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. 2016 Feb 26;6:21.
doi: 10.3389/fcimb.2016.00021. eCollection 2016.

Phenotypic Characterization of a Novel Virulence-Factor Deletion Strain of Burkholderia Mallei That Provides Partial Protection Against Inhalational Glanders in Mice

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Free PMC article

Phenotypic Characterization of a Novel Virulence-Factor Deletion Strain of Burkholderia Mallei That Provides Partial Protection Against Inhalational Glanders in Mice

Joel A Bozue et al. Front Cell Infect Microbiol. .
Free PMC article

Abstract

Burkholderia mallei (Bm) is a highly infectious intracellular pathogen classified as a category B biological agent by the Centers for Disease Control and Prevention. After respiratory exposure, Bm establishes itself within host macrophages before spreading into major organ systems, which can lead to chronic infection, sepsis, and death. Previously, we combined computational prediction of host-pathogen interactions with yeast two-hybrid experiments and identified novel virulence factor genes in Bm, including BMAA0553, BMAA0728 (tssN), and BMAA1865. In the present study, we used recombinant allelic exchange to construct deletion mutants of BMAA0553 and tssN (ΔBMAA0553 and ΔTssN, respectively) and showed that both deletions completely abrogated virulence at doses of >100 times the LD50 of the wild-type Bm strain. Analysis of ΔBMAA0553- and ΔTssN-infected mice showed starkly reduced bacterial dissemination relative to wild-type Bm, and subsequent in vitro experiments characterized pathogenic phenotypes with respect to intracellular growth, macrophage uptake and phagosomal escape, actin-based motility, and multinucleated giant cell formation. Based on observed in vitro and in vivo phenotypes, we explored the use of ΔTssN as a candidate live-attenuated vaccine. Mice immunized with aerosolized ΔTssN showed a 21-day survival rate of 67% after a high-dose aerosol challenge with the wild-type Bm ATCC 23344 strain, compared to a 0% survival rate for unvaccinated mice. However, analysis of histopathology and bacterial burden showed that while the surviving vaccinated mice were protected from acute infection, Bm was still able to establish a chronic infection. Vaccinated mice showed a modest IgG response, suggesting a limited potential of ΔTssN as a vaccine candidate, but also showed prolonged elevation of pro-inflammatory cytokines, underscoring the role of cellular and innate immunity in mitigating acute infection in inhalational glanders.

Keywords: Burkholderia mallei; aerosol; glanders; live-attenuated vaccine; virulence factor.

Figures

Figure 1
Figure 1
Extracellular and intracellular growth. Bacterial growth, as measured by optical density at 600 nm (OD600), in nutrient-rich LB media +4% glycerol (LB) and nutrient-limited minimal media (M9) are shown over 24 h for deletion strains ΔBMAA0553 (A) and ΔTssN (B) compared with the parental strain GRS 23344. Bacterial growth in RAW 264.7 macrophage-like cells is shown after 3 and 24-h incubation for GRS 23344 and ΔBMAA0553 at an MOI of 4 (C), and GRS 23344 and ΔTssN at an MOI of 8 (D). Growth was measured in terms of colony forming unit (CFU) recovery using gentamicin protection assays. Error bars represent standard errors from mean values determined from 2 to 4 replicates/assay. An “**” corresponds to a significant difference at p < 0.001.
Figure 2
Figure 2
Confocal analysis of the interaction of Burkholderia mallei (Bm) with RAW 264.7 cells. Macrophage-like cells were incubated with GRS 23344 (blue), ΔBMAA0553 (orange), or ΔTssN (red), stained and then visualized by confocal microscopy. The colocalization of the respective Bm strains with LAMP-1 (A,B) or actin tails (C,D) was quantified at the times indicated postinfection. The graphs are based on three independent experiments. The number of bacilli counted was 686 for (A), 724 for (B), 673 for (C), and 818 for (D). The columns represent the mean, and bars represent the standard errors. An “**” indicates a significant difference at p < 0.001.
Figure 3
Figure 3
Multinucleated giant cell (MNGC) formation in macrophage-like cells. RAW 264.7 cells were incubated with GRS 23344 (left), ΔBMAA0553 (middle), or ΔTssN (right) at an MOI of 1 for 6 h (top) and 24 h (bottom) before being stained for actin (white), macrophage nuclei (red) and Bm (green) and then visualized by confocal microscopy. The yellow arrows indicate the presence of a MNGC. Images are representative of three separate experiments. Scale bar = 20 μm.
Figure 4
Figure 4
Survival of BALB/c mice after aerosol exposure. Groups of mice (n = 10) were challenged by aerosol exposure with GRS 23344 (blue), ΔBMAA0553 (orange), or ΔTssN (red), and survival was measured over 21 days. The measured inhaled dose was 4.7 × 104, 3.3 × 104, and 2.1 × 105 CFU, for GRS 23344, ΔBMAA0553, and ΔTssN, respectively.
Figure 5
Figure 5
Cytokine expression profile. The levels of 20 cytokines/chemokines present in spleen extract supernatants were examined in mice after aerosol exposure to ΔBMA0553 (A) or ΔTssN (B) and compared to control mice 21 days postinfection. Only the expression levels of cytokines/chemokines that were at least two-fold above that of naïve mice are shown. All differences shown are statistically significant at p < 0.001.
Figure 6
Figure 6
Bacterial dissemination after infection in the lung and spleen in mice. Recovered CFUs from the lung (A) and spleen (B) are shown for time points ranging from 6 h to 6 days after aerosol exposure with parental strain GRS 23344 (blue), or the deletion strains ΔBMAA0553 (orange), and ΔTssN (red). In mice challenged with ΔBMAA0553, none had >10 CFUs recovered from the spleen.
Figure 7
Figure 7
ΔTssN vaccine efficacy against aerosol challenge with high-dose ATCC 23344 in BALB/c mice. Mice were vaccinated with ΔTssN via aerosol delivery on day 0 and 21 and challenged along with naïve mice via aerosol exposure on day 42. Survival curves of naïve and vaccinated mice after aerosol challenge with 43 LD50 of Bm ATCC 23344 over 21 days (A). Body weights of vaccinated surviving mice at prechallenge, 2 week postchallenge, and 3 week postchallenge (B). Recovered CFUs from five of the vaccinated surviving mice at 21 days postchallenge from the lung, liver, and spleen (C).
Figure 8
Figure 8
Histopathology of vaccinated and challenged mice. Histopathology of the lung (A–C), liver (D–F), and spleen (G–I) for mice 21 days after primary and boost vaccinations with ΔTssN, and after challenge with ATCC 23344. Sites of inflammation (filled arrow) and necrosis (hollow arrow) are noted, as well as relevant bronchioles (B) and blood vessels (V). All images are at × 200 magnification.

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