Analysis of the Prevalence, Secretion and Function of a Cell Cycle-Inhibiting Factor in the Melioidosis Pathogen Burkholderia pseudomallei

Abstract

Enteropathogenic and enterohaemorrhagic Escherichia coli express a cell cycle-inhibiting factor (Cif), that is injected into host cells via a Type III secretion system (T3SS) leading to arrest of cell division, delayed apoptosis and cytoskeletal rearrangements. A homologue of Cif has been identified in Burkholderia pseudomallei (CHBP; Cif homologue in B. pseudomallei; BPSS1385), which shares catalytic activity, but its prevalence, secretion and function are ill-defined. Among 43 available B. pseudomallei genome sequences, 33 genomes (76.7%) harbor the gene encoding CHBP. Western blot analysis using antiserum raised to a synthetic CHBP peptide detected CHBP in 46.6% (7/15) of clinical B. pseudomallei isolates from the endemic area. Secretion of CHBP into bacterial culture supernatant could not be detected under conditions where a known effector (BopE) was secreted in a manner dependent on the Bsa T3SS. In contrast, CHBP could be detected in U937 cells infected with B. pseudomallei by immunofluorescence microscopy and Western blotting in a manner dependent on bsaQ. Unlike E. coli Cif, CHBP was localized within the cytoplasm of B. pseudomallei-infected cells. A B. pseudomallei chbP insertion mutant showed a significant reduction in cytotoxicity and plaque formation compared to the wild-type strain that could be restored by plasmid-mediated trans-complementation. However, there was no defect in actin-based motility or multinucleated giant cell formation by the chbP mutant. The data suggest that the level or timing of CHBP secretion differs from a known Bsa-secreted effector and that CHBP is required for selected virulence-associated phenotypes in vitro.

Melioidosis: molecular aspects of pathogenesis

“…CHBP secretion was shown to occur following infection of human U937 macrophages with B. pseudomallei, require a functional T3SS-3, and was postulated to be host cell contact dependent [54]. A chbP mutant survived at wild type levels in U937 cells, retained the ability to form actin tails, and induced MNGC formation in J774A.1 macrophages [54].…”

“…CHBP secretion was shown to occur following infection of human U937 macrophages with B. pseudomallei, require a functional T3SS-3, and was postulated to be host cell contact dependent [54]. A chbP mutant survived at wild type levels in U937 cells, retained the ability to form actin tails, and induced MNGC formation in J774A.1 macrophages [54]. Interestingly, following infection of HeLa cells the plaque forming efficiency of the chbP mutant was significantly decreased, cytotoxicity was reduced, and smaller plaques were consistently observed in comparison to the wild type [54].…”

“…A chbP mutant survived at wild type levels in U937 cells, retained the ability to form actin tails, and induced MNGC formation in J774A.1 macrophages [54]. Interestingly, following infection of HeLa cells the plaque forming efficiency of the chbP mutant was significantly decreased, cytotoxicity was reduced, and smaller plaques were consistently observed in comparison to the wild type [54]. When ectopically expressed in HEK293T and HeLa cells, CHBP induced actin stress fiber formation and arrested the cell cycle at the G2/M boundary by deamidation of Gln 40 in ubiquitin and ubiquitin-like proteins [55-57].…”

Effects of sodium chloride on heat resistance, oxidative susceptibility, motility, biofilm and plaque formation of Burkholderia pseudomallei

“…Plaque‐forming efficiency was assessed as previously described (Pumirat et al., ). HeLa cells were infected with B. pseudomallei at a multiplicity of infection of 20 and incubated at 37°C with 5% CO 2 for 2 hr.…”

Type III Secretion in the Melioidosis Pathogen Burkholderia pseudomallei

“…Interestingly, CHBP is not secreted under standard growth conditions, but B. pseudomallei secretes CHBP in U937 cells in a bsaQ –dependent manner (Pumirat et al, 2014). A B. pseudomallei K96243 chbP insertion mutant was impaired in its ability to form plaques in HeLa cells at 24 h and demonstrated lower cytotoxicity at 6 h as assessed by LDH release assays (Pumirat et al, 2014).…”