RoaM negatively regulates expression of a limited number of rickettsial genes.

UNLABELLED

The recently described rickettsial protein RoaM (regulator of actin-based motility) negatively regulates the production of actin tails, and its abrogation induces hyper-spreading behavior in many laboratory-adapted strains of . RoaM is not surface exposed; thus, its mechanism of regulating actin-based motility is unclear. Using strains derived from the virulent Sheila Smith strain that express varying levels of , an RNA-seq experiment was performed. We found that -overexpressing strains downregulate expression of at least six genes which may link the regulatory effects of RoaM to the phenotypic effect on motility. Genes regulated by RoaM were confirmed by RT-qPCR. Among the genes regulated is the secreted effector RarP2, which disrupts the trans-Golgi network. Two of the hypothetical proteins were shown to be secreted via fusion to a glycogen synthase kinase tag, which when phosphorylated reveals exposure to the host-cell cytosol. Taken together, these data support the hypothesis that RoaM affects transcription, downregulating rickettsial genes important for pathogenicity in the mammalian host but which are perhaps otherwise detrimental within the tick vector. To determine how RoaM activity may itself be regulated, we investigated a role of temperature in transcription. RoaM expression itself is not temperature dependent, but many other rickettsial genes are, including some also regulated by RoaM. This suggests that rickettsiae utilize multiple mechanisms to control gene expression in response to environmental signals.

IMPORTANCE

RoaM was previously shown to repress the production of actin tails by unknown mechanisms. The gene is negatively selected for in cell culture resulting in hyper-spreading mutants. This work reveals that rather than specifically regulating motility in , a set of rickettsial genes is downregulated that includes the type IV secreted effector, , as well as two other secreted, putative effectors. Relatively few secreted effectors have been identified in . RoaM appears to be part of a larger biological program encompassing active spreading in mammalian cells and may be a critical component for to transition from arthropod to mammalian host.