Identification of OmpA, a Coxiella burnetii Protein Involved in Host Cell Invasion, by Multi-Phenotypic High-Content Screening

Abstract

Coxiella burnetii is the agent of the emerging zoonosis Q fever. This pathogen invades phagocytic and non-phagocytic cells and uses a Dot/Icm secretion system to co-opt the endocytic pathway for the biogenesis of an acidic parasitophorous vacuole where Coxiella replicates in large numbers. The study of the cell biology of Coxiella infections has been severely hampered by the obligate intracellular nature of this microbe, and Coxiella factors involved in host/pathogen interactions remain to date largely uncharacterized. Here we focus on the large-scale identification of Coxiella virulence determinants using transposon mutagenesis coupled to high-content multi-phenotypic screening. We have isolated over 3000 Coxiella mutants, 1082 of which have been sequenced, annotated and screened. We have identified bacterial factors that regulate key steps of Coxiella infections: 1) internalization within host cells, 2) vacuole biogenesis/intracellular replication, and 3) protection of infected cells from apoptosis. Among these, we have investigated the role of Dot/Icm core proteins, determined the role of candidate Coxiella Dot/Icm substrates previously identified in silico and identified additional factors that play a relevant role in Coxiella pathogenesis. Importantly, we have identified CBU_1260 (OmpA) as the first Coxiella invasin. Mutations in ompA strongly decreased Coxiella internalization and replication within host cells; OmpA-coated beads adhered to and were internalized by non-phagocytic cells and the ectopic expression of OmpA in E. coli triggered its internalization within cells. Importantly, Coxiella internalization was efficiently inhibited by pretreating host cells with purified OmpA or by incubating Coxiella with a specific anti-OmpA antibody prior to host cell infection, suggesting the presence of a cognate receptor at the surface of host cells. In summary, we have developed multi-phenotypic assays for the study of host/pathogen interactions. By applying our methods to Coxiella burnetii, we have identified the first Coxiella protein involved in host cell invasion.

Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development

“…Larvae were injected in the upper right proleg with 10 6 genome equivalents (GE)/mL of WT Coxiella, Tn1832, or the cvpB mutant Tn1146, and the survival rate was determined over 10 d. Larvae injected with PBS solution were used as controls. As previously reported (14,20), Galleria larvae were efficiently killed by WT Coxiella or Tn1832; however, the injection of the cvpB mutant strain resulted in the killing of 50% of the larvae over the same time course (SI Appendix, Fig. S3C).…”

“…Among others, this approach identified the icmS::Tn mutant as well as all six transposon insertions in cvpB (CBU_0021; SI Appendix, Fig. S2A) previously identified in our screen as affecting the size of intracellular Coxiella colonies (14) and recently reported as important for the homotypic fusion of CCVs (8). The axenic and intracellular replication of six independent cvpB transposon mutants (Tn1146, Tn396, Tn239, Tn170, Tn2579, and Tn2032) was thus analyzed.…”

“…Building on our recent screen of a Coxiella transposon library (14,15), here we use image-based supervised machine learning to address the mechanisms regulating the fusogenicity of CCVs. We show that the Coxiella effector CvpB localizes at CCVs and early endosomes by interacting with phosphatidylinositol 3-phosphate [PI(3)P] and phosphatidylserine (PS).…”

“…In the context of a multiparametric high-content screen of a Coxiella transposon mutant library, we have recently reported the seemingly multivacuolar phenotype associated with a transposon insertion in the dot/icm gene icmS (14). The Legionella pneumophila IcmS homolog is a chaperone that mediates the translocation of specific effectors (16,17), suggesting that Coxiella IcmS may regulate the translocation of effectors involved in the homotypic fusion of CCVs.…”

“…S1C, Right), which presented several tight-fitting vacuoles enclosing few bacteria. To determine whether the multivacuolar phenotype results from the defective secretion of Coxiella effectors involved in CCV homotypic fusion, we used image-based supervised machine learning to mine the database generated during the screen of our Coxiella transposon library (14) and identify transposon insertions resulting in multivacuolar phenotypes. Among others, this approach identified the icmS::Tn mutant as well as all six transposon insertions in cvpB (CBU_0021; SI Appendix, Fig.…”