Melanie Burette

Matteo Bonazzi
email: matteo.bonazzi@irim.cnrs.fr

From neglected to dissected: how technological advances are leading the way to the study of Coxiella burnetii pathogenesis

published or not. The documents may come

Introducing Coxiella burnetii

Coxiella burnetii is a Gram negative obligate intracellular pathogen, and the causative agent of Q fever, a worldwide zoonotic disease [START_REF] Eldin | From Q Fever to Coxiella burnetii Infection: a Paradigm Change[END_REF]. Animal coxiellosis is mostly associated with abortion, stillbirths, and weak offspring (Fig. 1).

Desiccation of placental materials, excretions of birthing fluids, urine, feces and milk from infected animals contribute to shed C. burnetii into the environment [START_REF] Eldin | From Q Fever to Coxiella burnetii Infection: a Paradigm Change[END_REF]. Transmission to humans results from exposure to contaminated aerosols and dust (Fig. 1). In humans, C. burnetii infections are often asymptomatic and self-limiting, however, 40% of individuals infected with C. burnetii develop an acute disease which is associated with a flu-like syndrome, pneumonia, hepatitis and chronic fatigue [START_REF] Eldin | From Q Fever to Coxiella burnetii Infection: a Paradigm Change[END_REF] (Fig. 1). Acute disease may convert into a chronic illness with severe complications, including endocarditis [START_REF] Eldin | From Q Fever to Coxiella burnetii Infection: a Paradigm Change[END_REF]. An important risk factor for Q fever outbreaks is the multiple zoonotic reservoirs of C. burnetii, which include domestic livestock and wild animals (mammals, reptiles, birds and ticks) [START_REF] Eldin | From Q Fever to Coxiella burnetii Infection: a Paradigm Change[END_REF]. C. burnetii is extremely infectious, with 1-to-10 bacteria being sufficient to cause disease [START_REF] Brooke | Human dose response relation for airborne exposure to Coxiella burnetii[END_REF]. The low infectious dose coupled to remarkable environmental stability contribute to the significant spreading of C. burnetii infections well away from the outbreak source and led to the classification of C. burnetii as a category B biothreat [START_REF] Madariaga | Q fever: a biological weapon in your backyard[END_REF].

C. burnetii is a stealth pathogen that actively escapes innate immune recognition by inhibiting the NF-kB pathway [START_REF] Mahapatra | Coxiella burnetii Employs the Dot/Icm Type IV Secretion System to Modulate Host NF-κB/RelA Activation[END_REF] and inflammasome activation [START_REF] Cunha | Inhibition of inflammasome activation by Coxiella burnetii type IV secretion system effector IcaA[END_REF]. Infected cells are also protected from apoptosis, thereby preserving the bacterial replicative niche over long periods [START_REF] Lührmann | Beginning to understand the role of the Type IV secretion system effector proteins in coxiella burnetii pathogenesis[END_REF]. C. burnetii enters macrophages by phagocytosis through the interaction with αVβ3 integrins [START_REF] Capo | Subversion of monocyte functions by coxiella burnetii: impairment of the cross-talk between alphavbeta3 integrin and CR3[END_REF]. In contrast, in non-phagocytic cells, internalization is facilitated by the invasin OmpA (Outer membrane protein A) [START_REF] Martinez | Identification of OmpA, a Coxiella burnetii Protein Involved in Host Cell Invasion, by Multi-Phenotypic High-Content Screening[END_REF] (Fig. 1). Following internalization, bacteria reside within early endosomal compartments, also called early CCVs, that passively mature along the endocytic pathway by successive fusion events with early and late endosomes and lysosomes (Fig. 1). Maturation is accompanied by the acidification of the CCV lumen [START_REF] Heinzen | Differential interaction with endocytic and exocytic pathways distinguish parasitophorous vacuoles of Coxiella burnetii and Chlamydia trachomatis[END_REF], which is required to activate bacterial metabolism [START_REF] Hackstadt | Biochemical stratagem for obligate parasitism of eukaryotic cells by Coxiella burnetii[END_REF] and the translocation of bacterial effector proteins by a Dot/Icm Type 4b

Secretion System (T4SS) [START_REF] Newton | Effector Protein Translocation by the Coxiella burnetii Dot/Icm Type IV Secretion System Requires Endocytic Maturation of the Pathogen-Occupied Vacuole[END_REF]. Thus, by 48 hours post-infection, cells display a single, large, mature CCV, where markers of multiple membrane compartments co-exist [START_REF] Dragan | Coxiella burnetii: international pathogen of mystery[END_REF], which is indicative of the capacity of C. burnetii to hijack multiple host membrane trafficking pathways (Fig. 1). Importantly, the biogenesis of these remarkable compartments is a two-step process requiring both cellular and bacterial factors. This review will focus on the recent technological advances that fostered remarkable progress in our understanding of the complex host/pathogen interplay that controls the generation of the C. burnetii replicative niche.

The long and winding road (to genetic manipulation)

Together with its high infectivity, the obligate intracellular nature of C. burnetii has imposed severe constraints on the study of its pathogenesis. An important step towards the development of tools to investigate C. burnetii infection was the isolation of a Phase II variant (NMII) of the Nine Mile Phase I strain (NMI), presenting a single deletion of 21 genes involved in lipopolysaccharide biosynthesis and displaying lossof-virulence phenotype in animal models [START_REF] Moos | Comparative virulence of intra-and interstrain lipopolysaccharide variants of Coxiella burnetii in the guinea pig model[END_REF][START_REF] Hoover | Chromosomal DNA Deletions Explain Phenotypic Characteristics of Two Antigenic Variants, Phase II and RSA 514 (Crazy), of the Coxiella burnetii Nine Mile Strain[END_REF]. Thus, NMII was allowed for manipulation in biosafety level-2 (BSL-2) environments (Fig. 2D), as opposed to other C. burnetii strains that require manipulation in biosafety level-3 (BSL-3) confinement. Importantly, loss-of-virulence of NMII has no impact either on intracellular growth kinetics or development of CCVs in cultured cells, making NMII a relevant model to study host-pathogen interactions [START_REF] Howe | Coxiella burnetii phase I and II variants replicate with similar kinetics in degradative phagolysosome-like compartments of human macrophages[END_REF].

Nevertheless, bacterial amplification of C. burnetii in embryonated eggs and/or cultured cells represented a real obstacle for its genetic manipulation. Indeed, transposon mutagenesis allowed the generation of C. burnetii mutants [START_REF] Beare | Characterization of a Coxiella burnetii ftsZ mutant generated by Himar1 transposon mutagenesis[END_REF]; however, amplification and isolation of these from infected cells ruled out the possibility of isolating mutations in virulence genes. Thus, until 2011, ftsZ was the only C. burnetii gene characterized using Himar1 transposon mutagenesis [START_REF] Beare | Characterization of a Coxiella burnetii ftsZ mutant generated by Himar1 transposon mutagenesis[END_REF] (Fig. 2F).

Whole-genome sequencing of C. burnetii NMI RSA493 in 2003 allowed the identification of typical features, including a Dot/Icm secretion system, which is highly homologous to that of L. pneumophila [START_REF] Seshadri | Complete genome sequence of the Q-fever pathogen Coxiella burnetii[END_REF] (Fig. 2C). This finding was pivotal to develop bioinformatics-based approaches to predict effector proteinscoding genes. These revealed that similar to L. pneumophila, effector proteins translocated by C. burnetii encode a C-terminal secretion signal called the E-block motif and are mostly under the control of a PmrA response regulator [START_REF] Zusman | The response regulator PmrA is a major regulator of the icm/dot type IV secretion system in Legionella pneumophila and Coxiella burnetii[END_REF]. Due to the genetic intractability of C. burnetii at that time, candidate effector proteins were tested for secretion using L. pneumophila as a surrogate model, exploiting the homologies between the Dot/Icm secretion systems of the two pathogens. This approach allowed the Roy laboratory to validate the translocation of four C. burnetii effector proteins encoding ankyrin repeat homology domains (ARHDs) [START_REF] Pan | Ankyrin repeat proteins comprise a diverse family of bacterial type IV effectors[END_REF] (Fig. 2E). Later work from the Samuel laboratory reported the first large-scale, bioinformatics-based identification of C. burnetii effector proteins and used the L. pneumophila system to validate the secretion of 32 new T4SS substrates [START_REF] Chen | Large-scale identification and translocation of type IV secretion substrates by Coxiella burnetii[END_REF] (Fig. 2H). Moreover, using a shuttle plasmid system for the expression of recombinant proteins in C. burnetii, Chen and colleagues also demonstrated that the functionality of the Dot/Icm secretion system [START_REF] Chen | Large-scale identification and translocation of type IV secretion substrates by Coxiella burnetii[END_REF].

One year later, a genetic screen of C. burnetii proteins carrying C-terminal secretion signals led to the identification of additional effector proteins [START_REF] Carey | The Coxiella burnetii Dot/Icm System Delivers a Unique Repertoire of Type IV Effectors into Host Cells and Is Required for Intracellular Replication[END_REF] (Fig. 2I). For this study, wild type (wt) L. pneumophila or the T4SSdefective DdotA mutant were transformed with a library containing adenylate cyclase enzyme (CyaA)-tagged random fragments of C. burnetii genome, leading to the validation of 7 additional C. burnetii effectors [START_REF] Carey | The Coxiella burnetii Dot/Icm System Delivers a Unique Repertoire of Type IV Effectors into Host Cells and Is Required for Intracellular Replication[END_REF]. These were further tested for their intracellular localization and function, which indicated a possible implication in the manipulation of host membrane trafficking. Of note, this is the first study to report a replication phenotype associated with a transposon insertion in the C. burnetii Dot/Icm gene icmL (icmL::Tn), thus demonstrating the importance of C. burnetii effector protein translocation for infection [START_REF] Carey | The Coxiella burnetii Dot/Icm System Delivers a Unique Repertoire of Type IV Effectors into Host Cells and Is Required for Intracellular Replication[END_REF] (Fig. 2I).

Today, the development of tailored bioinformatics algorithms for the identification of T4SS effector proteins led to the identification of over 140 candidate C. burnetii T4SS effectors [START_REF] Voth | The Coxiella burnetii ankyrin repeat domain-containing protein family is heterogeneous, with C-terminal truncations that influence Dot/Icm-mediated secretion[END_REF][START_REF] Chen | Large-scale identification and translocation of type IV secretion substrates by Coxiella burnetii[END_REF][START_REF] Carey | The Coxiella burnetii Dot/Icm System Delivers a Unique Repertoire of Type IV Effectors into Host Cells and Is Required for Intracellular Replication[END_REF][START_REF] Maturana | Refining the plasmid-encoded type IV secretion system substrate repertoire of Coxiella burnetii[END_REF][START_REF] Weber | Identification of Coxiella burnetii type IV secretion substrates required for intracellular replication and Coxiella-containing vacuole formation[END_REF]. Recent genome comparison of the C. burnetii strains NMII RSA493, Henzerling RSA331, G Q212, K Q154 and Dugway 5J108-111, revealed that many genes encoding candidate effector proteins are either pseudogenized or missing altogether, leaving only 44 out of the 143 identified effector protein-coding genes intact across all strains [START_REF] Larson | Right on Q: Genetics begin to unravel Coxiella burnetii host cell interactions[END_REF]. Despite these significant advances, functional analysis of C. burnetii genes involved in virulence was still limited by the genetic intractability of this pathogen.

A game-changer in the study of C. burnetii infections has been the development of a synthetic medium (ACCM for Acidified Citrate Cysteine Medium), followed by the development of ACCM-2, allowing the extracellular culture of this obligate intracellular bacterium [START_REF] Omsland | Host cell-free growth of the Q fever bacterium Coxiella burnetii[END_REF]Omsland et al., 2011) (Fig. 2G). ACCM-2 development stems from metabolic requirement studies using microarrays, genomic reconstruction of metabolic pathways and metabolite typing [START_REF] Omsland | Host cell-free growth of the Q fever bacterium Coxiella burnetii[END_REF]Omsland et al., 2011). These studies highlighted that C. burnetii requires acid activation buffer (pH 4.75), to reproduce the CCV microenvironment [START_REF] Heinzen | Differential interaction with endocytic and exocytic pathways distinguish parasitophorous vacuoles of Coxiella burnetii and Chlamydia trachomatis[END_REF] and increase its metabolic potential as well as low oxygen levels (2.5%) to facilitate the bacterial micro-aerophilic respiration [START_REF] Omsland | Host cell-free growth of the Q fever bacterium Coxiella burnetii[END_REF]Omsland et al., 2011). This scientific milestone finally enabled genetic manipulation of C. burnetii, leading to a new era in our understanding of its pathogenesis (Fig. 2).

From genes to function: characterization of C. burnetii effectors involved in vacuole biogenesis

With the development of axenic culture, targeted deletion of C. burnetii was formally achievable (albeit remaining extremely challenging), and the role of C. burnetii effector proteins in infection could be tested directly. The Heinzen laboratory first exploited axenic culture with the generation of a dotA deletion mutant [START_REF] Beare | Two systems for targeted gene deletion in Coxiella burnetii[END_REF] (Fig. 2J) and with the identification and characterization of CvpA (for Coxiella vacuolar protein A). This effector protein localizes at CCVs and reroutes recycling endosomes to this compartment by interacting with the clathrin adaptor protein AP2 [START_REF] Larson | Coxiella burnetii effector protein subverts clathrin-mediated vesicular trafficking for pathogen vacuole biogenesis[END_REF] (Fig. 1). In a follow-up study, the Heinzen laboratory identified four additional members of what constitutes today the Cvp sub-class of C. burnetii effector proteins (CvpA, CvpB/Cig2, and CvpC-to-E, Fig. 1) [START_REF] Larson | Coxiella burnetii Effector Proteins That Localize to the Parasitophorous Vacuole Membrane Promote Intracellular Replication[END_REF]. Accordingly, targeted deletion of C. burnetii vacuolar proteins severely affects the biogenesis of CCVs [START_REF] Larson | Coxiella burnetii effector protein subverts clathrin-mediated vesicular trafficking for pathogen vacuole biogenesis[END_REF][START_REF] Larson | Coxiella burnetii Effector Proteins That Localize to the Parasitophorous Vacuole Membrane Promote Intracellular Replication[END_REF].

If targeted deletion of C. burnetii genes remains challenging, Himar1-based transposon mutagenesis has been extensively applied to the generation of libraries of C. burnetii mutants. Thus, by 2013, the Samuel laboratory reported the first study that combined bioinformatics-mediated identification of candidate effector proteins with transposon mutagenesis [START_REF] Weber | Identification of Coxiella burnetii type IV secretion substrates required for intracellular replication and Coxiella-containing vacuole formation[END_REF] (Fig. 2N). Searching the C. burnetii genome for T4SS features (PmrA consensus sequences, E-block motifs, and homologies with known effectors), led to the identification of 234 genes encoding putative C. burnetii effector proteins. b-lactamase translocation assay in L. pneumophila validated 53 T4SS substrates, most of which were never reported before. Transposon mutagenesis showed that ten effector proteins were involved in the biogenesis of CCVs and bacterial replication [START_REF] Weber | Identification of Coxiella burnetii type IV secretion substrates required for intracellular replication and Coxiella-containing vacuole formation[END_REF] (Fig. 2N).

The following year, two independent studies reported the large-scale identification of C. burnetii virulence determinants based on the generation of two libraries of C. burnetii transposon mutants [START_REF] Martinez | Identification of OmpA, a Coxiella burnetii Protein Involved in Host Cell Invasion, by Multi-Phenotypic High-Content Screening[END_REF][START_REF] Newton | A Screen of Coxiella burnetii Mutants Reveals Important Roles for Dot/Icm Effectors and Host Autophagy in Vacuole Biogenesis[END_REF] (Fig. 2O,R). The Bonazzi library consisted of 3000 GFP-tagged mutants, among which over 1000 were sequenced, annotated, and screened using quantitative, multiparametric image analysis to identify bacterial factors involved in host cell invasion, intracellular replication and persistence [START_REF] Martinez | Identification of OmpA, a Coxiella burnetii Protein Involved in Host Cell Invasion, by Multi-Phenotypic High-Content Screening[END_REF][START_REF] Martinez | Generation and multi-phenotypic high-content screening of Coxiella burnetii transposon mutants[END_REF]. This approach allowed to validate the function of 16 out of the 22 genes constituting the C. burnetii Dot/Icm secretion system, characterize the phenotype associated with transposon insertions in 31 genes encoding effector proteins and identify the first C. burnetii invasin OmpA (for Outer membrane protein A), which is necessary and sufficient to trigger internalization by non-phagocytic cells [START_REF] Martinez | Identification of OmpA, a Coxiella burnetii Protein Involved in Host Cell Invasion, by Multi-Phenotypic High-Content Screening[END_REF]) (Fig. 1 &2P). This study was also the first to report the use of the insect model Galleria mellonella to investigate C. burnetii virulence in vivo [START_REF] Martinez | Identification of OmpA, a Coxiella burnetii Protein Involved in Host Cell Invasion, by Multi-Phenotypic High-Content Screening[END_REF].

The Roy laboratory used a modified-Himar1 expressing mCherry fluorescent protein to generate over 3200 mutants. These were visually screened in HeLa cells to identify genes important in the biogenesis of CCVs and bacterial replication using the lysosomal marker LAMP-1 to visualize the membranes of the C. burnetii replicative compartment [START_REF] Newton | A Screen of Coxiella burnetii Mutants Reveals Important Roles for Dot/Icm Effectors and Host Autophagy in Vacuole Biogenesis[END_REF] (Fig. 2O). This approach allowed the isolation of mutants characterized by different intracellular phenotypes, including defects in intracellular replication and homotypic fusion of independent CCVs or the appearance of filamentous bacteria. Of note, mutants carrying a transposon insertion in gene encoding CBU1751 (cig57) displayed a severe vacuole biogenesis defect [START_REF] Newton | A Screen of Coxiella burnetii Mutants Reveals Important Roles for Dot/Icm Effectors and Host Autophagy in Vacuole Biogenesis[END_REF]. The Newton laboratory has further characterized the role of this effector protein in the development of CCVs and intracellular replication, showing that Cig57 subverts clathrin-mediated traffic by interacting with FCHO2, an accessory protein of clathrin-coated pits [START_REF] Latomanski | The Effector Cig57 Hijacks FCHO-Mediated Vesicular Trafficking to Facilitate Intracellular Replication of Coxiella burnetii[END_REF] (Fig. 1). More recently, CTLC (clathrin heavy chain) has been observed at CCVs, where it plays an essential role in vacuole expansion [START_REF] Latomanski | Interaction between autophagic vesicles and the Coxiella-containing vacuole requires CLTC (clathrin heavy chain)[END_REF]. Importantly, CTLC recruitment to the CCV is related to autophagy, and conversely, the fusion of autophagosomes with CCVs is dependent on CTLC [START_REF] Latomanski | Interaction between autophagic vesicles and the Coxiella-containing vacuole requires CLTC (clathrin heavy chain)[END_REF]. The Roy visual screen also highlighted a multivacuolar phenotype associated with transposon insertions in the gene CBU0021 (cig2), identical to that previously observed following the knockdown of autophagy-related genes [START_REF] Mcdonough | Host Pathways Important for Coxiella burnetii Infection Revealed by Genome-Wide RNA Interference Screening[END_REF], suggesting a functional link between a bacterial effector protein and the manipulation of a specific host cell function (Fig. 2O). This hypothesis was further investigated, demonstrating that Cig2 contributes to the recruitment of the autophagy machinery to CCVs (Fig. 1), thus facilitating their homotypic fusion and contributing to an enhanced bacterial virulence in the Galleria mellonella infection model [START_REF] Kohler | Effector Protein Cig2 Decreases Host Tolerance of Infection by Directing Constitutive Fusion of Autophagosomes with the Coxiella -Containing Vacuole[END_REF].

It takes two to tango: role of host cell pathways in C. burnetii vacuole biogenesis

If on one hand effector proteins translocation is critical for pathogenesis, host cell proteins, lipids, and membrane trafficking pathways also play a significant role in C. burnetii infections. As mentioned above, the maturation of CCVs along the endocytic pathway is a pre-requisite for effector proteins translocation [START_REF] Newton | Effector Protein Translocation by the Coxiella burnetii Dot/Icm Type IV Secretion System Requires Endocytic Maturation of the Pathogen-Occupied Vacuole[END_REF](Fig. 2L); thus, Rab GTPases of the endocytic pathway play a key role in C. burnetii intracellular replication (Fig. 1) [START_REF] Beron | Coxiella burnetii Localizes in a Rab7-Labeled Compartment with Autophagic Characteristics[END_REF][START_REF] Romano | The autophagic pathway is actively modulated by phase II Coxiella burnetii to efficiently replicate in the host cell[END_REF][START_REF] Campoy | The Early Secretory Pathway Contributes to the Growth of the Coxiella -Replicative Niche[END_REF]. Indeed, silencing of either Rab5 or Rab7 correlates with effector protein translocation defects [START_REF] Newton | Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing[END_REF] (Fig. 2R).

A comprehensive characterization of the host cell components required for the biogenesis of CCVs was provided for the first time by a genome-wide screen using siRNA targeting eukaryotic genes in C. burnetii-infected HeLa cells [START_REF] Mcdonough | Host Pathways Important for Coxiella burnetii Infection Revealed by Genome-Wide RNA Interference Screening[END_REF] (Fig. 2M). Host determinants required for C. burnetii infection were identified by the analysis of the number and size of CCVs, revealing the importance of several eukaryotic pathways in critical infection events. As expected, the silencing of genes encoding pH-regulating proteins CLN3 and CLCN5, as well as components of the vacuolar ATPase resulted in a defect in the biogenesis of CCVs (McDonough et al., 2013). Besides, the depletion of the retromer cargo complex VPS26-VPS29-VPS35 leads to defective in bacterial replication, revealing a role for retrograde membrane trafficking in C. burnetii infections [START_REF] Mcdonough | Host Pathways Important for Coxiella burnetii Infection Revealed by Genome-Wide RNA Interference Screening[END_REF]. Interestingly, seminal work from the Colombo laboratory in the past decade demonstrated that the induction of autophagy favors the biogenesis of CCVs and that C. burnetii actively manipulates autophagy during infections [START_REF] Gutierrez | Autophagy induction favours the generation and maturation of the Coxiella-replicative vacuoles[END_REF][START_REF] Romano | The autophagic pathway is actively modulated by phase II Coxiella burnetii to efficiently replicate in the host cell[END_REF]. However, the precise role of autophagy in CCVs' development remained to be characterized.

The genome-wide screening approach of the Roy laboratory revealed that knockdown of autophagy proteins syntaxin-17, ATG5 and ATG12 results in the formation of multiple CCVs of smaller size as compared to control cells [START_REF] Mcdonough | Host Pathways Important for Coxiella burnetii Infection Revealed by Genome-Wide RNA Interference Screening[END_REF].

It was later demonstrated that this multivacuolar phenotype is the result of the defective homotypic fusion of CCVs [START_REF] Newton | A Screen of Coxiella burnetii Mutants Reveals Important Roles for Dot/Icm Effectors and Host Autophagy in Vacuole Biogenesis[END_REF][START_REF] Martinez | Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development[END_REF].

The Voth and Heinzen laboratories have further explored the complex interplay between C. burnetii and autophagy by. The autophagy-associated cargo receptor p62 is actively recruited at CCVs, independently of LC3-interacting domains [START_REF] Winchell | Coxiella burnetii Subverts p62/Sequestosome 1 and Activates Nrf2 Signaling in Human Macrophages[END_REF]. Interestingly, C. burnetii infections seem to preserve p62 from degradation upon induction of autophagy [START_REF] Winchell | Coxiella burnetii Subverts p62/Sequestosome 1 and Activates Nrf2 Signaling in Human Macrophages[END_REF]. Accordingly, C. burnetii infections inhibit mTORC1, a master regulator of autophagy, by a non-canonical mechanism that does not result in accelerated autophagy, nor a block of the autophagic flux [START_REF] Larson | Noncanonical Inhibition of mTORC1 by Coxiella burnetii Promotes Replication within a Phagolysosome-Like Vacuole[END_REF]. Finally, autophagy is also involved in repairing the membranes of CCVs during expansion, which are subject to transitory damage and loss-of-acidification (Mansilla Pareja et al., 2017) (Fig. 2T).

Besides host cell proteins, lipids also play a significant role in the development of CCVs, which are rich in sterols [START_REF] Gilk | Coxiella burnetii expresses a functional Δ24 sterol reductase[END_REF]. Indeed, cholesterol homeostasis regulates CCVs biogenesis and intracellular survival of C. burnetii [START_REF] Gilk | Bacterial Colonization of Host Cells in the Absence of Cholesterol[END_REF][START_REF] Mulye | Elevated cholesterol in the Coxiella burnetii intracellular niche is bacteriolytic[END_REF] (Fig. 2K). Accordingly, C. burnetii actively manipulates cholesterol metabolism via a eukaryotic-like D24 sterol reductase [START_REF] Gilk | Coxiella burnetii expresses a functional Δ24 sterol reductase[END_REF]. Furthermore, an image-based supervised machine learning approach led to the identification of CvpB/Cig2 as the first C. burnetii effector protein to bind phosphoinositides and manipulate their metabolism [START_REF] Martinez | Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development[END_REF] (Fig. 2S). Indeed, CvpB/Cig2 binds phosphatidylinositol-3-phosphate (PI(3)P) and phosphatidylserine (PS) and perturbs the activity of the PI3-kinase PIKFYVE. This inhibition results in an enrichment of PI(3)P at CCVs, which is essential for their homotypic fusion [START_REF] Martinez | Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development[END_REF].

Importantly, despite the apparent defect in the biogenesis of CCVs, cvpB transposon mutants were unaffected in their capacity of replicating within infected cells. However, cvpB mutants are attenuated in the in vivo models Galleria mellonella [START_REF] Martinez | Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development[END_REF] and SCID mice ( [START_REF] Schaik | The SCID mouse model for identifying virulence determinants in Coxiella burnetii[END_REF], demonstrating that the biogenesis of CCVs can modulate C. burnetii virulence, independently of bacterial replication.

To infinity and beyond (conclusions)

As of today, C. burnetii stands as the sole example of an obligate intracellular bacterial pathogen for which a specific axenic culture medium has been developed.

Consequently, research on this dangerous and complex pathogen has bloomed during this decade, with the development of genetic tools and phenotypic screening approaches to better understand the complex interactions established between C. burnetii and its host. The generation of libraries of C. burnetii transposon mutants combined with the development of multi-parametric screening approaches have allowed the rapid and unbiased identification of microbial genes involved in specific steps of the infectious process. This has been facilitated by the fact that the majority of C. burnetii transposon mutants isolated to date display a significant phenotype during infection [START_REF] Martinez | Identification of OmpA, a Coxiella burnetii Protein Involved in Host Cell Invasion, by Multi-Phenotypic High-Content Screening[END_REF], suggesting a milder functional gene redundancy as compared to the closely related pathogen L. pneumophila. Accordingly, bioinformatics predicted ~ 150 effector proteins compared to the 300 translocated by Legionella.

Nevertheless, understanding how these newly identified virulence determinants manipulate host cell functions remains exceptionally challenging. Probably due to the complex nature of this compartment, a large proportion of the C. burnetii effector proteins identified and characterized to date are involved in the biogenesis of the CCV; however, proteins involved in the manipulation of other signaling pathways including apoptosis and inflammation have been identified and partially characterized. With the development of advanced bioinformatics approaches such as unsupervised machine learning, hierarchical clustering and Bayesian network analysis, we can now compare microbial-targeted and host-targeted phenotypic screens, to identify sets of bacterial and eukaryotic genes predicted to be involved in the same biological process during infection.

Despite the giant leaps taken since the development of axenic culture, many technical barriers still exist today. Directed mutagenesis remains challenging, limiting our studies to the mutants available in the transposon libraries hosted in the laboratories that undertook this endeavor. Nevertheless, our knowledge of this onceneglected pathogen is ever increasing. This will allow, in the near future, to address burning questions on C. burnetii pathogenesis, including a characterization of the strategies used to evade immune recognition, cell-to-cell spread, and, consequently, dissemination to distant organs (heart and liver) following infection of alveolar macrophages.

Omsland, A., Beare, P.A., Hill, J., Cockrell, D.C., Howe, D., Hansen, B., et al. (2011) Isolation from Animal Tissue and Genetic Transformation of Coxiella burnetii Are Facilitated by an Improved Axenic Growth Medium. Appl Environ Microbiol 77: 3720-

Figure 1 .

 1 Figure 1. Overview of C. burnetii infections. C. burnetii is an obligate intracellular

Acknowledgements

Work in our laboratory is supported by the Agence Nationale de la Recherche (ANR; ANR-14-CE14-0012-01, project AttaQ and ANR-17-CE15-0021, project QPID) and by the ERA-NET Infect-ERA (ANR-13-IFEC-0003, project EUGENPATH). We apologize to all colleagues whose work was not discussed in this review.

Conflict of interest

The authors declare no conflict of interest.