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. 2017 Sep 20;85(10):e00468-17.
doi: 10.1128/IAI.00468-17. Print 2017 Oct.

Entry, Intracellular Survival, and Multinucleated-Giant-Cell-Forming Activity of Burkholderia pseudomallei in Human Primary Phagocytic and Nonphagocytic Cells

Liam Whiteley  1 , Teresa Meffert  1 , Maria Haug  1 , Christopher Weidenmaier  1 , Verena Hopf  2 , Katharina Bitschar  3 , Birgit Schittek  3 , Christian Kohler  2 , Ivo Steinmetz  2 , T Eoin West  4 , Sandra Schwarz  5
Affiliations

Entry, Intracellular Survival, and Multinucleated-Giant-Cell-Forming Activity of Burkholderia pseudomallei in Human Primary Phagocytic and Nonphagocytic Cells

Liam Whiteley et al. Infect Immun. .

Abstract

The human pathogen Burkholderia pseudomallei and the related species Burkholderia thailandensis are facultative intracellular bacteria characterized by the ability to escape into the cytosol of the host cell and to stimulate the formation of multinucleated giant cells (MNGCs). MNGC formation is induced via an unknown mechanism by bacterial type VI secretion system 5 (T6SS-5), which is an essential virulence factor in both species. Despite the vital role of the intracellular life cycle in the pathogenesis of the bacteria, the range of host cell types permissive for initiation and completion of the intracellular cycle is poorly defined. In the present study, we used several different types of human primary cells to evaluate bacterial entry, intracellular survival, and MNGC formation. We report the capacity of B. pseudomallei to enter, efficiently replicate in, and mediate MNGC formation of vein endothelial and bronchial epithelial cells, indicating that the T6SS-5 is important in the host-pathogen interaction in these cells. Furthermore, we show that B. pseudomallei invades fibroblasts and keratinocytes and survives inside these cells as well as in monocyte-derived macrophages and neutrophils for at least 17 h postinfection; however, MNGC formation is not induced in these cells. In contrast, infection of mixed neutrophils and RAW264.7 macrophages with B. thailandensis stimulated the formation of heterotypic MNGCs in a T6SS-5-dependent manner. In summary, the ability of the bacteria to enter and survive as well as induce MNGC formation in certain host cells may contribute to the pathogenesis observed in B. pseudomallei infection.

Keywords: Burkholderia pseudomallei; primary cells; type VI secretion system.

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Figures

FIG 1
FIG 1
(A) Entry and intracellular survival of B. pseudomallei wild type (wt) and T6SS-5 mutant (ΔT6SS-5) in human primary cells (HUVEC, human vein endothelial cells; NHBE, bronchial epithelial cells; HGF, gingival fibroblasts; KC, undifferentiated keratinocytes; hMSC, mesenchymal stem cells; MDM, monocyte-derived macrophages; PMN, neutrophils). Entry and intracellular survival were determined at 3 h and 17 to 24 h postinfection, respectively, using antibiotic protection assays. The MOI and time of infection were optimized for each cell and are listed in Table 1. (B) Entry and intracellular survival of B. thailandensis wild type (wt) and T6SS-5 mutant (ΔT6SS-5) in human epithelial cell lines derived from different tissues (SW 13, adrenal gland cell line; 5637, bladder cell line; HuTu-80, duodenal cell line; SK-OV-3, ovary cell line; PANC-1, pancreatic cell line; DU 146, prostate cell line). Entry and intracellular survival were measured at 3 h and 14 to 40 h postinfection, respectively, using antibiotic protection assays. The MOI and time of infection were optimized for each cell line and are listed in Table 1. Shown are mean values + standard deviation. a, not detected.
FIG 2
FIG 2
(A) Human primary cells infected with B. pseudomallei wild type (wt) and T6SS-5 mutant (ΔT6SS-5) for 17 to 24 h using antibiotic protection assays and stained with Giemsa. The same MOI and time of infection were used as for the determination of intracellular survival of B. pseudomallei in these cells and are listed in Table 1. MNGC formation was observed in NHBE and HUVEC infected with wild-type B. pseudomallei. (B) Human epithelial cell lines infected with B. thailandensis wild type (wt) and T6SS-5 mutant (ΔT6SS-5) for 14 to 40 h using antibiotic protection assays and stained with Giemsa (see legend to Fig. 1 for abbreviations of cell lines). The same MOI and time of infection were used as for the determination of intracellular survival of B. thailandensis in these cell lines and are listed in Table 1. Wild-type B. thailandensis stimulated MNGC formation in all cell lines tested.
FIG 3
FIG 3
MNGC formation in primary endothelial (HUVEC) and bronchial epithelial cells (NHBE) induced by B. thailandensis. (A) Giemsa stain of NHBE and HUVEC infected with B. thailandensis wild type and ΔT6SS-5 mutant at MOIs of 200 and 50, respectively, for approximately 20 h. (B) Quantification of MNGC formation efficiency under the same conditions. (C) Invasion and intracellular survival of B. thailandensis in HUVEC and NHBE at 3 h and 20 h postinfection, respectively. Shown are mean values + standard deviation. a, not detected; b, not determined.
FIG 4
FIG 4
(A) Quantification of MNGC formation in primary cells (see legend to Fig. 1 for abbreviations) infected with B. pseudomallei wild type (wt) and T6SS-5 mutant (ΔT6SS-5) or left uninfected. The same MOI and time of infection as for the determination of intracellular survival of B. pseudomallei in these cells were used and are listed in Table 1. (B) MNGC formation in epithelial cell lines infected with B. thailandensis wild type (wt) or ΔT6SS-5 or left uninfected (see legend to Fig. 1 for abbreviations of cell lines). The same MOI and time of infection were used as for the determination of intracellular survival of B. thailandensis in these cells and are listed in Table 1. Shown are mean values + standard deviation. a, not detected; b, not determined.
FIG 5
FIG 5
Intracellular loads of wild-type B. thailandensis in relation to the extent of MNGC formation. The diagram shows average values of final intracellular CFU of wild-type B. thailandensis plotted against average values of MNGC formation (%) for each cell line (black symbols), primary endothelial cells (HUVEC), and epithelial cells (NHBE) (gray symbols).
FIG 6
FIG 6
Confocal fluorescence microscopy z-stack imaging of mixed PMN and RAW264.7 macrophages infected with wild-type B. thailandensis (A) and B. thailandensis ΔT6SS-5 (B) for 22 h at an MOI of 50. z-stack images were collected at 0.6-μm sections, and the red and green lines indicate the orthogonal planes of the y-z and x-z projections, respectively. Heterotypic MNGC containing macrophage (single-lobed) and neutrophil (multilobed) nuclei were detected upon infection with wild-type B. thailandensis. Shown are representative images of two experiments performed in triplicate. Plasma membrane was stained with wheat germ agglutinin Alexa Fluor 594 conjugate (red), and DNA was stained with DAPI (blue). Arrows mark multilobed nuclei. Bars, 10 μm.
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