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Q fever is a highly infectious disease with a worldwide distribution. Its causative agent, the intracellular bacterium Coxiella burnetii, infects a variety of vertebrate species, including humans. Its evolutionary origin remains almost entirely unknown and uncertainty persists regarding the identity and lifestyle of its ancestors. A few tick species were recently found to harbor maternally-inherited Coxiella-like organisms engaged in symbiotic interactions, but their relationships to the Q fever pathogen remain unclear. Here, we extensively sampled ticks, identifying new and atypical Coxiella strains from 40 of 58 examined species, and used this data to infer the evolutionary processes leading to the emergence of C. burnetii. Phylogenetic analyses of multi-locus typing and whole-genome sequencing data revealed that Coxiella-like organisms represent an ancient and monophyletic group allied to ticks. Remarkably, all known C. burnetii strains originate within this group and are the descendants of a Coxiella-like progenitor hosted by ticks. Using both colony-reared and field-collected gravid females, we further establish the presence of highly efficient maternal transmission of these Coxiella-like organisms in four examined tick species, a pattern coherent with an endosymbiotic lifestyle. Our laboratory culture assays also showed that these Coxiella-like organisms were not amenable to culture in the vertebrate cell environment, suggesting different metabolic requirements compared to C. burnetii. Altogether, this corpus of data demonstrates that C. burnetii recently evolved from an inherited symbiont of ticks which succeeded in infecting vertebrate cells, likely by the acquisition of novel virulence factors.

The obligate intracellular bacterium Coxiella burnetii establishes an intracellular replicative niche termed the Coxiella -containing vacuole (CCV), which has been characterised as a bacterially modified phagolysosome. How C. burnetii withstands the acidic and degradative properties of this compartment is not well understood. We demonstrate that the key lysosomal protease cathepsin B is actively and selectively removed from C. burnetii- infected cells through a mechanism involving the Dot/Icm type IV-B secretion system effector CvpB. Overexpression of cathepsin B leads to defects in CCV biogenesis and bacterial replication, indicating that removal of this protein represents a strategy to reduce the hostility of the intracellular niche. In addition, we show that C. burnetii infection of mammalian cells induces the secretion of a wider cohort of lysosomal proteins, including cathepsin B, to the extracellular milieu via a mechanism dependent on retrograde traffic. This study reveals that C. burnetii is actively modulating the hydrolase cohort of its replicative niche to promote intracellular success and demonstrates that infection incites the secretory pathway to maintain lysosomal homoeostasis. The zoonotic pathogen Coxiella burnetii establishes a unique intracellular niche within a lysosome-derived vacuole. Here Bird et al. undertook proteomic, cell biology and microbiology approaches to characterise this niche and the strategies employed by C. burnetii to maintain a balance between intracellular success and maintaining host cell homoeostasis.

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.

Summary Manipulation of host cell apoptosis is a virulence property shared by many intracellular pathogens to ensure productive replication. For the obligate intracellular pathogen Coxiella burnetii anti‐apoptotic activity, which depends on a functional type IV secretion system (T4SS), has been demonstrated. Accordingly, the C. burnetii T4SS effector protein AnkG was identified to inhibit pathogen‐induced apoptosis, possibly by binding to the host cell mitochondrial protein p32 (gC1qR). However, it was unknown whether AnkG alone is sufficient for apoptosis inhibition or if additional effector proteins are required. Here, we identified two T4SS effector proteins CaeA and CaeB (C. burnetii anti‐apoptotic effector) that inhibit the intrinsic apoptotic pathway. CaeB blocks apoptosis very efficiently, while the anti‐apoptotic activity of CaeA is weaker. Our data suggest that CaeB inhibits apoptosis at the mitochondrial level, but does not bind to p32. Taken together, our results demonstrate that C. burnetii harbours several anti‐apoptotic effector proteins and suggest that these effector proteins use different mechanism(s) to inhibit apoptosis.

Coxiella burnetii infection has a worldwide distribution, although the incidence and clinical manifestations vary between and within countries. There are the following four basic forms: asymptomatic infection, acute Q fever, chronic Q fever, and post-Q fever fatigue syndrome. The aim of this review is to provide a comprehensive overview of the important aspects of its pathogenesis and pathophysiology. First, we provide a brief update of the taxonomic aspects, basic structures, and genotypes of C. burnetii necessary for the proper interpretation of the following sections. Routes of infection, different stages of pathogenesis (respiratory entry of C. burnetii; penetration into alveolar macrophages, life cycle, and effects; systemic dissemination), and innate, acquired humoral and cell-mediated immune responses in different forms of infection are described in detail. The pathophysiology and clinical manifestations of Q fever, such as the main mechanisms of injury, in isolation and in combination, are reviewed. The clinical and biological manifestations of the two main forms of Q fever (acute and chronic) are outlined, with a brief definition and mention of the mechanisms of post-Q fever fatigue syndrome.

Coxiella burnetii causes human Q fever and is a potential bioterrorism threat. In humans, C. burnetii evades host cell killing and establishes a prolonged replication cycle within AMs, which is a critical step toward presentation of acute or chronic disease symptoms. While macrophage metabolism fuels antibacterial activity, we identified key metabolic processes that C. burnetii manipulates to sustain a pro-bacterial growth niche. Currently, few infection models capture C. burnetii interaction with disease-relevant human cells. Here, we used the established primary human AM infection system to characterize bacterial modulation of macrophage metabolism. Our findings advance understanding of C. burnetii -AM interactions and lay the foundation for future therapeutic exploration.

, the etiologic agent of Q fever, is a Gram-negative obligate intracellular bacterium. It has been previously described that both the endocytic and autophagic pathways contribute to the replicative vacuole (CRV) generation. Galectins are β-galactoside-binding lectins that accumulate in the cytosol before being secreted via a non-conventional secretory pathway. It has been shown that Galectin-3, -8, -9 monitor bacteria vacuolar rupture and endosomal and lysosomal loss of membrane integrity through binding of host glycans exposed in the cytoplasm after membrane damage. Using microinjection of fluorescence-coupled dextrans, a FRET assay, and galectins distribution, we demonstrate that infection actually result in transient phagosomal/CRV membrane damage in a Dot/Icm-dependent manner. We also show the association of different adaptor molecules involved in autophagy and of LC3 to the limiting membrane of the CRV. Moreover, we show that upon autophagy inhibition, the proportion of CRVs labeled with galectins and less acidified increases which is associated with bacteria replication impairment. Based on these observations, we propose that autophagy can facilitate resealing of intracellular damaged membranes.

Q fever is a zoonosis caused by Coxiella burnetii. Infection with C burnetii can be acute or chronic, and exhibits a wide spectrum of clinical manifestations. The extreme infectivity of the bacterium results in large outbreaks and makes it a potential bioweapon. In the past decade, the complete genome sequencing of C burnetii, the exploration of bacterial interactions with the host, and the description of the natural history of the disease in human beings and in experimental models have all added to our knowledge about this fascinating disease. Advances in understanding the pathophysiology and natural history of Q fever are reviewed.

Summary Coxiella burnetii, the causative agent of the human disease Q fever, is a unique intracellular bacterial pathogen. Coxiella replicates to high numbers within a pathogen‐derived lysosome‐like vacuole, thriving within a low pH, highly proteolytic and oxidative environment. In 2009, researchers developed means to axenically culture Coxiella paving the way for the development of tools to genetically manipulate the organism. These advances have revolutionized our capacity to examine the pathogenesis of Coxiella. In recent years, targeted and random mutant strains have been used to demonstrate that the Dot/Icm type IV secretion system is essential for intracellular replication of Coxiella. Current research is focused towards understanding the unique cohort of over 130 effector proteins that are translocated into the host cell. Mutagenesis screens have been employed to identify effectors that play important roles for the biogenesis of the Coxiella‐containing vacuole and intracellular replication of Coxiella. A surprisingly high number of effector mutants demonstrate significant intracellular growth defects, and future studies on the molecular function of these effectors will provide great insight into the pathogenesis of Coxiella. Already, this expanse of new data implicates many eukaryotic processes that are targeted by the arsenal of Coxiella effectors including autophagy, apoptosis and vesicular trafficking.

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.