BPSS1504 , a Cluster 1 Type VI Secretion Gene, Is Involved in Intracellular Survival and Virulence of Burkholderia pseudomallei

Abstract

Burkholderia pseudomallei is a Gram-negative rod and the causative agent of melioidosis, an emerging infectious disease of tropical and subtropical areas worldwide. B. pseudomallei harbors a remarkable number of virulence factors, including six type VI secretion systems (T6SS). Using our previously described plaque assay screening system, we identified a B. pseudomallei transposon mutant defective in the BPSS1504 gene that showed reduced plaque formation. The BPSS1504 locus is encoded within T6SS cluster 1 (T6SS1), which is known to be involved in the pathogenesis of B. pseudomallei in mammalian hosts. For further analysis, a B. pseudomallei BPSS1504 deletion (Bp⌬BPSS1504) mutant and complemented mutant strain were constructed. B. pseudomallei lacking the BPSS1504 gene was highly attenuated in BALB/c mice, whereas the in vivo virulence of the complemented mutant strain was fully restored to the wild-type level. The Bp⌬BPSS1504 mutant showed impaired intracellular replication and formation of multinucleated giant cells in macrophages compared with wild-type bacteria, whereas the induction of actin tail formation within host cells was not affected. These observations resembled the phenotype of a mutant lacking hcp1, which is an integral component of the T6SS1 apparatus and is associated with full functionality of the T6SS1. Transcriptional expression of the T6SS components vgrG, tssA, and hcp1, as well as the T6SS regulators virAG, bprC, and bsaN, was not dependent on BPSS1504 expression. However, secretion of Hcp1 was not detectable in the absence of BPSS1504. Thus, BPSS1504 seems to serve as a T6SS component that affects Hcp1 secretion and is therefore involved in the integrity of the T6SS1 apparatus.

Melioidosis: molecular aspects of pathogenesis

“…Notably, hcp1 (BPSS1498) deletion mutants exhibited attenuated intracellular growth in RAW 264.7 cells, defects in actin-based motility, an inability to stimulate MNGC formation and a corresponding decrease in macrophage cytotoxicity [61]. Similar phenotypes were observed with tssF (BPSS1504) and tssK (BPSS1509) mutants which displayed impaired MNGC formation in macrophages, reduced plaque formation in Ptk2 cells, and attenuated virulence in BALB/c mice [65,66]. …”

Recent Advances in Burkholderia mallei and B. pseudomallei Research

“…In addition to abrogated Hcp1 secretion, the mutant exhibited decreased virulence in vivo and in vitro. These findings suggest that BPSS1504 may play a role in the functionality of the T6SS-1 apparatus [15]. …”

Characterization of the role of the Burkholderia pseudomallei type 3 secretion system using in vivo imaging.

“…T6SS5 system mutants are attenuated >3 log in a systemic hamster intraperitoneal model [118] and are attenuated ~2-3 log in intranasal murine models [119,120]. In this current study, the role for T6SS5 in respiratory melioidosis using IMIT delivery does not appear to be a prominent role given that we were unable to demonstrate significant attenuation in single strain challenge studies, which is in contrast to what has been previously reported for the intranasal models.…”

“…And in vitro a tag-AB mutant exhibits an intracellular fitness defect in both RAW264.7 cells and the human lung epithelial cell line A549 [71]. Importantly, inactivation of tag-AB leads to a pronounced defect in Hcp5 export, strongly suggesting that tag-AB is critical for expression of functional T6SS5 machinery.…”

“…A mutant in the tag-AB gene, encoding a hypothetical protein located in the T6SS5 locus, is highly attenuated in an intranasal BALB/c model of respiratory melioidosis [71]. And in vitro a tag-AB mutant exhibits an intracellular fitness defect in both RAW264.7 cells and the human lung epithelial cell line A549 [71].…”