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Legionella spp. can cause a life threatening form of pneumonia, which is observed world-wide. Outbreaks of the disease are, unfortunately, not a rare event, despite the introduction of government regulations which enforce the mandatory testing of cooling towers to ensure that they contain levels of the organism which are regarded as being within safe limits. Therefore, cooling towers should be monitored for Legionella spp. by using a biosensor. These could potentially save the community from a great deal of morbidity and mortality due to legionellosis. This study identified and investigated novel outer membrane proteins in L. pneumophila, and analysed their potential for use in a Legionella biosensor. A combination of bioinformatics and laboratory investigations was used to identify the Omp87, an outer membrane protein of L. pneumophila which had not been previously described in this organism. Sequence analysis of the protein showed that it shares similarity with various other members of the Omp85 protein family, including the D15 antigen of Haemophilus influenzae and the Oma87 of Pseudomonas aeruginosa. The omp87 gene of L. pneumophila was amplified and cloned, and was found to encode a protein of 786 amino acids, with a molecular weight of 87 kDa. Distribution studies revealed that the gene is present in most, but not all species and serogroups of Legionella. To investigate the function of the Omp87 protein in L. pneumophila, the omp87 gene was insertionally inactivated with the use of a kanamycin resistance gene. Amplicons of this disrupted gene were then introduced into L. pneumophila, and a double-cross over event occurred, integrating the inactivated gene into the genome of the organism. This resulted in non-viable cells, indicating that the gene is essential in L. pneumophila. The expression vector pRSETA was used to express the Omp87 protein in E. coli, and four truncates of varying sizes were designed, through the use of different PCR primers. Two of the protein truncates were then expressed and purified by gravity flow chromatography using columns packed with Ni-NTA sepharose resin. Following analysis of the proteins by SDS-PAGE and Western blotting, polyclonal antibodies were raised against the truncates. Distribution studies were then performed using the antiserum with different strains and species of Legionella. This study demonstrated that most serogroups of L. pneumophila, and most other Legionella species reacted with the polyclonal anti-Omp87 L. pneumophila antisera. Cross-reactivity was also observed with most other Legionella related organisms tested. The results presented in this thesis demonstrated that the Omp87 protein or the omp87 gene can be used to construct a biosensor. In addition other novel outer membrane proteins were identified which could also serve as potential targets for a biosensor. These biosensors will be able to identify Legionella spp. in water reservoirs and in clinical samples and hopefully reduce the number of infections and deaths caused by this organism.

Master of Science / Department of Biomedical Sciences / Sanjeev K. Narayanan / Fusobacterium necrophorum is an anaerobic Gram-negative non spore forming rod shaped bacteria that is a normal inhabitant of the alimentary tract of humans and animals. Two subspecies of F. necrophorum have been recognized- subspecies necrophorum and subspecies funduliforme. Subspecies necrophorum is an opportunistic pathogen in animals causing diseases such as bovine hepatic abscesses and sheep foot rot while as subspecies funduliforme is linked with human oral and hepatic infections such as sore throats, Lemierre’s syndrome and hepatic abscesses. The pathogenic mechanisms of F. necrophorum are complex and are not well understood or defined. Several virulence factors such as leukotoxin, haemolysin, haemagglutinin and adhesin have been described. One of the most important factors in F. necrophorum bacterial pathogenesis is the adhesion of the bacteria to the host cell. The adhesion of the bacteria to the host cell helps it colonize the host tissue and this is followed by intracellular multiplication with dissemination to other tissues, which could ultimately lead to septicemia and death. Bacteria use adhesins which are proteins found in the outer membrane which help them bind with host receptors and this helps with the adhesion of the bacteria to the host cell. Not much is known about F. necrophorum adhesins. Here, we describe and characterize a novel adhesin.

This thesis reports the characterisation of a novel outer membrane protein (OMP) from M. catarrhalis, designated M35, with a molecular mass of 36.1 kDa. This protein is structurally homologous to classic Gram-negative porins, such as OMP C from E. coli and OMP K36 from K. pneumoniae, with a predicted structure of 8 surface loops connecting 16 antiparallel -sheets. Comparison of the DNA sequences of the M35 genes from 18 diverse clinical isolates showed that the gene was highly conserved (99.6-100 % of nucleotides) with only one isolate (ID78LN266) having base variations that resulted in amino acid substitutions. A single amino acid mutation in the 3rd external loop of M35 in isolate ID78LN266 significantly affected antibody recognition, indicating that loop 3 contains an immunodominant B-cell epitope. The reduction in antibody-binding to M35 from ID78LN266 was similar to that caused by complete removal of loop 3. Since loop 3 folds into the porin channel in the classic structure, the antibody specificity to loop 3 was hypothesised to be a potential mechanism for evasion of host immune responses targeted to M35, potentially explaining the high degree of conservation across isolates. A series of recombinant proteins were constructed to analyse the binding to M35 of antibodies specificity for loop 3 or the remainder of the protein. It was found that loop 3- specific antibodies were not able to bind to M35 on the surface of M. catarrhalis and that this corresponds both with a lack of ability to enhance opsonophagocytosis in vitro and bacterial clearance in vivo. Additionally, antibodies raised against a version of M35 lacking loop 3 and M35 from the variant isolate ID78LN266 were both no less effective than the full consensus M35 by both these measures. It therefore appears that while the majority of antibodies raised against M35 are specific for loop 3 these antibodies do not mediate anti-M. catarrhalis actions. Two deletion mutant strains of M. catarrhalis that do not contain the outer membrane protein M35 were created by insertional inactivation of the M35 gene. Growth comparisons between these mutant strains and their wildtype parent strains initially led to the hypothesis that M35 is necessary for efficient glutamic acid uptake by M. catarrhalis, however this hypothesis was later shown to be incorrect. Efficient uptake of glutamic acid seemed to be mediated by a novel 40 kDa protein that was up-regulated in the deletion mutant strains, presumably to compensate for the lack of M35. M35 was also found to be essential for in vivo survival of M. catarrhalis in the nasal cavities of mice, indicating that it is an essential functional protein for colonisation of the mucosal surface.

Anaplasma phagocytophilum and A. marginale are the etiologic agents of human granulocytic anaplasmosis and bovine anaplasmosis, respectively. Both diseases can be severe, even fatal, and protective vaccines for each are lacking. We recently identified A. phagocytophilum outer membrane protein A (ApOmpA) as being critical for cellular invasion and is expressed during infection of mammalian but not tick cells. Disrupting ApOmpA-host cell interactions significantly inhibits A. phagocytophilum entry into host cells. ApOmpA and its A. marginale ortholog, AM854 (A. marginale OmpA; AmOmpA) exhibit 44% amino acid identity. The ApOmpA invasin domain is highly conserved between both proteins. In this study, we investigated the differential expression of AmOmpA in mammalian versus tick cell lines; the serological cross-reactivity between AmOmpA and ApOmpA; the potential role of AmOmpA in mediating interactions with mammalian host cells; and if inhibiting the AmOmpA-host cell interaction impairs A. marginale cellular invasion. AmOmpA is expressed throughout infection of mammalian, but not tick cells. Sera from A. marginale infected cows recognized both AmOmpA and ApOmpA. Sera from cows immunized with an A. marginale OM complex that conferred protection also recognized both proteins. Thus, ApOmpA and AmOmpA share cross-reactive B-cell epitopes. To determine if AmOmpA plays a role in promoting A. marginale infection, we assessed the abilities of recombinant AmOmpA to competitively inhibit infection of mammalian host cells. To examine the cross-reactive properties of OmpA, we showed that preincubation of host cells with GST-ApOmpA and pretreatment of A. marginale with anti-GST-ApOmpA significantly inhibit A. marginale infection of host cells; and that pretreatment of A. phagocytophilum with serum from cows immunized with an A. marginale OM complex reduces its infection of host cells. These studies advance understanding of conservation of OmpA-mediated cellular invasion between Anaplasma species and highlight the potential of OmpA as a vaccinogen that could offer protection against human and veterinary anaplasmoses.

Pseudomonas aeruginosais an opportunistic pathogen that infects cystic fibrosis (CF) patients contributing to their high morbidity and mortality. P. aeruginosaundergoes a phenotypic conversion in the CF lung, from nonmucoid to mucoid, by constitutively producing a polysaccharide called alginate. These mucoid strains often revert to nonmucoid in vitrodue to second-site suppressor mutations. We hypothesized that mapping these mutations would lead to the identification of novel genes involved in alginate production. In a previous study, a mucoid strain, PDO300 (PAOmucA22), was used to isolate suppressors of alginate phenotype (sap). One of the uncharacterized nonmucoid revertants, sap27, is the subject of this study. The mucoid phenotype in sap27was restored by pMO012217 from a minimal tiling path cosmid library. The cosmid pMO012217 harbors 18 P. aeruginosaopen reading frames (ORF). The cosmid was mutagenized with a transposon to map the contributing gene. It was mapped tolptD(PA0595) encoding lipopolysaccharide transport protein. E. coliLptD transports lipopolysaccharide to the outer leaflet of the outer membrane. The Alg+phenotype was restored upon complementation with P. aeruginosa lptDalone, suggesting that sap27likely harbor a chromosomal mutation inlptD. Sequencing analysis of sap27showed the presence of a mutation not in lptDbut in algO, which encodes a periplasmic protease protein. This suggests LptD is able to bypass analgO mutation by positively regulating alginate production. The lptD is a part of a three-gene operon lptD-surA-pdxA. SurA is an essential protein for survival in starvation and a major chaperone protein for all outer membrane proteins and PdxA is a NAD-dependent dehydrogenase and is involved in the vitamin B6biosynthetic pathway. Pyridoxal 5’-phosphate (PLP) is the active form of vitamin B6.P. aeruginosagrown in a media supplemented with PLP increased production of pyocyanin, a virulence factor. The PLP and aromatic amino acids are synthesized from a common precursor chorismic acid. We demonstrated an increase in pyocyanin production when the bacteria were cultured supplemented by the aromatic amino acids phenylalanine. We concluded that the lptDoperon plays a role in the P. aeruginosavirulence by regulating alginate and pyocyanin production.

Rare lipoprotein A (RlpA) has been studied previously only in Escherichia coli, where it localizes to the septal ring and scattered foci along the lateral wall, but mutants have no phenotypic change. In this thesis, we show rlpA mutants of Pseudomonas aeruginosa form chains of short, fat cells when grown in media of low osmotic strength. These morphological defects indicate RlpA is needed for efficient separation of daughter cells and maintenance of rod shape. Analysis of peptidoglycan sacculi from a ΔrlpA mutant revealed increased tetra and hexasaccharides that lack stem peptides (hereafter called "naked glycans"). Incubation of these sacculi with purified RlpA resulted in release of naked glycans containing 1,6-anhydro N-acetylmuramic acid ends. RlpA did not degrade sacculi from wild-type cells unless the sacculi were subjected to a limited digestion with an amidase to remove some of the stem peptides. Collectively, these findings indicate RlpA is a lytic transglycosylase with a strong preference for naked glycan strands. We propose that RlpA activity is regulated in vivo by substrate availability, and that amidases and RlpA work in tandem to degrade peptidoglycan in the division septum and lateral wall. Our discovery that RlpA from P. aeruginosa is a lytic transglycosylase motivated us to reinvestigate RlpA from E. coli. We confirmed predictions that RlpA of E. coli is an outer membrane protein and determined its abundance to be about 600 molecules per cell. However, multiple efforts to demonstrate that E. coli RlpA is a lytic transglycosylase were unsuccessful and the function of this protein in E. coli remains obscure.

Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular pathogen. Recent studies show that the complete genome sequence of Orientia tsutsugamushi have been determined. However, the early signaling events involved in the entry of O.tsutsugamushi into mammalian cells remains a challenge. In this study, we demonstrate that adherence ability and comparison of three major outer membrane protein TSA56, TSA47 and TSA22 of O.tsutsugamushi. Through expression and purification of three type-specific antigen 56-kDa (include TSA56-antigen domain I, TSA56-antigen domain III), 47-kDa and 22-kDa of O. tsutsugamushi , antiserum immunoblots from 22 clinical O. tsutsugamushi-infected patients and in vitro adhesion assay of E.coli overexpression outer membrane protein of O. tsutsugamushi , the antiserum titer and adherence ability of bacterial outer membrane proteins are determined. The data show that antiserum titer against three major outer membrane proteins of O. tsutsugamushi was markedly higher in TSA56 compared to TSA47 and TSA22. In adhesion assay, adhesion of host cells by TSA56 was readily than TSA47 and TSA22. Furthermore, adhesion experiment and antiserum titer against antigen-domain I (ADI) region (19-114 aa) in the extracellular domain of TSA56 was also significantly higher than previously reported antigen-domain III(ADIII) region (237-366 aa) which facilitates the invasion of O. tsutsugamushi through interaction with fibronectin .Taken together, these results clearly indicate that O. tsutsugamushi exploits TSA56-mediated bacterial adhesion, abundant antiserum titer and ADI region of TSA56 may draw another adhesion site (except for previously reported ADIII) to invade eukaryotic host cells.

Syphilis is a global public health concern with 36.4 million cases worldwide and 11 million new infections per year. It is a chronic multistage disease caused by the spirochete bacterium Treponema pallidum and is transmitted by sexual contact, direct contact with lesions or vertically from an infected mother to her fetus. T. pallidum is a highly invasive pathogen that rapidly penetrates tight junctions of endothelial cells and disseminates rapidly via the bloodstream to establish widespread infection. Previous investigations conducted in our laboratory identified the surface-exposed adhesin, pallilysin, as a metalloprotease that degrades the host components laminin (major component of the basement membrane lining blood vessels) and fibrinogen (primary component of the coagulation cascade), as well as fibrin clots (function to entrap bacteria and prevent disseminated infection). Furthermore, pallilysin expressed on the surface of the non-invasive spirochete Treponema phagedenis conferred upon this bacterium the ability to degrade fibrin clots. It was hypothesized that pallilysin is integral to the process of T. pallidum dissemination, and interference with its functioning will prevent spread throughout the host and establishment of chronic infection. To test this hypothesis, a two-pronged approach was undertaken during my thesis research. Bioinformatics analyses were used to trace the evolutionary history of pallilysin in an attempt to gain further insight into its role in the pathogenesis of T. pallidum. The sequence conservation of pallilysin was analyzed in the context of its homologues. The bioinformatics analyses revealed homologues in three spirochete genera, namely Treponema, Spirochaeta, and Borrelia, presented in decreasing order of the degree of sequence conservation. The HEXXH motif, part of the active site of the pallilysin metalloprotease, was fully conserved only in T. pallidum and T. paraluiscuniculi, both of which are systemic pathogens. However, the flanking sequences showed a high degree of conservation, especially in the Treponema and Spirochaeta genera. The minimum laminin-binding region of pallilysin identified previously was partially conserved among the treponema and spirochaeta homologues with the highest degree of conservation observed with the homologues from T. paraluiscuniculi and T. phagedenis, as well as among the homologues from the human oral pathogens. In vitro dissemination studies were performed to investigate the dissemination capacity of T. phagedenis heterologously expressing pallilysin. Human Umbilical Vein Endothelial Cells were seeded and grown to confluence on permeable inserts coated with growth factor-reduced Matrigel to create an artificial endothelial barrier. Wild type T. phagedenis, and T. phagedenis transformed either with the pallilysin open reading frame or its empty shuttle vector, were incubated with the barriers under anaerobic conditions. Dissemination across the barrier was assessed as percent traversal by both dark-field microscopic counts of treponemes and real-time quantitative PCR of genomic DNA extracted from the treponemes. The results were inconclusive. However, a traversal trend suggested heterologous expression of pallilysin may facilitate traversal of T. phagedenis across the artificial endothelial barrier. This study presented the first step towards elucidating the role of pallilysin in endothelial monolayer traversal and provided supporting evidence for the role of pallilysin in the widespread dissemination of T. pallidum in vivo. / Graduate

Chlamydia trachomatis infections are among the most frequently reported causes of human sexually transmitted infection. In Australia, the reported rate of infection in 2004 reached 175 per 100,000 population, the highest rate since surveillance of the condition began in 1991. Severe adverse sequelae that commonly occur following progression of the infection from the lower to the upper genital tract include pelvic inflammatory disease, infertility and ectopic pregnancy. However the frequent prevalance of asymptomatic infection makes diagnosis and treatment often late and therefore ineffective against upper genital tract complications. Hence there is a great need to develop a vaccine to protect against the sexual transmission of C.trachomatis. Despite many years of research investigating potential vaccine strategies to prevent sexually transmitted C.trachomatis infections, there remains no commercially available C.trachomatis vaccine. Early research showed that the use of live, attenuated or inactivated whole Chlamydia as a vaccine was not a viable option due to adverse effects caused by immunopathogenic cellular components. The early human vaccine trials that utilized whole chlamydial cells and resulted in exacerbated disease when immunized individuals were re-exposed to Chlamydia have led to the investigation of chlamydial subunit components as potential vaccine antigens. The most widely investigated vaccine candidate antigen is the major outer membrane protein (MOMP) as it is known to be immunogenic and surface exposed. Much research using this antigen has been undertaken with the antigen being delivered as a protein, peptide or DNA, via many mucosal and systemic routes of immunization, and in combination with various vaccine adjuvants. However, at best only partial protection against a chlamydial genital tract infection has been achieved. Only a few alternative candidate antigens have been investigated as potential vaccine targets to protect against chlamydial infections. These include the outer membrane porin PorB, the large cysteine rich outer membrane protein Omp2 and the heat shock proteins DnaK and GroEL. Although other candidate antigens have been predicted in various models of chlamydial infection (Finco et al., 2005; Stemke-Hale et al., 2005; Li et al., 2006), few have been tested for their protective efficacy. The aim of this study was to use expression library immunization to screen the whole C.muridarum genome for novel vaccine candidates capable of protecting against a chlamydial genital tract infection. C.muridarum was selected as the disease model for chlamydial genital tract infection as it has similarities to C.trachomatis in pathogenesis, immune response to infection and gene content and order. Once protective antigens had been isolated from an expression library, these were screened individually for immunogenicity and protective efficacy in the C.muridarum model of infection. An expression library containing over 21,000 recombinant C.muridarum clones was constructed and divided into pools of clones. DNA was extracted from these pools and used to immunize mice through gene gun technology, delivering 1μg of DNA to the abdomen of mice. Following the immunization regime, mice were challenged intra-vaginally with live C.muridarum as this route of infection best resembles the natural route of infection that is responsible for the sexual transmission of C.trachomatis in humans. Four in vivo screens of the C.muridarum expression library, each time using reduced numbers of clones, resulted in the identification of seven novel vaccine antigens that conferred protection against a genital tract challenge infection in mice. These warrant further investigation as vaccine antigens in the development of a vaccine against C.trachomatis infection. The identified antigens include antigens not conventionally believed to be potential vaccine candidates such as hypothetical proteins and housekeeping genes, including a DNA gyrase subunit, TC0462, and the ATP-dependent Clp protease, ATP-binding subunit ClpC, TC0559. Other antigens identified were more traditional, surface exposed vaccine targets that have not been previously investigated as vaccine targets, including a novel outer membrane protein, TC0512, a polymorphic membrane protein, TC0693, and TC0850, a protein of the type three secretion system, a family of proteins that allow gram-negative bacteria to inject virulence related proteins into the cytoplasm of a host cell. All antigens were shown to be partially protective with the putative outer membrane protein TC0512 showing an overall reduction in chlamydial burden of 55% and other antigens showing overall reductions in chlamydial burden of 26 - 44%. These antigens were also either capable of stimulating an immune response, or predicted to contain epitopes that may stimulate strong immune responses and so warrant further investigation as vaccine antigens to protect against chlamydial genital tract infections. The results of this research demonstrate that it is possible to identify novel vaccine targets through screening an expression library in a disease model. This study has identified several novel vaccine targets that are partially-protective against a C.muridarum infection and that are thought to be capable of stimulating strong immune responses. These antigens have high homology with C.trachomatis sequences, indicating that they have potential as vaccine candidates capable of protecting against the serovars of C.trachomatis that cause sexually transmitted infections in humans. Although the protection observed in this study was only partial, the immunization strategy utilised only fragments of the genes, an immunization mechanism known to elicit Th2 type immune responses, and no adjuvant to enhance the immunogenicity of the antigens. Through different immunization routes and in conjunction with adjuvants that stimulate Th1 type immune responses, complete protection against chlamydial genital tract infections may be achieved.