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 Oct;79(10):4010-8.
doi: 10.1128/IAI.05044-11. Epub 2011 Aug 1.

The Burkholderia pseudomallei Δasd mutant exhibits attenuated intracellular infectivity and imparts protection against acute inhalation melioidosis in mice

Affiliations

The Burkholderia pseudomallei Δasd mutant exhibits attenuated intracellular infectivity and imparts protection against acute inhalation melioidosis in mice

Michael H Norris et al. Infect Immun. 2011 Oct.

Abstract

Burkholderia pseudomallei, the cause of serious and life-threatening diseases in humans, is of national biodefense concern because of its potential use as a bioterrorism agent. This microbe is listed as a select agent by the CDC; therefore, development of vaccines is of significant importance. Here, we further investigated the growth characteristics of a recently created B. pseudomallei 1026b Δasd mutant in vitro, in a cell model, and in an animal model of infection. The mutant was typified by an inability to grow in the absence of exogenous diaminopimelate (DAP); upon single-copy complementation with a wild-type copy of the asd gene, growth was restored to wild-type levels. Further characterization of the B. pseudomallei Δasd mutant revealed a marked decrease in RAW264.7 murine macrophage cytotoxicity compared to the wild type and the complemented Δasd mutant. RAW264.7 cells infected by the Δasd mutant did not exhibit signs of cytopathology or multinucleated giant cell (MNGC) formation, which were observed in wild-type B. pseudomallei cell infections. The Δasd mutant was found to be avirulent in BALB/c mice, and mice vaccinated with the mutant were protected against acute inhalation melioidosis. Thus, the B. pseudomallei Δasd mutant may be a promising live attenuated vaccine strain and a biosafe strain for consideration of exclusion from the select agent list.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Mini-Tn7-bar-rfp, single-copy tagging vector based on phosphinothricin resistance, harboring rfp driven by the PS12 promoter. After insertion aided by pTNS3-asdEc (29), the non-antibiotic resistance marker, which is flanked by identical FRTs, can be removed by Flp-mediated excision. Abbreviations: bar, gene encoding bialaphos/phosphinothricin resistance; FRT, Flp recombination target sequences; oriT, RP4 conjugal origin of transfer; PCS12, promoter of the Burkholderia cenocepacia rpsL gene; PS12, promoter of the B. pseudomallei rpsL gene; R6Kγori, π protein-dependent R6K origin of replication; Tn7L/Tn7R, left and right transposase recognition sequences; ToT1, transcriptional terminator.
Fig. 2.
Fig. 2.
Growth curve experiments performed with B. pseudomallei strains. (A) B. pseudomallei strains were grown in the absence of DAP. The wild-type strain and the Δasd strain complemented with a single copy of the asd gene on a site-specific transposon exhibited the same growth rates and final optical densities, while the Δasd mutant exhibited a typical DAP-dependent phenotype. (B) The B. pseudomallei Δasd mutant was tested in different concentrations of DAP, ranging from 0 μg/ml to 500 μg/ml. Compared to wild type, the Δasd mutant exhibited absence of growth without DAP. All other concentrations of DAP afforded a partial growth rate recovery and final optical density, albeit only after a lag in growth.
Fig. 3.
Fig. 3.
Infection of HeLa and RAW264.7 cells by B. pseudomallei and the Δasd strain. HeLa (A) and RAW264.7 (B) cell monolayers were infected at an MOI of 10:1. The complemented Δasd strain showed no decrease in its ability to invade and replicate within either cell line. However, the Δasd mutant in the absence of DAP could not sustain an infection in either cell line, denoted by an overall drop in bacterial numbers.
Fig. 4.
Fig. 4.
(A) Cytotoxicity of B. pseudomallei strains to the RAW264.7 murine macrophage cell line. RAW264.7 cells were infected with the Δasd mutant (in the presence or absence of DAP), the complemented mutant strain, and wild-type strain. Between 2 h and 6 h postinfection there was a slight increase in cytotoxicity associated with infection by the complemented and wild-type strains compared to the Δasd mutant-infected monolayers. By 12 h postinfection, cytotoxicities of the complement- and wild-type-infected monolayers were more obvious, while cytotoxicity caused by the Δasd mutant remained similar to the noninfected control. At 24 h postinfection, the complement- and wild-type-infected monolayers exhibited maximal cytotoxicity. (B) Microscopy and time course of the cytopathic effects of B. pseudomallei Δasd infection. Monolayers were infected at an MOI of 10:1 and then analyzed for red fluorescence at 2 h and 24 h postinfection. Differential interference contrast (DIC) images were overlaid with the red fluorescent channel. Red fluorescence indicates the presence of B. pseudomallei. Note the high bacterial levels in the complement- and wild-type-infected monolayers at 24 h and the confluent MNGC formation. This coincides with high levels of cytotoxicity at 24 h postinfection. Abbreviations: CT, noninfected control; Δasd, B. pseudomallei Δasd/rfp; Δasd + DAP, B. pseudomallei Δasd/rfp in the presence of 200 μg/ml of DAP; Δasd + complement, B. pseudomallei Δasd/complement/rfp; WT, B. pseudomallei wt/rfp. Error bars represent the SEM of three experiments. Statistical significance was determined by the two-tailed unpaired t test (***, P < 0.0005).
Fig. 5.
Fig. 5.
Intracellular replication of B. pseudomallei. RAW264.7 murine macrophage monolayers were visualized using a combination of differential interference contrast and red fluorescence microscopy 24 h postinfection with the B. pseudomallei Δasd/complement/rfp strain (A and B) and with B. pseudomallei wt/rfp strain (C and D). All macrophages in the field of view are interacting with MNGCs and are filled with intracellular bacteria about to burst into the extracellular milieu. Images in panels A and C were captured at ×ばつ magnification, while images in panels B and D are zoomed-in images of the regions denoted in panels A and C, respectively. Note the large number of bacteria projecting out of the remaining macrophages in panels C and D. There were no bacteria and there was an absence of protrusions as well as MNGCs at all time points in monolayers infected with the Δasd mutant (data not shown).
Fig. 6.
Fig. 6.
(A) The B. pseudomallei 1026b Δasd mutant is avirulent in mice. Mice (n = 5 animals per group) were challenged i.n. with either 4,500 CFU B. pseudomallei 1026b (wild type) or 1 ×ばつ 107 CFU B. pseudomallei 1026b Δasd mutant, and survival was monitored. Statistical differences in survival times were determined by Kaplan-Meier curves followed by log-rank test (**, P < 0.01 for B. pseudomallei 1026b wt versus B. pseudomallei 1026b Δasd mutant). (B) Intranasal vaccination with the B. pseudomallei 1026b Δasd mutant protects mice from lethal B. pseudomallei challenge. Mice (n = 10 animals per group) were primed i.n. with 1 ×ばつ 107 CFU B. pseudomallei 1026b Δasd and boosted in the same manner 3 weeks later. Two weeks postboost, mice were challenged i.n. with 4 ×ばつ 103 CFU wild-type B. pseudomallei 1026b. Survival was monitored, and statistical differences in survival times were determined by Kaplan-Meier curves followed by a log-rank test (***, P < 0.0001 for vaccinated versus nonvaccinated mice). Data represen two individual pooled experiments.

References

    1. Atkins T., et al. 2002. Characterisation of an acapsular mutant of Burkholderia pseudomallei identified by signature tagged mutagenesis. J. Med. Microbiol. 51:539–553 - PubMed
    1. Barrett A. R., et al. 2008. Genetics tools for allelic-replacement in Burkholderia species. Appl. Environ. Microbiol. 74:4498–4508 - PMC - PubMed
    1. Belyakov I. M., Ahlers J. D. 2009. What role does the route of immunization play in the generation of protective immunity against mucosal pathogens? J. Immunol. 183:6883–6892 - PubMed
    1. Breitbach K., Kohler J., Steinmetz I. 2008. Induction of protective immunity against Burkholderia pseudomallei using attenuated mutants with defects in the intracellular life cycle. Trans. R. Soc. Trop. Med. Hyg. 2008:S89–S94 - PubMed
    1. Buckley A. M., et al. 2010. Evaluation of live-attenuated Salmonella vaccines expressing Campylobacter antigens for control of C. jejuni in poultry. Vaccine 28:1094–1105 - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources

Full text links
Atypon full text link Atypon Free PMC article
Cite

AltStyle によって変換されたページ (->オリジナル) /