TlyC, a conserved hemolysin in , contributes to spotted fever pathogenesis in mice.

Unlabelled: circulates between mammalian hosts and hematophagous arthropod vectors by exploiting their intracellular environment. With advances in rickettsial genetic tools, recent studies have identified novel molecular mechanisms involved in -host-vector interactions. However, a significant knowledge gap exists in understanding how modulates its virulence functions to survive in two drastically different environments (mammalian hosts vs arthropod vectors). Bacterial hemolysins play a crucial role in neutralizing innate immune functions through pore-forming activities and direct interactions with cognate receptors. Prior work suggested that typhus group (e.g., and ), but not spotted fever group (e.g., and ), can induce hemolytic activities upon direct interactions with red blood cells. Here, we demonstrate that typhus and spotted fever groups exhibit comparable hemolytic activities. Furthermore, by characterizing an transposon insertional variant (HK27), we document that TlyC, a factor conserved in , is responsible for pH-, temperature-, and host species-dependent hemolytic activities. Our biochemical and genetic studies confirmed that the first 10 amino acids are critical in facilitating hemolytic activities without affecting TlyC localization to the outer membrane. Compared to wild-type , the HK27 variant showed reduced intracellular survival in primary endothelial cells and attenuated virulence in mice. These findings suggest a functional role for a conserved hemolysin in rickettsial pathogenesis.

Importance: Rickettsiosis is a vector-borne disease that causes systemic and potentially fatal vasculitis if not diagnosed promptly and treated with antibiotics. Pathogenic species, such as , preferentially infect vascular endothelial cells with extensive abilities to survive in the cytoplasm of professional phagocytes. With the development of genetic tools for , recent studies have highlighted the biological roles of unique and conserved factors involved in rickettsial pathogenesis and vector transmission. However, additional studies are warranted to uncover essential molecular mechanisms that can be exploited to generate vaccines or therapeutics. The significance of our research is the identification of a conserved hemolysin exhibiting unconventional hemolytic activities and its contribution to rickettsial pathogenesis. Our research establishes a concrete foundation for studying protein secretion pathways that translocate effector proteins in , understanding how controls host cell membrane disruption, and identifying factors that support the rickettsial lifecycle between arthropod vectors and mammalian hosts.