Research Associate, University of Technology Sydney/ ithree institute
My research has been focused on elucidating novel virulence mechanisms of M. hyopneumoniae
My research has been focused on the highly infectious respiratory pathogen Mycoplasma hyopneumoniae (Mhp) that is an ongoing cause of major economic losses to the swine industry globally; pork being the most consumed meat product globally. Mhp is a major concern for swine farmers as infected pigs are slow to reach the desired market weight, in addition to creating an avenue for secondary infections. Little is known about how Mhp persists within its host and previous vaccines have largely been ineffective; leading to an over-reliance on antibiotics that have contributed to the incidence of antibiotic resistance. It is estimated that 10 million deaths per annum will be attributed to infections arising from antibiotic-resistance bacteria by 2050; exemplifying the need for novel intervention strategies to slow the rise of antibiotic-resistance.
My work concerning Mhp has led to a paradigm shift in the future study of this bacterium by identifying two novel life stages that it adopts to persist within its host: biofilms and invasion. Historically thought of as a strict extracellular pathogen, I demonstrated for the first time that Mhp possess an intracellular life stage. The ability to invade host cells is a sophisticated strategy utilised by many bacterial pathogens to hide from the host immune system as well as the lytic effects of antibiotics. Specifically, I showed that Mhp survives host mechanisms intended to kill these cells, called lysosomes. As mentioned above, my work also demonstrated for the first time, that Mhp has the ability to form biofilms within the swine respiratory tract. Biofilms are impenetrable bacterial communities that are highly resistant to the host immune response and antibiotics, and are often implicated in chronic infections. This is likely one of the primary ways in which Mhp persists within its host for prolonged periods.
Overall, my work has aided in further understanding novel mechanisms that this pathogen uses to escape the host immune system and antimicrobials. These findings have significant implications for future Mhp vaccine studies that have largely been ineffective. This work also has far-reaching influence on the alleviation of antibiotic overuse in the meat industry.
Abstract: Mycoplasma hyopneumoniae colonizes the ciliated epithelial lining of the upper respiratory tract of swine and results in chronic infection. Previously, we have observed that members of P97 and P102 paralog families of cilium adhesins undergo endoproteolytic processing on the surface of M. hyopneumoniae. We show that P159 (MHJ_0494), an epithelial cell adhesin unrelated to P97 and P102 paralog families, is a cilium adhesin that undergoes dominant cleavage events at S/T-X-F↓X-D/E-like motifs located at positions (233)F↓Q(234) and (981)F↓Q(982), generating P27, P110, and P52. An unrelated cleavage site (738)L-K-V↓G-A-A(743) in P110 shows sequence identity with a cleavage site (L-N-V↓A-V-S) identified in the P97 paralog, Mhp385, and generates 76 (P76) and 35 kDa (P35) fragments. LC-MS/MS analysis of biotinylated surface proteins identified six peptides with a biotin moiety on their N-terminus indicating novel, low abundance neo-N-termini. LC-MS/MS of proteins separated by 2D-PAGE, 2D immunoblotting using monospecific antiserum raised against recombinant fragments spanning P159 (F1(P159)-F4(P159)), and proteins that bound to heparin-agarose were all used to map P159 cleavage fragments. P159 is the first cilium adhesin not belonging to the P97/P102 paralog families and is extensively processed in a manner akin to ectodomain shedding in eukaryotes.
Pub.: 08 Nov '13, Pinned: 31 Aug '17
Abstract: Mycoplasma hyopneumoniae, the aetiological agent of porcine enzootic pneumonia, regulates the presentation of proteins on its cell surface via endoproteolysis, including those of the cilial adhesin P123 (MHJ_0194). These proteolytic cleavage events create functional adhesins that bind to proteoglycans and glycoproteins on the surface of ciliated and non-ciliated epithelial cells and to the circulatory host molecule plasminogen. Two dominant cleavage events of the P123 preprotein have been previously characterized; however, immunoblotting studies suggest that more complex processing events occur. These extensive processing events are characterized here. The functional significance of the P97 cleavage fragments is also poorly understood. Affinity chromatography using heparin, fibronectin and plasminogen as bait and peptide arrays were used to expand our knowledge of the adhesive capabilities of P123 cleavage fragments and characterize a novel binding motif in the C-terminus of P123. Further, we use immunohistochemistry to examine in vivo, the biological significance of interactions between M. hyopneumoniae and fibronectin and show that M. hyopneumoniae induces fibronectin deposition at the site of infection on the ciliated epithelium. Our data supports the hypothesis that M. hyopneumoniae possesses the molecular machinery to influence key molecular communication pathways in host cells.
Pub.: 09 Oct '14, Pinned: 31 Aug '17
Abstract: The plasminogen (Plg) system plays an important homeostatic role in the degradation of fibrin clots, extracellular matrices and tissue barriers important for cellular migration, as well as the promotion of neurotransmitter release. Plg circulates in plasma at physiologically high concentrations (150-200μg ml(-1)) as an inactive proenzyme. Proteins enriched in lysine and other positively charged residues (histidine and arginine) as well as glycosaminoglycans and gangliosides bind Plg. The binding interaction initiates a structural adjustment to the bound Plg that facilitates cleavage by proteases (plasminogen activators tPA and uPA) that activate Plg to the active serine protease plasmin. Both pathogenic and commensal bacteria capture Plg onto their cell surface and promote its conversion to plasmin. Many microbial Plg-binding proteins have been described underpinning the importance this process plays in how bacteria interact with their hosts. Bacteria exploit the proteolytic capabilities of plasmin by (i) targeting the mammalian fibrinolytic system and degrading fibrin clots, (ii) remodeling the extracellular matrix and generating bioactive cleavage fragments of the ECM that influence signaling pathways, (iii) activating matrix metalloproteinases that assist in the destruction of tissue barriers and promote microbial metastasis and (iv) destroying immune effector molecules. There has been little focus on the exploitation of the fibrinolytic system by veterinary pathogens. Here we describe several pathogens of veterinary significance that possess adhesins that bind plasmin(ogen) onto their cell surface and promote its activation to plasmin. Cumulative data suggests that these attributes provide pathogenic and commensal bacteria with a means to colonize and persist within the host environment.
Pub.: 06 May '15, Pinned: 31 Aug '17