Characterising putative effector proteins of burkholderia pseudomallei
thesisposted on 17.02.2017 by D'Cruze, Tanya
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Burkholderia pseudomallei, the aetiological agent of melioidosis, is classified as a category B bioterrorism agent (Stevens et al., 2004) by the United States Centre for Disease Control and Prevention. B. pseudomallei is an aerobic gram-negative saprophytic bacterium (Stevens et al., 2002), capable of dwelling in soil, stagnant water and rice paddies (Jones et al., 1996), in tropical climates. The bacterium is therefore endemic in parts of Thailand, northern Australia, Malaysia, Singapore, Vietnam and Burma (Wiersinga et al., 2006). Melioidosis was first described in Burma some 100 years ago (Whitmore and Krishnaswami, 1912). Infection is usually acquired through cutaneous inoculation – wounds and existing skin lesions (Wiersinga et al., 2006), or by inhalation or aspiration of contaminated water (Harland et al., 2007). An epidemiological survey carried out between 1997 and 2006 in north-east Thailand estimated the incidence of melioidosis to be 21.31 cases per 100,000, per annum in endemic regions, with the mortality rate being 40.5% (Limmathurotsakul et al., 2010). This high mortality rate places B. pseudomallei infection as the third most common cause of death in north-east Thailand, after HIV/AIDs and tuberculosis (Limmathurotsakul et al., 2010). Despite the severity of the disease, there are no available vaccines, and limited antibiotic options, as B. pseudomallei exhibits resistance to traditionally used antibiotics. Further study into the pathogenesis and virulence of B. pseudomallei is essential in working towards an effective therapy for melioidosis. Several B. pseudomallei virulence factors have been identified, including the capsule, pili, flagella, LPS, quorum sensing molecules, and Type Six and Type Three Secretory Systems (T6/T3SS). Similar to other gram-negative bacteria such as Salmonella typhimurium, Shigella flexneri and Yersinia enterocolitica, B. pseudomallei employs its TTSS (Sun et al., 2005) to translocate virulence effectors into the host cell (Roversi et al., 2007). B. pseudomallei has three TTSS gene clusters (designated TTSS1, TTSS2 and TTSS3) each of these clusters is present on the small chromosome. Previous work has shown that TTSS1 and TTSS2 are not involved in B. pseudomallei virulence in a hamster infection model (Warawa and Woods, 2005). TTSS3 contains the Burkholderia Secretion Apparatus (bsa) locus which shares homology with the S. typhimurium inv/spa/prg TTSS (Sun et al., 2005), and the S. flexneri ipa/mxi/spa TTSS (Ogawa and Sasakawa, 2006b). B. pseudomallei has been shown to invade either non-phagocytic (Stevens and Galyov, 2004), or phagocytic strains (Kespichayawattana et al., 2000). Prior to host cell invasion, the bacterium employs its TTSS3 to inject effector proteins into the host cytoplasm. Within 15 m of being phagocytosed into a host cell, bacteria escape from their phagosomes into the host cell cytoplasm by lysing the phagosomal membrane using the pre-secreted effector proteins (Wiersinga et al., 2006), and continue its infectivity cycle. Invading bacteria, viruses, fungi and parasites can initiate the autophagic response in mammalian cells to eliminate the intracellular pathogens and liberate metabolites that may have been utilised during pathogen infection, thus promoting cell survival (Orvedahl and Levine, 2008). Following infection, microbes such as B. pseudomallei, Listeria monocytogenes and S. flexneri have been found to escape the phagosome and then avoid autophagic capture, replicate and spread (Ogawa and Sasakawa, 2006a). The first component of this study was to conduct a literature search to identify virulence factors employed by bacteria to escape from their confining phagosomes. Initially, a list of 22 virulence factors was compiled. Bioinformatic analysis identified five of the corresponding 22 open reading frames (ORF) as sharing significant homology with a sequence within the B. pseudomallei genome, namely: BPSS1531 (bipC), BPSS1532 (bipB), BPSL0670, BPSS1394 (bpscN) and BPSS0670. The available reports at that time also identified mutants defective in the BPSS1529 (bipD) and BPSS1539 genes as being confined to the phagosome. Hence these two genes along with the five above mentioned ORFs were selected for characterisation. Based on reports using yeast to screen for putative bacterial effectors, the initial phase of characterisation involved amplification by PCR of each of the seven sequences encoding a B. pseudomallei ORF for expression in Saccharomyces cerevisiae. The primary screen was to observe any morphological or physiological changes conferred which arise under nutrient starvation (autophagic) conditions. Yeast strains also expressed either a cytosolic, mitochondrial or nucleus targeted Rosella biosensor. Each biosensor was composed of a pH-sensitive variant of green fluorescent protein (GFP) linked to a pH-stable variant of the red fluorescent protein, DsRed. When cells are subjected to autophagic conditions, delivery of the specific target organelle (for the relevant Rosella biosensor) to the vacuole results in accumulation of red fluorescence in the vacuole. The results indicated that each of the B. pseudomallei proteins, when expressed in yeast under nutrient starvation, was able to produce a multiple vacuolar morphology. The deficiency of red fluorescence in the vacuoles during starvation, suggested that the autophagy of cells expressing either the cytosolic or mitochondrial of those components was impaired. On the contrary, nucleus uptake during starvation, was identified through the accumulation of red fluorescence in the vacuoles. These results suggested that expression of each B. pseudomallei ORF had effects on membrane events in yeast cells. Given their influence on vacuole morphology and organelle turnover in S. cerevisiae, inactivation of each of these genes in the B. pseudomallei genome was attempted in order to determine any phenotype of mutant bacteria on infection of mammalian cells. Using the double cross-over allelic exchange method, three deletion mutants were successfully constructed in the B. pseudomallei K96243 strain, namely BPSS1532 (bipB), BPSL0670 and BPSS1394 (bpscN). Despite concerted efforts, deletion mutations in the other four genes could not be constructed. The BipB protein is a structural component of the TTSS needle. Using the in vivo relative growth assay, the BPSS1532 mutant appeared to be only partially attenuated for virulence in mice. Subsequent analysis of bacterial survival and replication in cultured murine RAW264.7 GFP-LC3 macrophage-like cells showed no significant difference to wild-type bacterium over the 6 h infection period. Consistent with this finding, the level of co-localisation between mutant bacteria and GFP-LC3 decreased over 6 h essentially in parallel to wild-type bacteria. These results suggest that mutant bacteria are able to escape the phagosome and evade intracellular killing as efficiently as wild-type bacteria. BPSL0670 is a putative cation transporter efflux protein. The BPSL0670 deletion mutant was also found to be partially attenuated for virulence in BALB/c mice and showed diminished survival and replication compared to wild-type bacteria following infection of cells in culture. A two-fold increase in the co-localisation of mutant bacteria with GFP-LC3 at 6 h p.i. (compared with wild-type bacteria) correlated with its decreased survival and replication. BpscN is a putative type three secretion associated protein and a hypothetical ATPase. The BPSS1394 – bpscN mutant encoded in TTSS1 was attenuated for virulence in a mouse model of infection. Additional studies using cultured RAW264.7 macrophage-like cells showed that while mutant bacteria were able to escape from phagosomes, they showed diminished survival and replication in RAW264.7 cells, and increased levels of co-localisation with the autophagy marker protein LC3. Complementation data indicated that intracellular survival and replication could be restored to about 50% of wild-type levels, confirming that the loss of function of bpscN strongly influences survival and replication. Collectively the data provide strong evidence that the TTSS1 bpscN gene plays an important role in B. pseudomallei pathogenesis. This is the first research crediting the TTSS1 for its role in B. pseudomallei virulence and justifies further research into TTSS1 effector proteins. This research aimed to identify and characterise potential virulence factors involved in the escape of B. pseudomallei from the phagosome in host cells. Deletion mutants were generated for the BPSS1532, BPSL0670 and BPSS1394 ORFs in the B. pseudomallei genome. The BPSS1532 and BPSL0670 mutants were found to be partially attenuated for virulence, whilst BPSS1394 was fully attenuated for virulence in the BALB/c melioidosis infection model.