BurkholderiaWide host range.

Burkholderia mallei (glanders) and Burkholderia pseudomallei (melioidosis) are Category B agents.

Germans used B. mallei as biological weapon during World War I.

“In a laboratory specially constructed for research on this organism, one-half of workers were infected within 1 yr. Besides Francisella tularensis, B. mallei is the most dangerous microorganism to handle in the laboratory.”

Burkholderia cepacia associate with lung disease in Cystic Fibrosis patients and also an agent of soft rot in onion.

Burkholderia fungorum are good biodegraders of polychlorinated biphenyls (PCBs)

Putative Virulence factors in Burkholderia

Type III secretion system, type II secretion system, type IV pili, quorum sensing system, autotransporter proteins, fimbriae. . .

And components involved in actin based motility.

Actin Polymerization

can occur spontaneously but inefficiently since limiting rate is nucleation.

VASPWasp/N-WASP/Scar/WaveARP2/3Proflin

Screen for Actin Motility FactorIn Burkholderia pseudomallei

• Breitbach et al., 2003 showed that WASP and Ena/VASP are not essential for actin-based motility and that overexpression of Scar1, an ARP2/3 binding protein, did not inhibit intracellular motility.

• Genome Analysis found no bacterial or viral homologues to ARP2/3 activators.

• IscA/Virg, an actin motility factor in Shigella, is autosecreted.

• Searched for B. pseudomallei genome for homologies to known autosecreted proteins and found 11 targets.

Fig. 1. Sequence analysis of BimA

Homology to Y. enterocoltica adhesin Yad A

Proline-rich motif (PRM1)

WASP homology domain 2 (WH2)

Glycosyl transferase

Fig. 2. Confocal microscopy of wt, BimA mutant, and complement

A. Wildtype

B. Bim A mutant

C. Bim A mutant complemented with pMEbimA

Suggests that Bim A is required for Actin-based motility

Fig. 3. Does a mutation in BimA affect TTSS and escape from endosomal vesicles?

A. Immunoblot Analysis. bsaZ encodes for a structural component of bsa TTSS; BopE is a TTSS effector.

=> Functional TTSS

B. Confocal microscopy of infected J774.2 cells.LAMP-1 (green); B. pseudo. (red).

=>ability to escape

endosomes/lysosome

C. Electron microscopy of infected J774.2 cells

=>Entry into cytoplasm

Fig. 4. Subcellular localization of BimA protein. J774.2 cells infected with :

A. wildtype.

B. BimA mutant.

A and B: BimA (green)B.ps LPS (red)

C. wildtype. B.ps (green) BimA (blue) F-actin (red)

=> suggests that BimA protein is localized at one pole at the site of actin nucleation.

Fig. 5.

A. Does BimA interact with Actin?

Incubate sepharose beads coated with GST-BimA48-384 with murine splenic lysate, wash, and elute. Probe for F-actin.

B. Does BimA directly interact with actin?Incubate sepharose beads coated with GST-BimA48-384 with with Rhodamine-actin containing either murine splenic lysate or PBS, and wash. Check for actin by confocal micro.

=>GST-BimA48-384 directly interacts with Actin

Fig. 6. Does BimA preferentially bind to G-Actin as opposed to F-Actin?

Performed cosedimentation assay.

Amount of G-actin retained in the supes increased in proportion to the amount of GST-BimA48-384.

=> BimA preferentially binds to monomeric actin

Fig. 7.

A. What are the effects of BimA expression on the actin cytoskeleton? Disruption of the actin cytoskeleton Co-localization of BimA and F-actin enriched sites => suggests that BimA may activate actin polymerization.

B. Can BimA initiate de novo actin polymerization?Not a clear assay. BimA may stimulate actin polymerization,

which results in filopodia-like structures but BimA may also be driven to filopodia.

Fig. 8 Pyrene-actin polymerization assay.

=>BimA weakly stimulate actin polymerization in concentration dependent manner but an Arp independent manner.

ConclusionBimA• Is required for actin-based motility.• Is localized at one pole of the bacterium.• Can weakly activate de novo actin polymerization in ARP2/3 independent

manner*

* However, Breitbach et al (2003) stated that the ARP2/3 is involved in actin-based motility of Burkholderia.

Fig. 1. Arp2/3 components are colocalized at actin tails. A. B. ps (red); actin (green)

D and E. B. ps (blue); actin (red); GFP-Arp3 (green)