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.     

Knockout and pullout recombineering for naturally transformable Burkholderia thailandensis and Burkholderia pseudomallei

Phage λ-Red proteins are powerful tools for pulling and knocking out chromosomal fragments but have been limited to the γ-proteobacteria. Procedures are described here to easily knock out (KO) and pull out (PO) chromosomal DNA fragments from naturally transformable Burkholderia thailandensis and Burkholderia pseudomallei. This system takes advantage of published compliant counterselectable and selectable markers (sacB, pheS, gat and the arabinose-utilization operon) and λ-Red mutant proteins. pheS-gat (KO) or oriT-ColE1ori-gat-ori1600-rep (PO) PCR fragments are generated with flanking 40- to 45-bp homologies to targeted regions incorporated on PCR primers. One-step recombination is achieved by incubation of the PCR product with cells expressing λ-Red proteins and subsequent selection on glyphosate-containing medium. This procedure takes ~10 d and is advantageous over previously published protocols: (i) smaller PCR products reduce primer numbers and amplification steps, (ii) PO fragments suitable for downstream manipulation in Escherichia coli are obtained and (iii) chromosomal KO increases flexibility for downstream processing.     

The factors dictating the codon usage variation among the genes in the genome of Burkholderia pseudomallei

Burkholderia pseudomallei is a recognized biothreat agent and the causative agent of melioidosis. Codon usage biases of all protein-coding genes (length greater than or equal to 300 bp) from the complete genome of B. pseudomallei K96243 have been analyzed. As B. pseudomallei is a GC-rich organism (68.5%), overall codon usage data analysis indicates that indeed codons ending in G and/or C are predominant in this organism. But multivariate statistical analysis indicates that there is a single major trend in the codon usage variation among the genes in this organism, which has a strong positively correlation with the expressivities of the genes. The majority of the lowly expressed genes are scattered towards the negative end of the major axis whereas the highly expressed genes are clustered towards the positive end. At the same time, from the results that there were two significant correlations between axis 1 coordinates and the GC, GC3s content at silent sites of each sequence, and clearly significant negatively correlations between the ‘Effective Number of Codons’ values and GC, GC3s content, we inferred that codon usage bias was affected by gene nucleotide composition also. In addition, some other factors such as the lengths of the genes as well as the hydrophobicity of genes also influence the codon usage variation among the genes in this organism in a minor way. At the same time, notably, 21 codons have been defined as ‘optimal codons’ of the B. pseudomallei. In summary, our work have provided a basic understanding of the mechanisms for codon usage bias and some more useful information for improving the expression of target genes in vivo and in vitro. Sheng Zhao and Qin Zhang contributed equally to this work.     

A high resolution four-locus multiplex single nucleotide repeat (SNR) genotyping system in Bacillus anthracis

The allelic identities of Single Nucleotide Repeat (SNR) markers in Bacillus anthracis are typically ascertained by DNA sequencing through the direct repeat. Here we describe a reproducible method for genotyping closely related isolates by using four SNR loci in a multiplex-PCR capillary electrophoresis system amenable to high-throughput analysis.     

Sequence- and Structure-Based Immunoreactive Epitope Discovery for Burkholderia pseudomallei Flagellin

Burkholderia pseudomallei is a Gram-negative bacterium responsible for melioidosis, a serious and often fatal infectious disease that is poorly controlled by existing treatments. Due to its inherent resistance to the major antibiotic classes and its facultative intracellular pathogenicity, an effective vaccine would be extremely desirable, along with appropriate prevention and therapeutic management. One of the main subunit vaccine candidates is flagellin of Burkholderia pseudomallei (FliC_Bp). Here, we present the high resolution crystal structure of FliC_Bp and report the synthesis and characterization of three peptides predicted to be both B and T cell FliC_Bp epitopes, by both structure-based in silico methods, and sequence-based epitope prediction tools. All three epitopes were shown to be immunoreactive against human IgG antibodies and to elicit cytokine production from human peripheral blood mononuclear cells. Furthermore, two of the peptides (F51-69 and F270-288) were found to be dominant immunoreactive epitopes, and their antibodies enhanced the bactericidal activities of purified human neutrophils. The epitopes derived from this study may represent potential melioidosis vaccine components.     

Development of a multiplex PCR assay for rapid identification of Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex

We have developed a multiplex PCR assay for rapid identification and differentiation of cultures for Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex. The assay is valuable for use in clinical and veterinary laboratories, and in a deployable laboratory during outbreaks.     

The wbiA locus is required for the 2-O-acetylation of lipopolysaccharides expressed by Burkholderia pseudomallei and Burkholderia thailandensis

Burkholderia pseudomallei and Burkholderia thailandensis express similar O-antigens (O-PS II) in which their 6-deoxy-alpha-L-talopyranosyl (L-6dTalp) residues are variably substituted with O-acetyl groups at the O-2 or O-4 positions. In previous studies we demonstrated that the protective monoclonal antibody, Pp-PS-W, reacted with O-PS II expressed by wild-type B. pseudomallei strains but not by a B. pseudomallei wbiA null mutant. In the present study we demonstrate that WbiA activity is required for the acetylation of the L-6dTalp residues at the O-2 position and that structural modification of O-PS II molecules at this site is critical for recognition by Pp-PS-W.     

Genetic diversity and microevolution of Burkholderia pseudomallei in the environment

Background

The soil dwelling Gram-negative pathogen Burkholderia pseudomallei is the cause of melioidosis. The diversity and population structure of this organism in the environment is poorly defined.

Methods and Findings

We undertook a study of B. pseudomallei in soil sampled from 100 equally spaced points within 237.5 m² of disused land in northeast Thailand. B. pseudomallei was present on direct culture of 77/100 sampling points. Genotyping of 200 primary plate colonies from three independent sampling points was performed using a combination of pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Twelve PFGE types and nine sequence types (STs) were identified, the majority of which were present at only a single sampling point. Two sampling points contained four STs and the third point contained three STs. Although the distance between the three sampling points was low (7.6, 7.9, and 13.3 meters, respectively), only two STs were present in more than one sampling point. Each of the three samples was characterized by the localized expansion of a single B. pseudomallei clone (corresponding to STs 185, 163, and 93). Comparison of PFGE and MLST results demonstrated that two STs contained strains with variable PFGE banding pattern types, indicating geographic structuring even within a single MLST-defined clone.

Conclusions

We discuss the implications of this extreme structuring of genotype and genotypic frequency in terms of micro-evolutionary dynamics and ecology, and how our results may inform future sampling strategies.     

Actin-binding proteins from Burkholderia mallei and Burkholderia thailandensis can functionally compensate for the actin-based motility defect of a Burkholderia pseudomallei bimA mutant

Recently we identified a bacterial factor (BimA) required for actin-based motility of Burkholderia pseudomallei. Here we report that Burkholderia mallei and Burkholderia thailandensis are capable of actin-based motility in J774.2 cells and that BimA homologs of these bacteria can restore the actin-based motility defect of a B. pseudomallei bimA mutant. While the BimA homologs differ in their amino-terminal sequence, they interact directly with actin in vitro and vary in their ability to bind Arp3.     

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

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.     

High resolution structure of BipD: an invasion protein associated with the type III secretion system of Burkholderia pseudomallei

Burkoldheria pseudomallei is a Gram-negative bacterium that possesses a protein secretion system similar to those found in Salmonella and Shigella. Recent work has indicated that the protein encoded by the BipD gene of B. pseudomallei is an important secreted virulence factor. BipD is similar in sequence to IpaD from Shigella and SipD from Salmonella and is therefore likely to be a translocator protein in the type-III secretion system of B. pseudomallei. The crystal structure of BipD has been solved at a resolution of 2.1 A revealing the detailed tertiary fold of the molecule. The overall structure is appreciably extended and consists of a bundle of antiparallel alpha-helical segments with two small beta-sheet regions. The longest helices of the molecule form a four-helix bundle and most of the remaining secondary structure elements (three helices and two three-stranded beta-sheets) are formed by the region linking the last two helices of the four-helix bundle. The structure suggests that the biologically active form of the molecule may be a dimer formed by contacts involving the C-terminal alpha-helix, which is the most strongly conserved part of the protein. Comparison of the structure of BipD with immunological and other data for IpaD indicates that the C-terminal alpha-helix is also involved in contacts with other proteins that form the translocon.     

类鼻疽伯克霍尔德菌感染与免疫逃逸机制的研究进展

类鼻疽是由类鼻疽伯克霍尔德菌(Burkholderia pseudomallei,B. pseudomallei)(简称类鼻疽菌)感染引起的一种热带医学疾病。该病临床表现复杂多样,严重感染时可快速发展为败血症,病死率高达40%。越来越多的证据表明,它是一种正在扩散的人兽共患传染病。本文就近年来关于类鼻疽菌感染的重要毒力因子以及其在免疫逃逸中的作用机制研究进展进行总结,以期了解类鼻疽菌的致病机制,为将来有效疫苗和治疗药物的研发提供理论指导。     

The mviN homolog in Burkholderia pseudomallei is essential for viability and virulence

The virulence factors of Burkholderia pseudomallei, the causative agent of melioidosis, are not fully understood. We have identified a gene with homology to the Salmonella typhimurium mouse virulence gene, mviN, a member of the mouse virulence factor family. Expression studies with an insertional mutant containing a lux operon demonstrated that the expression of the gene is influenced by free-iron availability in the media and by growth phase. The mutant displayed an increased LD50 value in the hamster infection model and a loss of the ability to invade human lung epithelial cells. The mutant has a slower growth rate than that of the wild type. Both defects were restored to various degrees when complemented in trans with the mviN gene. The mutant contains an insertion at 1229 bp of the 1548 bp gene, resulting in a truncated protein that is presumably responsible for the defects. Deletion mutants of the entire B. pseudomallei mviN gene were obtained only in the presence of the complement vector. This result and the inability of the complemented deletion mutant to lose the plasmid in the absence of antibiotic selection suggest that the gene is essential to B. pseudomallei.     

Epidemiological tracking and population assignment of the non-clonal bacterium, Burkholderia pseudomallei

Rapid assignment of bacterial pathogens into predefined populations is an important first step for epidemiological tracking. For clonal species, a single allele can theoretically define a population. For non-clonal species such as Burkholderia pseudomallei, however, shared allelic states between distantly related isolates make it more difficult to identify population defining characteristics. Two distinct B. pseudomallei populations have been previously identified using multilocus sequence typing (MLST). These populations correlate with the major foci of endemicity (Australia and Southeast Asia). Here, we use multiple Bayesian approaches to evaluate the compositional robustness of these populations, and provide assignment results for MLST sequence types (STs). Our goal was to provide a reference for assigning STs to an established population without the need for further computational analyses. We also provide allele frequency results for each population to enable estimation of population assignment even when novel STs are discovered. The ability for humans and potentially contaminated goods to move rapidly across the globe complicates the task of identifying the source of an infection or outbreak. Population genetic dynamics of B. pseudomallei are particularly complicated relative to other bacterial pathogens, but the work here provides the ability for broad scale population assignment. As there is currently no independent empirical measure of successful population assignment, we provide comprehensive analytical details of our comparisons to enable the reader to evaluate the robustness of population designations and assignments as they pertain to individual research questions. Finer scale subdivision and verification of current population compositions will likely be possible with genotyping data that more comprehensively samples the genome. The approach used here may be valuable for other non-clonal pathogens that lack simple group-defining genetic characteristics and provides a rapid reference for epidemiologists wishing to track the origin of infection without the need to compile population data and learn population assignment algorithms.     

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.