Bacterial genome adaptation to niches: Divergence of the potential virulence genes in three Burkholderia species of different survival strategies

BACKGROUND: Two closely related species Burkholderia mallei (Bm) and Burkholderia pseudomallei (Bp) are serious human health hazards and are potential bio-warfare agents, whereas another closely related species Burkholderia thailandensis (Bt) is a non-pathogenic saprophyte. To investigate the genomic factors resulting in such a dramatic difference, we first identified the Bm genes responsive to the mouse environment, and then examined the divergence of these genes in Bp and Bt. RESULTS: The genes down-expressed, which largely encode cell growth-related proteins, are conserved well in all three species, whereas those up-expressed, which include potential virulence genes, are less well conserved or absent notably in Bt. However, a substantial number of up-expressed genes is still conserved in Bt. Bm and Bp further diverged from each other in a small number of genes resulting from unit number changes in simple sequence repeats (ssr) in the homologs. CONCLUSION: Our data suggest that divergent evolution of a small set of genes, rather than acquisition or loss of pathogenic islands, is associated with the development of different life styles in these bacteria of similar genomic contents. Further divergence between Bm and Bp mediated by ssr changes may reflect different adaptive processes of Bm and Bp fine-tuning into their host environments

Genetic and phenotypic diversity in Burkholderia: contributions by prophage and phage-like elements

<p>Abstract</p> <p>Background</p> <p><it>Burkholderia </it>species exhibit enormous phenotypic diversity, ranging from the nonpathogenic, soil- and water-inhabiting <it>Burkholderia thailandensis </it>to the virulent, host-adapted mammalian pathogen <it>B. mallei</it>. Genomic diversity is evident within <it>Burkholderia </it>species as well. Individual isolates of <it>Burkholderia pseudomallei </it>and <it>B. thailandensis</it>, for example, carry a variety of strain-specific genomic islands (GIs), including putative pathogenicity and metabolic islands, prophage-like islands, and prophages. These GIs may provide some strains with a competitive advantage in the environment and/or in the host relative to other strains.</p> <p>Results</p> <p>Here we present the results of analysis of 37 prophages, putative prophages, and prophage-like elements from six different <it>Burkholderia </it>species. Five of these were spontaneously induced to form bacteriophage particles from <it>B. pseudomallei </it>and <it>B. thailandensis </it>strains and were isolated and fully sequenced; 24 were computationally predicted in sequenced <it>Burkholderia </it>genomes; and eight are previously characterized prophages or prophage-like elements. The results reveal numerous differences in both genome structure and gene content among elements derived from different species as well as from strains within species, due in part to the incorporation of additional DNA, or 'morons' into the prophage genomes. Implications for pathogenicity are also discussed. Lastly, RNAseq analysis of gene expression showed that many of the genes in ϕ1026b that appear to contribute to phage and lysogen fitness were expressed independently of the phage structural and replication genes.</p> <p>Conclusions</p> <p>This study provides the first estimate of the relative contribution of prophages to the vast phenotypic diversity found among the <it>Burkholderiae</it>.</p

A high-content imaging assay for the quantification of the Burkholderia pseudomallei induced multinucleated giant cell (MNGC) phenotype in murine macrophages

BACKGROUND: Burkholderia pseudomallei (Bp), a Gram-negative, motile, facultative intracellular bacterium is the causative agent of melioidosis in humans and animals. The Bp genome encodes a repertoire of virulence factors, including the cluster 3 type III secretion system (T3SS-3), the cluster 1 type VI secretion system (T6SS-1), and the intracellular motility protein BimA, that enable the pathogen to invade both phagocytic and non-phagocytic cells. A unique hallmark of Bp infection both in vitro and in vivo is its ability to induce cell-to-cell fusion of macrophages to form multinucleated giant cells (MNGCs), which to date are semi-quantitatively reported following visual inspection. RESULTS: In this study we report the development of an automated high-content image acquisition and analysis assay to quantitate the Bp induced MNGC phenotype. Validation of the assay was performed using T6SS-1 (∆hcp1) and T3SS-3 (∆bsaZ) mutants of Bp that have been previously reported to exhibit defects in their ability to induce MNGCs. Finally, screening of a focused small molecule library identified several Histone Deacetylase (HDAC) inhibitors that inhibited Bp-induced MNGC formation of macrophages. CONCLUSIONS: We have successfully developed an automated HCI assay to quantitate MNGCs induced by Bp in macrophages. This assay was then used to characterize the phenotype of the Bp mutants for their ability to induce MNGC formation and identify small molecules that interfere with this process. Successful application of chemical genetics and functional reverse genetics siRNA approaches in the MNGC assay will help gain a better understanding of the molecular targets and cellular mechanisms responsible for the MNGC phenotype induced by Bp, by other bacteria such as Mycobacterium tuberculosis, or by exogenously added cytokines

Genome sequence alterations detected upon passage of Burkholderia mallei ATCC 23344 in culture and in mammalian hosts

BACKGROUND: More than 12,000 simple sequence repeats (SSRs) have been identified in the genome of Burkholderia mallei ATCC 23344. As a demonstrated mechanism of phase variation in other pathogenic bacteria, these may function as mutable loci leading to altered protein expression or structure variation. To determine if such alterations are occurring in vivo, the genomes of various single-colony passaged B. mallei ATCC 23344 isolates, one from each source, were sequenced from culture, a mouse, a horse, and two isolates from a single human patient, and the sequence compared to the published B. mallei ATCC 23344 genome sequence. RESULTS: Forty-nine insertions and deletions (indels) were detected at SSRs in the five passaged strains, a majority of which (67.3%) were located within noncoding areas, suggesting that such regions are more tolerant of sequence alterations. Expression profiling of the two human passaged isolates compared to the strain before passage revealed alterations in the mRNA levels of multiple genes when grown in culture. CONCLUSION: These data support the notion that genome variability upon passage is a feature of B. mallei ATCC23344, and that within a host B. mallei generates a diverse population of clones that accumulate genome sequence variation at SSR and other loci

Molecular Epidemiology of Glanders, Pakistan

We collected epidemiologic and molecular data from Burkholderia mallei isolates from equines in Punjab, Pakistan from 1999 through 2007. We show that recent outbreaks are genetically distinct from available whole genome sequences and that these genotypes are persistent and ubiquitous in Punjab, probably due to human-mediated movement of equines

Continuing evolution of Burkholderia mallei through genome reduction and large-scale rearrangements

Burkholderia mallei (Bm), the causative agent of the predominately equine disease glanders, is a genetically uniform species that is very closely related to the much more diverse species Burkholderia pseudomallei (Bp), an opportunistic human pathogen and the primary cause of melioidosis. To gain insight into the relative lack of genetic diversity within Bm, we performed whole-genome comparative analysis of seven Bm strains and contrasted these with eight Bp strains. The Bm core genome (shared by all seven strains) is smaller in size than that of Bp, but the inverse is true for the variable gene sets that are distributed across strains. Interestingly, the biological roles of the Bm variable gene sets are much more homogeneous than those of Bp. The Bm variable genes are found mostly in contiguous regions flanked by insertion sequence (IS) elements, which appear to mediate excision and subsequent elimination of groups of genes that are under reduced selection in the mammalian host. The analysis suggests that the Bm genome continues to evolve through random IS-mediated recombination events, and differences in gene content may contribute to differences in virulence observed among Bm strains. The results are consistent with the view that Bm recently evolved from a single strain of Bp upon introduction into an animal host followed by expansion of IS elements, prophage elimination, and genome rearrangements and reduction mediated by homologous recombination across IS elements

Tandem repeat regions within the Burkholderia pseudomallei genome and their application for high resolution genotyping

BACKGROUND: The facultative, intracellular bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals. We identified and categorized tandem repeat arrays and their distribution throughout the genome of B. pseudomallei strain K96243 in order to develop a genetic typing method for B. pseudomallei. We then screened 104 of the potentially polymorphic loci across a diverse panel of 31 isolates including B. pseudomallei, B. mallei and B. thailandensis in order to identify loci with varying degrees of polymorphism. A subset of these tandem repeat arrays were subsequently developed into a multiple-locus VNTR analysis to examine 66 B. pseudomallei and 21 B. mallei isolates from around the world, as well as 95 lineages from a serial transfer experiment encompassing ~18,000 generations. RESULTS: B. pseudomallei contains a preponderance of tandem repeat loci throughout its genome, many of which are duplicated elsewhere in the genome. The majority of these loci are composed of repeat motif lengths of 6 to 9 bp with 4 to 10 repeat units and are predominately located in intergenic regions of the genome. Across geographically diverse B. pseudomallei and B.mallei isolates, the 32 VNTR loci displayed between 7 and 28 alleles, with Nei's diversity values ranging from 0.47 and 0.94. Mutation rates for these loci are comparable (>10(-5 )per locus per generation) to that of the most diverse tandemly repeated regions found in other less diverse bacteria. CONCLUSION: The frequency, location and duplicate nature of tandemly repeated regions within the B. pseudomallei genome indicate that these tandem repeat regions may play a role in generating and maintaining adaptive genomic variation. Multiple-locus VNTR analysis revealed extensive diversity within the global isolate set containing B. pseudomallei and B. mallei, and it detected genotypic differences within clonal lineages of both species that were identical using previous typing methods. Given the health threat to humans and livestock and the potential for B. pseudomallei to be released intentionally, MLVA could prove to be an important tool for fine-scale epidemiological or forensic tracking of this increasingly important environmental pathogen

Characterization of Clinically-Attenuated Burkholderia mallei by Whole Genome Sequencing: Candidate Strain for Exclusion from Select Agent Lists

is an understudied biothreat agent responsible for glanders which can be lethal in humans and animals. Research with this pathogen has been hampered in part by constraints of Select Agent regulations for safety reasons. Whole genomic sequencing (WGS) is an apt approach to characterize newly discovered or poorly understood microbial pathogens. genome. Therefore, the strain by itself is unlikely to revert naturally to its virulent phenotype. There were other genes present in one strain and not the other and vice-versa. was both avirulent in the natural host ponies, and did not possess T3SS associated genes may be fortuitous to advance biodefense research. The deleted virulence-essential T3SS is not likely to be re-acquired naturally. These findings may provide a basis for exclusion of SAVP1 from the Select Agent regulation or at least discussion of what else would be required for exclusion. This exclusion could accelerate research by investigators not possessing BSL-3 facilities and facilitate the production of reagents such as antibodies without the restraints of Select Agent regulation

Genes That Influence Swarming Motility and Biofilm Formation in Variovorax paradoxus EPS

Variovorax paradoxus is an aerobic soil bacterium associated with important biodegradative processes in nature. We use V. paradoxus EPS to study multicellular behaviors on surfaces.We recovered flanking sequence from 123 clones in a Tn5 mutant library, with insertions in 29 different genes, selected based on observed surface behavior phenotypes. We identified three genes, Varpa_4665, Varpa_4680, and Varpa_5900, for further examination. These genes were cloned into pBBR1MCS2 and used to complement the insertion mutants. We also analyzed expression of Varpa_4680 and Varpa_5900 under different growth conditions by qPCR.The 29 genes we identified had diverse predicted functions, many in exopolysaccharide synthesis. Varpa_4680, the most commonly recovered insertion site, encodes a putative N-acetyl-L-fucosamine transferase similar to WbuB. Expression of this gene in trans complemented the mutant fully. Several unique insertions were identified in Varpa_5900, which is one of three predicted pilY1 homologs in the EPS genome. No insertions in the two other putative pilY1 homologs present in the genome were identified. Expression of Varpa_5900 altered the structure of the wild type swarm, as did disruption of the chromosomal gene. The swarming phenotype was complemented by expression of Varpa_5900 from a plasmid, but biofilm formation was not restored. Both Varpa_4680 and Varpa_5900 transcripts were downregulated in biofilms and upregulated during swarming when compared to log phase culture. We identified a putative two component system (Varpa_4664-4665) encoding a response regulator (shkR) and a sensor histidine kinase (shkS), respectively. Biofilm formation increased and swarming was strongly delayed in the Varpa_4665 (shkS) mutant. Complementation of shkS restored the biofilm phenotype but swarming was still delayed. Expression of shkR in trans suppressed biofilm formation in either genetic background, and partially restored swarming in the mutant.The data presented here point to complex regulation of these surface behaviors

Expression and Function of Macrophage Migration Inhibitory Factor (MIF) in Melioidosis

Melioidosis is a severe tropical infection caused by the bacterium Burkholderia pseudomallei. B. pseudomallei is the major cause of community-acquired septicemia in northeast Thailand with a mortality rate in severe cases of around 40% Little is known, however, about the mechanisms of the host defense to B. pseudomallei infection. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that has emerged as an important mediator of the host defense in severe bacterial infections. In this article, we studied the expression and function of MIF both in patients with melioidosis and in mice during experimental melioidosis. We found that MIF concentrations were elevated in patients with melioidosis. Furthermore, high MIF concentrations are associated with poor outcome in patients with melioidosis. Also, in mice with experimentally induced melioidosis, we observed an upregulation of MIF concentrations. Furthermore, mice with melioidosis that were treated with a MIF blocking treatment showed lower bacterial counts in their lungs during infection. In conclusion, MIF seems to impair host defense mechanisms during melioidosis