Characterization of clinically-attenuated Burkholderia mallei by whole genome sequencing: candidate strain for exclusion from Select Agent lists.

Department of Medicine, University of Medicine and Dentistry-New Jersey Medical School, Newark, New Jersey, United States of America.
PLoS ONE (Impact Factor: 3.53). 02/2008; 3(4):e2058. DOI: 10.1371/journal.pone.0002058
Source: PubMed

ABSTRACT Burkholderia mallei 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.
We performed WGS on a strain of B. mallei, SAVP1, previously pathogenic, that was experimentally infected in 6 equids (4 ponies, 1 mule, 1 donkey), natural hosts, for purposes of producing antibodies. Multiple high inocula were used in some cases. Unexpectedly SAVP1 appeared to be avirulent in the ponies and mule, and attenuated in the donkey, but induced antibodies. We determined the genome sequence of SAVP1 and compared it to a strain that was virulent in horses and a human. In comparison, this phenotypic avirulent SAVP1 strain was missing multiple genes including all the animal type III secretory system (T3SS) complex of genes demonstrated to be essential for virulence in mice and hamster models. The loss of these genes in the SAVP1 strain appears to be the consequence of a multiple gene deletion across insertion sequence (IS) elements in the B. mallei 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.
The discovery that this strain of B. mallei 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.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A comparative analysis of 60 complete Burkholderia genomes was conducted to obtain insight in the evolutionary history behind the diversity and pathogenicity at species level. A concatenated multiprotein phyletic pattern and a dataset with Burkholderia clusters of orthologous genes (BuCOGs) were constructed. The extent of horizontal gene transfer (HGT) was assessed using a Markov based probabilistic method. A reconstruction of the gene gains and losses history shows that more than half of the Burkholderia genes families are inferred to have experienced HGT at least once during their evolution. Further analysis revealed that the number of gene gain and loss was correlated with the branch length. Genomic islands (GEIs) analysis based on evolutionary history reconstruction not only revealed that most genes in ancient GEIs were gained but also suggested that the fraction of the genome located in GEIs in the small chromosomes is higher than in the large chromosomes in Burkholderia. The mapping of coexpressed genes onto biological pathway schemes revealed that pathogenicity of Burkholderia strains is probably mainly determined by the gained genes in its ancestor. Taken together, our results strongly support that gene gain and loss especially in ancient evolutionary history play an important role in strain divergence, pathogenicity determinants of Burkholderia and GEIs formation.
    Evolutionary bioinformatics online 01/2011; 7:191-200. · 1.17 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Yersinia pestis is the causative agent of the plague. Y. pestis KIM 10+ strain was passaged and selected for loss of the 102 kb pgm locus, resulting in an attenuated strain, KIM D27. In this study, whole genome sequencing was performed on KIM D27 in order to identify any additional differences. Initial assemblies of 454 data were highly fragmented, and various bioinformatic tools detected between 15 and 465 SNPs and INDELs when comparing both strains, the vast majority associated with A or T homopolymer sequences. Consequently, Illumina sequencing was performed to improve the quality of the assembly. Hybrid sequence assemblies were performed and a total of 56 validated SNP/INDELs and 5 repeat differences were identified in the D27 strain relative to published KIM 10+ sequence. However, further analysis showed that 55 of these SNP/INDELs and 3 repeats were errors in the KIM 10+ reference sequence. We conclude that both 454 and Illumina sequencing were required to obtain the most accurate and rapid sequence results for Y. pestis KIMD27. SNP and INDELS calls were most accurate when both Newbler and CLC Genomics Workbench were employed. For purposes of obtaining high quality genome sequence differences between strains, any identified differences should be verified in both the new and reference genomes.
    PLoS ONE 04/2011; 6(4):e19054. · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Type III secretion systems are present in many pathogenic bacteria and mediate the translocation of bacterial effectors into host cells. Identification of host targets of these effectors is crucial for understanding bacterial virulence. IcsB, a type III secretion effector, helps Shigella to evade the host autophagy defense system by binding to the autophagy protein, Atg5. Here, we show that IcsB is able to interact specifically with cholesterol. The cholesterol binding domain (CBD) of IcsB is located between residues 288 and 351. Specific mutations of single tyrosine residues Y297 or Y340 of IcsB by phenylalanine (F) slightly reduced cholesterol binding, whereas deletion of the entire CBD or double mutation Y297F-Y340F strongly abolished interactions with cholesterol. To determine whether Shigella expressing IcsB variants could evade autophagy as effectively as the wild-type Shigella, we infected MDAMC cells stably expressing the autophagy marker LC3 fused to GFP and bacterial autophagosome formation was quantified using fluorescence microscopy. Mutation Y297F or Y340F slightly impaired IcsB function, whereas complete removal of CBD or mutation Y297F-Y340F significantly impaired autophagy evasion. Furthermore, we report that BopA, the counterpart of IcsB in Burkholderia pseudomallei with similar autophagy-evading properties, contains the CBD domain and is also able to bind cholesterol.
    Microbes and Infection 11/2010; 12(12-13):956-66. · 2.73 Impact Factor

Full-text (2 Sources)

Available from
May 31, 2014