Clostridium difficile is the most common cause of nosocomial diarrhoea today. Through the changing epidemiology of C. difficile infection, the emergence and decline of different strains of varying virulence and a broad spectrum of disease from asymptomatic carriage and mild infection to severe pseudomembranous colitis have been observed. The main aim of this three-part thesis was to identify bacterial factors that might explain these variations by comparing five C. difficile strains - strain 630, an historic strain, strain VPI 10463, a reference strain, the hypervirulent ribotype 027 and the current locally endemic ribotypes 001 and 106. The first study focussed on the growth-related phenotypic and genotypic expression of virulence factors in C. difficile. Growth was studied over twenty-four hours, with simultaneous assessment of toxin and spore production. Total toxin production was measured by a commercial ELISA, while a quantitative ELISA for toxin A and a quantitative cytotoxicity assay for toxin B were developed for individual toxin levels, and spores were enumerated by viable counts. Ribotype 027 produced large amounts of toxin A and toxin B and was the second highest spore producer after ribotype 106. Growth may not affect virulence, but the ability to produce more toxins and spores could. To study the transcription of the genes involved in these processes, a real-time RT-PCR was developed. The transcription of the pathogenicity locus (tcdA-E) that regulates toxin production in C. difficile, and of spo0A, the initiator of sporulation, was studied. There were three key observations: firstly, the transcription of tcdC, the negative regulator of toxin production, did not decrease over time, suggesting it has a modulatory rather than repressive effect on the process. Secondly, tcdE expression was highest in ribotype 027, which might explain its hypertoxicity by greater toxin release. Thirdly, there was almost steady state expression of spo0A during the exponential growth phase in ribotypes 106 and 027, the highest spore producers, suggesting prolonged activation of sporulation. Thus, distinct inter-strain differences exist between C. difficile strains in vitro, which could mirror their virulence in vivo, and several traits contribute synergistically to the hypervirulence of ribotype 027. The second study aimed to identify suitable laboratory disinfectants against C. difficile. The efficacy of four commonly-used disinfectants and one decontaminant was tested; one disinfectant was a chlorine-based agent commonly used in hospitals. In conventional susceptibility tests, all five agents were effective against vegetative cells and spores of C. difficile. However, only the chlorine-based disinfectant was effective against spores dried onto surfaces, but this too required more than two minutes of treatment. The presence of organic matter significantly impaired the efficacy of the non-chlorine agents. The spores of epidemic strains were destroyed less effectively and exposure to sub-MIC levels of disinfectant increased sporulation, especially in ribotype 001, a common outbreak strain. Environmental sampling of the laboratory and surrounding areas showed considerable dissemination of C. difficile, highlighting the need for effective decontamination in conjunction with basic hygiene methods like hand-washing. The third study examined the biological activity of C. difficile. Macrophages were challenged in vitro with S-layer proteins, flagella, heat-shock proteins and culture supernatants of the five strains and cytokine production was measured by specially developed ELISAs. No significant inter-strain differences were observed, although the epidemic strains generally elicited a slightly greater cytokine response. Using epithelial cell lines it was observed that epidemic strains showed greater adherence; from inhibition assays, flagella and S-layer proteins were found to contribute equally to this. Through these studies, inter-strain differences between epidemic and historic isolates were identified with respect to virulence factors, survival in the environment and possible behaviour within the host. A sum of these observations suggests increased virulence in contemporary versus historical C. difficile strains. Finally, a supplementary study characterising a collection of ribotype 027 strains isolated in Scotland and the Netherlands by typing schemes, gene sequencing, susceptibility testing and phenotypic studies was performed. In agreement with other studies, the clonality of these hypervirulent strains was observed.

Staphylococcus aureus is a major pathogen, which causes a wide range of infections. Despite its obvious clinical importance, little is known about the mechanisms of pathogenesis. An in vitro model mimicking infection was developed in order to identify putative virulence determinants. The model involves the growth of S. aureus in serum under microaerobic conditions. All known virulence factors tested were shown not to be required for growth, or preferentially expressed, in serum. Tn917 transposon libraries of S. aureus were screened to identify genes preferentially expressed in serum, compared to a nutrient-rich growth medium. 73 clones were identified and the transposon insertion site was characterised for 23 of these clones. Analysis of sequence flanking the transposon insertion revealed the identity of the mutated loci. 10 out of 23 sequenced clones, contained transposons inserted within genes involved in the biosynthesis of the aspartate family of amino acids (lysine. threonine, methionine and isoleucine). These were: the two common pathway enzymes; aspartokinase (lysC) , and aspartate semi aldehyde dehydrogenase (asd) , along with; dihydrodipicolinate dehydrogenase (dapA), and cystathionine y-synthase (yjcf) , involved in the biosynthesis oflysine and methionine respectively. Analysis of methionine biosynthesis indicated that S. aureus possesses only a single pathway, which proceeds via cystathionine. Several genes encoding methionine biosynthetic enzymes were found clustered on the S. aureus chromosome. The genes lyse, asd and dapA were found to be encoded by the first three genes of an eight gene operon, which also contains three other genes involved in lysine biosynthesis. This operon named the dap operon, is the major lysine biosynthetic operon of S. aureus. lysC, asd and dapA were all found to be repressed at the transcriptional level primarily by lysine, although factors other than the availability of lysine may be responsible for the regulation of lysine biosynthetic gene expression in serum. lysC, asd and dapA were all found to be expressed in vivo, in a murine pyelonephritis model using both RT-PCR and TaqMan techniques. However, these genes were not found to be important in three murine pathogenicity models. Finally, in addition to the development of a model mimicking infection, and the identification of genes with a potentially important role in vivo, this thesis has enhanced our understanding of both methionine and lysine biosynthesis in S. aureus.

This thesis describes attempts to investigate the regulation of the Candida albicans hyphal-specific gene HYR1 by a functional dissection of the HYR1 promoter, protein localisation studies and analysis of HYR1 expression in C. albicans morphological mutants. Sequencing of the HYR1 promoter revealed several putative cis-acting elements within 700 bp of the determined HYR1 transcriptional start site. The possibility of using the LAC4 gene from Kluyveromyces lactis as a reporter for dissection of the HYR1 promoter in C. albicans was investigated. Expression of LAC4 in S. cerevisiae and C. albicans was driven by the C. albicans ADH1 promoter. LAC4 expression was carbon-source-dependent in Saccharomyces cerevisiae as shown by a plate assay and -galactosidase assay, and was confirmed by northern analyses which showed high levels of LAC4 mRNA. However, -galactosidase activity was not detectable in C. albicans transformants using the plate assay or the enzyme assay, and this lack of LAC4 expression was confirmed by northern analysis of the LAC4 mRNA. Preliminary Southern analysis revealed that the LAC4 sequences in S. cerevisae and C. albicans are maintained at approximately equal copy numbers between transformants. Hence LAC4 was not sufficiently sensitive to act as reporter of HYR1 expression and therefore the recently developed yEGFP gene was used for a preliminary HYR1 promoter dissection. However, the HYR1 promoter-yEGFP fusions failed to confirm a role for these elements in the regulation of HYR1 expression . Nevertheless, the hyphal-specific nature of HYR1 expression was confirmed by analysis of the HYR1-yEGFP mRNA by northern analysis. The yEGFP reporter also proved to be too insensitive for use as a reporter of HYR1 expression in C. albicans. To investigate the proposed localisation of the Hyr1p, an in-frame Hyr1-yEGFP fusion was created and expressed in C. albicans and S. cerevisiae. However, this work was inconclusive and the status of Hyr1p as a component of the hyphal cell wall remains to be confirmed.

Staphylococcus aureus is a significant cause of morbidity and mortality world-wide. This opportunistic pathogen is capable of causing several severe diseases that are exacerbated by its diverse and widespread antibiotic resistance profile. Therefore it is necessary to identify novel therapeutic targets to effectively treat S. aureus disease. Lorenz et al first described the Immunodominant Surface Antigen B, IsaB, because it was 1 of 4 unique proteins immunogenic during septicemia and not colonization, suggesting that IsaB may be a virulence factor and a possible novel therapeutic target. Interestingly, IsaB has no homology to proteins of known function and appears to be found only in Staphylococci. We sought to characterize the function of IsaB in S. aureus. We began our studies by determining how isaB was regulated by known S. aureus regulators and environmental stimuli. It was observed that the transcriptional regulator SarA represses expression of isaB, while serum and acidic pH induce expression. We found that IsaB is an extracellular nucleic acid binding protein, able to bind to dsDNA, ssDNA, and RNA and leads significant accumulation of eDNA on the cell surface. We employed multiple virulence models to ascertain the role of IsaB in virulence. Excitingly, we found that IsaB significantly protects S. aureus from antimicrobial peptides and Neutrophil Extracellular Traps, both components of the innate immune system. Another virulence mechanism of S. aureus is the ability to form biofilms. While recent studies show a significant role for eDNA in S. aureus biofilms, we found that IsaB actually had a negative affect on biofilms under certain growth conditions. Finally, to group IsaB into a known functional class, we successfully expressed and purified mature IsaB for structural determination by Nuclear Magnetic Resonance, which is currently underway. Our studies show that IsaB is a novel virulence factor of S. aureus, able to bind eDNA and significantly protect from AMPs and NETs, and could therefore play a key role in immune evasion.

Bacterial infections are an increasing cause for concern as resistance spreads to the majority of our front line antibiotics. To counter antibiotic resistance, new treatment regimens and drug targets are being investigated, including directly targeting bacterial virulence (pathogen-induced harm to the host), and therapies which target resistance mechanisms. The outcome of successful treatment with these compounds is not always killing or halting growth of bacteria, therefore selection for resistance to these types of therapeutics is complex. This complexity is increased by the secretion of many virulence factors, meaning their effects are shared with neighbouring individuals. In addition virulence factors show high phenotypic plasticity due to regulation by processes like quorum sensing (QS), which further complicates treatments targeting virulence, or the regulatory processes themselves. Using the example of quorum sensing inhibitors this study shows the importance of understanding the function and ecology of targeted virulence factors, to predict the selection for resistance to anti-virulence drugs. Later chapters elaborate on this to show how quorum sensing control affects selection on secreted virulence factors. The use of anti-virulence drugs as adjuvants is discussed, with a study showing that the interaction between QS inhibition and translation inhibitors is dependent on the environment. The selection for resistance to combinations of antibiotics and adjuvants is investigated using co-amoxiclav as an example, showing that treatment with high doses of adjuvant are robust to the evolution of resistance.

The pathogenic actinomycete Rhodococcus equi is a facultative intracellular parasite that replicates within macrophages. This ability is dependent on the pVAP virulence plasmid, and more specifically, on the laterally acquired vap pathogenicity island (vap PAI) carried by it. R. equi has two contrasting lifestyles as a soil-dwelling microbe and as an inhabitant of the intracellular macrophage compartment. In the first part of this thesis work we analysed the nature of the signals recognised by R. equi to adapt the expression of the virulence genes of the plasmid during the transition from soil saprotroph to intracellular parasite. The expression profile of virulence plasmid genes in response to temperature and pH in vitro and to the macrophage environment was investigated by microarray analysis. A shift to 37ºC was the main stimulus involved in vap PAI gene activation and macrophage-derived signals did not further modulate the expression of the PAI genes contrary to previous suggestions. In a second part of the thesis we investigated the role of a horizontally acquired island encoding exopolysaccharide biosynthesis in the R. equi saprotroph-intracellular parasite dual lifestyle. Mutational analysis of this locus showed that it is responsible for the typical mucoid colony morphology of R. equi and the ability to produce a polysaccharide capsule. Mutations in the capsule locus favoured macrophage uptake but had no effect on intracellular proliferation and in vivo survival in mice. However, the capsule mutants showed significantly increased susceptibility to desiccation, ultraviolet radiation and heat and were outcompeted by capsulated wild-type R. equi in dry soil. Thus, while having a minor role in virulence, the R. equi capsule appears to be primarily required for survival in soil and to act as a transmission factor. The third part of this work followed the identification of a horizontally acquired locus that encodes pili appendages that promote association with macrophages and colonization of the mouse lung. The ability of a component of this structure, the RplB pilin subunit, to act as a vaccine antigen was investigated in mice and horses. Vaccinated mice produced high levels of anti-RplB IgG and showed significant protection against pulmonary challenge with virulent R. equi. The experimental RplB subunit vaccine proved also to be immunogenic in horses, eliciting a strong IgG response in pregnant mares and foals. We also demonstrated passive transfer of high levels of maternal anti-RplB antibodies from the mares to the foals via colostrum. Our results indicate that the RplB pilin subunit is a promising novel candidate R. equi vaccine antigen.

Staphylococcus aureus secretes two cysteine proteases, Staphopain A (scpA) and Staphopain B (sspB). We hypothesized that ScpA will exhibit a distinct activation mechanism, and a different or complementary substrate specificity compared to SspB. A Cys>Ala active site substitution led to the accumulation of unprocessed 40-kDa proScpA, confirming that ScpA undergoes autocatalytic activation. A temporal analysis of ScpA expression revealed that activation was initiated by processing at Lys171 and Glu176, producing an intermediate that was rapidly converted to several isoforms of mature protease by processing after Thr202, Lys209, Thr214 and Asn216. Consistent with broad specificity, mature ScpA was sensitive to autocatalytic degradation. ScpA demonstrated activity towards elastin, fibrinogen and indicated evidence for binding to heparin. Elastinolytic activity was uniquely associated with strains belonging to CC30, and was correlated with ScpA expression. Therefore, although ScpA and SspB share both sequence and structural similarity, they exhibited very different substrate specificities and activation mechanisms.

Staphylococcus aureus secretes two cysteine proteases, Staphopain A (scpA) and Staphopain B (sspB). We hypothesized that ScpA will exhibit a distinct activation mechanism, and a different or complementary substrate specificity compared to SspB. A Cys>Ala active site substitution led to the accumulation of unprocessed 40-kDa proScpA, confirming that ScpA undergoes autocatalytic activation. A temporal analysis of ScpA expression revealed that activation was initiated by processing at Lys171 and Glu176, producing an intermediate that was rapidly converted to several isoforms of mature protease by processing after Thr202, Lys209, Thr214 and Asn216. Consistent with broad specificity, mature ScpA was sensitive to autocatalytic degradation. ScpA demonstrated activity towards elastin, fibrinogen and indicated evidence for binding to heparin. Elastinolytic activity was uniquely associated with strains belonging to CC30, and was correlated with ScpA expression. Therefore, although ScpA and SspB share both sequence and structural similarity, they exhibited very different substrate specificities and activation mechanisms.

Thesis (Ph. D.)--Lehigh University, 2004. / Includes vita. Includes bibliographical references (leaves 102-126).

MP1, a gene previously identified in P. marneffei by cDNA library screening, encodes a secreted cell wall mannoprotein Mp1p. Thirteen MP1 homologues named MPLP1 to 13 were previously identified in P. marneffei by BLAST analysis. Two MP1 homologues namely AFMP1 and AFMP2 which encodes Afmp1p and Afmp2p were previously identified by expressed sequence tag library screening in Aspergillus fumigatus – an important fungal pathogen closely related to P. marneffei. Mp1p, Afmp1p and Afmp2p have previously been reported to be immunogenic. Mp1p was also reported to bind fatty acid and was suggested to contribute to virulence in a MP1 knockout P. marneffei strain in a mouse model and a cell line model although the Koch’s postulates has yet been met to establish MP1 as a novel virulence factor. With reference to sequence identity of Afmp proteins to Mp1p, Afmp proteins were speculated to have functions similar to Mp1p. BLAST searches against the A. fumigatus genome identified two novel AFMPs namely AFMP3 and AFMP4. Sequence analysis of Afmp3p and Afmp4p revealed the presence of putative N-terminal signal peptide and substantial sequence identity to Mp1p, Afmp1p and Afmp2p. Two MP1 knockdown P. marneffei mutants were constructed to demonstrate suppression of MP1 expression alone can result in loss of virulence and also the dosage effect of MP1 expression on P. marneffei virulence towards mice. Subsequent mice challenge experiments using MP1 like protein (MPLP) knockdown strains suggested MP1 to be the most important virulence factor among all its homologues in P. marneffei. Histopathology examinations of organs from challenged mice suggested survival disadvantages in mice for P. marneffei mutants with knockdown of MP1 and effect of MP1 on granuloma formation in infected mice. Mice challenge experiments using AFMP1 to 4 knockdown A. fumgiatus mutants suggested significant decrease in virulence of A. fumigatus upon AFMP4 knockdown and complete protection of challenged mice upon knockdown of AFMP1 to 4. Histopathology examinations of organs from challenged mice suggested survival disadvantages in mice for A. fumigatus mutants with knockdown of AFMPs and effect of AFMPs on granuloma formation in infected mice. Mice experiments using Pichia pastoris expressing MP1 or AFMP4 suggested the effects of MP1 and AFMP4 on virulence are not caused by factors specific to P. marneffei or A. fumigatus. It was shown using a human peripheral blood mononuclear cells model that Mp1p and Afmp4p confer intracellular survival advantage to P. marneffei and A. fumigatus upon infection. Expression of Mp1p or Afmp4p in P. pastoris also confers survival advantage to this nonpathogenic yeast in human peripheral blood mononuclear cells. Reduction in proinflammatory prostaglandin E2 production were noticed in human peripheral blood mononuclear cells infected by P. marneffei, A. fumigatus or P. pastoris strains that expressed Mp1p or Afmp4p. Such reduction in eicosanoids production also coincides with the inhibition of apoptosis as shown by enzyme activity of caspase-8, caspase-9 and caspase-3 in human peripheral blood mononuclear cells. These findings suggest two novel virulence factors – Mp1p and Afmp4p, which confer survival advantages to P. marneffei and A. fumigates respectively. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy