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.
Highlights
Burkholderia pseudomallei is a facultative intracellular pathogen that causes melioidosis, a severe invasive disease of humans that may involve subacute and latent phases
A 1.5 kb deletion of chbP between the predicted transposase genes bpss1384 and bpss1385a was detected in the draft genome sequence of the virulent strain 10276 used to identify the bsa locus, and was confirmed by polymerase chain reaction (PCR) with flanking primers
There was no evidence of any truncations in the chbP sequences that may ablate function as described previously from analysis of E. coli cycle-inhibiting factor (Cif) sequences [8]
Summary
Burkholderia pseudomallei is a facultative intracellular pathogen that causes melioidosis, a severe invasive disease of humans that may involve subacute and latent phases. The basis of entry and persistence of B. pseudomallei in host cells is ill-defined, but the bsaencoded Inv/Mxi-Spa-like Type III secretion system (T3SS-3) has been identified as a key virulence factor [1,2]. A small number of effectors have been confirmed to be substrates of the Bsa T3SS in B. pseudomallei, including BopC [4] and the guanine nucleotide exchange factor BopE [5]. A homologue of an E. coli Type III secreted effector termed Cif (cycle-inhibiting factor) was identified in B. pseudomallei and exhibits 21% amino acid identity and 40% similarity [8], but no evidence has yet been presented that it is secreted via the Bsa apparatus or that it influences pathogenesis during melioidosis
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