Burkholderia Morphotype Genome Project
Burkholderia pseudomallei, the causative agent of the infectious disease melioidosis, is a CDC Category B biological threat agent, and occurs as a soil organism in Southeast Asia and Northern Australia. Incidents of melioidosis are generally confined to these endemic areas. Recent surveys, however, show that the organism is much more prevalent worldwide than previously believed, and isolation of B. pseudomallei from the environment and in clinical situations in parts of Africa, the Middle East, Europe and in Central and South America has been documented.
NIH-NIAID has funded this genome project to sequence nine phenotypically characterized strains of B. pseudomallei, as well as 25 B. pseudomallei bacteriophage genomes isolated from 48 different B. pseudomallei strains from various geographic and clinical sources. Variable horizontal gene acquisition by B. pseudomallei is an important feature of recent genetic evolution, and has resulted in a genetically diverse bacterial species. The goal of this project is to identify the specific nucleotide sequences and/or single nucleotide polymorphisms that are correlated with expression of virulence and disease via comparative genomic analysis between B. pseudomallei strains as well as between various bacteriophages harbored within B. pseudomallei. All data will be released immediately in the public domain. A repository of well-characterized strains will be made publicly available for further studies. This work will increase our knowledge of the virulence of this understudied biothreat pathogen and provide a foundation for the development of diagnostic and therapeutic countermeasures and a protective vaccine for melioidosis.
Molecular characterization of morphotype switching in Burkholderia pseudomallei
It has been observed for nearly 80 years that Bps can change colony morphology, and recent studies have shown an association between morphotypes and specificity for body compartments, ability to adhere and penetrate epithelial cells or macrophages, and expression of virulence factors. MLST and PFGE analyses show that the morphotypes arise from a clonal population and that they are not the result of co-infections with different strains. Similar observations were noted at the Health Protection Agency (HPA) in the UK, where different morphotypes were observed from lung samples in mice infected with Bps K96243. To date, the molecular mechanisms underlying this morphotype switching remain unstudied. A study in a close relative, Burkholderia thailandensis (Bth), showed that a quorum sensing mutant had marked differences in colony morphology, but other genes involved in exopolysaccharide production could also be necessary for morphotype switching. However, no such studies have been done with Bps, and the universality of the findings in Bth are not known.
To understand the mechanisms that lead to morphotype switching, several different morphotypes of Bps will be sequenced and their genomes will be scanned for differences as part of the GSC contract. One of these types is from the positive control of B. pseudomallei K96243 created for use in the enumeration procedure and is therefore typical of the morphologies seen to date. Two other types were isolated from two animals which had received a low dose stationary phase aerosol infection. With Bps K96243 these morphotypes were atypical of the morphologies seen to date. All three isolates were characterized as Gram negative rods and are PCR positive for B. pseudomallei (lpxO gene target). Since the three colonies are isogenic, differences between them could explain the observed morphological discrepancies. An additional 6 isolates with varying morphotypes retrieved from the UK HPA reference library will also be sequenced and compared for further identification of mutations that may lead to morphology differences. These six isolates were all collected across different time periods from a single individual who suffered from cystic fibrosis. The phenotypes and characteristics of each strain are presented in the table below.
White Paper Access
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|Strain or Isolate||Source||Morphology||Other Information|
|Bps K96243||HPA reference library||Wild-type, 5-8mm, creamy, shiny, smooth, raised||Same as NCTC 13392|
|Isolate 2||Mouse Study||Atypical, 2-5mm. As type 1 but more domed than raised||Low dose, aerosol infection with Bps K96243|
|Isolate 7||Mouse Study||Atypical, 3mm, creamy/white, shiny, smooth, raised||Low dose, aerosol infection with Bps K96243|
|Bp1||HPA reference library||Similar to wild-type, circular, smooth, shiny||Originally isolated from an individual with Cystic Fibrosis|
|Bp10M||HPA reference library||Rare, large, white, mucoid||Isolated from same individual different time period|
|BpEW||HPA reference library||Atypical, circular, wrinkled (umbonate), yellow, flat, dry||Isolated from same individual different time period|
|BpK||HPA reference library||Atypical, white, irregular, undulate||Isolated from same individual different time period|
|BpPW||HPA reference library||Atypical, small, flat, circular, undulate||Isolated from same individual different time period|
|BpP||HPA reference library||Rare, small, flat, shiny||Isolated from same individual different time period|
This project has been funded in whole or part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services under contract numbers N01-AI30071 and/or HHSN272200900007C.