Rickettsia rickettsii RoaM negatively regulates expression of a limited number of rickettsial genes

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 Rickettsia rickettsii. RoaM is not surface exposed thus its mechanism of regulating actin-based motility is unclear. Using R. rickettsii strains derived from the virulent Sheila Smith strain that express varying levels of roaM, an RNA-seq experiment was performed. We found that roaM-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 roaM 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. This study includes two bulk RNA-seq experiments on Rickettsia rickettsii Sheila Smith. One experiment compares the expression of different genotypes to investigate the role of the gene RoaM. For this experiment, four genotypes of R. rickettsii Shelia Smith (wildtype, truncated RoaM, RoaM knockout, and RoaM overexpression) were grown in identical conditions. The other experiment compares the expression of R. rickettsia Shelia Smith wildtype and RoaM deletion strains at two temperatures, 34ºC (standard temperature) and 22ºC.