title: Identification of motifs of Burkholderia pseudomallei BimA required for intracellular motility, actin binding, and actin polymerization.
abstract: Actin-based motility of the melioidosis pathogen Burkholderia pseudomallei requires BimA (Burkholderia intracellular motility A). The mechanism by which BimA mediates actin assembly at the bacterial pole is ill-defined. Toward an understanding of the regions of B. pseudomallei BimA required for intracellular motility and the binding and polymerization of actin, we constructed plasmid-borne bimA variants and glutathione-S-transferase fusion proteins with in-frame deletions of specific motifs. A 13-amino-acid direct repeat and IP₇ proline-rich motif were dispensable for actin binding and assembly in vitro, and expression of the mutated proteins in a B. pseudomallei bimA mutant restored actin-based motility in J774.2 murine macrophage-like cells. However, two WASP homology 2 (WH2) domains were found to be required for actin binding, actin assembly, and plaque formation. A tract of five PDASX direct repeats influenced the polymerization of pyrene-actin monomers in vitro and was required for actin-based motility and intercellular spread, but not actin binding. None of the mutations impaired surface expression or polar targeting of BimA. The number of PDASX repeats varied in natural isolates from two to seven. Such repeats acted additively to promote pyrene-actin polymerization in vitro, with stepwise increases in the rate of polymerization as the number of repeats was increased. No differences in the efficiency of actin tail formation could be discerned between strains expressing BimA variants with two, five, or seven PDASX repeats. The data provide valuable new insights into the role of conserved and variable motifs of BimA in actin-based motility and intercellular spread of B. pseudomallei.
authors.affiliation: Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkoknoi, Bangkok 10700, Thailand.
authors.forename: Chayada
authors.initials: C
authors.lastname: Sitthidet
authors.forename: Sunee
authors.initials: S
authors.lastname: Korbsrisate
authors.forename: Abigail N
authors.initials: AN
authors.lastname: Layton
authors.forename: Terence R
authors.initials: TR
authors.lastname: Field
authors.forename: Mark P
authors.initials: MP
authors.lastname: Stevens
authors.forename: Joanne M
authors.initials: JM
authors.lastname: Stevens
mesh_terms: D000199:Actins / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D016957:Burkholderia pseudomallei / Q000502:physiology*; D002460:Cell Line; D008124:Locomotion; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D000199:Actins; C503173:BimA protein, Burkholderia pseudomallei; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins