Microbiology & Immunology - Research Publications
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ItemPI3K Activation in Neural Stem Cells Drives Tumorigenesis which can be Ameliorated by Targeting the cAMP Response Element Binding (CREB) Protein(Oxford University Press, 2018-10)BACKGROUND: Hyperactivation of phosphoinositide 3-kinase (PI3K) signaling is common in cancers, but the precise role of the pathway in glioma biology remains to be determined. Some understanding of PI3K signaling mechanisms in brain cancer comes from studies on neural stem/progenitor cells (NSPCs), where signals transmitted via the PI3K pathway cooperate with other intracellular pathways and downstream transcription factors to regulate critical cell functions. METHODS: To investigate the role of the PI3K pathway in glioma initiation and development, we generated a mouse model targeting the inducible expression of a PIK3CAH1047A oncogenic mutant and deletion of the PI3K negative regulator, phosphatase and tensin homolog (PTEN), to NSPCs. RESULTS: Expression of a Pik3caH1047A was sufficient to generate tumors with oligodendroglial features, but simultaneous loss of PTEN was required for the development of invasive, high-grade glioma. Pik3caH1047A-PTEN mutant NSPCs exhibited enhanced neurosphere formation which correlated with increased Wnt signaling, while loss of cAMP response element binding protein (CREB) in Pik3caH1047A-Pten mutant tumors led to longer symptom-free survival in mice. CONCLUSION: Taken together, our findings present a novel mouse model for glioma demonstrating that the PI3K pathway is important for initiation of tumorigenesis and that disruption of downstream CREB signaling attenuates tumor expansion.
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ItemInvestigating Neural Stem Cell and Glioma Stem Cell Self-renewal Potential Using Extreme Limiting Dilution Analysis (ELDA)(BIO-PROTOCOL, 2018-09-05)Glioma stem cells (GSC) grown as neurospheres exhibit similar characteristics to neural stem cells (NSC) grown as neurospheres, including the ability to self-renew and differentiate. GSCs are thought to play a role in cancer initiation and progression. Self-renewal potential of GSCs is thought to reflect many characteristics associated with malignancy, including tumor recurrence following cytotoxic therapy due to their proliferative dormancy and capacity to allow for the development of resistant tumor cell sub-clones due to mutations acquired during their differentiation. Here, we demonstrate that using extreme limiting dilution analysis (ELDA), subtle differences in the frequency of sphere-forming potential between PI3K-mutant oncogenic NSCs and non-oncogenic NSCs can be measured, in vitro. We further show how ELDA can be used on cells, before and after forced differentiation to amplify inherent differences in sphere-forming potential between mutant and control NSCs. Ultimately, ELDA exploits a difference in the ability of a single or a few seeded stem cells to self-renew, divide and form neurospheres. Importantly, the assay also allows a comparison between genetically distinct cells or between the same cells under different conditions, where the impact of target-specific drugs or other novel cancer stem cell therapies can be tested.
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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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ItemThe structure and activity of the glutathione reductase from Streptococcus pneumoniae(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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ItemDefining the Role of the Streptococcus agalactiae Sht-Family Proteins in Zinc Acquisition and Complement Evasion(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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ItemTracing the environmental footprint of the Burkholderia pseudomallei lipopolysaccharide genotypes in the tropical "Top End" of the Northern Territory, Australia(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.
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ItemMelioidosis fatalities in captive slender-tailed meerkats (Suricata suricatta): combining epidemiology, pathology and whole-genome sequencing supports variable mechanisms of transmission with one health implications(BMC, 2019-12-19)BACKGROUND: Melioidosis is a tropical infectious disease which is being increasingly recognised throughout the globe. Infection occurs in humans and animals, typically through direct exposure to soil or water containing the environmental bacterium Burkholderia pseudomallei. Case clusters of melioidosis have been described in humans following severe weather events and in exotic animals imported into melioidosis endemic zones. Direct transmission of B. pseudomallei between animals and/or humans has been documented but is considered extremely rare. Between March 2015 and October 2016 eight fatal cases of melioidosis were reported in slender-tailed meerkats (Suricata suricatta) on display at a Wildlife Park in Northern Australia. To further investigate the melioidosis case cluster we sampled the meerkat enclosure and adjacent park areas and performed whole-genome sequencing (WGS) on all culture-positive B. pseudomallei environmental and clinical isolates. RESULTS: WGS confirmed that the fatalities were caused by two different B. pseudomallei sequence types (STs) but that seven of the meerkat isolates were highly similar on the whole-genome level. Used concurrently with detailed pathology data, our results demonstrate that the seven cases originated from a single original source, but routes of infection varied amongst meerkats belonging to the clonal outbreak cluster. Moreover, in some instances direct transmission may have transpired through wounds inflicted while fighting. CONCLUSIONS: Collectively, this study supports the use of high-resolution WGS to enhance epidemiological investigations into transmission modalities and pathogenesis of melioidosis, especially in the instance of a possible clonal outbreak scenario in exotic zoological collections. Such findings from an animal outbreak have important One Health implications.
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ItemOpportunistic pathogens and large microbial diversity detected in source-to-distribution drinking water of three remote communities in Northern Australia(PUBLIC LIBRARY SCIENCE, 2019-09)In the wet-dry tropics of Northern Australia, drinking water in remote communities is mostly sourced from bores accessing groundwater. Many aquifers contain naturally high levels of iron and some are shallow with surface water intrusion in the wet season. Therefore, environmental bacteria such as iron-cycling bacteria promoting biofilm formation in pipes or opportunistic pathogens can occur in these waters. An opportunistic pathogen endemic to northern Australia and Southeast Asia and emerging worldwide is Burkholderia pseudomallei. It causes the frequently fatal disease melioidosis in humans and animals. As we know very little about the microbial composition of drinking water in remote communities, this study aimed to provide a first snapshot of the microbiota and occurrence of opportunistic pathogens in bulk water and biofilms from the source and through the distribution system of three remote water supplies with varying iron levels. Using 16s-rRNA gene sequencing, we found that the geochemistry of the groundwater had a substantial impact on the untreated microbiota. Different iron-cycling bacteria reflected differences in redox status and nutrients. We cultured and sequenced B. pseudomallei from bores with elevated iron and from a multi-species biofilm which also contained iron-oxidizing Gallionella, nitrifying Nitrospira and amoebae. Gallionella are increasingly used in iron-removal filters in water supplies and more research is needed to examine these interactions. Similar to other opportunistic pathogens, B. pseudomallei occurred in water with low organic carbon levels and with low heterotrophic microbial growth. No B. pseudomallei were detected in treated water; however, abundant DNA of another opportunistic pathogen group, non-tuberculous mycobacteria was recovered from treated parts of one supply. Results from this study will inform future studies to ultimately improve management guidelines for water supplies in the wet-dry tropics.
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ItemA cluster of melioidosis infections in hatchling saltwater crocodiles (Crocodylus porosus) resolved using genome-wide comparison of a common north Australian strain of Burkholderia pseudomallei(MICROBIOLOGY SOC, 2019-08)Burkholderia pseudomallei is a Gram-negative saprophytic bacillus and the aetiological agent of melioidosis, a disease of public-health importance throughout Southeast Asia and northern Australia. Infection can occur in humans and a wide array of animal species, though zoonotic transmission and case clusters are rare. Despite its highly plastic genome and extensive strain diversity, fine-scale investigations into the population structure of B. pseudomallei indicate there is limited geographical dispersal amongst sequence types (STs). In the 'Top End' of northern Australia, five STs comprise 90 % of the overall abundance, the most prevalent and widespread of which is ST-109. In May 2016, ST-109 was implicated in two fatal cases of melioidosis in juvenile saltwater crocodiles at a wildlife park near Darwin, Australia. To determine the probable source of infection, we sampled the crocodile enclosures and analysed the phylogenetic relatedness of crocodile and culture-positive ST-109 environmental park isolates against an additional 135 ST-109 B. pseudomallei isolates from the Top End. Collectively, our whole-genome sequencing (WGS) and pathology findings confirmed B. pseudomallei detected in the hatchling incubator as the likely source of infection, with zero SNPs identified between clinical and environmental isolates. Our results also demonstrate little variation across the ST-109 genome, with SNPs in recombinogenic regions and one suspected case of ST homoplasy accounting for nearly all observed diversity. Collectively, this study supports the use of WGS for outbreak source attribution in highly recombinogenic pathogens, and confirms the epidemiological and phylogenetic insights that can be gained from high-resolution sequencing platforms.
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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.