The mechanistic basis of the membrane-permeabilizing activities of the virulence-associated protein A (VapA) from Rhodococcus equi.

Pathogenic Rhodococcus equi release the virulence-associated protein A (VapA) within macrophage phagosomes. VapA permeabilizes phagosome and lysosome membranes and reduces acidification of both compartments. Using biophysical techniques, we found that VapA interacts with model membranes in four steps: (i) binding, change of mechanical properties, (ii) formation of specific membrane domains, (iii) permeabilization within the domains, and (iv) pH-specific transformation of domains. Biosensor data revealed that VapA binds to membranes in one step at pH 6.5 and in two steps at pH 4.5 and decreases membrane fluidity. The integration of VapA into lipid monolayers was only significant at lateral pressures <20 mN m-1 indicating preferential incorporation into membrane regions with reduced integrity. Atomic force microscopy of lipid mono- and bilayers showed that VapA increased the surface heterogeneity of liquid disordered domains. Furthermore, VapA led to the formation of a new microstructured domain type and, at pH 4.5, to the formation of 5 nm high domains. VapA binding, its integration and lipid domain formation depended on lipid composition, pH, protein concentration and lateral membrane pressure. VapA-mediated permeabilization is clearly distinct from that caused by classical microbial pore formers and is a key contribution to the multiplication of Rhodococcus equi in phagosomes.

Differential Effects of Rhodococcus equi Virulence-Associated Proteins on Macrophages and Artificial Lipid Membranes.

Virulence-associated protein A (VapA) of Rhodococcus equi is a pathogenicity factor required for the multiplication of virulent R. equi strains within spacious macrophage vacuoles. The production of VapA is characteristic for R. equi isolates from pneumonic foals. VapB and VapN proteins in R. equi isolates from infected pig (VapB) and cattle (VapN) have amino acid sequences very similar to VapA and consequently have been assumed to be its functional correlates. Using model membrane experiments, phagosome pH acidification analysis, lysosome size measurements, protein partitioning, and degradation assays, we provide support for the view that VapA and VapN promote intracellular multiplication of R. equi by neutralizing the pH of the R. equi-containing vacuole. VapB does not neutralize vacuole pH, is not as membrane active as VapA, and does not support intracellular multiplication. This study also shows that the size of the sometimes enormous R. equi-containing vacuoles or the partitioning of purified Vaps into organic phases are not features that have predictive value for virulence of R. equi, whereas the ability of Vaps to increase phagosome pH is coupled to virulence. IMPORTANCE Rhodococcus equi is a major cause of life-threatening pneumonia in foals and occasionally in immunocompromised persons. Virulence-associated protein A (VapA) promotes R. equi multiplication in lung macrophages, which are the major host cells during foal infection. In this study, we compare cellular, biochemical, and biophysical phenotypes associated with VapA to those of VapB (typically produced by isolates from pigs) or VapN (isolates from cattle). Our data support the hypothesis that only some Vaps support multiplication in macrophages by pH neutralization of the phagosomes that R. equi inhabit.

Specific preadaptations of Rhodococcus equi cooperate with its Virulence-associated protein A during macrophage infection.

Gram-positive Rhodococcus equi (Prescotella equi) is a lung pathogen of foals and immunocompromised humans. Intra-macrophage multiplication requires production of the bacterial Virulence-associated protein A (VapA) which is released into the phagosome lumen. VapA pH-neutralizes intracellular compartments allowing R. equi to multiply in an atypical macrophage phagolysosome. Here, we show that VapA does not support intra-macrophage growth of several other bacterial species demonstrating that only few bacteria have the specific preadaptations needed to profit from VapA. We show that the closest relative of R. equi, environmental Rhodococcus defluvii (Prescotella defluvii), does not multiply in macrophages at 37°C even when VapA is present because of its thermosensitivity but it does so once the infection temperature is lowered providing rare experimental evidence for 'thermal restriction'. Using growth experiments with isolated macrophage lysosomes and modified infection schemes we provide evidence that R. equi resists the attack by phagolysosome contents at low pH for several hours. During this time, R. equi produces and secretes VapA which enables it to grow at the expense of lysosome constituents. We present arguments that, under natural infection conditions, R. equi is VapA-less during the initial encounter with the host. This has important implications for vaccine development.