The core and accessory genomes of Burkholderia pseudomallei: implications for human melioidosis

Natural isolates of Burkholderia pseudomallei (Bp), the causative agent of melioidosis, can exhibit significant ecological flexibility that is likely reflective of a dynamic genome. Using whole-genome Bp microarrays, we examined patterns of gene presence and absence across 94 South East Asian strains isolated from a variety of clinical, environmental, or animal sources. 86% of the Bp K96243 reference genome was common to all the strains representing the Bp "core genome", comprising genes largely involved in essential functions (eg amino acid metabolism, protein translation). In contrast, 14% of the K96243 genome was variably present across the isolates. This Bp accessory genome encompassed multiple genomic islands (GIs), paralogous genes, and insertions/deletions, including three distinct lipopolysaccharide (LPS)-related gene clusters. Strikingly, strains recovered from cases of human melioidosis clustered on a tree based on accessory gene content, and were significantly more likely to harbor certain GIs compared to animal and environmental isolates. Consistent with the inference that the GIs may contribute to pathogenesis, experimental mutation of BPSS2053, a GI gene, reduced microbial adherence to human epithelial cells. Our results suggest that the Bp accessory genome is likely to play an important role in microbial adaptation and virulence.     

The BpsIR quorum-sensing system of Burkholderia pseudomallei

BpsIR, a LuxIR quorum-sensing homolog, is required for optimal expression of virulence and secretion of exoproducts in Burkholderia pseudomallei. Cell density-dependent expression of bpsI and bpsR, the positive regulation of bpsIR expression by BpsR, and the synthesis of N-octanoyl-homoserine lactone (C8HSL) by BpsI are described in this report.     

Membrane-active mechanism of LFchimera against Burkholderia pseudomallei and Burkholderia thailandensis

LFchimera, a construct combining two antimicrobial domains of bovine lactoferrin, lactoferrampin265–284 and lactoferricin17–30, possesses strong bactericidal activity. As yet, no experimental evidence was presented to evaluate the mechanisms of LFchimera against Burkholderia isolates. In this study we analyzed the killing activity of LFchimera on the category B pathogen Burkholderia pseudomallei in comparison to the lesser virulent Burkholderia thailandensis often used as a model for the highly virulent B. pseudomallei. Killing kinetics showed that B. thailandensis E264 was more susceptible for LFchimera than B. pseudomallei 1026b. Interestingly the bactericidal activity of LFchimera appeared highly pH dependent; B. thailandensis killing was completely abolished at and below pH 6.4. FITC-labeled LFchimera caused a rapid accumulation within 15 min in the cytoplasm of both bacterial species. Moreover, freeze-fracture electron microscopy demonstrated extreme effects on the membrane morphology of both bacterial species within 1 h of incubation, accompanied by altered membrane permeability monitored as leakage of nucleotides. These data indicate that the mechanism of action of LFchimera is similar for both species and encompasses disruption of the plasma membrane and subsequently leakage of intracellular nucleotides leading to cell dead.     

Development of a diagnostic system for Burkholderia pseudomallei infections

Monoclonal antibodies were generated against whole cell lysate of Burkholderia pseudomallei. Two out of 6 monoclonal antibodies were found specific and exhibited high affinity against B. pseudomallei, one of which, was utilized to develop sandwich ELISA for detection of specific B. pseudomallei antigen. Immunoassays were found to be specific as no reaction was observed with closely related Burkholderia and Pseudomonas species. Blood samples from experimentally infected mice were found positive for isolation till 4 days post infection (DPI) and ELISA till 10 DPI. One out of 40 sick animal serum samples tested in Thailand was found positive by sandwich ELISA that was earlier confirmed by isolation of B. pseudomallei. The results indicate the potentiality of the assay for its applicability in specific diagnosis of septicaemic melioidosis.     

Identification of differentially expressed proteins from Burkholderia pseudomallei isolated during primary and relapsing melioidosis

Burkholderia pseudomallei causes septicemic melioidosis with a high rate of relapse, however microbial determinants of relapse are unknown. Proteins were analyzed from sequential B. pseudomallei isolates from primary and relapsing melioidosis. Analysis by isotope tagging for relative and absolute quantitation revealed that factors required for nitric oxide detoxification (HmpA) and necessary for anaerobic growth (ArcA, ArcC and ArcB) were highly expressed in the relapse isolate. Two-dimensional gel electrophoresis revealed up-regulation of a putative hemolysin-coregulated protein in the primary isolate, and flagellin and HSP20/alpha crystalline in the relapse isolate. These observations provide targets for further analysis of latency and virulence of melioidosis.     

Burkholderia mallei and Burkholderia pseudomallei. Study of immuno- and pathogenesis of glanders and melioidosis. Heterologous vaccines]

Parameters of the infectious activity of B.mallei and B.pseudomallei for animals of various species were determined. Pathomorphological characteristics of the process of malleus and melioidosis were studied on golden hamsters, mice, guinea pigs, rats and monkeys. Tularemia, plague and salmonellosis vaccines were shown to have protective effects in experimental malleus and melioidosis. An insignificant cross immune response between the malleus and melioidosis pathogens was observed.     

The molecular and cellular basis of pathogenesis in melioidosis: how does Burkholderia pseudomallei cause disease?

Melioidosis, a febrile illness with disease states ranging from acute pneumonia or septicaemia to chronic abscesses, was first documented by Whitmore & Krishnaswami (1912) . The causative agent, Burkholderia pseudomallei , was subsequently identified as a motile, gram-negative bacillus, which is principally an environmental saprophyte. Melioidosis has become an increasingly important disease in endemic areas such as northern Thailand and Australia ( Currie et al. , 2000 ). This health burden, plus the classification of B. pseudomallei as a category B biological agent ( Rotz et al. , 2002 ), has resulted in an escalation of research interest. This review focuses on the molecular and cellular basis of pathogenesis in melioidosis, with a comprehensive overview of the current knowledge on how B. pseudomallei can cause disease. The process of B. pseudomallei movement from the environmental reservoir to attachment and invasion of epithelial and macrophage cells and the subsequent intracellular survival and spread is outlined. Furthermore, the diverse assortment of virulence factors that allow B. pseudomallei to become an effective opportunistic pathogen, as well as to avoid or subvert the host immune response, is discussed. With the recent increase in genomic and molecular studies, the current understanding of the infection process of melioidosis has increased substantially, yet, much still remains to be elucidated.     

Development of protective immunity in a murine model of melioidosis is influenced by the source of Burkholderia pseudomallei antigens

Melioidosis is a potentially fatal disease caused by the bacterium, Burkholderia pseudomallei. The current study was carried out to determine the mechanisms involved in the development of protective immunity in a murine model of melioidosis. Following intravenous infection with B. pseudomallei, both C57BL/6 and BALB/c mice demonstrated delayed-type hypersensitivity responses and lymphocyte proliferation towards B. pseudomallei antigens, indicating the generation of B. pseudomallei-specific lymphocytes. Adoptive transfer of these lymphocytes to na?ve C57BL/6 mice was demonstrated by a delayed-type hypersensitivity response. Mice were not protected from a subsequent lethal challenge with a highly virulent strain of B. pseudomallei, suggesting that a single intravenous dose of the bacterium is insufficient to induce a protective adaptive immune response. Attempts to induce resistance in susceptible BALB/c mice used repetitive low-dose exposure to live B. pseudomallei. Immune responses and resistance following subcutaneous immunization with live B. pseudomallei were compared with exposure to heat-killed, culture filtrate and sonicated B. pseudomallei antigens. Compared to heat-killed B. pseudomallei, significant protection was generated in BALB/c mice following immunization with live bacteria. Our studies also demonstrate that the type of immune response generated in vivo is influenced by the antigenic preparation of B. pseudomallei used for immunization.     

BLF1, the first Burkholderia pseudomallei toxin, connects inhibition of host protein synthesis with melioidosis

Melioidosis is a disease caused by infection with Burkholderia pseudomallei. The molecular basis for the pathogenicity of B. pseudomallei is poorly understood. However, recent work has identified the first toxin from this bacterium and shown that it inhibits host protein synthesis. Here, we review the illness that is potentially associated with biological warfare, the pathogen and its deadly molecular mechanism of action, as well as therapeutic developments that may follow.     

Burkholderia multivorans acts as an antagonist against the growth of Burkholderia pseudomallei in soil

In this study, it was demonstrated, by using agar diffusion tests and a Transwell system, that Burkholderia multivorans NKI379 has an antagonistic effect against the growth of B. pseudomallei. Bacterial representatives were isolated from agricultural crop soil and mixed to construct a partial bacterial community structure that was based on the results of reproducible patterns following PCR-denaturing gradient gel electrophoresis analysis of total soil chromosomes. The antagonistic effect of B. multivorans on B. pseudomallei was observed in this imitate community. In a field study of agricultural crop soil, the presence of B. pseudomallei was inversely related to the presence of the antagonistic strains B. multivorans or B. cenocepacia. B. multivorans NKI379 can survive in a broader range of pH, temperatures and salt concentrations than B. pseudomallei, suggesting that B. multivorans can adapt to extreme environmental changes and therefore predominates over B. pseudomallei in natural environments.     

Roles and interactions of Burkholderia pseudomallei BpsIR quorum-sensing system determinants

The Burkholderia pseudomallei quorum-sensing system (QSS), designated BpsIR, is encoded by five bpsR genes and three bpsI genes. This study investigated the roles and interactions of the QSS determinants in terms of gene regulation and protein interaction. We report two novel findings, that BpsR can function as an activator and a repressor for bpsI expression and that BpsR may form homodimers and heterodimers.     

In silico analysis of Burkholderia pseudomallei genome sequence for potential drug targets

Recent advances in DNA sequencing technology have enabled elucidation of whole genome information from a plethora of organisms. In parallel with this technology, various bioinformatics tools have driven the comparative analysis of the genome sequences between species and within isolates. While drawing meaningful conclusions from a large amount of raw material, computer-aided identification of suitable targets for further experimental analysis and characterization, has also led to the prediction of non-human homologous essential genes in bacteria as promising candidates for novel drug discovery. Here, we present a comparative genomic analysis to identify essential genes in Burkholderia pseudomallei. Our in silico prediction has identified 312 essential genes which could also be potential drug candidates. These genes encode essential proteins to support the survival of B. pseudomallei including outer-inner membrane and surface structures, regulators, proteins involved in pathogenenicity, adaptation, chaperones as well as degradation of small and macromolecules, energy metabolism, information transfer, central/intermediate/miscellaneous metabolism pathways and some conserved hypothetical proteins of unknown function. Therefore, our in silico approach has enabled rapid screening and identification of potential drug targets for further characterization in the laboratory.     

Out of the ground: aerial and exotic habitats of the melioidosis bacterium Burkholderia pseudomallei in grasses in Australia

Melioidosis is an emerging infectious disease of humans and animals in the tropics caused by the soil bacterium Burkholderia pseudomallei. Despite high fatality rates, the ecology of B.pseudomallei remains unclear. We used a combination of field and laboratory studies to investigate B.pseudomallei colonization of native and exotic grasses in northern Australia. Multivariable and spatial analyses were performed to determine significant predictors for B.pseudomallei occurrence in plants and soil collected longitudinally from field sites. In plant inoculation experiments, the impact of B.pseudomallei upon these grasses was studied and the bacterial load semi-quantified. Fluorescence in situ hybridization and confocal laser scanning microscopy were performed to localize the bacteria in plants. Burkholderia pseudomallei was found to inhabit not only the rhizosphere and roots but also aerial parts of specific grasses. This raises questions about the potential spread of B.pseudomallei by grazing animals whose droppings were found to be positive for these bacteria. In particular, B.pseudomallei readily colonized exotic grasses introduced to Australia for pasture. The ongoing spread of these introduced grasses creates new habitats suitable for B.pseudomallei survival and may be an important factor in the evolving epidemiology of melioidosis seen both in northern Australia and elsewhere globally.     

Genomic islands as a marker to differentiate between clinical and environmental Burkholderia pseudomallei

Burkholderia pseudomallei, as a saprophytic bacterium that can cause a severe sepsis disease named melioidosis, has preserved several extra genes in its genome for survival. The sequenced genome of the organism showed high diversity contributed mainly from genomic islands (GIs). Comparative genome hybridization (CGH) of 3 clinical and 2 environmental isolates, using whole genome microarrays based on B. pseudomallei K96243 genes, revealed a difference in the presence of genomic islands between clinical and environmental isolates. The largest GI, GI8, of B. pseudomallei was observed as a 2 sub-GI named GIs8.1 and 8.2 with distinguishable %GC content and unequal presence in the genome. GIs8.1, 8.2 and 15 were found to be more common in clinical isolates. A new GI, GI16c, was detected on chromosome 2. Presences of GIs8.1, 8.2, 15 and 16c were evaluated in 70 environmental and 64 clinical isolates using PCR assays. A combination of GIs8.1 and 16c (positivity of either GI) was detected in 70% of clinical isolates and 11.4% of environmental isolates (P<0.001). Using BALB/c mice model, no significant difference of time to mortality was observed between K96243 isolate and three isolates without GIs under evaluation (P>0.05). Some virulence genes located in the absent GIs and the difference of GIs seems to contribute less to bacterial virulence. The PCR detection of 2 GIs could be used as a cost effective and rapid tool to detect potentially virulent isolates that were contaminated in soil.     

Alanine racemase mutants of Burkholderia pseudomallei and Burkholderia mallei and use of alanine racemase as a non-antibiotic-based selectable marker

Burkholderia pseudomallei and Burkholderia mallei are category B select agents and must be studied under BSL3 containment in the United States. They are typically resistant to multiple antibiotics, and the antibiotics used to treat B. pseudomallei or B. mallei infections may not be used as selective agents with the corresponding Burkholderia species. Here, we investigated alanine racemase deficient mutants of B. pseudomallei and B. mallei for development of non-antibiotic-based genetic selection methods and for attenuation of virulence. The genome of B. pseudomallei K96243 has two annotated alanine racemase genes (bpsl2179 and bpss0711), and B. mallei ATCC 23344 has one (bma1575). Each of these genes encodes a functional enzyme that can complement the alanine racemase deficiency of Escherichia coli strain ALA1. Herein, we show that B. pseudomallei with in-frame deletions in both bpsl2179 and bpss0711, or B. mallei with an in-frame deletion in bma1575, requires exogenous D-alanine for growth. Introduction of bpsl2179 on a multicopy plasmid into alanine racemase deficient variants of either Burkholderia species eliminated the requirement for D-alanine. During log phase growth without D-alanine, the viable counts of alanine racemase deficient mutants of B. pseudomallei and B. mallei decreased within 2 hours by about 1000-fold and 10-fold, respectively, and no viable bacteria were present at 24 hours. We constructed several genetic tools with bpsl2179 as a selectable genetic marker, and we used them without any antibiotic selection to construct an in-frame ΔflgK mutant in the alanine racemase deficient variant of B. pseudomallei K96243. In murine peritoneal macrophages, wild type B. mallei ATCC 23344 was killed much more rapidly than wild type B. pseudomallei K96243. In addition, the alanine racemase deficient mutant of B. pseudomallei K96243 exhibited attenuation versus its isogenic parental strain with respect to growth and survival in murine peritoneal macrophages.     

Structure of a Burkholderia pseudomallei Trimeric Autotransporter Adhesin Head

Background

Pathogenic bacteria adhere to the host cell surface using a family of outer membrane proteins called Trimeric Autotransporter Adhesins (TAAs). Although TAAs are highly divergent in sequence and domain structure, they are all conceptually comprised of a C-terminal membrane anchoring domain and an N-terminal passenger domain. Passenger domains consist of a secretion sequence, a head region that facilitates binding to the host cell surface, and a stalk region.

Methodology/Principal Findings

Pathogenic species of Burkholderia contain an overabundance of TAAs, some of which have been shown to elicit an immune response in the host. To understand the structural basis for host cell adhesion, we solved a 1.35 Å resolution crystal structure of a BpaA TAA head domain from Burkholderia pseudomallei, the pathogen that causes melioidosis. The structure reveals a novel fold of an intricately intertwined trimer. The BpaA head is composed of structural elements that have been observed in other TAA head structures as well as several elements of previously unknown structure predicted from low sequence homology between TAAs. These elements are typically up to 40 amino acids long and are not domains, but rather modular structural elements that may be duplicated or omitted through evolution, creating molecular diversity among TAAs.

Conclusions/Significance

The modular nature of BpaA, as demonstrated by its head domain crystal structure, and of TAAs in general provides insights into evolution of pathogen-host adhesion and may provide an avenue for diagnostics.