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. Infectious diseases are among the major causes of mortality worldwide. Pathogens‚ invasion, colonization and persistence within their hosts depend on a tightly orchestrated cascade of events that are commonly referred to as host/pathogen interactions. These interactions are extremely diversified and every pathogen is characterized by its unique way of co-opting and manipulating host functions to its advantage. Understanding host/pathogen interactions is the key to face the threats imposed by infectious diseases and find alternative strategies to fight the emergence of multi-drug resistant pathogens. In this study, we have setup and validated a protocol for the rapid and unbiased identification of bacterial factors that regulate host/pathogen interactions. We have applied this method to the study of Coxiella burnetii, the etiological agent of the emerging zoonosis Q fever. We have isolated, sequenced and screened over 1000 bacterial mutations and identified genes important for Coxiella invasion and replication within host cells. Ultimately, we have characterized the first Coxiella invasin, which mediates bacterial internalization within non-phagocytic cells. Most importantly, our finding may lead to the development of a synthetic vaccine against Q fever.

Significance: The biogenesis of a replicative vacuole is an essential step of Coxiella burnetii infections and involves the hijack of several host membrane trafficking pathways. Here we describe Coxiella vacuolar protein B (CvpB) as a Coxiella effector that interacts with phosphoinositides on host cell membranes and manipulates phosphatidylinositol 3-phosphate [PI(3)P] metabolism for optimal Coxiella -containing vacuole (CCV) development. This is achieved by perturbing the activity of the phosphatidylinositol 5-kinase PIKfyve, leading to an enrichment of PI(3)P on CCV membranes, which is required for the autophagy machinery to mediate CCV homotypic fusion. The importance of this process is highlighted by a homotypic fusion defect between CCVs in cells infected with CvpB Coxiella mutants, which translates into an attenuated virulence in the insect model Galleria mellonella .

Invasion of macrophages and replication within an acidic and degradative phagolysosome-like vacuole are essential for disease pathogenesis by Coxiella burnetii, the bacterial agent of human Q fever. Previous experimental constraints imposed by the obligate intracellular nature of Coxiella limited knowledge of pathogen strategies that promote infection. Fortunately, new genetic tools facilitated by axenic culture now allow allelic exchange and transposon mutagenesis approaches for virulence gene discovery. Phenotypic screens have illuminated the critical importance of Coxiella's type 4B secretion system in host cell subversion and discovered genes encoding translocated effector proteins that manipulate critical infection events. Here, we highlight the cellular microbiology and genetics of Coxiella and how recent technical advances now make Coxiella a model organism to study macrophage parasitism.

Mussels were translocated from a shell-fish breeding area (Sète, on the French Mediterranean coast) to sites exposed to trace element inputs in April 2000. They were recovered 3 months later. Whole soft tissues from all of the sites (n = 97) were analysed for arsenic, cadmium, chromium, copper, mercury, nickel, lead and zinc. Metallothioneins (MTs) were also measured in the digestive gland and in the remaining tissues (allowing calculation of whole soft tissue concentrations) at 22 of the 97 sites. MT concentrations in the digestive gland and the whole soft tissues were strongly correlated. The condition index varied with food availability at different sites. This did not influenced MT concentrations in the whole soft tissues, whereas the condition index was negatively correlated to trace element concentrations. A model is proposed to minimize this influence of condition. Metal concentrations adjusted using this model showed significant correlations with MT levels for those metals (cadmium, copper, nickel and zinc) that are known to bind to this protein, with the exception of mercury. Even in moderately contaminated sites, measurement of the MT level in the soft tissues of mussels was generally able to discriminate between different levels of contamination, allowing the use of a simplified procedure compared with dissection of the digestive gland. It is recommended to avoid translocation and sampling during the reproductive period, which is well documented for commercial species such as Mytilus sp.

Significance: Coxiella burnetii is a stealth pathogen that evades innate immune recognition by inhibiting the NF-κB signaling pathway. This process is mediated by the bacterial Dot/Icm secretion system; however, the bacterial effector/s, as well as the molecular mechanism involved in this process have remained unknown to date. Here, by investigating C. burnetii proteins with eukaryotic-like features (EUGENs), we discovered a new effector protein, NopA (nucleolar protein A), which localizes at nucleoli of infected cells and perturbs nucleocytoplasmic transport by manipulating the intracellular gradients of the GTPase Ran. In doing so, NopA reduces the nuclear levels of transcription factors involved in the innate immune sensing of pathogens and single-handedly down-modulates the expression of a panel of cytokines.

For uptake of ferrichrome into bacterial cells, FhuA, a TonB-dependent outer membrane receptor of Escherichia coli, is required. The periplasmic protein FhuD binds and transfers ferrichrome to the cytoplasmic membrane-associated permease FhuB/C. We exploited phage display to map protein-protein interactions in the E. coli cell envelope that contribute to ferrichrome transport. By panning random phage libraries against TonB and against FhuD, we identified interaction surfaces on each of these two proteins. Their interactions were detected in vitro by dynamic light scattering and indicated a 1:1 TonB-FhuD complex. FhuD residue Thr-181, located within the siderophorebinding site and mapping to a predicted TonB-interaction surface, was mutated to cysteine. FhuD T181C was reacted with two thiol-specific fluorescent probes; addition of the siderophore ferricrocin quenched fluorescence emissions of these conjugates. Similarly, quenching of fluorescence from both probes confirmed binding of TonB and established an apparent KD of approximately 300 nM. Prior saturation of the siderophorebinding site of FhuD with ferricrocin did not alter affinity of TonB for FhuD. Binding, further characterized with surface plasmon resonance, indicated a higher affinity complex with KD values in the low nanomolar range. Addition of FhuD to a preformed TonB-FhuA complex resulted in formation of a ternary complex. These observations led us to propose a novel mechanism in which TonB acts as a scaffold, directing FhuD to regions within the periplasm where it is poised to accept and deliver siderophore.