Type II
protein secretion plays a role in a wide variety of functions that are important for the ecology and pathogenesis of Legionella pneumophila. Perhaps most dramatic is the critical role that this secretion pathway has in L. pneumophila intracellular
infection of aquatic protozoa. Recently, we showed that virulent L. pneumophila strain 130b secretes
RNase activity through its
type II secretion system. We now report the cloning and mutational analysis of the gene (srnA) encoding that novel type of secreted activity. The SrnA
protein was defined as being a member of the T2 family of secreted RNases. Supernatants from mutants inactivated for srnA completely lacked
RNase activity, indicating that SrnA is the major secreted
RNase of L. pneumophila. Although srnA mutants grew normally in bacteriological media and human U937 cell macrophages, they were impaired in their ability to grow within Hartmannella vermiformis amoebae. This finding represents the second identification of a L. pneumophila type II effector being necessary for optimal intracellular
infection of amoebae, with the first being the ProA
zinc metalloprotease. Newly constructed srnA proA double mutants displayed an even larger
infection defect that appeared to be the additive result of losing both SrnA and ProA. Overall, these data represent the first demonstration of a secreted
RNase promoting an intracellular
infection event, and support our long-standing hypothesis that the
infection defects of L. pneumophila type II secretion mutants are due to the loss of multiple secreted effectors.