Identification of OmpA, a Coxiella burnetii Protein Involved in Host Cell Invasion, by Multi-Phenotypic High- Content Screening Eric Martinez 1,2,3 , Franck Cantet 1,2,3 , Laura Fava 1,2,3 , Isobel Norville 4 , Matteo Bonazzi 1,2,3 * 1 CNRS, UMR5236, CPBS, Montpellier, France, 2 Universite ´ Montpellier 1, CPBS, Montpellier, France, 3 Universite ´ Montpellier 2, CPBS, Montpellier, France, 4 Defence Science and Technology Laboratory, Porton Down, United Kingdom 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. Citation: Martinez E, Cantet F, Fava L, Norville I, Bonazzi M (2014) Identification of OmpA, a Coxiella burnetii Protein Involved in Host Cell Invasion, by Multi- Phenotypic High-Content Screening. PLoS Pathog 10(3): e1004013. doi:10.1371/journal.ppat.1004013 Editor: Raphael H. Valdivia, Duke University, United States of America Received August 30, 2013; Accepted February 4, 2014; Published March 20, 2014 Copyright: ß 2014 Martinez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by an ATIP/Avenir Grant to MB and a Marie Curie CIG to EM (grant n. 293731). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: matteo.bonazzi@cpbs.cnrs.fr Introduction Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible of the worldwide neglected zoonosis Q fever [1,2]. Acute forms of the disease are characterized by a febrile illness associated with severe headache, pneumonia and hepatitis. In a small percentage (2–5%) of cases, acute Q fever develops into a chronic infection that may lead to endocarditis and chronic fatigue syndrome [1,3]. Coxiella resists environmental stress by generating small cell variants (SCVs) that facilitate its airborne dissemination; during infections, this pathogen converts into a metabolically active large cell variant (LCV) with a unique resistance to the degradative machinery of host cells [4,5]. These factors contribute to the extreme infectivity of this microbe, making of Coxiella a serious health concern, especially in rural areas where outbreaks are likely to occur and are accompanied by heavy economic burdens [6,7]. Moreover, the development of Coxiella as a potential bioweapon during and since World War II, has ascribed this pathogen among class B biothreats [7]. Coxiella has two antigenic phases: phase I organisms, isolated from natural sources of infection, are extremely virulent. Phase II bacteria originate from spontaneous mutations after several in vitro passages of phase I organisms and present a truncated lipopolysaccharide (LPS) [8]. These non-reversible mutations result in a strong attenuation of virulence in vivo [9,10]. Phase II Coxiella organisms are internalized more efficiently than phase I organisms by both professional macrophages and non-phagocytic cells [9,11], how- ever, once internalized, both antigenic phases replicate within host cells with similar kinetics. A phase II clone (Nine Mile phase II clone 4 or NMIIC4), which has been authorized for biosafety level 2 (BSL-2) manipulation, represents therefore an optimal model to study Coxiella infections [2,5]. In natural infections, Coxiella has a tropism for alveolar macrophages [1,2], however, infection of epithelial and endothelial cells has also been reported [12,13]. Indeed, in vitro, Coxiella invades and replicates in a wide variety of phagocytic and non-phagocytic cells [5]. Coxiella internalization within host cells is a passive, endocytic process, which involves the remodeling of the host cell actin cytoskeleton [14,15] and a V b 3 integrins have been reported as Coxiella receptors in THP-1 cells [11]. However the Coxiella factors that mediate interactions with PLOS Pathogens | www.plospathogens.org 1 March 2014 | Volume 10 | Issue 3 | e1004013