OpiA, a Type Six Secretion System Substrate, localizes to the cell pole and plays a role in bacterial growth and viability in Francisella tularensis LVS.

Abstract

Francisella tularensis is an intracellular pathogen and the causative agent of tularemia. The F. tularensis type six secretion system (T6SS) is required for a number of host-pathogen interactions including phagolysosomal escape and invasion of erythrocytes. One known effector of the T6SS, OpiA, has recently been shown to be a phosphatidylinositol-3 kinase. To investigate the role of OpiA in erythrocyte invasion, we constructed an opiA-null mutant in the live vaccine strain, F. tularensis LVS. OpiA was not required for erythrocyte invasion, however, deletion of opiA affected growth of F. tularensis LVS in broth cultures in a medium-dependent manner. We also found that opiA influenced cell size, gentamicin sensitivity, bacterial viability, and the lipid content of F. tularensis. A fluorescently tagged OpiA (OpiA-EmGFP) accumulated at the cell poles of F. tularensis which is consistent with the location of the T6SS. However, OpiA-EmGFP also exhibited a highly dynamic localization and this fusion protein was detected in erythrocytes and THP-1 cells in vitro further supporting that OpiA is secreted. Similar to previous reports using F. novicida, our data demonstrated that opiA had a minimal effect on intracellular replication of F. tularensis in host immune cells in vitro. However, THP-1 cells infected with the opiA mutant produced modestly (but significantly) higher levels of the pro-inflammatory cytokine TNF-α compared to these host cells infected with wild-type bacteria. Together, our results support previous observations that implicate opiA in infection and virulence, but also reveal an additional role for opiA central to the biology of F. tularensis bacteria. (This research was made possible by NASA West Virginia Space Grant Consortium Training Grant #NNX15A101H and by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence).