Pseudomonas aeruginosa evasion of phagocytosis is mediated by loss of swimming motility and is independent of flagellum expression

Abstract

Pseudomonas aeruginosa is a pathogenic Gram-negative bacterium that causes severe opportunistic infections in immunocompromised individuals; in particular, severity of infection with P. aeruginosa positively correlates with poor prognosis in cystic fibrosis (CF) patients. Establishment of chronic infection by this pathogen is associated with downregulation of flagellar expression and of other genes that regulate P. aeruginosa motility. The current paradigm is that loss of flagellar expression enables immune evasion by the bacteria due to loss of engagement by phagocytic receptors that recognize flagellar components and loss of immune activation through flagellin-mediated Toll-like receptor (TLR) signaling. In this work, we employ bacterial and mammalian genetic approaches to demonstrate that loss of motility, not the loss of the flagellum per se, is the critical factor in the development of resistance to phagocytosis by P. aeruginosa. We demonstrate that isogenic P. aeruginosa mutants deficient in flagellar function, but retaining an intact flagellum, are highly resistant to phagocytosis by both murine and human phagocytic cells at levels comparable to those of flagellum-deficient mutants. Furthermore, we show that loss of MyD88 signaling in murine phagocytes does not recapitulate the phagocytic deficit observed for either flagellum-deficient or motility-deficient P. aeruginosa mutants. Our data demonstrate that loss of bacterial motility confers a dramatic resistance to phagocytosis that is independent of both flagellar expression and TLR signaling. These findings provide an explanation for the well-documented observation of nonmotility in clinical P. aeruginosa isolates and for how this phenotype confers upon the bacteria an advantage in the context of immune evasion.

FIG. 1.

P. aeruginosa mutants deficient for flagellum or flagellar stator proteins are resistant to phagocytosis by BMDCs in vitro. (A) C57BL/6 BMDCs were analyzed by FACS for relative association with GFP-transformed PA14, the pilus-deficient pilG mutant, or the flagellum-deficient flgK and fliN mutants. (B) C57BL/6 BMDCs were assayed by gentamicin protection assay for relative phagocytic uptake of PA14 or flagellum-deficient flgK and fliN mutants. The graph is plotted on a logarithmic scale. (C) FACS analysis (left) and gentamicin protection assay (right) of C57BL/6 BMDCs coincubated with either WT PA14 or the ΔmotAB ΔmotCD flagellar stator mutant. (D) FACS histogram of total BMDC cellular association with GFP-transformed P. aeruginosa strains after 45 min of coincubation. (E) Fluorescence microscopy of RFP-expressing BMDCs coincubated with GFP-transformed PA14 or the ΔmotAB ΔmotCD mutant viewed with DIC overlay at ×65 magnification. For all graphs, phagocytic uptake levels were normalized as percentages of the mean WT phagocytosis. For all genotypes, n is ≥9; means, standard deviations, and statistical significance (asterisks) are shown.

FIG. 2.

Phagocytic resistance of nonswimming P. aeruginosa is not due to bactericidal activity or differential flagellar expression. (A) WT PA14 (PA14) and the ΔmotAB ΔmotCD (mot) strain express similar total levels of flagellin as assessed by Western analysis for the FliC protein (right). Coomassie blue staining of parallel lanes is shown as a control for protein load (left). (B) To confirm that the ΔmotAB ΔmotCD mutant strain has intact extracellular flagella, the flagella were mechanically sheared from the bacteria and assessed by Coomassie blue staining (left) and Western analysis (right). (C) The BMDC killing rate of P. aeruginosa was assayed by gentamicin protection assay. Following a 1-h coincubation of PA14 with BMDCs, gentamicin was added, and at the indicated time points following gentamicin addition, aliquots were harvested and lysed to assess the death of internalized bacteria over the course of the assay. CFU were plotted relative to initial recovery. For each time point, n is ≥6; standard deviations are shown.

FIG. 3.

P. aeruginosa mutants deficient for flagellum or flagellar motor proteins are resistant to phagocytosis by human macrophages in vitro. Peripheral blood mononuclear cell (PBMC)-derived human macrophages were cultured and assayed by gentamicin protection assay for relative in vitro uptake of PA14, pilus-deficient pilB and pilG mutants, flagellum-deficient flgK and fliN mutants, and the flagellar stator protein-deficient ΔmotAB ΔmotCD mutant. Phagocytic uptake levels were normalized as percentages of the mean WT phagocytosis. Statistical significance (P < 0.05) of differences from wild-type levels is indicated (asterisks). Graphs are plotted on a log scale. For all genotypes, n is ≥9; means and standard deviations are shown.

FIG. 4.

The phagocytic deficiency for nonmotile P. aeruginosa strains is independent of MyD88 signaling. (A) WT or MyD88^−/− BMDCs were assayed for relative in vitro cellular association of GFP-PA14, GFP-flgK, or GFP-ΔmotAB ΔmotCD P. aeruginosa strains by FACS analysis. Data are represented as the mean fluorescence intensity (MFI) of each experimental group. (B) WT or MyD88^−/− BMDCs were assayed for in vitro phagocytosis of PA14 or flgK or ΔmotAB ΔmotCD P. aeruginosa strains by gentamicin protection assay. Phagocytic uptake levels were normalized as percentages of mean WT PA14 phagocytosis. The graph is plotted on a logarithmic scale. For all genotypes, n is ≥6 and means, standard deviations, and statistical significance (asterisks) are shown.

FIG. 5.

Motor protein-deficient P. aeruginosa strains are resistant to phagocytosis by ex vivo primary phagocytic cells from the peritoneum and the lung. (A) C57BL/6 peritoneal exudate cells were harvested by lavage and assayed by FACS ex vivo for relative cellular association with the GFP-expressing bacterial strains indicated. MFI, mean fluorescence intensity. (B) Peritoneal phagocytes were assayed for relative phagocytosis of WT PA14, flgK, and ΔmotAB ΔmotCD bacteria by gentamicin protection assay. (C) C57BL/6 lung phagocytes were harvested via bronchoalveolar lavage and subsequently assayed by FACS ex vivo for relative cellular association with GFP-transformed bacteria as indicated. (D) Lung phagocytes were assayed by gentamicin protection assay ex vivo for relative uptake of WT, flagellum-deficient mutant flgK, and flagellar motor protein-deficient mutant ΔmotAB ΔmotCD bacteria. For all graphs, n is ≥4 and means, standard deviations, and statistical significance (asterisks) are shown.

FIG. 6.

Flagellum-deficient and stator protein-deficient P. aeruginosa strains are resistant to phagocytosis in vivo. (A) (Top) An in vivo gentamicin protection assay was used to determine relative uptake of PA14, flgK, and ΔmotAB ΔmotCD bacterial strains by peritoneal phagocytes. Phagocytic uptake levels were normalized as percentages of the mean WT phagocytosis levels. (Bottom) Total peritoneal cells were quantified from lavage samples of mice treated with PA14, flgK, or ΔmotAB ΔmotCD bacteria, to control for differential immune cell recruitment by the different bacterial strains. (B) Gentamicin protection assay results following in vivo oropharyngeal aspiration of WT or ΔmotAB ΔmotCD PA14 are consistent with the data shown in panel A, with nonmotile PA14 being ∼10-fold more resistant to phagocytosis than motile PA14. Individual data points (mice), means, and statistical significance (asterisks) are shown.