Burkholderia pseudomalleitype III secreted protein BipC: role in actin modulation and translocation activities required for the bacterial intracellular lifecycle

Abstract

Melioidosis, an infection caused by the facultative intracellular pathogen Burkholderia pseudomallei, has been classified as an emerging disease with the number of patients steadily increasing at an alarming rate. B. pseudomalleipossess various virulence determinants that allow them to invade the host and evade the host immune response, such as the type III secretion systems (TTSS). The products of this specialized secretion system are particularly important for the B. pseudomallei infection. Lacking in one or more components of the TTSS demonstrated different degrees of defects in the intracellular lifecycle of B. pseudomallei. Further understanding the functional roles of proteins involved in B. pseudomallei TTSS will enable us to dissect the enigma of B. pseudomallei-host cell interaction. In this study, BipC (a translocator), which was previously reported to be involved in the pathogenesis of B. pseudomallei, was further characterized using the bioinformatics and molecular approaches. The bipCgene, coding for a putative invasive protein, was first PCR amplified from B. pseudomallei K96243 genomic DNA and cloned into an expression vector for overexpression in Escherichia coli. The soluble protein was subsequently purified and assayed for actin polymerization and depolymerization. BipC was verified to subvert the host actin dynamics as demonstrated by the capability to polymerize actin in vitro. Homology modeling was also attempted to predict the structure of BipC. Overall, our findings identified that the protein encoded by the bipC gene plays a role as an effector involved in the actin binding activity to facilitate internalization of B. pseudomalleiinto the host cells.

BipC, a Predicted Burkholderia pseudomallei Type 3 Secretion System Translocator Protein with Actin Binding Activity

“…Here we have demonstrated that GST-BipC has the ability to bind actin directly, in the absence of other bacterial or cellular proteins. We acknowledge that whilst our study was in review, Kang et al ( 2016 ) published similar observations to those presented in our manuscript. However, upon closer inspection there is only a small amount of “overlap” with our data regarding the function of BipC as an actin binding and polymerizing factor.…”

“…At the time of submission of this manuscript for peer review we believed this to be the first evidence that BipC is an actin-binding protein. However, we have since become aware of a similar study by Kang et al ( 2016 ), which also demonstrates that B. pseudomallei BipC is an actin-binding protein.…”

“…In contrast the GST-BipC protein showed no significant F-actin stabilizing activity in this assay, demonstrating that this protein, unlike its homolog SipC, does not have intrinsic F-actin bundling activity (Figure 2C ). Since submission of this manuscript for peer review, a study by Kang et al ( 2016 ) suggested that a His-tagged BipC protein exhibited F-actin stabilizing activity in a similar assay. However, it is notable that much higher concentrations of BipC protein were required in these experiments than used in our study, or that of McGhie et al ( 2001 ).…”

Melioidosis in Malaysia: Incidence, Clinical Challenges, and Advances in Understanding Pathogenesis

“…This study was extended to further evaluate the role of the cell invasion protein, BipC, in pathogenesis of B. pseudomallei . BipC, an immunoreactive protein, is involved in actin binding to facilitate internalization of B. pseudomallei into host cells, as a bipC mutant was impaired in adherence, invasion, and intracellular survival in epithelial cells, and BipC protein is required for full virulence in a murine model of melioidosis [ 78 , 79 ]. Recently, Vadivelu et al [ 80 ] showed that B. pseudomallei localized within the nuclear compartment of host cells, suggesting that the nucleus may play a role as an occult or transient niche for persistence of intracellular pathogens, potentially leading to recurrent episodes or recrudescence of infection.…”

Type III Secretion in the Melioidosis Pathogen Burkholderia pseudomallei

“…Internalization of B. pseudomallei can be blocked by the actin polymerization inhibitor cytochalasin D (Jones et al, 1996 ), indicating the importance of actin cytoskeletal rearrangements in the uptake of B. pseudomallei . Similarly to SipC, BipC is also able to polymerise actin in vitro and stabilizes F-actin indicating possible actin bundling activity (Kang et al, 2016a and our own unpublished observations). Interestingly, Salmonella SipA protein enhances the ability of SipC to nucleate and bundle F-actin (McGhie et al, 2001 ), but a homolog of SipA is not encoded by the genome of B. pseudomallei .…”