Microbiology & Immunology - Research Publications
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ItemPatrolling monocytes promote intravascular neutrophil activation and glomerular injury in the acutely inflamed glomerulus(NATL ACAD SCIENCES, 2016-08-30)Nonclassical monocytes undergo intravascular patrolling in blood vessels, positioning them ideally to coordinate responses to inflammatory stimuli. Under some circumstances, the actions of monocytes have been shown to involve promotion of neutrophil recruitment. However, the mechanisms whereby patrolling monocytes control the actions of neutrophils in the circulation are unclear. Here, we examined the contributions of monocytes to antibody- and neutrophil-dependent inflammation in a model of in situ immune complex-mediated glomerulonephritis. Multiphoton and spinning disk confocal intravital microscopy revealed that monocytes patrol both uninflamed and inflamed glomeruli using β2 and α4 integrins and CX3CR1. Monocyte depletion reduced glomerular injury, demonstrating that these cells promote inappropriate inflammation in this setting. Monocyte depletion also resulted in reductions in neutrophil recruitment and dwell time in glomerular capillaries and in reactive oxygen species (ROS) generation by neutrophils, suggesting a role for cross-talk between monocytes and neutrophils in induction of glomerulonephritis. Consistent with this hypothesis, patrolling monocytes and neutrophils underwent prolonged interactions in glomerular capillaries, with the duration of these interactions increasing during inflammation. Moreover, neutrophils that interacted with monocytes showed increased retention and a greater propensity for ROS generation in the glomerulus. Also, renal patrolling monocytes, but not neutrophils, produced TNF during inflammation, and TNF inhibition reduced neutrophil dwell time and ROS production, as well as renal injury. These findings show that monocytes and neutrophils undergo interactions within the glomerular microvasculature. Moreover, evidence indicates that, in response to an inflammatory stimulus, these interactions allow monocytes to promote neutrophil recruitment and activation within the glomerular microvasculature, leading to neutrophil-dependent tissue injury.
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ItemNo Preview AvailableIncidence and seroprevalence of dengue virus infections in Australian travellers to Asia(SPRINGER, 2012-06)The purpose of this study was to estimate the incidence density and prevalence of dengue virus infection in Australian travellers to Asia. We conducted a multi-centre prospective cohort study of Australian travellers over a 32-month period. We recruited 467 travellers (≥ 16 years of age) from three travel clinics who intended to travel Asia, and 387 (82.9%) of those travellers completed questionnaires and provide samples pre- and post-travel for serological testing for dengue virus infection. Demographic data, destination countries and history of vaccinations and flavivirus infections were obtained. Serological testing for dengue IgG and IgM by enzyme-linked immunosorbent assay (ELISA) (PanBio assay) was performed. Acute seroconversion for dengue infection was demonstrated in 1.0% of travellers, representing an incidence of 3.4 infections per 10,000 days of travel (95% confidence interval [CI]: 0.9-8.7). The seroprevalence of dengue infection was 4.4% and a greater number of prior trips to Asia was a predictor for dengue seroprevalence (p = 0.019). All travellers experienced subclinical dengue infections and had travelled to India (n = 3) and China (n = 1). This significant attack rate of dengue infection can be used to advise prospective travellers to dengue-endemic countries.
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ItemNo Preview AvailableLow Risk of Japanese Encephalitis in Short-Term Australian Travelers to Asia(WILEY-BLACKWELL, 2013)The risk of Japanese encephalitis (JE) in travelers is unknown. In this prospective study, we investigated the incidence of JE in 387 short-term Australian travelers visiting Asia over a 32-month period from August 2007 to February 2010 by performing pre- and post-travel antibody testing. No travelers were infected with JE virus during travel, indicating a low risk of infection for short-term travelers.
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ItemBiosynthesis, Localization, and Macromolecular Arrangement of the Plasmodium falciparum Translocon of Exported Proteins (PTEX)(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2012-03-09)To survive within its host erythrocyte, Plasmodium falciparum must export hundreds of proteins across both its parasite plasma membrane and surrounding parasitophorous vacuole membrane, most of which are likely to use a protein complex known as PTEX (Plasmodium translocon of exported proteins). PTEX is a putative protein trafficking machinery responsible for the export of hundreds of proteins across the parasitophorous vacuole membrane and into the human host cell. Five proteins are known to comprise the PTEX complex, and in this study, three of the major stoichiometric components are investigated including HSP101 (a AAA(+) ATPase), a protein of no known function termed PTEX150, and the apparent membrane component EXP2. We show that these proteins are synthesized in the preceding schizont stage (PTEX150 and HSP101) or even earlier in the life cycle (EXP2), and before invasion these components reside within the dense granules of invasive merozoites. From these apical organelles, the protein complex is released into the host cell where it resides with little turnover in the parasitophorous vacuole membrane for most of the remainder of the following cell cycle. At this membrane, PTEX is arranged in a stable macromolecular complex of >1230 kDa that includes an ∼600-kDa apparently homo-oligomeric complex of EXP2 that can be separated from the remainder of the PTEX complex using non-ionic detergents. Two different biochemical methods undertaken here suggest that PTEX components associate as EXP2-PTEX150-HSP101, with EXP2 associating with the vacuolar membrane. Collectively, these data support the hypothesis that EXP2 oligomerizes and potentially forms the putative membrane-spanning pore to which the remainder of the PTEX complex is attached.
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ItemPhylogeographic, genomic, and meropenem susceptibility analysis of Burkholderia ubonensis(PUBLIC LIBRARY SCIENCE, 2017-09)The bacterium Burkholderia ubonensis is commonly co-isolated from environmental specimens harbouring the melioidosis pathogen, Burkholderia pseudomallei. B. ubonensis has been reported in northern Australia and Thailand but not North America, suggesting similar geographic distribution to B. pseudomallei. Unlike most other Burkholderia cepacia complex (Bcc) species, B. ubonensis is considered non-pathogenic, although its virulence potential has not been tested. Antibiotic resistance in B. ubonensis, particularly towards drugs used to treat the most severe B. pseudomallei infections, has also been poorly characterised. This study examined the population biology of B. ubonensis, and includes the first reported isolates from the Caribbean. Phylogenomic analysis of 264 B. ubonensis genomes identified distinct clades that corresponded with geographic origin, similar to B. pseudomallei. A small proportion (4%) of strains lacked the 920kb chromosome III replicon, with discordance of presence/absence amongst genetically highly related strains, demonstrating that the third chromosome of B. ubonensis, like other Bcc species, probably encodes for a nonessential pC3 megaplasmid. Multilocus sequence typing using the B. pseudomallei scheme revealed that one-third of strains lack the "housekeeping" narK locus. In comparison, all strains could be genotyped using the Bcc scheme. Several strains possessed high-level meropenem resistance (≥32 μg/mL), a concern due to potential transmission of this phenotype to B. pseudomallei. In silico analysis uncovered a high degree of heterogeneity among the lipopolysaccharide O-antigen cluster loci, with at least 35 different variants identified. Finally, we show that Asian B. ubonensis isolate RF23-BP41 is avirulent in the BALB/c mouse model via a subcutaneous route of infection. Our results provide several new insights into the biology of this understudied species.
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ItemMembrane-Wrapping Contributions to Malaria Parasite Invasion of the Human Erythrocyte(CELL PRESS, 2014-07-01)The blood stage malaria parasite, the merozoite, has a small window of opportunity during which it must successfully target and invade a human erythrocyte. The process of invasion is nonetheless remarkably rapid. To date, mechanistic models of invasion have focused predominantly on the parasite actomyosin motor contribution to the energetics of entry. Here, we have conducted a numerical analysis using dimensions for an archetypal merozoite to predict the respective contributions of the host-parasite interactions to invasion, in particular the role of membrane wrapping. Our theoretical modeling demonstrates that erythrocyte membrane wrapping alone, as a function of merozoite adhesive and shape properties, is sufficient to entirely account for the first key step of the invasion process, that of merozoite reorientation to its apex and tight adhesive linkage between the two cells. Next, parasite-induced reorganization of the erythrocyte cytoskeleton and release of parasite-derived membrane can also account for a considerable energetic portion of actual invasion itself, through membrane wrapping. Thus, contrary to the prevailing dogma, wrapping by the erythrocyte combined with parasite-derived membrane release can markedly reduce the expected contributions of the merozoite actomyosin motor to invasion. We therefore propose that invasion is a balance between parasite and host cell contributions, evolved toward maximal efficient use of biophysical forces between the two cells.
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ItemDrug resistance and genetic profile of bacterial species associated with Buruli ulcer wound infections in two districts of Ghana(BMJ, 2017-02)Background: We identified secondary infection of Buruli ulcer (BU) wounds as a cause of healing delay. In order to contribute to the improvement of wound management and reduction of healing delay, we initiated a study to gain understanding of the possible routes of infection and also characterised the resistant profiles of Gram negative bacteria isolated from the wounds of patients attending two health facilities in Ghana. Methods: Staphylococcus aureus isolates were characterised by the spa gene, mecA and the Pantone Valentine Leukocidin (PVL) toxin followed by spa sequencing and whole genome sequencing of a subset of isolates. Phenotypic antibiotic susceptibility testing of Gram negative clinical isolates was performed and multidrug-resistant Pseudomonas aeruginosa identified. The Enterobacteriaceae were further investigated for ESBL and carbapenem production, and some resistance conferring genes were analysed by PCR. Results: Twenty-four isolates were identified as methicillin-resistant S. aureus (MRSA), and lukFS genes encoding PVL were identified in 67 isolates. Typing and sequencing of the spa gene from 91 isolates identified 29 different spa types with t355 (ST152), t186 (ST88), and t346 dominating. While many distinct strains were isolated from both health centres, genotype clustering was identified within centres pointing to possible health care-associated transmission. Phylogenomic analysis confirmed these clusters. Among the GNB, phenotype screening showed widespread resistance to ampicillin, chloramphenicol, ticarcillin-clavulanic acid, cefuroxime and sulphamethoxazole-trimethoprim. ESBL production was confirmed in 15 isolates phenotypically while 61.5% of screen-positive isolates harboured at least one ESBL-conferring gene. Carbapenem encoding genes were detected in 41% of the isolates. Conclusions: Our findings indicate that the health-care environment likely contributes to superinfection of BU wounds and calls for training in wound management and infection control techniques. The observed frequency of ESBL and carbapenem resistance indicates the need to set up surveillance networks and strictly enforce policies which guide the rational use of antibiotics.
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ItemCorrelation of Multi-drug Resistance, Integron and blaESBL Gene Carriage With Genetic Fingerprints of Extended-Spectrum β-Lactamase Producing Klebsiella pneumoniae(AHVAZ JUNDISHAPUR UNIV MED SCI, 2014)BACKGROUND: Some genetic and phenotypic variables are associated among distinct microbial populations. OBJECTIVES: The associations between multi-drug resistance (MDR) phenotypes, prevalence of antibiotic resistance integrons (ARIs), bla SHV, bla TEM and bla CTX-M gene carriage and genetic fingerprints of random amplified polymorphic DNA (RAPD), confirmed by pulsed field gel electrophoresis (PFGE), were investigated among extended-spectrum β-lactamases (ESBL)-producing nosocomial isolates of Klebsiella pneumoniae. MATERIALS AND METHODS: Susceptibility of 35 ESBL-producing K. pneumoniae nosocomial isolates to 22 antimicrobial agents was determined. Integron carriage was detected using specific primers for intI1, intI2 and intI3 genes by PCR. RESULTS: All isolates were resistant to piperacillin and susceptible to imipenem. MDR phenotype was observed in 91.4% of the isolates. Class 1 integrons were detected in 21 (60%) and class 2 integrons in 3 (8.57%) of the isolates. Two of the isolates carried both classes and none harbored class 3 integrons. Significant correlations were observed between resistance to aminoglycosides, fluoroquinolones and sulfonamides, and between genotype groups with carriage of ARIs, MDR phenotype and bla SHV gene carriage. ARI carriage was also significantly associated with MDR phenotype. CONCLUSIONS: Our findings suggest the possible co-carriage of some bla SHV genes and ARIs on the same plasmids harboring the MDR genes. Possible role of integrons in dissemination of ESBL-encoding bla SHV genes among ESBL-producing K. pneumoniae nosocomial isolates may be inferred.
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ItemMevalonate kinase deficiency leads to decreased prenylation of Rab GTPases(NATURE PUBLISHING GROUP, 2016-11)Mevalonate kinase deficiency (MKD) is caused by mutations in a key enzyme of the mevalonate-cholesterol biosynthesis pathway, leading to recurrent autoinflammatory disease characterised by enhanced release of interleukin-1β (IL-1β). It is currently believed that the inflammatory phenotype of MKD is triggered by temperature-sensitive loss of mevalonate kinase activity and reduced biosynthesis of isoprenoid lipids required for the prenylation of small GTPase proteins. However, previous studies have not clearly shown any change in protein prenylation in patient cells under normal conditions. With lymphoblast cell lines from two compound heterozygous MKD patients, we used a highly sensitive in vitro prenylation assay, together with quantitative mass spectrometry, to reveal a subtle accumulation of unprenylated Rab GTPases in cells cultured for 3 days or more at 40 °C compared with 37 °C. This included a 200% increase in unprenylated Rab7A, Rab14 and Rab1A. Inhibition of sterol regulatory element-binding protein (SREBP) activation by fatostatin led to more pronounced accumulation of unprenylated Rab proteins in MKD cells but not parent cells, suggesting that cultured MKD cells may partially overcome the loss of isoprenoid lipids by SREBP-mediated upregulation of enzymes required for isoprenoid biosynthesis. Furthermore, while inhibition of Rho/Rac/Rap prenylation promoted the release of IL-1β, specific inhibition of Rab prenylation by NE10790 had no effect in human peripheral blood mononuclear cells or human THP-1 monocytic cells. These studies demonstrate for the first time that mutations in mevalonate kinase can lead to a mild, temperature-induced defect in the prenylation of small GTPases, but that loss of prenylated Rab GTPases is not the cause of enhanced IL-1β release in MKD.