Microbiology & Immunology - Research Publications

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    Patrolling monocytes promote intravascular neutrophil activation and glomerular injury in the acutely inflamed glomerulus
    Finsterbusch, M ; Hall, P ; Li, A ; Devi, S ; Westhorpe, CLV ; Kitching, AR ; Hickey, MJ (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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    The structure and activity of the glutathione reductase from Streptococcus pneumoniae
    Sikanyika, M ; Aragao, D ; McDevitt, CA ; Maher, MJ (INT UNION CRYSTALLOGRAPHY, 2019-01)
    The glutathione reductase (GR) from Streptococcus pneumoniae is a flavoenzyme that catalyzes the reduction of oxidized glutathione (GSSG) to its reduced form (GSH) in the cytoplasm of this bacterium. The maintenance of an intracellular pool of GSH is critical for the detoxification of reactive oxygen and nitrogen species and for intracellular metal tolerance to ions such as zinc. Here, S. pneumoniae GR (SpGR) was overexpressed and purified and its crystal structure determined at 2.56 Å resolution. SpGR shows overall structural similarity to other characterized GRs, with a dimeric structure that includes an antiparallel β-sheet at the dimer interface. This observation, in conjunction with comparisons with the interface structures of other GR enzymes, allows the classification of these enzymes into three classes. Analyses of the kinetic properties of SpGR revealed a significantly higher value for Km(GSSG) (231.2 ± 24.7 µM) in comparison to other characterized GR enzymes.
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    Defining the Role of the Streptococcus agalactiae Sht-Family Proteins in Zinc Acquisition and Complement Evasion
    Moulin, P ; Rong, V ; Ribeiro E Silva, A ; Pederick, VG ; Camlade, E ; Mereghetti, L ; McDevitt, CA ; Hiron, A ; Federle, MJ (American Society for Microbiology, 2019-04)
    Streptococcus agalactiae is not only part of the human intestinal and urogenital microbiota but is also a leading cause of septicemia and meningitis in neonates. Its ability to cause disease depends upon the acquisition of nutrients from its environment, including the transition metal ion zinc. The primary zinc acquisition system of the pathogen is the Adc/Lmb ABC permease, which is essential for viability in zinc-restricted environments. Here, we show that in addition to the AdcCB transporter and the three zinc-binding proteins, Lmb, AdcA, and AdcAII, S. agalactiae zinc homeostasis also involves two streptococcal histidine triad (Sht) proteins. Sht and ShtII are required for zinc uptake via the Lmb and AdcAII proteins with apparent overlapping functionality and specificity. Both Sht-family proteins possess five-histidine triad motifs with similar hierarchies of importance for Zn homeostasis. Independent of its contribution to zinc homeostasis, Sht has previously been reported to bind factor H leading to predictions of a contribution to complement evasion. Here, we investigated ShtII to ascertain whether it had similar properties. Analysis of recombinant Sht and ShtII reveals that both proteins have similar affinities for factor H binding. However, neither protein aided in resistance to complement in human blood. These findings challenge prior inferences regarding the in vivo role of the Sht proteins in resisting complement-mediated clearance. IMPORTANCE This study examined the role of the two streptococcal histidine triad (Sht) proteins of Streptococcus agalactiae in zinc homeostasis and complement resistance. We showed that Sht and ShtII facilitate zinc homeostasis in conjunction with the metal-binding proteins Lmb and AdcAII. Here, we show that the Sht-family proteins are functionally redundant with overlapping roles in zinc uptake. Further, this work reveals that although the Sht-family proteins bind to factor H in vitro this did not influence survival in human blood.
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    Incidence and seroprevalence of dengue virus infections in Australian travellers to Asia
    Ratnam, I ; Black, J ; Leder, K ; Biggs, B-A ; Matchett, E ; Padiglione, A ; Woolley, I ; Panagiotidis, T ; Gherardin, T ; Pollissard, L ; Demont, C ; Luxemburger, C ; Torresi, J (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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    Low Risk of Japanese Encephalitis in Short-Term Australian Travelers to Asia
    Ratnam, I ; Leder, K ; Black, J ; Biggs, B-A ; Matchett, E ; Padiglione, A ; Woolley, I ; Panagiotidis, T ; Gherardin, T ; Luxemburger, C ; Torresi, J (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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    Biosynthesis, Localization, and Macromolecular Arrangement of the Plasmodium falciparum Translocon of Exported Proteins (PTEX)
    Bullen, HE ; Charnaud, SC ; Kalanon, M ; Riglar, DT ; Dekiwadia, C ; Kangwanrangsan, N ; Torii, M ; Tsuboi, T ; Baum, J ; Ralph, SA ; Cowman, AF ; de Koning-Ward, TF ; Crabb, BS ; Gilson, PRD (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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    Conservation of a Glycine-rich Region in the Prion Protein Is Required for Uptake of Prion Infectivity
    Harrison, CF ; Lawson, VA ; Coleman, BM ; Kim, Y-S ; Masters, CL ; Cappai, R ; Barnham, KJ ; Hill, AF (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010-06-25)
    Prion diseases are associated with the misfolding of the endogenously expressed prion protein (designated PrP(C)) into an abnormal isoform (PrP(Sc)) that has infectious properties. The hydrophobic domain of PrP(C) is highly conserved and contains a series of glycine residues that show perfect conservation among all species, strongly suggesting it has functional and evolutionary significance. These glycine residues appear to form repeats of the GXXXG protein-protein interaction motif (two glycines separated by any three residues); the retention of these residues is significant and presumably relates to the functionality of PrP(C). Mutagenesis studies demonstrate that minor alterations to this highly conserved region of PrP(C) drastically affect the ability of cells to uptake and replicate prion infection in both cell and animal bioassay. The localization and processing of mutant PrP(C) are not affected, although in vitro and in vivo studies demonstrate that this region is not essential for interaction with PrP(Sc), suggesting these residues provide conformational flexibility. These data suggest that this region of PrP(C) is critical in the misfolding process and could serve as a novel, species-independent target for prion disease therapeutics.
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    Determinants of the Specificity of Rotavirus Interactions with the α2β1 Integrin
    Fleming, FE ; Graham, KL ; Takada, Y ; Coulson, BS (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2011-02-25)
    The human α2β1 integrin binds collagen and acts as a cellular receptor for rotaviruses and human echovirus 1. These ligands require the inserted (I) domain within the α2 subunit of α2β1 for binding. Previous studies have identified the binding sites for collagen and echovirus 1 in the α2 I domain. We used CHO cells expressing mutated α2β1 to identify amino acids involved in binding to human and animal rotaviruses. Residues where mutation affected rotavirus binding were located in several exposed loops and adjacent regions of the α2 I domain. Binding by all rotaviruses was eliminated by mutations in the activation-responsive αC-α6 and αF helices. This is a novel feature that distinguishes rotavirus from other α2β1 ligands. Mutation of residues that co-ordinate the metal ion (Ser-153, Thr-221, and Glu-256 in α2 and Asp-130 in β1) and nearby amino acids (Ser-154, Gln-215, and Asp-219) also inhibited rotavirus binding. The importance of most of these residues was greatest for binding by human rotaviruses. These mutations inhibit collagen binding to α2β1 (apart from Glu-256) but do not affect echovirus binding. Overall, residues where mutation affected both rotavirus and collagen recognition are located at one side of the metal ion-dependent adhesion site, whereas those important for collagen alone cluster nearby. Mutations eliminating rotavirus and echovirus binding are distinct, consistent with the respective preference of these viruses for activated or inactive α2β1. In contrast, rotavirus and collagen utilize activated α2β1 and show an overlap in α2β1 residues important for binding.
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    Phylogeographic, genomic, and meropenem susceptibility analysis of Burkholderia ubonensis
    Price, EP ; Sarovich, DS ; Webb, JR ; Hall, CM ; Jaramillo, SA ; Sahl, JW ; Kaestli, M ; Mayo, M ; Harrington, G ; Baker, AL ; Sidak-Loftis, LC ; Settles, EW ; Lummis, M ; Schupp, JM ; Gillece, JD ; Tuanyok, A ; Warner', J ; Busch, JD ; Keim, P ; Currie, BJ ; Wagner, DM ; Torres, AG (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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    Tracing the environmental footprint of the Burkholderia pseudomallei lipopolysaccharide genotypes in the tropical "Top End" of the Northern Territory, Australia
    Webb, JR ; Rachlin, A ; Rigas, V ; Sarovich, DS ; Price, EP ; Kaestli, M ; Ward, LM ; Mayo, M ; Currie, BJ ; Torres, AG (PUBLIC LIBRARY SCIENCE, 2019-07)
    The Tier 1 select agent Burkholderia pseudomallei is an environmental bacterium that causes melioidosis, a high mortality disease. Variably present genetic markers used to elucidate strain origin, relatedness and virulence in B. pseudomallei include the Burkholderia intracellular motility factor A (bimA) and filamentous hemagglutinin 3 (fhaB3) gene variants. Three lipopolysaccharide (LPS) O-antigen types in B. pseudomallei have been described, which vary in proportion between Australian and Asian isolates. However, it remains unknown if these LPS types can be used as genetic markers for geospatial analysis within a contiguous melioidosis-endemic region. Using a combination of whole-genome sequencing (WGS), statistical analysis and geographical mapping, we examined if the LPS types can be used as geographical markers in the Northern Territory, Australia. The clinical isolates revealed that LPS A prevalence was highest in the Darwin and surrounds (n = 660; 96% being LPS A and 4% LPS B) and LPS B in the Katherine and Katherine remote and East Arnhem regions (n = 79; 60% being LPS A and 40% LPS B). Bivariate logistics regression of 999 clinical B. pseudomallei isolates revealed that the odds of getting a clinical isolate with LPS B was highest in East Arnhem in comparison to Darwin and surrounds (OR 19.5, 95% CI 9.1-42.0; p<0.001). This geospatial correlation was subsequently confirmed by geographically mapping the LPS type from 340 environmental Top End strains. We also found that in the Top End, the minority bimA genotype bimABm has a similar remote region geographical footprint to that of LPS B. In addition, correlation of LPS type with multi-locus sequence typing (MLST) was strong, and where multiple LPS types were identified within a single sequence type, WGS confirmed homoplasy of the MLST loci. The clinical, sero-diagnostic and vaccine implications of geographically-based B. pseudomallei LPS types, and their relationships to regional and global dispersal of melioidosis, require global collaborations with further analysis of larger clinically and geospatially-linked datasets.