Virulence and cellular interactions of Burkholderia multivorans in chronic granulomatous disease

Abstract

Chronic granulomatous disease (CGD) patients are susceptible to life-threatening infections by the Burkholderia cepacia complex. We used leukocytes from CGD and healthy donors and compared cell association, invasion, and cytokine induction by Burkholderia multivorans strains. A CGD isolate, CGD1, showed higher cell association than that of an environmental isolate, Env1, which correlated with cell entry. All B. multivorans strains associated significantly more with cells from CGD patients than with those from healthy donors. Similar findings were observed with another CGD pathogen, Serratia marcescens, but not with Escherichia coli. In a mouse model of CGD, strain CGD1 was virulent while Env1 was avirulent. B. multivorans organisms were found in the spleens of CGD1-infected mice at levels that were 1,000 times higher than those found in Env1-infected mice, which was coincident with higher levels of the proinflammatory cytokine interleukin-1beta. Taken together, these results may shed light on the unique susceptibility of CGD patients to specific pathogens.

FIG. 1.

Cell association and cell invasion of B. multivorans with normal human monocytes. (A) Percent of normal human elutriated monocytes with associated bacteria. Cell monolayers on coverslips were exposed to bacteria for 2 h (MOI = 4). Coverslips were stained with Giemsa reagent and then examined at ×400 and ×1,000 magnifications to determine the percentages of cells with associated bacteria. These results were averaged from 10 independent experiments. P < 0.001. (B to D) Cell association was assessed by the Giemsa staining of monocytes infected with strain CGD1 (B) or Env1 (C and D); magnification, ×1,000. Cell invasion by GFP-expressing bacteria CGD1 (E) or Env1 (F and G) was assessed by DIC images collected simultaneously with the fluorescence images at the mid-plane of the cell nucleus. Arrows show cells with bacteria.

FIG. 2.

Cytokine production (TNF-α [A], IL-6 [B], and IL-8 [C]) by normal human elutriated monocytes infected with B. multivorans. Culture supernatants from monocytes exposed for 2 h to different strains (MOI = 4) were recovered and assayed through a multiplex bead-based assay (Bio-Rad Laboratories). NS, nonstimulated cells. Cytokines are expressed in picograms/milliliter. Results were averaged from 10 independent experiments. P > 0.05 for cytokine levels induced by CGD1 compared to those induced by Env1.

FIG. 3.

Cell association of bacteria with normal and CGD leukocytes. Shown are the associations of five B. multivorans strains with normal and CGD PBMCs (A) and neutrophils (C) and CFU of B. multivorans associated with normal or CGD PBMCs (B). Cell monolayers on coverslips were exposed to bacteria for 2 h (MOI = 4). Coverslips were washed extensively and either stained with Giemsa reagent (A and C) or treated with Triton X-100 and serially diluted with HBSS for microbial cultures (B). Stained coverslips were examined at a ×400 and ×1,000 magnification under light microscopy to determine the percentage of cells with associated bacteria. Quantitative microbial cultures were performed on TSA with 5% sheep blood plates.

FIG. 4.

Cell association of S. marcescens and E. coli with normal and CGD PBMCs. Cell monolayers on coverslips were exposed to bacteria for 2 h (MOI = 4). Coverslips were washed extensively, fixed with methanol and stained with Giemsa reagent, and then examined at ×400 and ×1,000 magnifications under light microscopy to determine the percentage of cells with associated bacteria.

FIG. 5.

TNF-α (A) and IL-6 (B) production by normal and CGD human PBMCs infected with B. multivorans strains. Culture supernatants from PBMCs exposed for 2 h to different strains (MOI = 4) were recovered and assayed through a multiplex bead-based assay (Bio-Rad Laboratories). NS, nonstimulated cells. Cytokines are expressed in picograms/milliliter. These results were averaged from 10 independent experiments. P > 0.05 for each pair of normal and CGD cells.

FIG. 6.

Kaplan-Meier survival curves of p47^phox−/− mice after intraperitoneal (i.p.) challenge with 4 × 10⁵ CFU of B. multivorans (A). Strain CGD1 or Env1 was inoculated into groups of six to eight animals. To assess in vivo bacterial loads (B) and serum levels of IL-1β (C), strain CGD1 or Env1 was inoculated in p47^phox−/− mice as described for the survival experiments. Three days postinoculation, blood samples were obtained by tail bleeding right before mice were sacrificed, and spleens were collected and processed for CFU analyses. These data summarize three independent experiments (P = 0.0008, 0.0169, and 0.0281 for A, B, and C, respectively).