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. Rhodococcus equi bacteria entering macrophages can face different fates: Avirulent R. equi are delivered to a phagolysosome (bottom right) and killed. When virulent R. equi are cultivated at 30°C, they do not express the major virulence factor VapA (yellow), and they spend hours in an acidified phagolysosome (blue) before they have produced enough VapA to pH‐neutralize the phagosome and grow. 37°C‐grown virulent R. equi produce VapA to start with and find themselves immediately in a privileged compartment (bottom left).