This site needs JavaScript to work properly. Please enable it to take advantage of the complete set of features!
Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log in
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Apr;79(4):1512-25.
doi: 10.1128/IAI.01218-10. Epub 2011 Feb 7.

The cluster 1 type VI secretion system is a major virulence determinant in Burkholderia pseudomallei

Affiliations

The cluster 1 type VI secretion system is a major virulence determinant in Burkholderia pseudomallei

Mary N Burtnick et al. Infect Immun. 2011 Apr.

Abstract

The Burkholderia pseudomallei K96243 genome encodes six type VI secretion systems (T6SSs), but little is known about the role of these systems in the biology of B. pseudomallei. In this study, we purified recombinant Hcp proteins from each T6SS and tested them as vaccine candidates in the BALB/c mouse model of melioidosis. Recombinant Hcp2 protected 80% of mice against a lethal challenge with K96243, while recombinant Hcp1, Hcp3, and Hcp6 protected 50% of mice against challenge. Hcp6 was the only Hcp constitutively produced by B. pseudomallei in vitro; however, it was not exported to the extracellular milieu. Hcp1, on the other hand, was produced and exported in vitro when the VirAG two-component regulatory system was overexpressed in trans. We also constructed six hcp deletion mutants (Δhcp1 through Δhcp6) and tested them for virulence in the Syrian hamster model of infection. The 50% lethal doses (LD(50)s) for the Δhcp2 through Δhcp6 mutants were indistinguishable from K96243 (<10 bacteria), but the LD(50) for the Δhcp1 mutant was >10(3) bacteria. The hcp1 deletion mutant also exhibited a growth defect in RAW 264.7 macrophages and was unable to form multinucleated giant cells in this cell line. Unlike K96243, the Δhcp1 mutant was only weakly cytotoxic to RAW 264.7 macrophages 18 h after infection. The results suggest that the cluster 1 T6SS is essential for virulence and plays an important role in the intracellular lifestyle of B. pseudomallei.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
SDS-PAGE of recombinant Hcp proteins encoded by six T6SS gene clusters in B. pseudomallei K96243. The recombinant Hcp proteins contain C-terminal ×ばつHis tags and were purified by immobilized metal affinity chromatography. Approximately 1 μg of protein was subjected to SDS-PAGE and stained with colloidal Coomassie blue. The relative migrations of molecular mass protein standards (lane M; in kDa) are indicated on the left. The recombinant proteins are designated Hcp1 (BPSS1498), Hcp2 (BPSS0518), Hcp3 (BPSS2098), Hcp4 (BPSS0171), Hcp5 (BPSS0099), and Hcp6 (BPSL3105).
FIG. 2.
FIG. 2.
Immunoblot analysis of B. pseudomallei recombinant Hcp proteins with human serum samples. A crude B. pseudomallei lysate (L) and the recombinant Hcp proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with pooled sera from 10 healthy donors (A) or pooled sera from 10 melioidosis patients (B). The relative migrations of molecular mass protein standards (lanes M; in kDa) are indicated on the left. The recombinant Hcp proteins are as described in the legend for Fig. 1.
FIG. 3.
FIG. 3.
Immunoblot analysis of B. pseudomallei Hcp6 production and export. B. pseudomallei wild-type and Δhcp6 cells were grown to logarithmic phase in LB, and the supernatant proteins (S) and cell-associated proteins (CA) were separated by SDS-PAGE, transferred to a polyvinylidenen difluoride membrane, and reacted with mouse polyclonal anti-Hcp6 sera. The relative migrations of molecular mass protein standards (M; in kDa) are indicated on the left. Purified recombinant Hcp6 served as a positive control for the experiment.
FIG. 4.
FIG. 4.
Immunoblot analysis of B. pseudomallei Hcp1 export. (A) B. pseudomallei strains were grown to logarithmic phase in LB, and the supernatant proteins were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and reacted with mouse polyclonal anti-Hcp1 sera. The Burkholderia strains used, AI (wild type), DDS1498 (Δhcp1), DDS1503-1 (ΔvgrG-5′), and DDS1503-2 (ΔvgrG1-3′), all harbored pBHR2 (VirAG) or pBHR2-virAG (VirAG+). The relative migrations of molecular mass protein standards (lane M; in kDa) are indicated on the left. (B) Schematic representation of native VgrG1 and VgrG1 derivatives generated by the ΔvgrG-5′ and ΔvgrG-3′ mutations. The proteins are represented as rectangles, and the domains are color coded black (tail spike domain) and dark gray ("evolved VgrG" domain). Light gray coloring represents amino acids not present in native VgrG1 resulting from a −2-bp frameshift in the coding sequence of the vgrG1 gene due to the ΔvgrG1-3′ mutation. The numbers under the proteins represent the number of amino acids in each protein.
FIG. 5.
FIG. 5.
Hepatic inflammatory cell infiltrates 2 days after infection with B. pseudomallei. Fixed and sectioned liver tissues from hamsters infected i.p. with wild-type (A and B) and Δhcp1 (C and D) strains were stained with H&E and viewed at ×ばつ (A and C) and ×ばつ (B and D) magnification. The livers from hamsters infected with the wild type contained areas with damaged hepatocytes (arrowheads) and necrotic infiltrates of neutrophils and macrophages. Necrosis and damaged hepatocytes were not observed in the hepatic inflammatory infiltrates from hamsters infected with the Δhcp1 mutant, suggesting a role for T6SS-1 in these processes.
FIG. 6.
FIG. 6.
The B. pseudomallei Δhcp1 mutant exhibits MNGC, growth, and cytotoxicity defects in RAW 264.7 cells. Monolayers infected with B. pseudomallei wild type (A and C) or Δhcp1 (B and D) cells harboring pBHR1-TG were fixed at 12 h (A and B) and 18 h (C and D) postinfection, stained, and examined by fluorescence microscopy. Bacteria expressing GFP are shown in green, host cell actin stained with Alexa 568 phalloidin is shown in red, and nuclei stained with DRAQ5 are shown in blue. Micrographs are representative of at least three independent experiments. Bars, 20 μm. (E) Monolayers infected with B. pseudomallei wild type (closed circles) or the Δhcp1 mutant (open circles) and uptake and survival were quantitated by utilizing a modified Km protection assay at 3, 6, 12, and 18 h postinfection. All assays were conducted on at least two separate occasions. The error bars represent standard deviations. *, P < 0.05; **, P < 0.01. (F) Filter-sterilized B. pseudomallei-infected RAW 264.7 cell supernatants were assayed for LDH release at 18 h postinfection. The error bars represent standard deviations. ***, P < 0.001; WT, wild type.

References

    1. Ashdown, L. R. 1979. Identification of Pseudomonas pseudomallei in the clinical laboratory. J. Clin. Pathol. 32:500-504. - PMC - PubMed
    1. Bernard, C. S., Y. R. Brunet, E. Gueguen, and E. Cascales. 2010. Nooks and crannies in type VI secretion regulation. J. Bacteriol. 192:3850-3860. - PMC - PubMed
    1. Boddey, J. A., et al. 2007. The bacterial gene lfpA influences the potent induction of calcitonin receptor and osteoclast-related genes in Burkholderia pseudomallei-induced TRAP-positive multinucleated giant cells. Cell. Microbiol. 9:514-531. - PubMed
    1. Bönemann, G., A. Pietrosiuk, and A. Mogk. 2010. Tubules and donuts: a type VI secretion story. Mol. Microbiol. 76:815-821. - PubMed
    1. Boyer, F., G. Fichant, J. Berthod, Y. Vandenbrouck, and I. Attree. 2009. Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources? BMC Genomics 10:104. - PMC - PubMed

Publication types

MeSH terms

Cite

AltStyle によって変換されたページ (->オリジナル) /