Step-wise loss of bacterial flagellar torsion confers progressive phagocytic evasion

Abstract

Phagocytosis of bacteria by innate immune cells is a primary method of bacterial clearance during infection. However, the mechanisms by which the host cell recognizes bacteria and consequentially initiates phagocytosis are largely unclear. Previous studies of the bacterium Pseudomonas aeruginosa have indicated that bacterial flagella and flagellar motility play an important role in colonization of the host and, importantly, that loss of flagellar motility enables phagocytic evasion. Here we use molecular, cellular, and genetic methods to provide the first formal evidence that phagocytic cells recognize bacterial motility rather than flagella and initiate phagocytosis in response to this motility. We demonstrate that deletion of genes coding for the flagellar stator complex, which results in non-swimming bacteria that retain an initial flagellar structure, confers resistance to phagocytic binding and ingestion in several species of the gamma proteobacterial group of Gram-negative bacteria, indicative of a shared strategy for phagocytic evasion. Furthermore, we show for the first time that susceptibility to phagocytosis in swimming bacteria is proportional to mot gene function and, consequently, flagellar rotation since complementary genetically- and biochemically-modulated incremental decreases in flagellar motility result in corresponding and proportional phagocytic evasion. These findings identify that phagocytic cells respond to flagellar movement, which represents a novel mechanism for non-opsonized phagocytic recognition of pathogenic bacteria.

Figure 1. Non-swimming gram-negative bacteria are resistant to phagocytosis.

Gentamicin protection assays were used to assess: (A) C57BL/6 BMDC phagocytosis of WT P. aeruginosa strain PA14, the independent mucoid clinical isolate FRD1, and increasing concentrations of non-flagellated mutant flgK and flagellated but non-swimming mutant motABmotCD (in PA14 background). (B) Murine BMDC phagocytosis of WT V. cholerae strain O395, and the flaA, motX, tcpA, and toxT mutants. (C) Murine BMDC phagocytosis of WT E. coli strain K12, and the flgK, fliC, and motA mutants. (D) Human THP-1 leukocyte phagocytosis of P. aeruginosa WT, flgK, and motABmotCD (left); or V. cholerae WT, flaA, and motX (right). Where indicated throughout this and the other figures, phagocytosis of WT strains (PA14, K12 and 0395 in this figure) has been normalized to 100% and the relative phagocytosis of the mutant strains shown as the percent of WT. N≥6, *p<0.05 compared to WT.

Figure 2. Fluorescence microscopy of phagocytic interactions with GFP-expressing bacteria.

(A) Confocal fluorescence microscopy of untreated murine peritoneal macrophages co-incubated at 37°C for 45 minutes with GFP-transformed V. cholerae O395 WT (left) or motX (right), washed, and subsequently stained on ice with Alexa647-conjugated wheat germ agglutinin (WGA). (B) Internalized bacteria, as in (A), were quantified on the basis of being within a contiguous WGA-decorated phagocyte plasma membrane and not co-localizing with WGA (co-localization seen as yellow, as at the plasma membrane or being external to a phagocytic cell). N≥6 images, *p<0.05.

Figure 3. The phagocytic resistance by P. aeruginosa motABmotCD is not due to resultant changes in bacterial secretions, extracellular protein expression, or PAMP regulation.

(A) BMDCs were co-incubated with a mixture of equal numbers of carbinicillin-resistant PA14 WT and carb-sensitive motABmotCD or, conversely, carb-resistant motABmotCD and carb-sensitive WT. Phagocytic susceptibility was assayed by gentamicin protection assay and plating on carbinicillin-treated LB agar. (B) Volcano plot of WT gene expression versus motABmotCD mutant gene expression. Red points indicate genes corresponding to likely immunogenic molecules (see Table S1). N≥7, *p<0.05.

Figure 4. Microbiocidal activity and limited bacteria-cell contact does not provide for decreased phagocytic clearance of non-swimming bacteria.

(A) Adherent murine peritoneal macrophages were co-incubated with PA14 WT or motABmotCD and cellular associated bacteria and non-associated bacteria were quantitatively assessed at the indicated time points. (B) BMDCs were co-incubated with WT or motABmotCD in the presence or absence of 0.01% Tween80, 0.01% beta-octyl glucoside, or 2% Survanta, and assayed by gentamicin protection assay for relative bacterial phagocytosis. (C, inset) GFP-expressing PA14 WT or motABmotCD were centrifuged onto BMDCs and immediately fixed and analyzed by FACs for cellular association. Phagocytic cells in the absence of bacteria are shown as background. (C) P. aeruginosa PA14 WT, flgK, or motABmotCD, or (D) V. cholerae O395 WT, flaA, or motX were centrifuged onto BMDCs or peritoneal macrophages, respectively, and assayed by gentamicin protection assay. N≥5, *p<0.05 as compared to WT.

Figure 5. Flagellar motility enhances both the association and the uptake of bacteria by phagocytes.

(A) P. aeruginosa PA14 WT, flgK, or motABmotCD that were incubated in parallel with adherent macrophages at 4°C exhibited similar binding (assessed by CFUs following washing and lysis of the macrophages) (left). However, the difference in relative bacterial association with macrophages dramatically changed when, following washing, the bound bacteria and cells were warmed to 37°C (middle); and this differential was even more substantial when assessed on the basis of phagocytosed (gentamicin-resistant) bacteria (right). Plots show total mean recovered CFUs accounting for input bacteria (left panel) and the number of recovered CFUs plotted relative to WT (right panel). (B) Murine peritoneal macrophages were treated with cytochalasin D prior to co-incubation with PA14 WT, flgK, or motABmotCD and were assayed for bacterial association and protection from gentamicin. (C) Fluorescence microscopy of cytochalasin D-treated macrophages co-incubated with GFP-expressing PA14 WT, GFP-expressing flgK, or GFP-expressing motABmotCD and subsequently stained with wheat germ agglutinin-conjugated Alexa647. 65x magnification. N≥5, *p<0.05.

Figure 6. Live imaging of murine peritoneal macrophage interactions with PA14 WT or motABmotCD bacteria in vitro.

Representative images of adherent macrophages treated with liquid culture of GFP-expressing PA14 WT (top) or GFP-expressing motABmotCD (bottom) under constant flow at Time  = 1 min, Time  = 15 min, and Time  = 30 min. Bacterial concentrations were equalized prior to imaging for comparative visualization of bacterial accumulation and retention (arrows) on phagocytes. Macrophages viewed by DIC, bacteria by fluorescence. See Videos S1 and S2.

Figure 7. Successive loss of flagellar functionality enables stepwise increases in phagocytic resistance.

(A) FACS analysis and (B) gentamicin protection assay of BMDCs co-incubated with PA14 WT, motAB, motCD, or motABmotCD. (C) FACS analysis of cytochalasin D treated BMDCs co-incubated with GFP-expressing PA14 WT, flgK, motAB, motCD, or motABmotCD. (D) Adherent macrophage assay of cells co-incubated with PA14 WT, motAB, motCD, or motABmotCD bacteria at 4°C, warmed to 37 °C, or treated with gentamicin after warming to 37 °C. (E) Gentamicin protection assay of murine peritoneal macrophages co-incubated as indicated with P. aeruginosa PA14 WT or V. cholerae O395 WT in 70 mM, 18 mM, 9 mM, or 4.5 mM NaCl buffers, or in 15 mM NaCl buffer with 135 mM choline chloride. Recovered CFUs are normalized against recovery in HBSS (138 mM NaCl). N≥5, *p<0.05.