VapA of Rhodococcus equi binds phosphatidic acid

Abstract

Rhodococcus equi is a multihost, facultative intracellular bacterial pathogen that primarily causes pneumonia in foals less than six months in age and immunocompromised people. Previous studies determined that the major virulence determinant of R. equi is the surface bound virulence associated protein A (VapA). The presence of VapA inhibits the maturation of R. equi-containing phagosomes and promotes intracellular bacterial survival, as determined by the inability of vapA deletion mutants to replicate in host macrophages. While the mechanism of action of VapA remains elusive, we show that soluble recombinant VapA^32-189 both rescues the intramacrophage replication defect of a wild type R. equi strain lacking the vapA gene and enhances the persistence of nonpathogenic Escherichia coli in macrophages. During macrophage infection, VapA was observed at both the bacterial surface and at the membrane of the host-derived R. equi containing vacuole, thus providing an opportunity for VapA to interact with host constituents and promote alterations in phagolysosomal function. In support of the observed host membrane binding activity of VapA, we also found that rVapA^32-189 interacted specifically with liposomes containing phosphatidic acid in vitro. Collectively, these data demonstrate a lipid binding property of VapA, which may be required for its function during intracellular infection.

Figure 1. Recombinant VapA^32-189 and VapK2^32-202 complement the replication defect of R. equi ΔvapA

Murine J774A.1 macrophage monolayers were incubated overnight with the indicated concentration of recombinant Vap protein (dashed lines with open symbols) or media (solid lines with closed symbols), then infected with the indicated R. equi strain. Each experimental condition was performed in triplicate per infection; n = 3. Symbols denote the mean number of bacteria observed and the bars denote the mean bacterial fold change. Error bars represent the standard deviation using a two-way analysis of variation (ANOVA) by way of the Holm-Sidak test. (A,C) Letters to the right of each curve denote statistical significance; the same letter signifies no statistical difference, while different letters signify statistical difference at P ≤ 0.05. (B,D) ns = not significant; (*) = P < 0.05; (**) = P < 0.01; (***) = P < 0.001.

Figure 2. Recombinant VapA^32-189 enhances intracellular persistence of nonpathogenic E. coli in J774A.1 cells

J774A.1 macrophage monolayers were incubated overnight with either media (triangles) or 150 nM recombinant Vap protein (VapA, circles; VapG, crosses), and infected with E. coli. Symbols delineate the mean bacterial number and bars denote the mean percent survival. The error bars represent the standard deviation calculated using a two-way ANOVA via the Holm-Sidak method. Each experimental condition was performed in triplicate per infection; n = 3. (A) Statistical significance is expressed as letters to the right of the curve, with same letters defining a lack of significant difference, and different letters defining significance with P ≤ 0.05. (B) Percent of viable E. coli cells detected, as compared to 1 hour post infection (HPI) is shown; ns = not significant; (***) = P < 0.001.

Figure 3. VapA associates with the RCV membrane during infection

Murine bone marrow-derived macrophages (BMDMs) were infected with R. equi 103S or ΔvapA strains harboring the GFP expression plasmid, pGFPmut2. Where indicated, 100 nM rVapA was added to the BMDM monolayer the night before the infection. R. equi (GFP, green), BMDM nucleus (DAPI, blue), and VapA (anti-VapA, red) were observed. (A) Representative confocal images of infection, bar = 5 μ, inset bar = 1 μ. Arrows indicate VapA detected at the RCV membrane. (B) Bacterial numbers per 200 macrophages were quantified by direct visualization at the indicated time points. (C) Macrophages containing ten or more bacteria, discerned via direct visualization. (B,C) Bars indicate the mean number of quantified bacteria or macrophages, while the error bars represent the standard deviation calculated by a two-way ANOVA using the Holm-Sidak method. n = 3; ns = not significant and (***) = P ≤ 0.001.

Figure 4. GFP-VapA binds to the yeast plasma membrane upon expression in vivo

(A) Saccharomyces cerevisiae strain BY4742 harboring either GFP (vector), GFP-VapA, GFP-VapA^32-189, GFP-VapG^27-172, GFP-VapB^35-197, or GFP-VapK2^32-202 plasmid constructs were grown overnight and proteins were extracted from equal amounts of cell pellets, separated via SDS-PAGE, and probed with polyclonal anti-VapA antisera. (B) Cells from (A), with the exception of the strain harboring GFP-VapG^27-172, were visualized for GFP localization via fluorescence microscopy. Bar = 5 μ. (C) Quantification of GFP localization from yeast strains imaged in (A); Cells with clear accumulation of GFP-Vap protein at the plasma membrane was counted as “plasma membrane”, diffuse GFP staining of the cytoplasm with no plasma membrane localization was counted as “cytoplasm;” all other morphologies were counted as “other.” 100 yeast cells were counted per experimental condition, bars denote the mean percent of cells displaying a particular Vap localization pattern. Error bars represent the standard deviation using a two-way analysis of variation (ANOVA) by way of the Holm-Sidak test, and significance pertaining to the difference in plasma membrane localization of GFP-Vap proteins is indicated. n = 3; ns = not significant and (***) = P ≤ 0.001.

Figure 5. rVapA binds phosphatidic acid containing liposomes

Liposomes of indicated compositions (PA, 20% phosphatidic acid; PC, 100% phosphatidylcholine; PE; 20% phosphatidylethanolamine; PS, 20% phosphatidylserine) were generated as in Experimental Procedures. Liposomes were incubated with recombinant Vap protein at a pH of either (A) 7.4 or (B) 5.5, and liposomes were isolated by flotation (Experimental Procedures). Equal fractions representing 10% of the total reaction (T) and either 900 nmol (A,B) or 1 μmol (C,D) total floated (F) liposomes were separated via SDS-PAGE and immunoblotted for VapA. (C) Liposomes containing increasing amounts of PA were assayed for VapA binding, as above in (5B). (D) Liposomes containing 20% PA were incubated with 1 μg indicated recombinant Vap protein, and assayed for binding, as above in (5B). All recombinant Vap proteins tested cross-react with VapA antiserum.

Figure 6. The presence of VapA prevents the accumulation of LAMP1 within the RCV 48 and 72 h post infection

(A) BMDMs were infected with either R. equi 103S or ΔvapA strains, and 100 nM rVapA^32-189 was added the night before the infection, where indicated. At indicated time points, cells were fixed, immunostained, and visualized via confocal microscopy. R. equi (GFP, green), BMDM nucleus (DAPI, blue), VapA (anti-VapA, red), and murine LAMP1 (anti-LAMP1, purple) were observed. n = 3, bar = 5 μ, inset bar = 1 μ. (B) Representative confocal image of 103S infection of BMDMs at T72, performed and stained as in (A). A large RCV containing replicating bacteria and RCV membrane-associated VapA (arrow) is shown in comparison to macrophages containing strongly LAMP1-positive compartments surrounding bacteria that lack detectable VapA at the RCV membrane (asterisks), bar = 5 μ.