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Journal of Clinical Microbiology, July 2008, p. 2418-2422, Vol. 46, No. 7
0095-1137/08/$08.00+0     doi:10.1128/JCM.00368-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Prevalence and Sequence Diversity of a Factor Required for Actin-Based Motility in Natural Populations of Burkholderia Species{triangledown} ,{dagger}

Chayada Sitthidet,1,{ddagger} Joanne M. Stevens,2,{ddagger} Narisara Chantratita,3 Bart J. Currie,4 Sharon J. Peacock,3 Sunee Korbsrisate,1* and Mark P. Stevens2

Department of Immunology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand,1 Division of Microbiology, Institute for Animal Health, Compton Laboratory, Berkshire RG20 7NN, United Kingdom,2 Mahidol Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Rajvithi Road, Bangkok 10400, Thailand,3 Menzies School of Health Research, Charles Darwin University, Darwin, Northern Territory, Australia4

Received 23 February 2008/ Returned for modification 15 April 2008/ Accepted 13 May 2008


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ABSTRACT
 
Actin-based motility of the melioidosis pathogen Burkholderia pseudomallei requires BimA. We report a high degree of conservation of bimA in 99 B. pseudomallei isolates from the area of endemicity. A geographically restricted subset of B. pseudomallei isolates harbored a B. mallei-like bimA allele (12.1%), confounding a differential diagnostic test based on amplification of species-specific bimA regions.


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TEXT
 
Burkholderia pseudomallei is a gram-negative facultative intracellular pathogen that causes melioidosis, a severe invasive disease of animals and humans that is endemic in southeast Asia and northern Australia (reviewed in reference 1). The organism can invade nonphagocytic cells, persist in phagocytes, escape endosomes, propel itself within and between cells by polar nucleation of actin, and induce cell fusion (reviewed in reference 32). B. pseudomallei is related to the etiological agent of glanders, and multilocus sequencing typing (MLST) (8), comparison of complete genome sequences (12, 18), and microarray analysis of gene content and transcription (15, 21) indicate that Burkholderia mallei is a clone of B. pseudomallei that has undergone gene decay. B. pseudomallei is a saprophyte, and environmental isolates were previously separated into two groups on the basis of their restriction and amplified-fragment profiles, ribotyping, and ability to assimilate arabinose. Those which assimilate arabinose (ara+) were later assigned to species Burkholderia thailandensis, a distinction supported by MLST (8). Those which do not assimilate arabinose are associated with melioidosis and are retained in the species B. pseudomallei. B. thailandensis exhibits a substantially elevated median lethal dose relative to B. pseudomallei in rodents and is less adherent and invasive in cell culture models (reviewed in reference 32).

The rapid deterioration and poor prognosis of patients with melioidosis and glanders highlight a requirement for early diagnosis and intervention. PCR-based methods for discrimination of B. pseudomallei and B. mallei from ara+ biotypes such as B. thailandensis have been developed based on the uneven conservation of type III secretion system genes (19, 28) and show promise in a clinical setting (16). A further PCR-based test to discriminate between B. pseudomallei and B. mallei that relies on variation in the bimA gene has been reported (30). BimA is required for actin-based motility of B. pseudomallei (27), and there are orthologues in B. mallei and B. thailandensis that can restore intracellular motility to a B. pseudomallei bimA mutant, despite divergence of their N-terminal sequences (26). BimA is not required for virulence of B. mallei in a Syrian hamster model of acute glanders (23); however, attenuation of a B. pseudomallei bimA mutant has been detected (M. Stevens and G. Bancroft, unpublished observations), consistent with the role of actin-based motility in the pathogenesis of Listeria and Shigella sp. infections (reviewed in reference 25).

Natural diversity exists in the prevalence and sequences of bacterial factors required for actin-dependent movement, including Listeria ActA (10, 17), Shigella and enteroinvasive Escherichia coli IcsA (4), Rickettsia sp. RickA (9, 14), and the Tir and TccP proteins of attaching and effacing E. coli (7, 20, 22). Such factors are not ubiquitous in all species of these genera, and the impact of polymorphisms on actin assembly, cell-to-cell spread, and pathogenesis is ill defined. The BimA orthologues described to date contain variable numbers and types of a proline-rich motif (26), and variation in the number of such motifs has been related to the efficiency of actin assembly by enterohemorrhagic E. coli TccP (6) and Listeria ActA (24). Further, the BimA protein from B. thailandensis was uniquely able to interact with Arp-3 (actin-related protein 3), consistent with the presence of a central and acidic domain that is absent in the BimA proteins from B. pseudomallei and B. mallei (26). Here we sampled the prevalence and sequence of BimA in clinical and environmental isolates of B. pseudomallei, both to evaluate the reliability of differential PCR tests based on bimA and to examine the conservation of motifs and domains predicted to be important for the subversion of actin dynamics by this factor.

To gain insight into the extent of variation of BimA and design a strategy for amplification of bimA genes, we first examined bimA sequences in the complete or partial genomes of 18 B. pseudomallei, 10 B. mallei, and 2 B. thailandensis strains available at the time of writing. In all cases, a single bimA open reading frame was predicted to give rise to a protein of approximately the expected size for the species and no pseudogenes were detected. ClustalW alignment (3) of predicted protein sequences revealed that the BimA orthologues previously described in B. pseudomallei (BimABp), B. mallei (BimABm), and B. thailandensis (BimABt) (26) are highly conserved in other representatives of the same species. However, an Australian isolate of B. pseudomallei (MSHR668) possessed a BimA sequence exhibiting only 54% identity to the BimA of the prototypic B. pseudomallei strain K96243. The BimA of strain MSHR668 is 95% identical to the BimA of the prototypic B. mallei strain ATCC 23344 and exhibits the same predicted domain organization. Of the BimA domains described previously (27), the membrane anchor and predicted actin monomer-binding Wiskott-Aldrich syndrome protein homology 2 (WH2) domains of the BimA from strains ATCC 23344 and MSHR668 are identical, as determined using MotifScan (5). However, differences existed in the predicted proline-rich domain, which in B. mallei ATCC 23344 BimA contains six PRM1 motifs (VP18 and five SP4 motifs) but in the BimA of strain MSHR668 contains only two PRM1 motifs (VP8 and SP4) (Fig. 1). PRM1 motifs interact with profilin, which in turn recruits actin monomers to sites of assembly (reviewed in reference 13); however, the impact of the polymorphisms on BimA function remains to be elucidated.


Figure 1
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  		FIG. 1. Alignment of B. mallei-like BimA sequences derived from three Australian B. pseudomallei isolates with BimA sequences of B. mallei ATCC 23344 and B. pseudomallei MSHR668. Amino acid sequences were aligned using ClustalW (3). Alignment scores representing the level of identity to the B. mallei ATCC 23344 BimA sequence are shown in parentheses. The proline-rich region is boxed.

Of the 17 predicted B. pseudomallei BimA sequences that resemble the prototypic BimABp from K96243, the predicted proline-rich motifs and tandem WH2 domains were conserved. The BimA of B. pseudomallei strain 14 contains only a single NIPVPPPMPGGGA motif, which is directly repeated in tandem in all other strains. The number of PDAST repeats (predicted phosphorylation sites for host cell casein kinase II) was also found to range from two (B. pseudomallei BCC215) to seven (B. pseudomallei MSHR305) (see Fig. S1 in the supplemental material). The central and acidic domain predicted in B. thailandensis E264 was found to be conserved in the only other BimABt sequence available but was absent in all other BimABp and BimABm sequences analyzed.

To determine if the prevalence and diversity of BimA encoded by sequenced genomes reflect those of BimA encoded by genomes of natural populations and to determine if the B. mallei-like BimA exists in other B. pseudomallei strains, bimA genes were amplified from a collection of 99 B. pseudomallei strains of clinical or environmental origin from the area of endemicity (Table 1). Genomic DNA was prepared by cetyltrimethylammonium bromide extraction and subjected to PCR with GoTaq Green Master Mix polymerase (Promega, Madison, WI) using the primers 5'-CTCGAATTCCATGCGTGCAATAGCTG-3' and 5'-CTTCTCGAGTGCTTACCATTGCCAGCTCAT-3', which amplify a 1.78-kb product representing the region 232 nucleotides upstream of the predicted start codon to 4 nucleotides downstream of the predicted stop codon of B. pseudomallei K96243 bimA. PCRs involved initial denaturation at 97°C for 3 min and 30 cycles at 97°C for 30 s and 68°C for 3 min, followed by a final extension for 10 min at 68°C. Amplicons were resolved by agarose gel electrophoresis and were obtained from all strains. Eighty-seven strains yielded an amplicon typical of the size of bimA from B. pseudomallei K96243 (87.9%), whereas 12 yielded a ca. 1.2-kb amplicon comparable in size to bimA from B. mallei ATCC 23344 (12.1%) (Table 1). Consistent with the findings of Ulrich et al. (30), the bimA-specific primers failed to amplify a specific product from the genomes of 53 strains representing nine genomovars of the Burkholderia cepacia complex (D. Kenna and J. Govan, unpublished data). Analysis of the sequenced genomes of B. cepacia complex strains also failed to identify bimA orthologues.


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  		TABLE 1. B. pseudomallei strains used in this study, their origin, MLST type, and YLF/BTFC type (where known)a

The bimA genes of three B. pseudomallei isolates that yielded 1.2-kb amplicons (strains MSHR33, MSHR172, and MSHR491) were amplified, restricted with EcoRI and XhoI, cloned under the control of a Ptac promoter into similarly restricted pME6032 (11), and sequenced by the Bio-Technology Service Unit, Thailand. The predicted BimA proteins of strains MSHR33, MSHR172, and MSHR491 vary slightly in length (369, 374, and 365 amino acids, respectively) and exhibit 54 to 55% identity to B. pseudomallei K96243 BimA yet 95 to 96% identity to the BimA of B. mallei ATCC 23344, indicating that they are B. mallei-like (Fig. 1). The BimABm-like BimA proteins from strains MSHR33, MSHR172, and MSHR491 differ from those of B. mallei ATCC 23344 and B. pseudomallei MSHR668 only in the proline-rich domain, where they show greater similarity to the region in B. pseudomallei MSHR668 BimA than the corresponding region in B. mallei ATCC 23344 (Fig. 1). The bimABp and bimABm alleles may reflect distinct horizontal gene transfer events, as recently described for Yersinia-like fimbriae (YLF) and B. thailandensis-like flagellum and chemotaxis (BTFC) clusters that define distinct and geographically restricted isolates of B. pseudomallei (29).

Remarkably, all 12 B. pseudomallei strains harboring bimABm-like genes were isolated from the Australian Northern Territory, consistent with the existence of a bimABm-like allele in the sequenced genome of the Australian B. pseudomallei isolate MSHR668. A further 23 B. pseudomallei strains originating from Australia (65.7%) yielded a 1.8-kb bimABp-like amplicon (Table 1). Previously, MLST indicated that Australian and Thai isolates of B. pseudomallei are distinct (2, 31). The MLST types of some of the strains tested are known (http://bpseudomallei.mlst.net) (8) (Table 1), and the sequence types of a further three B. pseudomallei isolates harboring the bimABm allele were determined herein. This was accomplished by sequencing internal fragments of the housekeeping genes ace, gltB, gmhD, lepA, lipA, narK, and ndh from strains MSHR33, MSHR42, and MSHR172 on both strands as described previously (8, 31). A phylogenetic analysis was performed to determine the relatedness of bimABm allele strains to each other and to the population of isolates from northern Australia. A neighbor-joining tree was constructed based on the concatenated sequences of the seven MLST genes for the B. pseudomallei strains with known sequence types in this study, together with all B. pseudomallei strains in the MLST database (http://www.mlst.net/) originating from northern Australia that were deposited by one of the coauthors (B. Currie). The tree generated from data for 606 isolates corresponding to 227 sequence types demonstrated that the bimABm allele strains were distributed throughout the B. pseudomallei population, with no obvious association with specific lineages. (see Fig. S2 in the supplemental material). All of the Australian isolates had sequence types distinct from that of B. mallei (ST40) (31), and there are no known reports of isolation of B. mallei in Australia. The bimA locus (BPSS1492) is not genetically linked with the YLF or BTFC clusters described by Tuanyok et al. (29), and the bimABm allele was found in strains of either the YLF or BTFC group by PCR using locus-specific primers as described previously (29) (Table 1). It will be interesting to determine whether strains of a given MLST sequence type can contain either a bimABp or bimABm allele. At this stage, it is not possible to state whether bimABm was derived by gene decay in B. pseudomallei or if B. mallei arose from a B. pseudomallei strain in which the bimABm allele was present, although the latter seems more plausible as MLST has demonstrated clear separation between Thai and Australian strains of B. pseudomallei (31). It has been suggested previously that B. pseudomallei may have originated in Australia and was carried by animals to southeast Asia via a land bridge that existed around 15 million years ago between Asia and the Australian-New Guinea continent (2). Further studies are under way to ascertain if the bimABm-like allele is associated with severity of disease or specific clinical presentations and whether this allele is present in B. pseudomallei from regions of endemicity other than the Northern Territory of Australia.

Ulrich et al. (30) previously reported the development of primers (AT4 and AT5) based on the sequence heterogeneity in the 5' end of bimA to generate a 250-bp B. mallei-specific amplicon spanning the translation initiation site. Although Ulrich et al. previously reported no amplification from B. pseudomallei isolates from Australia, all 12 B. pseudomallei isolates harboring the bimABm-like allele from this study yielded a 250-bp amplicon with these primers (data not shown). There is robust evidence from API 20NE, arabinose assimilation, and latex agglutination tests that these 12 strains are indeed B. pseudomallei, indicating that the AT4 and AT5 primers are not suitable for distinguishing B. pseudomallei from B. mallei, at least for strains from this geographical area.

The data presented herein indicate that all B. pseudomallei isolates examined contain an intact bimA gene and reveal that an allele detected in the genome of B. mallei is not widespread (~12%) in B. pseudomallei. This allele was present in isolates from Northern Territory, Australia, but not Thailand and was not restricted to a specific sequence type. The data have important implications for the design and reliability of bimA-based tests to differentiate between Burkholderia species and highlight the existence of polymorphisms that have the potential to influence actin binding and assembly by a key factor required for intercellular spread and virulence.

Nucleotide sequence accession numbers. The nucleotide sequences determined in this study have been registered in GenBank accession numbers EU382734, EU437409, and EU437410.


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ACKNOWLEDGMENTS
 
We gratefully acknowledge the support of the Thailand Research Fund and Commission of Higher Education (grant RMU5080015; S.K.), the Royal Thai Golden Jubilee studentship scheme (grant PHD0188/2547; C.S.), the Biotechnology and Biological Sciences Research Council UK (grant E021212; M.P.S.), and the Australian National Health and Medical Research Council (grant 383504; B.J.C.). S.J.P. is supported by the Wellcome Trust.

We thank A. Tuanyok for providing the YLF/BTFC typing results and V. Wuthiekanun, S. Tumapa, S. Tandhavanant, and E. Singsuksawat for their assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Immunology, Faculty of Medicine, Siriraj Hospital, Mahidol University, 2 Pran-nok Road, Bangkoknoi, Bangkok 10700, Thailand. Phone: 66-2-418-0569. Fax: 66-2-418-1636. E-mail: grsks{at}mahidol.ac.th Back

{triangledown} Published ahead of print on 21 May 2008. Back

{dagger} Supplemental material for this article may be found at http://jcm.asm.org/. Back

{ddagger} These authors contributed equally to this study. Back


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Journal of Clinical Microbiology, July 2008, p. 2418-2422, Vol. 46, No. 7
0095-1137/08/$08.00+0     doi:10.1128/JCM.00368-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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