors. (C) 1997 Academic Press Limited.

Destructive periodontitis lesions are determined by the nature of the lymphocytic response

It is now 35 years since Brandtzaeg and Kraus (1965) published their seminal work entitled Autoimmunity and periodontal disease. Initially, this work led to the concept that destructive periodontitis was a localized hypersensitivity reaction involving immune complex formation within the tissues. In 1970, Ivanyi and Lehner highlighted a possible role for cell-mediated immunity, which stimulated a flurry of activity centered on the role of lymphokines such as osteoclast-activating factor (OAF), macrophage-activating factor (MAF), macrophage migration inhibition factor (MIF), and myriad others. In the late 1970s and early 1980s, attention focused on the role of polymorphonuclear neutrophils, and it was thought that periodontal destruction occurred as a series of acute exacerbations. As well, at this stage doubt was being cast on the concept that there was a neutrophil chemotactic defect in periodontitis patients. Once it was realized that neutrophils were primarily protective and that severe periodontal destruction occurred in the absence of these cells, attention swung back to the role of lymphocytes and in particular the regulatory role of T-cells. By this time in the early 1990s, while the roles of interleukin (IL)-1, prostaglandin (PG) E-2, and metalloproteinases as the destructive mediators in periodontal disease were largely understood, the control and regulation of these cytokines remained controversial. With the widespread acceptance of the Th1/Th2 paradigm, the regulatory role of T-cells became the main focus of attention, Two apparently conflicting theories have emerged. One is based on direct observations of human lesions, while the other is based on animal model experiments and the inability to demonstrate IL-4 mRNA in gingival extracts. As part of the Controversy series, this review is intended to stimulate debate and hence may appear in some places provocative. In this context, this review will present the case that destructive periodontitis is due to the nature of the lymphocytic infiltrate and is not due to periodic acute exacerbations, nor is it due to the so-called virulence factors of putative periodontal pathogens.

Quorum sensing is not required for twitching motility in Pseudomonas aeruginosa

It has been reported that mutations in the quorum-sensing genes lasI and rhlI in Pseudomonas aeruginosa result in, among many other things, loss of twitching motility (A. Glessner, R. S. Smith, B. H. Iglewski, and J. B. Robinson, J. Bacteriol. 181:1623-1629, 1999). We constructed knockouts of lasI and rhlI and the corresponding regulatory genes lasR and rhlR and found no effect on twitching motility. However, twitching-defective variants accumulated during culturing of lasI and rhlI mutants. Further analysis showed that the stable twitching-defective variants of lasI and rhlI mutants had arisen as a consequence of secondary mutations in vfr and algR, respectively, both of which encode key regulators affecting a variety of phenotypes, including twitching motility. In addition, when grown in shaking broth culture, lasI and rhlI mutants, but not the wild-type parent, also accumulated unstable variants that lacked both twitching motility and swimming motility and appeared to be identical in phenotype to the S1 and S2 variants that were recently reported to occur at high frequencies in P. aeruginosa strains grown as a biofilm or in static broth culture (E. Deziel, Y. Comeau, and R. Villemur, J. Bacteriol. 183:1195-1204, 2001). These results indicate that mutations in one regulatory system may create distortions that select during subsequent culturing for compensatory mutations in other regulatory genes within the cellular network. This problem may have compromised some past studies of regulatory hierarchies controlled by quorum sensing and of bacterial regulatory systems in general.

Purification of post-translationally modified proteins from bacteria: homologous expression and purification of histidine-tagged pilin from Neisseria meningitidis

Until recently, glycosylation of proteins in prokaryotes was regarded as uncommon and thought to be limited to special cases such as S-layer proteins and some archeal outer membrane proteins. Now, there are an increasing number of reports of bacterial proteins that are glycosylated. Pilin of pathogenic Neisseria is one of the best characterised post-translation ally modified bacterial proteins, with four different types of modifications reported, including a novel glycosylation. Pilin monomers assemble to form pilus fibres, which are long protein filaments that protrude from the surface of bacterial cells and are key virulence factors. To aid in the investigation of these modifications, pure pilin is required. A number of pilin purification methods have been published, but none are appropriate for the routine purification of pilin from many different isolates. This study describes a novel, rapid, and simple method of pilin purification from Neisseria meningitidis C311#3, which facilitates the production of consistent quantities of pure, native pilin. A 6 x histidine tag was fused to the C-terminus of the pilin subunit structural gene, pilE, via homologous recombination placing the 6 x histidine-tagged allele in the chromosome of N. meningitidis C311#3. Pilin was purified under non-denaturing conditions via a two-step process using immobilised metal affinity chromatography (IMAC), followed by dye affinity chromatography. Analysis of the purified pilin confirmed that it retained both of the post-translational modifications examined. This novel approach may prove to be a generally applicable method for purification and analysis of post-translationally modified proteins in bacteria. (C) 2003 Elsevier Science (USA). All rights reserved.

Proteome analysis of extracellular proteins regulated by the las and rhl quorum sensing systems in Pseudomonas aeruginosa PAO1

The las and rhl quorum sensing (QS) systems regulate the expression of several genes in response to cell density changes in Pseudomonas aeruginosa. Many of these genes encode surface-associated or secreted virulence factors. Proteins from stationary phase culture supernatants were collected from wild-type and P. aeruginosa PAO1 mutants deficient in one or more of the lasRI, rhIRI and vfr genes and analysed using two-dimensional gel electrophoresis. All mutants released significantly lower amounts of protein than the wild-type. Protein spot patterns from each strain were compared using image analysis and visible spot differences were identified using mass spectrometry. Several previously unknown OS-regulated proteins were characterized, including an aminopeptidase (PA2939), an endoproteinase (PrpL) and a unique 'hypothetical' protein (PA0572), which could not be detected in the culture supernatants of Delta/as mutants, although they were unaffected in Deltarhl mutants. Chitin-binding protein (CbpD) and a hypothetical protein (PA4944) with similarity to host factor I (HF-1) could not be detected when any of the lasRI or rhIRI genes were disrupted. Fourteen proteins were present at significantly greater levels in the culture supernatants of OS mutants, suggesting that QS may also negatively control the expression of some genes. Increased levels of two-partner secretion exoproteins (PA0041 and PA4625) were observed and may be linked to increased stability of their cognate transporters in a CS-defective background. Known QS-regulated extracellular proteins, including elastase (lasB), LasA protease (lasA) and alkaline metalloproteinase (aprA) were also detected.

Characterization of a complex chemosensory signal transduction system which controls twitching motility in Pseudomonas aeruginosa

Virulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of a wide range of virulence factors including type IV pili which are required for colonization of host tissues and are associated with a form of surface translocation termed twitching motility. Twitching motility in P. aeruginosa is controlled by a complex signal transduction pathway which shares many modules in common with chemosensory systems controlling flagella rotation in bacteria and which is composed, in part, of the previously described proteins PilG, PilH, PilI, PilJ and PilK. Here we describe another three components of this pathway: ChpA, ChpB and ChpC, as well as two downstream genes, ChpD and ChpE, which may also be involved. The central component of the pathway, ChpA, possesses nine potential sites of phosphorylation: six histidine-containing phosphotransfer (HPt) domains, two novel serine- and threonine-containing phosphotransfer (SPt, TPt) domains and a CheY-like receiver domain at its C-terminus, and as such represents one of the most complex signalling proteins yet described in nature. We show that the Chp chemosensory system controls twitching motility and type IV pili biogenesis through control of pili assembly and/or retraction as well as expression of the pilin subunit gene pilA. The Chp system is also required for full virulence in a mouse model of acute pneumonia.

Receptor binding studies disclose a novel class of high-affinity inhibitors of the Escherichia coli FimH adhesin

Mannose-binding type 1 pili are important virulence factors for the establishment of Escherichia coli urinary tract infections (UTIs). These infections are initiated by adhesion of uropathogenic E. coli to uroplakin receptors in the uroepithelium via the FimH adhesin located at the tips of type 1 pili. Blocking of bacterial adhesion is able to prevent infection. Here, we provide for the first time binding data of the molecular events underlying type 1 fimbrial adherence, by crystallographic analyses of the FimH receptor binding domains from a uropathogenic and a K-12 strain, and affinity measurements with mannose, common mono- and disaccharides, and a series of alkyl and aryl mannosides. Our results illustrate that the lectin domain of the FimH adhesin is a stable and functional entity and that an exogenous butyl alpha- D-mannoside, bound in the crystal structures, exhibits a significantly better affinity for FimH (K-d = 0.15 muM) than mannose (K-d = 2.3 muM). Exploration of the binding affinities of alpha-D-mannosides with longer alkyl tails revealed affinities up to 5 nM. Aryl mannosides and fructose can also bind with high affinities to the FimH lectin domain, with a 100-fold improvement and 15-fold reduction in affinity, respectively, compared with mannose. Taken together, these relative FimH affinities correlate exceptionally well with the relative concentrations of the same glycans needed for the inhibition of adherence of type 1 piliated E. coli. We foresee that our findings will spark new ideas and initiatives for the development of UTI vaccines and anti-adhesive drugs to prevent anticipated and recurrent UTIs.

Sub-inhibitory concentrations of ceftazidime and tobramycin reduce the quorum sensing signals of Pseudomonas aeruginosa

Aim: Concentrations of antimicrobials below minimum inhibitory concentration (subMIC) may reduce the production by Pseudomonas aeruginosa of virulence factors such as elastase. We sought to determine whether the reduction in elastase production may be mediated by a reduction in acyl-homoserine lactones. Methods: Pseudomonas aeruginosa in broth was exposed to three conditions for ceftazidime and tobramycin: control, 6% MIC and 25% MIC. Elastase was assayed using elastin congo red. N-(3-Oxododecanoyl)-homoserine lactone (C12-HSL) and N-butyryl-homoserine lactone (C4-HSL) were assayed using biosensor Escherichia coli. Results: Elastase was unchanged with ceftazidime. Elastase was reduced by 16% at 6% MIC tobramycin and reduced by 70% at 25% MIC tobramycin (P