1,321 research outputs found

    A Machine Learning-Based Raman Spectroscopic Assay for the Identification of Burkholderia mallei and Related Species

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    Burkholderia (B.) mallei, the causative agent of glanders, and B. pseudomallei, the causative agent of melioidosis in humans and animals, are genetically closely related. The high infectious potential of both organisms, their serological cross-reactivity, and similar clinical symptoms in human and animals make the differentiation from each other and other Burkholderia species challenging. The increased resistance against many antibiotics implies the need for fast and robust identification methods. The use of Raman microspectroscopy in microbial diagnostic has the potential for rapid and reliable identification. Single bacterial cells are directly probed and a broad range of phenotypic information is recorded, which is subsequently analyzed by machine learning methods. Burkholderia were handled under biosafety level 1 (BSL 1) conditions after heat inactivation. The clusters of the spectral phenotypes and the diagnostic relevance of the Burkholderia spp. were considered for an advanced hierarchical machine learning approach. The strain panel for training involved 12 B. mallei, 13 B. pseudomallei and 11 other Burkholderia spp. type strains. The combination of top- and sub-level classifier identified the mallei-complex with high sensitivities (>95%). The reliable identification of unknown B. mallei and B. pseudomallei strains highlighted the robustness of the machine learning-based Raman spectroscopic assay

    Characterization of the role of the Burkholderia pseudomallei type 3 secretion system using in vivo imaging.

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    Melioidosis is a fatal infectious disease caused by the Tier 1 Select Agent Burkholderia pseudomallei. Hallmarks of melioidosis include pneumonic disease and prominent septicaemic spread. Both forms of disease are contingent upon the bacterium’s intracellular life cycle and particularly on its ability to escape from host cell phagosomes. Upon encountering a host cell, B. pseudomallei is internalized into membrane-bound vacuoles from which the bacterium must rapidly escape to the cytoplasm in order to replicate and promote its survival. In the host cytoplasm, B. pseudomallei is capable of polymerizing actin for intracellular and intercellular motility and spread, lysing the host cell and perpetuating the cycle of infection. Commonly used intranasal and aerosol models to study respiratory melioidosis result in significant upper respiratory tract colonization, dramatically altering disease progression. Accordingly, we developed an improved lung-specific instillation approach to deliver bacteria directly into mice lungs, coupled with in vivo optical imaging and observed the development of disease that closely resembles human melioidosis in mice. We found that in the absence of upper respiratory tract infection, a capsular polysaccharide (CPS) mutant is only 6.8-fold attenuated. This mutant is unable to spread to secondary sites of infection, consistent with the role of capsule in protecting the bacterium from host antimicrobial activity. Similarly, a type 3 secretion system cluster 3 (T3SS3) structural mutant is spread deficient, yet this mutant is attenuated 290-fold, strongly suggesting that T3SS3 is critical for respiratory melioidosis. Having a strong platform for studying the pathogenesis of B. pseudomallei in a mouse model of lung-specific melioidosis, we used transposon mutagenesis to comprehensively identify virulence factors required for B. pseudomallei lung colonization and spread to the liver and spleen. Notably, T3SS3, capsular polysaccharide and type 6 secretion system cluster 5 (T6SS5) were the major genetic loci required for respiratory melioidosis. A T6SS5 mutant is not attenuated by LD50 estimations using our lung-specific melioidosis mouse model. Yet by competition analysis T6SS5, T3SS3 and CPS mutants were attenuated, substantiating the requirement of these factors for B. pseudomallei infection as previously reported. These results highlight the importance of competition analysis for studying the fitness of distinct virulence determinants. Importantly, T3SS3 was the only virulence determinant attenuated by both LD50 analysis and competition studies, corroborating the critical requirement of this virulence system for respiratory melioidosis. B. pseudomallei is a facultative intracellular cytosolic bacterium and its ability to survive intracellularly is fundamental to mammalian host infection. Upon B. pseudomallei internalization into host cell vacuoles, the bacterium rapidly escapes this compartment by action of the T3SS3. The T3SS3 is required by B. pseudomallei for intracellular survival by translocating protein effectors to the cytoplasm of host cells and mediating the rapid escape of the bacterium from phagosomes of these cells. We hypothesized that effectors act in concert to mediate B. pseudomallei’s rapid escape from phagosomes of host cells by manipulating host signaling pathways and promoting bacterial survival. Using a high-resolution, high-throughput in vivo imaging screening approach we profiled the contributions of five of the six B. pseudomallei putative type 3 effectors to the bacterium’s rapid phagosome escape. Effector mutants exhibited distinct temporal differences in escape, with bopA inactivation resulting in the most pronounced delay in vacuolar rupture, strongly suggesting that BopA directly mediates escape of B. pseudomallei from endocytic vesicles. We confirmed that a previously identified BopA host target, the trafficking particle protein C8 (TRAPPC8), colocalized with Burkholderia containing vacuoles. Small interfering RNA knockdown of this protein strongly suggests that TRAPPC8 is required for vacuolar membrane stabilization. These findings substantiate the significant role of the T3SS3 and provide a paradigm to study B. pseudomallei natural infection processes and potential vaccine and therapeutic targets

    Structure analysis of biologically important prokaryotic glycopolymers

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    Of the many post-translational modifications organisms can undertake, glycosylation is the most prevalent and the most diverse. The research in this thesis focuses on the structural characterisation of glycosylation in two classes of glycopolymer (lipopolysaccharide (LPS) and glycoprotein) in two domains of life (bacteria and archaea). The common theme linking these subprojects is the development and application of high sensitivity analytical techniques, primarily mass spectrometry (MS), for studying prokaryotic glycosylation. Many prokaryotes produce glycan arrangements with extraordinary variety in composition and structure. A further challenge is posed by additional functionalities such as lipids whose characterisation is not always straightforward. Glycosylation in prokaryotes has a variety of different biological functions, including their important roles in the mediation of interactions between pathogens and hosts. Thus enhanced knowledge of bacterial glycosylation may be of therapeutic value, whilst a better understanding of archaeal protein glycosylation will provide further targets for industrial applications, as well as insight into this post- translational modification across evolution and protein processing under extreme conditions. The first sub-project focused on the S-layer glycoprotein of the halophilic archeaon Haloferax volcanii, which has been reported to be modified by both glycans and lipids. Glycoproteomic and associated MS technologies were employed to characterise the N- and O-linked glycosylation and to explore putative lipid modifications. Approximately 90% of the S-layer was mapped and N-glycans were identified at all the mapped consensus sites, decorated with a pentasaccharide consisting of two hexoses, two hexuronic acids and a methylated hexuronic acid. The O-glycans are homogeneously identified as a disaccharide consisting of galactose and glucose. Unexpectedly it was found that membrane-derived lipids were present in the S- layer samples despite extensive purification, calling into question the predicted presence of covalently linked lipid. The H. volcanii N-glycosylation is mediated by the products of the agl gene cluster and the functional characterisation of members of the agl gene cluster was investigated by MS analysis of agl-mutant strains of the S-layer. Burkholderia pseudomallei is the causative agent of melioidosis, a serious and often fatal disease in humans which is endemic in South-East Asia and other equatorial regions. Its LPS is vital for serum resistance and the O-antigen repeat structures are of interest as vaccine targets. B. pseudomallei is reported to produce several polysaccharides, amongst which the already characterised ‘typical’ O-antigen of K96243 represents 97% of the strains. The serologically distinct ‘atypical’ strain 576 produces a different LPS, whose characterisation is the subject of this research project. MS strategies coupled with various hydrolytic and chemical derivatisation methodologies were employed to define the composition and potential sequences of the O-antigen repeat unit. These MS strategies were complemented by a novel NMR technique involving embedding of the LPS into micelles. Taken together the MS and NMR data have revealed a highly unusual O-antigen structure for atypical LPS which is remarkably different from the typical O-antigen. The development of structural analysis tools in MS and NMR applicable to the illustrated types of glycosylation in these prokaryotes will give a more consistent approach to sugar characterisation and their modifications thus providing more informative results for pathogenicity and immunological studies as well as pathway comparisons.Open Acces

    Accurate and Rapid Identification of the Burkholderia pseudomallei Near-Neighbour, Burkholderia ubonensis, Using Real-Time PCR

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    Burkholderia ubonensis is an environmental bacterium belonging to the Burkholderia cepacia complex (Bcc), a group of genetically related organisms that are associated with opportunistic but generally nonfatal infections in healthy individuals. In contrast, the near-neighbour species Burkholderia pseudomallei causes melioidosis, a disease that can be fatal in up to 95% of cases if left untreated. B. ubonensis is frequently misidentified as B. pseudomallei from soil samples using selective culturing on Ashdown’s medium, reflecting both the shared environmental niche and morphological similarities of these species. Additionally, B. ubonensis shows potential as an important biocontrol agent in B. pseudomallei-endemic regions as certain strains possess antagonistic properties towards B. pseudomallei. Current methods for characterising B. ubonensis are laborious, time-consuming and costly, and as such this bacterium remains poorly studied. The aim of our study was to develop a rapid and inexpensive real-time PCR-based assay specific for B. ubonensis. We demonstrate that a novel B. ubonensis-specific assay, Bu550, accurately differentiates B. ubonensis from B. pseudomallei and other species that grow on selective Ashdown’s agar. We anticipate that Bu550 will catalyse research on B. ubonensis by enabling rapid identification of this organism from Ashdown’s-positive colonies that are not B. pseudomallei

    Melioidosis Vaccines: A Systematic Review and Appraisal of the Potential to Exploit Biodefense Vaccines for Public Health Purposes

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    The designation of Burkholderia pseudomallei as a category B select agent has resulted in considerable research funding to develop a protective vaccine. This bacterium also causes a naturally occurring disease (melioidosis), an important cause of death in many countries including Thailand and Australia. In this study, we explored whether a vaccine could be used to provide protection from melioidosis. An economic evaluation based on its use in Thailand indicated that a vaccine could be a cost-effective intervention if used in high-risk populations such as diabetics and those with chronic kidney or lung disease. A literature search of vaccine studies in animal models identified the current candidates, but noted that models failed to take account of the common routes of infection in natural melioidosis and major risk factors for infection, primarily diabetes. This review highlights important areas for future research if biodefence-driven vaccines are to play a role in reducing the global incidence of melioidosis

    Type three secretion system-mediated escape of Burkholderia pseudomallei into the host cytosol is critical for the activation of NFκB.

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    BackgroundBurkholderia pseudomallei is the causative agent of melioidosis, a potentially fatal disease endemic in Southeast Asia and Northern Australia. This Gram-negative pathogen possesses numerous virulence factors including three "injection type" type three secretion systems (T3SSs). B. pseudomallei has been shown to activate NFκB in HEK293T cells in a Toll-like receptor and MyD88 independent manner that requires T3SS gene cluster 3 (T3SS3 or T3SSBsa). However, the mechanism of how T3SS3 contributes to NFκB activation is unknown.ResultsKnown T3SS3 effectors are not responsible for NFκB activation. Furthermore, T3SS3-null mutants are able to activate NFκB almost to the same extent as wildtype bacteria at late time points of infection, corresponding to delayed escape into the cytosol. NFκB activation also occurs when bacteria are delivered directly into the cytosol by photothermal nanoblade injection.ConclusionsT3SS3 does not directly activate NFκB but facilitates bacterial escape into the cytosol where the host is able to sense the presence of the pathogen through cytosolic sensors leading to NFκB activation

    Tracing melioidosis back to the source: using whole-genome sequencing to investigate an outbreak originating from a contaminated domestic water supply

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    Melioidosis, a disease of public health importance in Southeast Asia and northern Australia, is caused by the Gram-negative soil bacillus Burkholderia pseudomallei. Melioidosis is typically acquired through environmental exposure, and case clusters are rare, even in regions where the disease is endemic. B. pseudomallei is classed as a tier 1 select agent by the Centers for Disease Control and Prevention; from a biodefense perspective, source attribution is vital in an outbreak scenario to rule out a deliberate release. Two cases of melioidosis within a 3-month period at a residence in rural northern Australia prompted an investigation to determine the source of exposure. B. pseudomallei isolates from the property's groundwater supply matched the multilocus sequence type of the clinical isolates. Whole-genome sequencing confirmed the water supply as the probable source of infection in both cases, with the clinical isolates differing from the likely infecting environmental strain by just one single nucleotide polymorphism (SNP) each. For the first time, we report a phylogenetic analysis of genomewide insertion/deletion (indel) data, an approach conventionally viewed as problematic due to high mutation rates and homoplasy. Our whole-genome indel analysis was concordant with the SNP phylogeny, and these two combined data sets provided greater resolution and a better fit with our epidemiological chronology of events. Collectively, this investigation represents a highly accurate account of source attribution in a melioidosis outbreak and gives further insight into a frequently overlooked reservoir of B. pseudomallei. Our methods and findings have important implications for outbreak source tracing of this bacterium and other highly recombinogenic pathogens

    Antibody interactions with the capsular polysaccharide of Burkholderia pseudomallei

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    Burkholderia pseudomallei is an important human pathogen that causes melioidosis. Infection is highly lethal and notoriously difficult to diagnose and treat. As such, it has tremendous bioterror potential and has been classified as a Tier 1 select agent by the Centers for Disease Control and the Department of Health & Human Services. One reason that B. pseudomallei is a successful pathogen is that it is surrounded by a high molecular weight capsular polysaccharide (CPS) comprised of mannoheptopyranose residues. CPS inhibits complement deposition, prevents phagocytosis, and greatly enhances virulence. Previous studies have indicated that antibodies targeting CPS have high therapeutic value and can be used to diagnose B. pseudomallei infection. The present work describes the development and characterization of 15 monoclonal antibodies (mAbs) in an effort to further the understanding of how antibodies interact with B. pseudomallei CPS. We have generated two complete Immunoglobulin G (IgG) subclass families; subclass families are antibodies that have identical variable regions, but different constant regions, and thus different effector functions. We have determined that some of these mAbs are protective in a murine model of pulmonary melioidosis in a subclass-independent manner. In this study, protection appears to be a function of mAb binding affinity. Additionally, we determined that non-IgG3 mAbs are best for diagnosing active infection. Isolating a high affinity IgG3 and generating a subclass-switch family yielded mAbs with low affinities that did not perform well in a diagnostic test format. Thus, immunization strategies should focus on eliciting alternative IgG immune responses. Using this information, we have updated a prototype Active Melioidosis Detect™ Lateral Flow Immunoassay (AMD LFI) by replacing the original IgG3 mAb with a high affinity IgG1 mAb. This updated AMD LFI has increased sensitivity, is highly specific, and rapid; it can detect B. pseudomallei CPS in multiple sample types in 15 minutes or less

    Whole-genome sequencing of a quarter-century melioidosis outbreak in temperate Australia uncovers a region of low-prevalence endemicity

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    This study was funded by the National Health and Medical Research Council via awards 1046812 and 1098337, and the Wellcome Trust Sanger Institute via award 098051. S.J.P. receives funding from the NIHR Cambridge Biomedical Research Centre.Melioidosis, caused by the highly recombinogenic bacterium Burkholderia pseudomallei, is a disease with high mortality. Tracing the origin of melioidosis outbreaks and understanding how the bacterium spreads and persists in the environment are essential to protecting public and veterinary health and reducing mortality associated with outbreaks. We used whole-genome sequencing to compare isolates from a historical quarter-century outbreak that occurred between 1966 and 1991 in the Avon Valley, Western Australia, a region far outside the known range of B. pseudomallei endemicity. All Avon Valley outbreak isolates shared the same multilocus sequence type (ST-284), which has not been identified outside this region. We found substantial genetic diversity among isolates based on a comparison of genome-wide variants, with no clear correlation between genotypes and temporal, geographical or source data. We observed little evidence of recombination in the outbreak strains, indicating that genetic diversity among these isolates has primarily accrued by mutation. Phylogenomic analysis demonstrated that the isolates confidently grouped within the Australian B. pseudomallei clade, thereby ruling out introduction from a melioidosis-endemic region outside Australia. Collectively, our results point to B. pseudomallei ST-284 being present in the Avon Valley for longer than previously recognized, with its persistence and genomic diversity suggesting long-term, low-prevalence endemicity in this temperate region. Our findings provide a concerning demonstration of the potential for environmental persistence of B. pseudomallei far outside the conventional endemic regions. An expected increase in extreme weather events may reactivate latent B. pseudomallei populations in this region.Publisher PDFPeer reviewe
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