2,214 research outputs found

    Burkholderia Hep_Hag autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis

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    Background The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei. Results Using bacteriophage-mediated immunoscreening we identified genes expressed in vivo during experimental equine glanders infection. A family of immunodominant antigens were identified that share protein domain architectures with hemagglutinins and invasins. These have been designated Burkholderia Hep_Hag autotransporter (BuHA) proteins. A total of 110/207 positive clones (53%) of a B. mallei expression library screened with sera from two infected horses belonged to this family. This contrasted with 6/189 positive clones (3%) of a B. pseudomallei expression library screened with serum from 21 patients with culture-proven melioidosis. Conclusion Members of the BuHA proteins are found in other Gram-negative bacteria and have been shown to have important roles related to virulence. Compared with other bacterial species, the genomes of both B. mallei and B. pseudomallei contain a relative abundance of this family of proteins. The domain structures of these proteins suggest that they function as multimeric surface proteins that modulate interactions of the cell with the host and environment. Their effect on the cellular immune response to B. mallei and their potential as diagnostics for glanders requires further study

    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

    Burkholderia pseudomallei Isolates from Sarawak, Malaysian Borneo, Are Predominantly Susceptible to Aminoglycosides and Macrolides

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    Melioidosis is a potentially fatal disease caused by the saprophytic bacterium Burkholderia pseudomallei. Resistance to gentamicin is generally a hallmark of B. pseudomallei, and gentamicin is a selective agent in media used for diagnosis of melioidosis. In this study, we determined the prevalence and mechanism of gentamicin susceptibility found in B. pseudomallei isolates from Sarawak, Malaysian Borneo. We performed multilocus sequence typing and antibiotic susceptibility testing on 44 B. pseudomallei clinical isolates from melioidosis patients in Sarawak district hospitals. Whole-genome sequencing was used to identify the mechanism of gentamicin susceptibility. A novel allelic-specific PCR was designed to differentiate gentamicin-sensitive isolates from wild-type B. pseudomallei. A reversion assay was performed to confirm the involvement of this mechanism in gentamicin susceptibility. A substantial proportion (86%) of B. pseudomallei clinical isolates in Sarawak, Malaysian Borneo, were found to be susceptible to the aminoglycoside gentamicin, a rare occurrence in other regions where B. pseudomallei is endemic. Gentamicin sensitivity was restricted to genetically related strains belonging to sequence type 881 or its single-locus variant, sequence type 997. Whole-genome sequencing identified a novel nonsynonymous mutation within amrB, encoding an essential component of the AmrAB-OprA multidrug efflux pump. We confirmed the role of this mutation in conferring aminoglycoside and macrolide sensitivity by reversion of this mutation to the wild-type sequence. Our study demonstrates that alternative B. pseudomallei selective media without gentamicin are needed for accurate melioidosis laboratory diagnosis in Sarawak. This finding may also have implications for environmental sampling of other locations to test for B. pseudomallei endemicity

    Phylogenetic analysis reveals an ancient gene duplication as the origin of the MdtABC efflux pump.

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    The efflux pumps from the Resistance-Nodulation-Division family, RND, are main contributors to intrinsic antibiotic resistance in Gram-negative bacteria. Among this family, the MdtABC pump is unusual by having two inner membrane components. The two components, MdtB and MdtC are homologs, therefore it is evident that the two components arose by gene duplication. In this paper, we describe the results obtained from a phylogenetic analysis of the MdtBC pumps in the context of other RNDs. We show that the individual inner membrane components (MdtB and MdtC) are conserved throughout the Proteobacterial species and that their existence is a result of a single gene duplication. We argue that this gene duplication was an ancient event which occurred before the split of Proteobacteria into Alpha-, Beta- and Gamma- classes. Moreover, we find that the MdtABC pumps and the MexMN pump from Pseudomonas aeruginosa share a close common ancestor, suggesting the MexMN pump arose by another gene duplication event of the original Mdt ancestor. Taken together, these results shed light on the evolution of the RND efflux pumps and demonstrate the ancient origin of the Mdt pumps and suggest that the core bacterial efflux pump repertoires have been generally stable throughout the course of evolution

    Landscape Changes Influence the Occurrence of the Melioidosis Bacterium Burkholderia pseudomallei in Soil in Northern Australia

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    Melioidosis is a severe disease affecting humans and animals in the tropics. It is caused by the bacterium Burkholderia pseudomallei, which lives in tropical soil and especially occurs in southeast Asia and northern Australia. Despite the recognition that melioidosis is an emerging infectious disease, little is known about the habitat of B. pseudomallei in the environment. We performed a survey in the Darwin area in tropical Australia, screening 809 soil samples for the presence of these bacteria using molecular methods. We found that environmental factors describing the habitat of these bacteria differed between environmentally undisturbed and disturbed sites. At undisturbed sites, B. pseudomallei was primarily found in close proximity to streams and in grass- and roots-rich areas. In disturbed soil, B. pseudomallei was associated with the presence of animals, farming or irrigation. Highest B. pseudomallei counts were retrieved from paddocks, pens and kennels holding livestock and dogs. This study contributes to the elucidation of the habitat of B. pseudomallei in northern Australia. It also raises concerns that B. pseudomallei may spread due to changes in land management

    No hints for abundance of Bacillus anthracis and Burkholderia pseudomallei in 100 environmental samples from Cameroon

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    Background: Little is known on the abundance of the pathogens Bacillus anthracis and Burkholderia pseudomallei in environmental samples in Cameroon. Therefore, 100 respective samples were assessed in a proof-of-principle assessment. Methods: DNA residuals from nucleic acid extractions of 100 environmental samples, which were collected between 2011 and 2013 in the Mape Basin of Cameroon, were screened for B. anthracis and B. pseudomallei by real-time PCR. The samples comprised soil samples with water contact (n = 88), soil samples without water contact (n = 6), plant material with water contact (n = 3), water (n = 2), and soil from a hospital dressing room (n = 1). Results: B. anthracis and B. pseudomallei were detected in none of the samples assessed. Conclusion: The results indicate that at least a quantitatively overwhelming, ubiquitous occurrence of B. anthracis and B. pseudomallei in the environment in Cameroon is highly unlikely. However, the number and choice of the assessed samples limit the interpretability of the results

    Burkholderia pseudomallei in a lowland rice paddy: seasonal changes and influence of soil depth and physico-chemical properties.

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    Melioidosis, a severe infection with the environmental bacterium Burkholderia pseudomallei, is being recognised increasingly frequently. What determines its uneven distribution within endemic areas is poorly understood. We cultured soil from a rice field in Laos for B. pseudomallei at different depths on 4 occasions over a 13-month period. We also measured physical and chemical parameters in order to identify associated characteristics. Overall, 195 of 653 samples (29.7%) yielded B. pseudomallei. A higher prevalence of B. pseudomallei was found at soil depths greater than the 30?cm currently recommended for B. pseudomallei environmental sampling. B. pseudomallei was associated with a high soil water content and low total nitrogen, carbon and organic matter content. Our results suggested that a sampling grid of 25 five metre square quadrats (i.e. 25?×?25?m) should be sufficient to detect B. pseudomallei at a given location if samples are taken at a soil depth of at least 60?cm. However, culture of B. pseudomallei in environmental samples is difficult and liable to variation. Future studies should both rely on molecular approaches and address the micro-heterogeneity of soil when investigating physico-chemical associations with the presence of B. pseudomallei

    Burkholderia pseudomallei: an update on disease, virulence and host interaction

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    Burkholderia pseudomallei is the etiological agent of melioidosis, a life-threatening disease of humans and animals that occurs primarily in Southeast Asia and northern Australia. The distribution of B. pseudomallei and occurrence of melioidosis globally is very much evident. This soil and water-dwelling saprophyte is resilient to various environments. This organism has gained further notoriety following the Centre for Disease Control’s classification of B. pseudomalleias a Tier 1 biological agent. Despite several decades of clinical research, the mortality rate for melioidosis remains high. Genomics-based studies have demonstrated the plasticity of the B. pseudomallei genome and the coding sequences consists of a myriad of functions that enable the bacteria to adapt to these hostile environments as well as various hosts. Diagnosis is mainly based on bacterial culture or serological assays whilst treatment is limited to third generation cephalosporins. To date, no vaccine is currently available as an immunoprophylaxis for melioidosis. By utilising the available genome sequence, a number of virulence factors have recently been identified and have provided more insight into the pathogenesis of B. pseudomallei. These factors include surface associated proteins as well as secreted effector proteins and their corresponding secretion systems. In addition, a number of recent studies on host-pathogen interaction have also demonstrated how the pathogen is able to subvert the host immune system and survive within the cell. This review presents an overview of the current understanding on B. pseudomallei pathogenesis and the disease melioidosis

    Identification of Burkholderia pseudomallei Near-Neighbor Species in the Northern Territory of Australia

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    Identification and characterization of near-neighbor species are critical to the development of robust molecular diagnostic tools for biothreat agents. One such agent, Burkholderia pseudomallei, a soil bacterium and the causative agent of melioidosis, is lacking in this area because of its genomic diversity and widespread geographic distribution. The Burkholderia genus contains over 60 species and occupies a large range of environments including soil, plants, rhizospheres, water, animals and humans. The identification of novel species in new locations necessitates the need to identify the true global distribution of Burkholderia species, especially the members that are closely related to B. pseudomallei. In our current study, we used the Burkholderia-specific recA sequencing assay to analyze environmental samples from the Darwin region in the Northern Territory of Australia where melioidosis is endemic. Burkholderia recA PCR negative samples were further characterized using 16s rRNA sequencing for species identification. Phylogenetic analysis demonstrated that over 70% of the bacterial isolates were identified as B. ubonensis indicating that this species is common in the soil where B. pseudomallei is endemic. Bayesian phylogenetic analysis reveals many novel branches within the B. cepacia complex, one novel B. oklahomensis-like species, and one novel branch containing one isolate that is distinct from all other samples on the phylogenetic tree. During the analysis with recA sequencing, we discovered 2 single nucleotide polymorphisms in the reverse priming region of B. oklahomensis. A degenerate primer was developed and is proposed for future use. We conclude that the recA sequencing technique is an effective tool to classify Burkholderia and identify soil organisms in a melioidosis endemic area
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