Cell death induction facilitates egress of Coxiella burnetii from infected host cells at late stages of infection.

Intracellular bacteria have evolved mechanisms to invade host cells, establish an intracellular niche that allows survival and replication, produce progeny, and exit the host cell after completion of the replication cycle to infect new target cells. Bacteria exit their host cell by (i) initiation of apoptosis, (ii) lytic cell death, and (iii) exocytosis. While bacterial egress is essential for bacterial spreading and, thus, pathogenesis, we currently lack information about egress mechanisms for the obligate intracellular pathogen C. burnetii, the causative agent of the zoonosis Q fever. Here, we demonstrate that C. burnetii inhibits host cell apoptosis early during infection, but induces and/or increases apoptosis at later stages of infection. Only at later stages of infection did we observe C. burnetii egress, which depends on previously established large bacteria-filled vacuoles and a functional intrinsic apoptotic cascade. The released bacteria are not enclosed by a host cell membrane and can infect and replicate in new target cells. In summary, our data argue that C. burnetii egress in a non-synchronous way at late stages of infection. Apoptosis-induction is important for C. burnetii egress, but other pathways most likely contribute.

The Coxiella burnetii T4SS effector protein AnkG hijacks the 7SK small nuclear ribonucleoprotein complex for reprogramming host cell transcription.

Inhibition of host cell apoptosis is crucial for survival and replication of several intracellular bacterial pathogens. To interfere with apoptotic pathways, some pathogens use specialized secretion systems to inject bacterial effector proteins into the host cell cytosol. One of these pathogens is the obligate intracellular bacterium Coxiella burnetii, the etiological agent of the zoonotic disease Q fever. In this study, we analyzed the molecular activity of the anti-apoptotic T4SS effector protein AnkG (CBU0781) to understand how C. burnetii manipulates host cell viability. We demonstrate by co- and RNA-immunoprecipitation that AnkG binds to the host cell DExD box RNA helicase 21 (DDX21) as well as to the host cell 7SK small nuclear ribonucleoprotein (7SK snRNP) complex, an important regulator of the positive transcription elongation factor b (P-TEFb). The co-immunoprecipitation of AnkG with DDX21 is probably mediated by salt bridges and is independent of AnkG-7SK snRNP binding, and vice versa. It is known that DDX21 facilitates the release of P-TEFb from the 7SK snRNP complex. Consistent with the documented function of released P-TEFb in RNA Pol II pause release, RNA sequencing experiments confirmed AnkG-mediated transcriptional reprogramming and showed that expression of genes involved in apoptosis, trafficking, and transcription are influenced by AnkG. Importantly, DDX21 and P-TEFb are both essential for AnkG-mediated inhibition of host cell apoptosis, emphasizing the significance of the interaction of AnkG with both, the DDX21 protein and the 7SK RNA. In line with a critical function of AnkG in pathogenesis, the AnkG deletion C. burnetii strain was severely affected in its ability to inhibit host cell apoptosis and to generate a replicative C. burnetii-containing vacuole. In conclusion, the interference with the activity of regulatory host cell RNAs mediated by a bacterial effector protein represent a novel mechanism through which C. burnetii modulates host cell transcription, thereby enhancing permissiveness to bacterial infection.

Blockade of the CD47/SIRPα checkpoint axis potentiates the macrophage-mediated anti-tumor efficacy of tafasitamab.

Macrophages are one of the key mediators of the therapeutic effects exerted by monoclonal antibodies, such as the anti-CD19 antibody tafasitamab, approved in combination with lenalidomide for the treatment of relapsed or refractory (r/r) diffuse large B cell lymphoma (DLBCL). However, antibody-dependent cellular phagocytosis (ADCP) in the tumor microenvironment can be counteracted by increased expression of the inhibitory receptor SIRPα on macrophages and its ligand, the immune checkpoint molecule CD47 on tumor cells. The aim of this study was to investigate the impact of the CD47-SIRPα axis on tafasitamabmediated phagocytosis and explore the potential of anti-CD47 blockade to enhance its antitumor activity. Elevated expression of both SIRPα and CD47 was observed in DLBCL patient-derived lymph node biopsies compared to healthy controls. CRISPR-mediated CD47 overexpression impacted tafasitamab-mediated ADCP in vitro and increased expression of SIRPα on macrophages correlated with decreased ADCP activity of tafasitamab against DLBCL cell lines. Combination of tafasitamab and an anti-CD47 blocking antibody enhanced ADCP activity of in vitro generated macrophages. Importantly, tafasitamab-mediated phagocytosis was elevated in combination with CD47 blockade using primary DLBCL cells and patient-derived lymphoma-associated macrophages (LAMs) in an autologous setting. Furthermore, lymphoma cells with low CD19 expression were efficiently eliminated by the combination treatment. Finally, combined treatment of tafasitamab and an anti-CD47 antibody resulted in enhanced tumor volume reduction and survival benefit in lymphoma xenograft mouse models. These findings provide evidence that CD47 blockade can enhance the phagocytic potential of tumor targeting immunotherapies such as tafasitamab and suggest there is value in exploring the combination in the clinic.

Interdisciplinary studies on Coxiella burnetii: From molecular to cellular, to host, to one health research.

The Q-GAPS (Q fever GermAn interdisciplinary Program for reSearch) consortium was launched in 2017 as a German consortium of more than 20 scientists with exceptional expertise, competence, and substantial knowledge in the field of the Q fever pathogen Coxiella (C.) burnetii. C. burnetii exemplifies as a zoonotic pathogen the challenges of zoonotic disease control and prophylaxis in human, animal, and environmental settings in a One Health approach. An interdisciplinary approach to studying the pathogen is essential to address unresolved questions about the epidemiology, immunology, pathogenesis, surveillance, and control of C. burnetii. In more than five years, Q-GAPS has provided new insights into pathogenicity and interaction with host defense mechanisms. The consortium has also investigated vaccine efficacy and application in animal reservoirs and identified expanded phenotypic and genotypic characteristics of C. burnetii and their epidemiological significance. In addition, conceptual principles for controlling, surveilling, and preventing zoonotic Q fever infections were developed and prepared for specific target groups. All findings have been continuously integrated into a Web-based, interactive, freely accessible knowledge and information platform (www.q-gaps.de), which also contains Q fever guidelines to support public health institutions in controlling and preventing Q fever. In this review, we will summarize our results and show an example of how an interdisciplinary consortium provides knowledge and better tools to control a zoonotic pathogen at the national level.

The Coxiella burnetii effector protein CaeB modulates endoplasmatic reticulum (ER) stress signalling and is required for efficient replication in Galleria mellonella.

The obligate intracellular pathogen Coxiella burnetii is the causative agent of the zoonosis Q fever. C. burnetii infection can have severe outcomes due to the development of chronic infection. To establish and maintain an infection, C. burnetii depends on a functional type IVB secretion system (T4BSS) and, thus, on the translocation of effector proteins into the host cell. Here, we showed that the C. burnetii T4BSS effector protein CaeB targets the conserved endoplasmatic reticulum (ER) stress sensor IRE1 during ER stress in mammalian and plant cells. CaeB-induced upregulation of IRE1 RNase activity was essential for CaeB-mediated inhibition of ER stress-induced cell death. Our data reveal a novel role for CaeB in ER stress signalling modulation and demonstrate that CaeB is involved in pathogenicity in vivo. Furthermore, we provide evidence that C. burnetii infection leads to modulation of the ER stress sensors IRE1 and PERK, but not ATF6 during ER stress. While the upregulation of the RNase activity of IRE1 during ER stress depends on CaeB, modulation of PERK is CaeB independent, suggesting that C. burnetii encodes several factors influencing ER stress during infection.

Mechanisms controlling bacterial infection in myeloid cells under hypoxic conditions.

Various factors of the tissue microenvironment such as the oxygen concentration influence the host-pathogen interaction. During the past decade, hypoxia-driven signaling via hypoxia-inducible factors (HIF) has emerged as an important factor that affects both the pathogen and the host. In this chapter, we will review the current knowledge of this complex interplay, with a particular emphasis given to the impact of hypoxia and HIF on the inflammatory and antimicrobial activity of myeloid cells, the bacterial responses to hypoxia and the containment of bacterial infections under oxygen-limited conditions. We will also summarize how low oxygen concentrations influence the metabolism of neutrophils, macrophages and dendritic cells. Finally, we will discuss the consequences of hypoxia and HIFα activation for the invading pathogen, with a focus on Pseudomonas aeruginosa, Mycobacterium tuberculosis, Coxiella burnetii, Salmonella enterica and Staphylococcus aureus. This includes a description of the mechanisms and microbial factors, which the pathogens use to sense and react to hypoxic conditions.

Coxiella burnetii-Infected NK Cells Release Infectious Bacteria by Degranulation.

Natural killer (NK) cells are critically involved in the early immune response against various intracellular pathogens, including Coxiella burnetii and Chlamydia psittaciChlamydia-infected NK cells functionally mature, induce cellular immunity, and protect themselves by killing the bacteria in secreted granules. Here, we report that infected NK cells do not allow intracellular multiday growth of Coxiella, as is usually observed in other host cell types. C. burnetii-infected NK cells display maturation and gamma interferon (IFN-γ) secretion, as well as the release of Coxiella-containing lytic granules. Thus, NK cells possess a potent program to restrain and expel different types of invading bacteria via degranulation. Strikingly, though, in contrast to Chlamydia, expulsed Coxiella organisms largely retain their infectivity and, hence, escape the cell-autonomous self-defense mechanism in NK cells.

Defying Death - How Coxiella burnetii Copes with Intentional Host Cell Suicide.

The obligate intracellular pathogen Coxiella burnetii is the causative agent of the worldwide zoonotic disease Q fever. This Gram-negative bacterium infects macrophages where it establishes a replicative niche in an acidic and phagolysosome-like vacuole. Establishing and maintaining the niche requires a functional type IV secretion system (T4SS) which translocates multiple effector proteins into the host cell. These effector proteins act by manipulating diverse cellular processes allowing the bacterium to establish an infection and complete its complex biphasic developmental cycle. The lengthy nature of this life cycle suggests that C. burnetii has to successfully deal with cellular defense processes. Cell death is one mechanism infected cells frequently utilize to control or to at least minimize the impact of an infection. To date, four effector proteins have been identified in C. burnetii, which interfere with the induction of cell death. Three, AnkG, CaeA, and CaeB, affect intrinsic apoptosis, CaeA additionally extrinsic apoptosis. The proteins target different steps of the apoptotic pathway and are not conserved among isolates suggesting redundancy as an important feature of cell death inhibition. The fourth effector protein, IcaA, interferes with the non-canonical pathway of pyroptosis, an important inflammatory cell death pathway for controlling infectious disease. Autophagy is relevant for the C. burnetii life-cycle, but to which extent autophagic cell death is a factor in bacterial survival and proliferation is still not clear. To convincingly understand how bacterial manipulation of autophagy affects cell death either directly or indirectly will require further experiments. Collectively, C. burnetii modulates the extrinsic and intrinsic apoptotic pathways and non-canonical pyroptosis to inhibit host cell death, thereby providing a stable, intracellular niche for the course of the pathogen's infectious cycle.

Coxiella burnetii as a useful tool to investigate bacteria-friendly host cell compartments.

Coxiella burnetii is an obligate intracellular and airborne pathogen which can cause the zoonotic disease Q fever. After inhalation of contaminated aerosols alveolar macrophages are taking up C. burnetii into a phagosome. This phagosome matures to a very large vacuole called the C. burnetii-containing vacuole (CCV). Host endogenous and bacterial driven processes lead to the biogenesis of this unusual compartment, which resembles partially a phagolysosome. However, there are several important differences to the classical phagolysosome, which are crucial for the ability of C. burnetii to replicate intracellularly and depend on a functional type IV secretion system (T4SS). The T4SS delivers effector proteins into the host cell cytoplasm to redirect intracellular processes, leading to the establishment of a microenvironment allowing bacterial replication. This article summarizes the current knowledge of the microenvironment permissive for C. burnetii replication.

Beginning to Understand the Role of the Type IV Secretion System Effector Proteins in Coxiella burnetii Pathogenesis.

Coxiella burnetii is the etiological agent of the zoonotic disease Q fever, which manifests in severe outbreaks and is associated with important health and economic burden. Moreover, C. burnetii belongs to the list of class B bioterrorism organisms, as it is an airborne and highly infective pathogen with remarkable resistance to environmental stresses. Detailed study of the host-pathogen interaction during C. burnetii infection has been hampered due to the obligate intracellular nature of this pathogen. However, the development of an axenic culture medium, together with the implementation of bioinformatics tools and high-content screening approaches, have significantly progressed C. burnetii research in the last decade. This has facilitated identification of the Dot/Icm type IV secretion system (T4SS) as an essential virulence factor. T4SS is used to deliver an arsenal of effector proteins into the cytoplasm of the host cell. These effectors mediate the survival of the host cell and the development of very large replicative compartments called Coxiella-containing vacuoles (CCVs). Biogenesis of the CCV relies on T4SS-dependent re-routing of numerous intracellular trafficking pathways to deliver membranes and nutrients that are essential for bacterial replication. This review aims to illustrate the key milestones that have contributed to ascribe C. burnetii as a model organism for the study of host/pathogen interactions as well as presenting an up-to-date description of our knowledge of the cell biology of C. burnetii infections.

Nuclear trafficking of the anti-apoptotic Coxiella burnetii effector protein AnkG requires binding to p32 and Importin-α1.

The obligate intracellular bacterium Coxiella burnetii causes the zoonotic disease Q-fever. Coxiella pathogenesis depends on a functional type IV secretion system (T4SS). The T4SS effector AnkG inhibits pathogen-induced host cell apoptosis, which is believed to be important for the establishment of a persistent infection. However, the mode of action of AnkG is not fully understood. We have previously demonstrated that binding of AnkG to p32 is crucial for migration of AnkG into the nucleus and that nuclear localization of AnkG is essential for its anti-apoptotic activity. Here, we compared the activity of AnkG from the C. burnetii strains Nine Mile and Dugway. Although there is only a single amino acid exchange at residue 11, we observed a difference in anti-apoptotic activity and nuclear migration. Mutation of amino acid 11 to glutamic acid, threonine or valine results in AnkG mutants that had lost the anti-apoptotic activity and the ability to migrate into the nucleus. We identified Importin-α1 to bind to AnkG, but not to the mutants and concluded that binding of AnkG to p32 and Importin-α1 is essential for migration into the nucleus. Also during Coxiella infection binding of AnkG to p32 and Importin-α1 is crucial for nuclear localization of AnkG.

LAMP proteins account for the maturation delay during the establishment of the Coxiella burnetii-containing vacuole.

The obligate intracellular pathogen Coxiella burnetii replicates in a large phagolysosomal-like vacuole. Currently, both host and bacterial factors required for creating this replicative parasitophorous C. burnetii-containing vacuole (PV) are poorly defined. Here, we assessed the contributions of the most abundant proteins of the lysosomal membrane, LAMP-1 and LAMP-2, to the establishment and maintenance of the PV. Whereas these proteins were not critical for uptake of C. burnetii, they influenced the intracellular replication of C. burnetii. In LAMP-1/2 double-deficient fibroblasts as well as in LAMP-1/2 knock-down cells, C. burnetii establishes a significantly smaller, yet faster maturing vacuole, which harboured more bacteria. The accelerated maturation of PVs in LAMP double-deficient fibroblasts, which was partially or fully reversed by ectopic expression of LAMP-1 or LAMP-2, respectively, was characterized by an increased fusion rate with endosomes, lysosomes and bead-containing phagosomes, but not by different fusion kinetics with autophagy vesicles. These findings establish that LAMP proteins are critical for the maturation delay of PVs. Unexpectedly, neither the creation of the spacious vacuole nor the delay in maturation was found to be prerequisites for the intracellular replication of C. burnetii.

Antiapoptotic activity of Coxiella burnetii effector protein AnkG is controlled by p32-dependent trafficking.

Intracellular bacterial pathogens frequently inhibit host cell apoptosis to ensure survival of their host, thereby allowing bacterial propagation. The obligate intracellular pathogen Coxiella burnetii displays antiapoptotic activity which depends on a functional type IV secretion system (T4SS). Accordingly, antiapoptotic T4SS effector proteins, like AnkG, have been identified. AnkG inhibits pathogen-induced apoptosis, possibly by binding to the host cell mitochondrial protein p32 (gC1qR). However, the molecular mechanism of AnkG activity remains unknown. Here, we demonstrate that ectopically expressed AnkG associates with mitochondria and traffics into the nucleus after apoptosis induction, although AnkG lacks a predicted nuclear localization signal. We identified the p32 interaction region in AnkG and constructed an AnkG mutant (AnkGR(22/23S)) unable to bind to p32. By using this mutant, we found that intracellular localization and trafficking of AnkG into the nucleus are dependent on binding to p32. Furthermore, we demonstrated that nuclear localization of AnkG but not binding to p32 is required for apoptosis inhibition. Thus, the antiapoptotic activity of AnkG is controlled by p32-mediated intracellular trafficking, which, in turn, seems to be regulated by host cell processes that sense stress.

The Coxiella burnetii type IV secretion system substrate CaeB inhibits intrinsic apoptosis at the mitochondrial level.

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.

The Coxiella burnetii Dot/Icm system delivers a unique repertoire of type IV effectors into host cells and is required for intracellular replication.

Coxiella burnetii, the causative agent of human Q fever, is an intracellular pathogen that replicates in an acidified vacuole derived from the host lysosomal network. This pathogen encodes a Dot/Icm type IV secretion system that delivers bacterial proteins called effectors to the host cytosol. To identify new effector proteins, the functionally analogous Legionella pneumophila Dot/Icm system was used in a genetic screen to identify fragments of C. burnetii genomic DNA that when fused to an adenylate cyclase reporter were capable of directing Dot/Icm-dependent translocation of the fusion protein into mammalian host cells. This screen identified Dot/Icm effectors that were proteins unique to C. burnetii, having no overall sequence homology with L. pneumophila Dot/Icm effectors. A comparison of C. burnetii genome sequences from different isolates revealed diversity in the size and distribution of the genes encoding many of these effectors. Studies examining the localization and function of effectors in eukaryotic cells provided evidence that several of these proteins have an affinity for specific host organelles and can disrupt cellular functions. The identification of a transposon insertion mutation that disrupts the dot/icm locus was used to validate that this apparatus was essential for translocation of effectors. Importantly, this C. burnetii Dot/Icm-deficient mutant was found to be defective for intracellular replication. Thus, these data indicate that C. burnetii encodes a unique subset of bacterial effector proteins translocated into host cells by the Dot/Icm apparatus, and that the cumulative activities exerted by these effectors enables C. burnetii to successfully establish a niche inside mammalian cells that supports intracellular replication.

Inhibition of pathogen-induced apoptosis by a Coxiella burnetii type IV effector protein.

Coxiella burnetii and Legionella pneumophila are evolutionarily related pathogens with different intracellular infection strategies. C. burnetii persists within and is transmitted by mammalian hosts, whereas, L. pneumophila is found primarily in the environment associated with protozoan hosts. Although a type IV secretion system encoded by the defect in organelle trafficking (dot) and intracellular multiplication (icm) genes is a virulence determinant that remains highly conserved in both bacteria, the two pathogens encode a different array of effector proteins that are delivered into host cells by the Dot/Icm machinery. This difference suggests that adaptations to evolutionarily distinct hosts may be reflected in the effector protein repertoires displayed by these two pathogens. Here we provide evidence in support of this hypothesis. We show that a unique C. burnetii effector from the ankyrin repeat (Ank) family called AnkG interferes with the mammalian apoptosis pathway. AnkG was found to interact with the host protein gC1qR (p32). Either the addition of AnkG to the repertoire of L. pneumophila effector proteins or the silencing of p32 in mouse dendritic cells resulted in a gain of function that allowed intracellular replication of L. pneumophila in these normally restrictive mammalian host cells by preventing rapid pathogen-induced apoptosis. These data indicate that p32 regulates pathogen-induced apoptosis and that AnkG functions to block this pathway. Thus, emergence of an effector protein that interferes with a proapoptotic signaling pathway directed against intracellular bacteria correlates with adaptation of a pathogen to mammalian hosts.

Antimicrobial resistance- and pathogen patterns in the fecal microbiota of sows and their offspring in German commercial pig farms.

Reducing antibiotic use is one of the biggest challenges in pig farming, as antibiotics have been used for years to control typical problems such as newborn or post-weaning diarrhea. The pressure a one health approach has created on animal production regarding antimicrobial resistance is an opportunity to find other strategies against enterobacterial pathogens in suckling and weaned piglets. A farm-specific approach could have a good success due to the individual farm structures in Germany and other countries. In this study, non-metric multidimensional scaling, hierarchical clustering, and latent class analysis were used to determine the impact of antibiotic use on antibiotic resistance patterns and pathogen prevalence in 20 German pig farms. This may help to develop individualized health strategies. 802 fresh fecal samples were collected from sows and piglets from 20 piglet production and rearing farms at different production times (sows antepartum and postpartum, suckling piglets, weaned piglets). In addition, the use of antibiotics was recorded. DNA extracts were subjected to quantitative real-time qPCR with primers specific for antibiotic resistance genes (int1, sul1-3, dfrA1, mcr-1, blaCTX-M), and virulence factors of relevant bacteria (C. difficile, C. perfringens, Salmonella, Escherichia/Shigella/Hafnia, E. coli). Linear and logistic regression models were used to analyze the relationship between different antibiotics and the major genes contributing to the clustering of observations for the different animal groups. Clustering revealed different farm clusters for sows, suckling piglets, and weaned piglets, with the most remarkable diversity in antibiotic use among weaned piglets. Amoxicillin, lincomycin, and enrofloxacin were identified as the most probable cause of increased odds of the presence of relevant antibiotic resistance genes (mcr1, dfrA1, blaCTX-M). Still, direct effects of a specific antibiotic on its associated resistance gene were rare. Enrofloxacin and florfenicol favored the occurrence of C. difficile in sows. The E. coli fimbriae genes were less affected by antibiotic use in sows and piglets, but the F4 fimbriae gene could be associated with the integrase 1 gene in piglets. The results confirm that multidrug-resistant enterobacteria are widespread in German pig farms and give awareness of the impact of current antibiotic use while searching for alternative health strategies.

Bovine blood derived macrophages are unable to control Coxiella burnetii replication under hypoxic conditions.

Background: Coxiella burnetii is a zoonotic pathogen, infecting humans, livestock, pets, birds and ticks. Domestic ruminants such as cattle, sheep, and goats are the main reservoir and major cause of human infection. Infected ruminants are usually asymptomatic, while in humans infection can cause significant disease. Human and bovine macrophages differ in their permissiveness for C. burnetii strains from different host species and of various genotypes and their subsequent host cell response, but the underlying mechanism(s) at the cellular level are unknown. Methods: C. burnetii infected primary human and bovine macrophages under normoxic and hypoxic conditions were analyzed for (i) bacterial replication by CFU counts and immunofluorescence; (ii) immune regulators by westernblot and qRT-PCR; cytokines by ELISA; and metabolites by gas chromatography-mass spectrometry (GC-MS). Results: Here, we confirmed that peripheral blood-derived human macrophages prevent C. burnetii replication under oxygen-limiting conditions. In contrast, oxygen content had no influence on C. burnetii replication in bovine peripheral blood-derived macrophages. In hypoxic infected bovine macrophages, STAT3 is activated, even though HIF1α is stabilized, which otherwise prevents STAT3 activation in human macrophages. In addition, the TNFα mRNA level is higher in hypoxic than normoxic human macrophages, which correlates with increased secretion of TNFα and control of C. burnetii replication. In contrast, oxygen limitation does not impact TNFα mRNA levels in C. burnetii-infected bovine macrophages and secretion of TNFα is blocked. As TNFα is also involved in the control of C. burnetii replication in bovine macrophages, this cytokine is important for cell autonomous control and its absence is partially responsible for the ability of C. burnetii to replicate in hypoxic bovine macrophages. Further unveiling the molecular basis of macrophage-mediated control of C. burnetii replication might be the first step towards the development of host directed intervention measures to mitigate the health burden of this zoonotic agent.

Macrophages inhibit Coxiella burnetii by the ACOD1-itaconate pathway for containment of Q fever.

Infection with the intracellular bacterium Coxiella (C.) burnetii can cause chronic Q fever with severe complications and limited treatment options. Here, we identify the enzyme cis-aconitate decarboxylase 1 (ACOD1 or IRG1) and its product itaconate as protective host immune pathway in Q fever. Infection of mice with C. burnetii induced expression of several anti-microbial candidate genes, including Acod1. In macrophages, Acod1 was essential for restricting C. burnetii replication, while other antimicrobial pathways were dispensable. Intratracheal or intraperitoneal infection of Acod1-/- mice caused increased C. burnetii burden, weight loss and stronger inflammatory gene expression. Exogenously added itaconate restored pathogen control in Acod1-/- mouse macrophages and blocked replication in human macrophages. In axenic cultures, itaconate directly inhibited growth of C. burnetii. Finally, treatment of infected Acod1-/- mice with itaconate efficiently reduced the tissue pathogen load. Thus, ACOD1-derived itaconate is a key factor in the macrophage-mediated defense against C. burnetii and may be exploited for novel therapeutic approaches in chronic Q fever.