Fig 7.

Inhibition of autophagy impairs R. rickettsii replication (A) R. rickettsii infection induced LC3B conjugation. HeLa cells infected with R. rickettsii at MOI = 1 were probed for LC3B and p62 by immunoblotting. Actin was probed as a loading control. (B) MTM counteracts the elevated level of autophagy induced by R. rickettsii infection. HeLa cells transfected to express GFP, GFP-MTM, or GFP-MTM_C375S for 14 h priority to infected with R. rickettsii at MOI = 1. The cell lysates at indicated timepoints of infection were probed for LC3B, PikA, and GFP. Actin was detected as a loading control. (C) Overexpression of MTM impairs intracellular bacterial growth. HeLa cells transfected to express GFP or GFP-MTM for 14 h were infected with R. rickettsii and treated with 1 µM wortmannin or DMSO (solvent control). Genome equivalent was determined 48 h after bacterial uptake. GE, genome equivalents/mL. Data (mean ± s.e.) shown one representative from three independent experiments each done in triplicate. (D) A model for autophagy modulation by PikA in R. rickettsii replication. PikA translocated into the host cell by R. rickettsii converts PI to PI3P, the effector also interacts with the host autophagosome initiation complex (Atg14L-Beclin 1-Vps15) to promote the formation of autophagosome. The PI3P phosphatase MTM reduced cellular PI3P, thus blocking R. rickettsii replication. ***, P < 0.001.