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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., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Coxiella burnetii is an intracellular pathogen responsible for causing Q fever in humans, a disease with varied presentations ranging from a mild flu-like sickness to a debilitating illness that can result in endocarditis. The intracellular lifestyle of C. burnetii is unique, residing in an acidic phagolysosome-like compartment within host cells. An understanding of the core molecular biology of C. burnetii will greatly increase our understanding of C. burnetii growth, survival and pathogenesis. We used transposon-directed insertion site sequencing (TraDIS) to reveal C. burnetii Nine Mile Phase II genes fundamental for growth and in vitro survival. Screening a transposon library containing >10 000 unique transposon mutants revealed 512 predicted essential genes. Essential routes of synthesis were identified for the mevalonate pathway, as well as peptidoglycan and biotin synthesis. Some essential genes identified (e.g. predicted type IV secretion system effector genes) are typically considered to be associated with C. burnetii virulence, a caveat concerning the axenic media used in the study. Investigation into the conservation of the essential genes identified revealed that 78 % are conserved across all C. burnetii strains sequenced to date, which probably play critical functions. This is the first report of a whole genome transposon screen in C. burnetii that has been undertaken for the identification of essential genes.

MicroRNAs (miRNAs), a class of small noncoding RNAs, are critical to gene regulation in eukaryotes. They are involved in modulating a variety of physiological processes, including the host response to intracellular infections. Little is known about miRNA functions during infection by Coxiella burnetii, the causative agent of human Q fever. This bacterial pathogen establishes a large replicative vacuole within macrophages by manipulating host processes such as apoptosis and autophagy. We investigated miRNA expression in C. burnetii-infected macrophages and identified several miRNAs that were down- or upregulated during infection. We further explored the functions of miR-143-3p, an miRNA whose expression is downregulated in macrophages infected with C. burnetii, and show that increasing the abundance of this miRNA in human cells results in increased apoptosis and reduced autophagy-conditions that are unfavorable to C. burnetii intracellular growth. In sum, this study demonstrates that C. burnetii infection elicits a robust miRNA-based host response, and because miR-143-3p promotes apoptosis and inhibits autophagy, downregulation of miR-143-3p expression during C. burnetii infection likely benefits the pathogen.

Genotyping of Coxiella burnetii using multispacer sequence typing (MST) and multiple locus variable number tandem repeat analysis (MLVA) was conducted from infected animals for the first time in the Republic of Korea. C. burnetii was detected by real-time PCR, and followed by MST and MLVA genotyping. The result showed that detected C. burnetii all had the same MLVA genotype, 6-13-2-7-9-10 for markers MS23-MS24-MS27-MS28-MS33-MS34, respectively, and genotype group 61 for MST. The same genotypes were previously identified in Poland. Importantly, this MLVA type was detected in humans in France, suggesting that the Korean strain can also potentially cause Q fever in humans. MST and MLVA were very useful tools for analyzing the molecular epidemiology of C. burnetii and helpful for interpreting the epidemiological relationship between isolates from domestic and international resources.

Coxiella burnetii is a bacterial pathogen that replicates within host cells by establishing a membrane-bound niche called the Coxiella -containing vacuole. Biogenesis of this compartment requires effectors of its Dot/Icm type IV secretion system. A large cohort of such effectors has been identified, but the function of most of them remain elusive. Here, by a cell-based functional screening, we identified the effector Cbu0513 (designated as CinF) as an inhibitor of NF-κB signaling. CinF is highly similar to a fructose-1,6-bisphosphate (FBP) aldolase/phosphatase present in diverse bacteria. Further study reveals that unlike its ortholog from Sulfolobus tokodaii , CinF does not exhibit FBP phosphatase activity. Instead, it functions as a protein phosphatase that specifically dephosphorylates and stabilizes IκBα. The IκBα phosphatase activity is essential for the role of CinF in C. burnetii virulence. Our results establish that C. burnetii utilizes a protein adapted from sugar metabolism to subvert host immunity., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Background: Coxiella burnetii is the causative agent of Q fever which is a highly infectious zoonotic disease. C. burnetii has become one of the most important causes of abortion in livestock, which can lead to widespread abortions in these animals. There are very limited studies on the prevalence of C. burnetii infection in cases of animal abortion in Iran. The aim of this study was to investigate the occurrence of C. burnetii in ruminant abortion samples in Iran., Methods: Abortion samples from cattle, sheep and goats were collected from different parts of Iran and were tested using Real-time PCR targeting the IS1111 element of C. burnetii., Results: In this study, 36 samples (24.7%) of the 146 collected samples were positive for C. burnetii. The prevalence of C. burnetii was 21.3% (20 of 94 samples) in sheep samples. Also, 10 of 46 cattle samples (21.7%) were positive. All six goat abortion samples were positive for C. burnetii., Conclusions: The findings of the study demonstrate that C. burnetii plays an important role in domestic ruminant abortions in Iran, suggesting that more attention should be paid to the role of C. burnetii in domestic animal abortions by veterinary organizations. The risk of transmitting the infection to humans due to abortion of animals should also be considered., Competing Interests: The authors have declared that no competing interests exist.

The gram-negative bacterium Coxiella burnetii is the causative agent of Query (Q) fever in humans and coxiellosis in livestock. Host genetics are associated with C. burnetii pathogenesis both in humans and animals; however, it remains unknown if specific genes are associated with severity of infection. We employed the Drosophila Genetics Reference Panel to perform a genome-wide association study to identify host genetic variants that affect host survival to C. burnetii infection. The genome-wide association study identified 64 unique variants (P < 10-5) associated with 25 candidate genes. We examined the role each candidate gene contributes to host survival during C. burnetii infection using flies carrying a null mutation or RNAi knockdown of each candidate. We validated 15 of the 25 candidate genes using at least one method. This is the first report establishing involvement of many of these genes or their homologs with C. burnetii susceptibility in any system. Among the validated genes, FER and tara play roles in the JAK/STAT, JNK, and decapentaplegic/TGF-β signaling pathways which are components of known innate immune responses to C. burnetii infection. CG42673 and DIP-ε play roles in bacterial infection and synaptic signaling but have no previous association with C. burnetii pathogenesis. Furthermore, since the mammalian ortholog of CG13404 (PLGRKT) is an important regulator of macrophage function, CG13404 could play a role in host susceptibility to C. burnetii through hemocyte regulation. These insights provide a foundation for further investigation regarding the genetics of C. burnetii susceptibility across a wide variety of hosts., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

Coxiella burnetii is a highly infectious zoonotic pathogen infecting wide range of mammals, including humans. In the present study, a total of 711 blood samples from bovines [cattle (n = 543) and buffaloes (n = 168)] from eight farms at different geographical locations in India were screened for C. burnetii targeting the IS1111 and the com1 genes. The anti-C. burnetii antibodies in serum samples were detected using indirect-ELISA kits. Also, a total of 21 parameters pertaining to animal health and farm management were identified to assess their role as possible risk factors for coxiellosis among the targeted farms. The apparent prevalence (positive for PCR and/or ELISA) for coxiellosis was reported to be 24.5% in cattle and 8.9% in buffaloes. In cattle, the detection rate of C. burnetii employing the IS1111 gene (8.5%) was found to be significantly higher (p<0.05) as compared to the com1 (6.5%) gene. The seropositivity by ELISA was higher among cattle (17.7%) than in buffaloes (8.3%). Further, on univariable analysis of risk factors, species (cattle) (OR:3.31; 95%CI:1.88-5.82), inadequate floor spacing (OR:1.64; 95%CI:1.10-2.43), mastitis (OR:2.35, 95%CI:1.45-3.81) and reproductive disorders (OR:2.54; 95%CI:1.67-3.85) were significantly (p<0.05) having high odds for coxiellosis. The multivariable logistic regression analysis of the animal level risk factors revealed that species and age were found to be significantly associated with coxiellosis. However, since the number of screened farms is limited; further research is needed with a higher number of animals to confirm the farm level odds ratio of risk factors. Quarantine and biosecurity measures including farm hygiene operations were observed to be inadequate and also the lack of awareness about coxiellosis among the farm workers. In absence of vaccination program for coxiellosis in India, robust surveillance, farm biosecurity measures and the awareness for the disease among risk groups can play an important role in the disease prevention and subsequent transmission of the pathogen., Competing Interests: The authors have declared that no competing interests exist.

Q-fever is a flu-like illness caused by Coxiella burnetii (Cb), a highly infectious intracellular bacterium. There is an unmet need for a safe and effective vaccine for Q-fever. Correlates of immune protection to Cb infection are limited. We proposed that analysis by longitudinal high dimensional immune (HDI) profiling using mass cytometry combined with other measures of vaccination and protection could be used to identify novel correlates of effective vaccination and control of Cb infection. Using a vaccine-challenge model in HLA-DR transgenic mice, we demonstrated significant alterations in circulating T-cell and innate immune populations that distinguished vaccinated from naïve mice within 10 days, and persisted until at least 35 days post-vaccination. Following challenge, vaccinated mice exhibited reduced bacterial burden and splenomegaly, along with distinct effector T-cell and monocyte profiles. Correlation of HDI data to serological and pathological measurements was performed. Our data indicate a Th1-biased response to Cb, consistent with previous reports, and identify Ly6C, CD73, and T-bet expression in T-cell, NK-cell, and monocytic populations as distinguishing features between vaccinated and naïve mice. This study refines the understanding of the integrated immune response to Cb vaccine and challenge, which can inform the assessment of candidate vaccines for Cb.

The Q fever agent Coxiella burnetii uses a defect in organelle trafficking/intracellular multiplication (Dot/Icm) type 4b secretion system (T4SS) to silence the host innate immune response during infection. By investigating C. burnetii effector proteins containing eukaryotic-like domains, here we identify NopA (nucleolar protein A), which displays four regulator of chromosome condensation (RCC) repeats, homologous to those found in the eukaryotic Ras-related nuclear protein (Ran) guanine nucleotide exchange factor (GEF) RCC1. Accordingly, NopA is found associated with the chromatin nuclear fraction of cells and uses the RCC-like domain to interact with Ran. Interestingly, NopA triggers an accumulation of Ran-GTP, which accumulates at nucleoli of transfected or infected cells, thus perturbing the nuclear import of transcription factors of the innate immune signaling pathway. Accordingly, qRT-PCR analysis on a panel of cytokines shows that cells exposed to the C. burnetii nopA ::Tn or a Dot/Icm-defective dotA ::Tn mutant strain present a functional innate immune response, as opposed to cells exposed to wild-type C. burnetii or the corresponding nopA complemented strain. Thus, NopA is an important regulator of the innate immune response allowing Coxiella to behave as a stealth pathogen., Competing Interests: The authors declare no competing interest.

The intracellular bacterial pathogen Coxiella burnetii is the etiological agent of the emerging zoonosis Q fever. Crucial to its pathogenesis is type 4b secretion system-mediated secretion of bacterial effectors into host cells that subvert host cell membrane trafficking, leading to the biogenesis of a parasitophorous vacuole for intracellular replication. The characterization of prokaryotic serine/threonine protein kinases in bacterial pathogens is emerging as an important strategy to better understand host-pathogen interactions. In this study, we investigated CstK (for Coxiella Ser/Thr kinase), a protein kinase identified in C. burnetii by in silico analysis. We demonstrate that this putative protein kinase undergoes autophosphorylation on Thr and Tyr residues and phosphorylates a classical eukaryotic protein kinase substrate in vitro This dual Thr-Tyr kinase activity is also observed for a eukaryotic dual-specificity Tyr phosphorylation-regulated kinase class. We found that CstK is translocated during infections and localizes to Coxiella -containing vacuoles (CCVs). Moreover, a CstK-overexpressing C. burnetii strain displayed a severe CCV development phenotype, suggesting that CstK fine-tunes CCV biogenesis during the infection. Protein-protein interaction experiments identified the Rab7 GTPase-activating protein TBC1D5 as a candidate CstK-specific target, suggesting a role for this host GTPase-activating protein in Coxiella infections. Indeed, CstK co-localized with TBC1D5 in noninfected cells, and TBC1D5 was recruited to CCVs in infected cells. Accordingly, TBC1D5 depletion from infected cells significantly affected CCV development. Our results indicate that CstK functions as a bacterial effector protein that interacts with the host protein TBC1D5 during vacuole biogenesis and intracellular replication., (© 2020 Martinez et al.)

Infection with Coxiella burnetii , the causative agent of Q fever, can result in life-threatening persistent infection. Reactogenicity hinders worldwide implementation of the only licensed human Q fever vaccine. We previously demonstrated long-lived immunoreactivity in individuals with past symptomatic and asymptomatic Coxiella infection (convalescents) to promiscuous HLA class II C. burnetii epitopes, providing the basis for a novel T-cell targeted subunit vaccine. In this study, we investigated in a cohort of 22 individuals treated for persistent infection (chronic Q fever) whether they recognize the same set of epitopes or distinct epitopes that could be candidates for a therapeutic vaccine or aid in the diagnosis of persistent infection. In cultured enzyme-linked immunosorbent spot (ELISpot) assays, individuals with chronic Q fever showed strong class II epitope-specific responses that were largely overlapping with the peptide repertoire identified previously for convalescents. Five additional peptides were recognized more frequently by chronic subjects, but there was no combination of epitopes uniquely recognized by or nonreactive in subjects with chronic Q fever. Consistent with more recent/prolonged exposure, we found, however, stronger ex vivo responses by direct ELISpot to both whole-cell C. burnetii and individual peptides in chronic patients than in convalescents. In conclusion, we have validated and expanded a previously published set of candidate epitopes for a novel T-cell targeted subunit Q fever vaccine in treated patients with chronic Q fever and demonstrated that they successfully mounted a T-cell response comparable to that of convalescents. Finally, we demonstrated that individuals treated for chronic Q fever mount a broader ex vivo response to class II epitopes than convalescents, which could be explored for diagnostic purposes., (Copyright © 2019 American Society for Microbiology.)

Most intracellular pathogens that reside in a vacuole prevent transit of their compartment to lysosomal organelles. Effector mechanisms induced by the pro-inflammatory cytokine Interferon-gamma (IFNγ) can promote the delivery of pathogen-occupied vacuoles to lysosomes for proteolytic degradation and are therefore important for host defense against intracellular pathogens. The bacterial pathogen Coxiella burnetii is unique in that, transport to the lysosome is essential for replication. The bacterium modulates membrane traffic to create a specialized autophagolysosomal compartment called the Coxiella-containing vacuole (CCV). Importantly, IFNγ signaling inhibits intracellular replication of C. burnetii, raising the question of which IFNγ-activated mechanisms restrict replication of a lysosome-adapted pathogen. To address this question, siRNA was used to silence a panel of IFNγ-induced genes in HeLa cells to identify genes required for restriction of C. burnetii intracellular replication. This screen demonstrated that Indoleamine 2,3-dioxygenase 1 (IDO1) contributes to IFNγ-mediated restriction of C. burnetii. IDO1 is an enzyme that catabolizes cellular tryptophan to kynurenine metabolites thereby reducing tryptophan availability in cells. Cells deficient in IDO1 function were more permissive for C. burnetii replication when treated with IFNγ, and supplementing IFNγ-treated cells with tryptophan enhanced intracellular replication. Additionally, ectopic expression of IDO1 in host cells was sufficient to restrict replication of C. burnetii in the absence of IFNγ signaling. Using differentiated THP1 macrophage-like cells it was determined that IFNγ-activation resulted in IDO1 production, and that supplementation of IFNγ-activated THP1 cells with tryptophan enhanced C. burnetii replication. Thus, this study identifies IDO1 production as a key cell-autonomous defense mechanism that limits infection by C. burnetii, which suggests that peptides derived from hydrolysis of proteins in the CCV do not provide an adequate supply of tryptophan for bacterial replication., Competing Interests: The authors have declared that no competing interests exist.

Cadherins switching is a hallmark of neoplasic processes. The E-cadherin (E-cad) subtype is one of the surface molecules regulating cell-to-cell adhesion. After its cleavage by sheddases, a soluble fragment (sE-cad) is released that has been identified as a pro-carcinogenic inflammatory signal in several bacteria-induced cancers. Recently we reported that Q fever, a disease due to Coxiella burnetii infection, can be complicated by occurrence of non-Hodgkin lymphoma (NHL). Therefore, we studied E-cad switching in Q fever. The sE-cad levels were found increased in the sera of acute and persistent Q fever patients, whereas they remained at the baseline in controls groups of healthy donors, people cured of Q fever, patients suffering from unrelated inflammatory diseases, and past Q fever patients who developed NHL. These results indicate that sE-cad can be considered as a new biomarker of C. burnetii infection rather than a marker of NHL-associated to Q fever. We wondered if changes in sE-cad reflected variations in the CDH1 gene transcription. The expression of E-cad mRNA and its intracellular ligand β-catenin was down-regulated in peripheral blood mononuclear cells (PBMCs) of patients with either acute or persistent forms of Q fever. Indeed, a lower cell-surface expression of E-cad was measured in a minority (<5%) subpopulation of HLADR + /CD16 + monocytes from patients with acute Q fever. However, a very strong increase in E-cad expression was observed on more than 30% of the HLADR + /CD16 + monocytes of persistent Q fever patients, a cell subpopulation known to be a target for C. burnetii in humans. An experimental in vitro infection of healthy donors' PBMCs with C. burnetii , was performed to directly evaluate the link between C. burnetii interaction with PBMCs and their E-cad expression. A significant increase in the percentage of HLADR + /CD16 + monocytes expressing E-cad was measured after PBMCs had been incubated for 8 h with C. burnetii Nine Mile strain. Altogether, these data demonstrate that C. burnetii severely impairs the E-cad expression in circulating cells of Q fever patients.

Coxiella burnetii, the agent causing Q fever, has been associated with B-cell non-Hodgkin lymphoma (NHL). To better clarify this link, we analysed the genetic transcriptomic profile of peripheral blood leukocytes from patients with C. burnetii infection to identify possible links to lymphoma. Microarray analyses revealed that 1189 genes were expressed differently (p <.001 and fold change ≥4) in whole blood of patients with C. burnetii infection compared to controls. In addition, 95 genes expressed in patients with non-Hodgkin lymphoma (NHL) and in patients with C. burnetii persistent infection have allowed us to establish the 'C. burnetii-associated NHL signature'. Among these, 33 genes previously found modulated in C. burnetii-associated -NHL by the microarray analysis were selected and their mRNA expression levels were measured in distinct C. burnetii-induced pathologies, namely, acute Q fever, focalized persistent infection, lymphadenitis and C.burnetii-associated NHL. Specific genes involved in anti-apoptotic process were found highly expressed in leukocytes from patients with C. burnetii associated-NHL: MIR17HG, REL and SP100. This signature differed from that found for NHL-control group. Patients with C. burnetii lymphadenitis presented significant elevated levels of BCL2 and ETS1 mRNAs. Altogether, we identified a specific transcriptionnal signature for NHL during C. burnetii infection reflecting the up-regulation of anti-apoptotic processes and the fact that lymphadenitis might constitute a critical step towards lymphomagenesis., Competing Interests: The authors have declared that no competing interests exist.

Background: Q fever fatigue syndrome (QFS) is a well-documented state of prolonged fatigue following around 20% of acute Q fever infections. It has been hypothesized that low grade inflammation plays a role in its aetiology. In this study, we aimed to identify transcriptome profiles that could aid to better understand the pathophysiology of QFS., Methods: RNA of monocytes was collected from QFS patients (n = 10), chronic fatigue syndrome patients (CFS, n = 10), Q fever seropositive controls (n = 10), and healthy controls (n = 10) who were age- (± 5 years) and sex-matched. Transcriptome analysis was performed using RNA sequencing., Results: Mitochondrial-derived peptide (MDP)-coding genes MT-RNR2 (humanin) and MT-RNR1 (MOTS-c) were differentially expressed when comparing QFS (- 4.8 log2-fold-change P = 2.19 × 10 -9 and - 4.9 log2-fold-change P = 4.69 × 10 -8 ), CFS (- 5.2 log2-fold-change, P = 3.49 × 10 -11 - 4.4 log2-fold-change, P = 2.71 × 10 -9 ), and Q fever seropositive control (- 3.7 log2-fold-change P = 1.78 × 10 -6 and - 3.2 log2-fold-change P = 1.12 × 10 -5 ) groups with healthy controls, resulting in a decreased median production of humanin in QFS patients (371 pg/mL; Interquartile range, IQR, 325-384), CFS patients (364 pg/mL; IQR 316-387), and asymptomatic Q fever seropositive controls (354 pg/mL; 292-393)., Conclusions: Expression of MDP-coding genes MT-RNR1 (MOTS-c) and MT-RNR2 (humanin) is decreased in CFS, QFS, and, to a lesser extent, in Q fever seropositive controls, resulting in a decreased production of humanin. These novel peptides might indeed be important in the pathophysiology of both QFS and CFS.

Objectives: Chronic Q fever is a persistent infection, mostly of aortic aneurysms, vascular prostheses or damaged heart valves, caused by the intracellular bacterium Coxiella burnetii. Only a fraction of C. burnetii-infected individuals at risk develop chronic Q fever. In these individuals, a defective innate immune response may contribute to the development of chronic Q fever. We assessed whether genetic variations in genes involved in the killing machinery for C. burnetii by macrophages, contribute to the progression to chronic Q fever., Methods: The prevalence of 66 single nucleotide polymorphisms (SNPs) in 31 genes pivotal in phagolysosomal maturation, bacterial killing and autophagy, was determined in 173 chronic Q fever patients and 184 controls with risk factors for chronic Q fever and serological evidence of a C. burnetii infection. Associations were detected with univariate logistic regression models. To assess the effect of these SNPs on innate responses to C. burnetii, the C. burnetii-induced cytokine production and basal reactive oxygen species production of healthy volunteers was determined., Results: RAB7A (rs13081864) and P2RX7 loss-of-function SNP (rs3751143) were more common in chronic Q fever patients than in controls. RAB5A (rs8682), P2RX7 gain-of-function SNP (rs1718119), MAP1LC3A (rs1040747) and ATG5 (rs2245214) were more common in controls. In healthy volunteers, RAB7A (rs13081864) and MAP1LC3A (rs1040747) influenced the C. burnetii-induced cytokine production. RAB7A (rs13081864) modulated basal reactive oxygen species production., Conclusions: RAB7A (rs13081864) and P2RX7 (rs3751143) are associated with the development of chronic Q fever, whereas RAB5A (rs8682), P2RX7 (rs1718119), MAP1LC3A (rs1040747) and ATG5 (rs2245214) may have protective effects., (Copyright © 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Although lysosomes perform a number of essential cellular functions, damaged lysosomes represent a potential hazard to the cell. Such lysosomes are therefore engulfed by autophagic membranes in the process known as lysophagy, which is initiated by recognition of luminal glycoprotein domains by cytosolic lectins such as Galectin-3. Here, we show that, under various conditions that cause injury to the lysosome membrane, components of the endosomal sorting complex required for transport (ESCRT)-I, ESCRT-II, and ESCRT-III are recruited. This recruitment occurs before that of Galectin-3 and the lysophagy machinery. Subunits of the ESCRT-III complex show a particularly prominent recruitment, which depends on the ESCRT-I component TSG101 and the TSG101- and ESCRT-III-binding protein ALIX Interference with ESCRT recruitment abolishes lysosome repair and causes otherwise reversible lysosome damage to become cell lethal. Vacuoles containing the intracellular pathogen Coxiella burnetii show reversible ESCRT recruitment, and interference with this recruitment reduces intravacuolar bacterial replication. We conclude that the cell is equipped with an endogenous mechanism for lysosome repair which protects against lysosomal damage-induced cell death but which also provides a potential advantage for intracellular pathogens., (© 2018 The Authors.)

Cytokine responses of chronic Q fever patients to the intracellular bacterium Coxiella burnetii have mostly been studied using ex vivo stimulation of immune cells with heat-killed C. burnetii due to the extensive measures needed to work with viable biosafety level 3 agents. Whether research with heat-killed C. burnetii can be translated to immune responses to viable C. burnetii is imperative for the interpretation of previous and future studies with heat-killed C. burnetii Peripheral blood mononuclear cells (PBMCs) of chronic Q fever patients ( n = 10) and healthy controls ( n = 10) were stimulated with heat-killed or viable C. burnetii of two strains, Nine Mile and the Dutch outbreak strain 3262, for 24 h, 48 h, and 7 days in the absence or presence of serum containing anti- C. burnetii antibodies. When stimulated with viable C. burnetii , PBMCs of chronic Q fever patients and controls produced fewer proinflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor alpha, and IL-1β) after 24 h than after stimulation with heat-killed C. burnetii In the presence of Q fever seronegative serum, IL-10 production was higher after stimulation with viable rather than heat-killed C. burnetii ; however, when incubating with anti- C. burnetii antibody serum, the effect on IL-10 production was reduced. Levels of adaptive, merely T-cell-derived cytokine (gamma interferon, IL-17, and IL-22) and CXCL9 production were not different between heat-killed and viable C. burnetii stimulatory conditions. Results from previous and future research with heat-killed C. burnetii should be interpreted with caution for innate cytokines, but heat-killed C. burnetii -induced adaptive cytokine production is representative of stimulation with viable bacteria., (Copyright © 2018 American Society for Microbiology.)

Coxiella burnetii is an obligate intracellular bacterium and the etiological agent of Q fever. Successful host cell infection requires the Coxiella type IVB secretion system (T4BSS), which translocates bacterial effector proteins across the vacuole membrane into the host cytoplasm, where they manipulate a variety of cell processes. To identify host cell targets of Coxiella T4BSS effector proteins, we determined the transcriptome of murine alveolar macrophages infected with a Coxiella T4BSS effector mutant. We identified a set of inflammatory genes that are significantly upregulated in T4BSS mutant-infected cells compared to mock-infected cells or cells infected with wild-type (WT) bacteria, suggesting that Coxiella T4BSS effector proteins downregulate the expression of these genes. In addition, the interleukin-17 (IL-17) signaling pathway was identified as one of the top pathways affected by the bacteria. While previous studies demonstrated that IL-17 plays a protective role against several pathogens, the role of IL-17 during Coxiella infection is unknown. We found that IL-17 kills intracellular Coxiella in a dose-dependent manner, with the T4BSS mutant exhibiting significantly more sensitivity to IL-17 than WT bacteria. In addition, quantitative PCR confirmed the increased expression of IL-17 downstream signaling genes in T4BSS mutant-infected cells compared to WT- or mock-infected cells, including the proinflammatory cytokine genes Il1a , Il1b , and Tnfa , the chemokine genes Cxcl2 and Ccl5 , and the antimicrobial protein gene Lcn2 We further confirmed that the Coxiella T4BSS downregulates macrophage CXCL2/macrophage inflammatory protein 2 and CCL5/RANTES protein levels following IL-17 stimulation. Together, these data suggest that Coxiella downregulates IL-17 signaling in a T4BSS-dependent manner in order to escape the macrophage immune response., (Copyright © 2018 American Society for Microbiology.)

Our previous study demonstrated that neutrophils play an important role in host defense against Coxiella burnetii infection in mice. In this study, avirulent strain C. burnetii Nine Mile phase II (NMII) was used to examine if C. burnetii can modulate mouse bone marrow-derived neutrophil apoptosis. The results indicated that NMII can inhibit neutrophil apoptosis. Western blotting demonstrated that caspase-3 cleavage was decreased in NMII-infected neutrophils, while phosphorylated mitogen-activated protein kinase (MAPK) p38 and extracellular signal-regulated kinase 1 (Erk1) were increased. Additionally, p38, Erk1/2, phosphoinositide 3-kinase (PI3K), or NF-κB inhibitors reduced the ability of NMII to inhibit neutrophil apoptosis. These results suggest that NMII-mediated inhibition of neutrophil apoptosis depends on its ability to activate neutrophil MAPK pathways. Antiapoptotic protein myeloid cell leukemia-1 (Mcl-1) was significantly increased in NMII-infected neutrophils, and an Mcl-1 inhibitor significantly reduced the ability of NMII to inhibit neutrophil apoptosis. Mcl-1 protein stability was enhanced by phosphorylation at Thr-163 by Erk, and the protein levels were regulated by p38, Erk, PI3K, and NF-κB. Furthermore, the observation that a type IV secretion system (T4SS)-deficient dotA mutant showed a significantly reduced ability to inhibit neutrophil apoptosis compared to wild-type (WT) NMII suggests that T4SS-secreted factors may be involved in NMII-induced inhibition of neutrophil apoptosis. Collectively, these results demonstrate that NMII inhibits neutrophil apoptosis through inhibition of caspase-3 cleavage and activation of MAPK survival pathways with subsequent expression and stabilization of antiapoptotic protein Mcl-1, a process that may partially require the T4SS., (Copyright © 2018 American Society for Microbiology.)

To understand the role of class I major histocompatibility complex (MHC-I) and class II MHC (MHC-II) antigen presentation pathways in host defense against Coxiella burnetii infection, we examined whether MHC-I or MHC-II deficiency in mice would significantly influence their susceptibility to virulent C. burnetii Nine Mile phase I (NMI) infection. The results indicate that NMI infection induced more severe disease in both MHC-I-deficient and MHC-II-deficient mice than in wild-type (WT) mice, while only MHC-I-deficient mice developed a severe persistent infection and were unable to control bacterial replication. These results suggest that both MHC-I-restricted CD8 + T cells and MHC-II-restricted CD4 + T cells contribute to host defense against primary C. burnetii infection, while MHC-I-restricted CD8 + T cells appear to play a more critical role in controlling bacterial replication. Additionally, although NMI infection induced more severe disease in TAP1-deficient mice than in their WT counterparts, TAP1 deficiency in mice did not significantly influence their ability to eliminate C. burnetii This suggests that C. burnetii antigen presentation to CD8 + T cells by the MHC-I classical pathway may depend only partially on TAP1. Furthermore, granzyme B deficiency in mice did not significantly alter their susceptibility to C. burnetii infection, but perforin-deficient mice were unable to control host inflammatory responses during primary C. burnetii infection. These results suggest that perforin, but not granzyme B, is required for C. burnetii antigen-specific cytotoxic CD8 + T cells to control primary C. burnetii infection., (Copyright © 2018 American Society for Microbiology.)

Background: Many emerging infectious pathogens are well known for existing in healthy blood donors and could be transmitted via blood transfusion or plasma derivatives usage. Therefore, there is an urgent need to discover the pathogens in qualified blood donation to avoid potential threats to blood safety., Study Design and Methods: The objective of this study was to investigate the microbiome that existed in pooled plasma from different manufacturers in Chengdu and Guiyang. Random polymerase chain reaction, large-scale clone sequencing, and bioinformatics were used to investigate the metagenomics and microbiome structure of pooled plasma. Among detected microbiomes, potential pathogens were subsequently identified., Result: After host DNA cleaning, 551 clones were classified as bacteria; 88 clones were classified as viruses, and four clones were considered to be parasites, respectively. Thirteen kinds of bacteria and two kinds of parasites that might potentially threaten blood safety were identified along with six kinds of nonpathogenic viruses. The infection status of one identified pathogen Coxiella burnetii was evaluated in 1638 plasma samples. The reactive rate of immunoglobulin (Ig)G1 was 1.10% (18/1638), the reactive rate of IgG2 was 0.85% (14/1638), and the reactive rate of IgM was 0.98% (16/1638)., Conclusion: Some pathogens that were already considered as threats to blood safety were discovered in those pooled plasma such as C. burnetii, Orientia tsutsugamushi, and Plasmodium sp. As a result, we should initiate some specific tests in the endemic area on plasma donors to enhance the blood safety in China., (© 2017 AABB.)

Coxiella burnetii is an intracellular pathogen that causes human Q fever, a disease that normally presents as a severe flu-like illness. Due to high infectivity and disease severity, the pathogen is considered a risk group 3 organism. Full-length lipopolysaccharide (LPS) is required for full virulence and disease by C. burnetii and is the only virulence factor currently defined by infection of an immunocompetent animal. Transition of virulent phase I bacteria with smooth LPS, to avirulent phase II bacteria with rough LPS, occurs during in vitro passage. Semi-rough intermediate forms are also observed. Here, the genetic basis of LPS phase conversion was investigated to obtain a more complete understanding of C. burnetii pathogenesis. Whole genome sequencing of strains producing intermediate and/or phase II LPS identified several common mutations in predicted LPS biosynthesis genes. After passage in broth culture for 30 weeks, phase I strains from different genomic groups exhibited similar phase transition kinetics and elevation of mutations in LPS biosynthesis genes. Targeted mutagenesis and genetic complementation using a new C. burnetii nutritional selection system based on lysine auxotrophy confirmed that six of the mutated genes were necessary for production of phase I LPS. Disruption of two of these genes in a C. burnetii phase I strain resulted in production of phase II LPS, suggesting inhibition of the encoded enzymes could represent a new therapeutic strategy for treatment of Q fever. Additionally, targeted mutagenesis of genes encoding LPS biosynthesis enzymes can now be used to construct new phase II strains from different genomic groups for use in pathogen-host studies at a risk group 2 level.

Background: In the aftermath of the largest Q fever outbreak in the world, diagnosing the potentially lethal complication chronic Q fever remains challenging. PCR, Coxiella burnetii IgG phase I antibodies, CRP and 18 F-FDG-PET/CT scan are used for diagnosis and monitoring in clinical practice. We aimed to identify and test biomarkers in order to improve discriminative power of the diagnostic tests and monitoring of chronic Q fever., Methods: We performed a transcriptome analysis on C. burnetii stimulated PBMCs of 4 healthy controls and 6 chronic Q fever patients and identified genes that were most differentially expressed. The gene products were determined using Luminex technology in whole blood samples stimulated with heat-killed C. burnetii and serum samples from chronic Q fever patients and control subjects., Results: Gene expression of the chemokines CXCL9, CXCL10, CXCL11 and CCL8 was strongly up-regulated in C. burnetii stimulated PBMCs of chronic Q fever patients, in contrast to healthy controls. In whole blood cultures of chronic Q fever patients, production of all four chemokines was increased upon C. burnetii stimulation, but also healthy controls and past Q fever individuals showed increased production of CXCL9, CXCL10 and CCL8. However, CXCL9 and CXCL11 production was significantly higher for chronic Q fever patients compared to past Q fever individuals. In addition, CXCL9 serum concentrations in chronic Q fever patients were higher than in past Q fever individuals., Conclusion: CXCL9 protein, measured in serum or as C. burnetii stimulated production, is a promising biomarker for the diagnosis of chronic Q fever.

Aim: Comparison of diagnostic capabilities of 2 variants of PCR for detection of Coxiella burneli persistence in dynamics of infectious process in patients with Q fever., Materials and Methods: 110 samples of clinical material, obtained from patients with Q fever in an endemic region for this infection (Astrakhan region), were studied. The samples were studied in a standard PCR (marker - 16S rRNA gene fragment) and in real-time. PCR (RT-PCR) (marker - groEL gene fragment)., Results: Both markers were established to be perspective for detection of C. burnetii DNA in clinical material, and RT-PCR detects positive result including late stages of the disease (illness day 21 - 31)., Conclusion: This study is the first Russian publication on comparison on different PCR variants for detection of C. burnetii in blood of Q fever patients in dynamics of the infectious process.

One of the largest Q fever outbreaks ever occurred in the Netherlands from 2007-2010, with 25 fatalities among 4,026 notified cases. Airborne dispersion of Coxiella burnetii was suspected but not studied extensively at the time. We investigated temporal and spatial variation of Coxiella burnetii in ambient air at residential locations in the most affected area in the Netherlands (the South-East), in the year immediately following the outbreak. One-week average ambient particulate matter < 10 μm samples were collected at eight locations from March till September 2011. Presence of Coxiella burnetii DNA was determined by quantitative polymerase chain reaction. Associations with various spatial and temporal characteristics were analyzed by mixed logistic regression. Coxiella burnetii DNA was detected in 56 out of 202 samples (28%). Airborne Coxiella burnetii presence showed a clear seasonal pattern coinciding with goat kidding. The spatial variation was significantly associated with number of goats on the nearest goat farm weighted by the distance to the farm (OR per IQR: 1.89, CI: 1.31-2.76). We conclude that in the year after a large Q fever outbreak, temporal variation of airborne Coxiella burnetii is suggestive to be associated with goat kidding, and spatial variation with distance to and size of goat farms. Aerosol measurements show to have potential for source identification and attribution of an airborne pathogen, which may also be applicable in early stages of an outbreak.

A hallmark of Coxiella burnetii, the bacterial cause of human Q fever, is a biphasic developmental cycle that generates biologically, ultrastructurally, and compositionally distinct large cell variant (LCV) and small cell variant (SCV) forms. LCVs are replicating, exponential phase forms while SCVs are non-replicating, stationary phase forms. The SCV has several properties, such as a condensed nucleoid and an unusual cell envelope, suspected of conferring enhanced environmental stability. To identify genetic determinants of the LCV to SCV transition, we profiled the C. burnetii transcriptome at 3 (early LCV), 5 (late LCV), 7 (intermediate forms), 14 (early SCV), and 21 days (late SCV) post-infection of Vero epithelial cells. Relative to early LCV, genes downregulated in the SCV were primarily involved in intermediary metabolism. Upregulated SCV genes included those involved in oxidative stress responses, arginine acquisition, and cell wall remodeling. A striking transcriptional signature of the SCV was induction (>7-fold) of five genes encoding predicted L,D transpeptidases that catalyze nonclassical 3-3 peptide cross-links in peptidoglycan (PG), a modification that can influence several biological traits in bacteria. Accordingly, of cross-links identified, muropeptide analysis showed PG of SCV with 46% 3-3 cross-links as opposed to 16% 3-3 cross-links for LCV. Moreover, electron microscopy revealed SCV with an unusually dense cell wall/outer membrane complex as compared to LCV with its clearly distinguishable periplasm and inner and outer membranes. Collectively, these results indicate the SCV produces a unique transcriptome with a major component directed towards remodeling a PG layer that likely contributes to Coxiella's environmental resistance.

Coxiella burnetii is a Gram-negative obligate parasitic bacterium that causes the disease Q-fever in humans. To establish its intracellular niche, it utilizes the Icm/Dot type IVB secretion system (T4BSS) to inject protein effectors into the host cell cytoplasm. The host targets of most cognate and candidate T4BSS-translocated effectors remain obscure. We used the yeast Saccharomyces cerevisiae as a model to express and study six C. burnetii effectors, namely AnkA, AnkB, AnkF, CBU0077, CaeA and CaeB, in search for clues about their role in C. burnetii virulence. When ectopically expressed in HeLa cells, these effectors displayed distinct subcellular localizations. Accordingly, GFP fusions of these proteins produced in yeast also decorated distinct compartments, and most of them altered cell growth. CaeA was ubiquitinated both in yeast and mammalian cells and, in S. cerevisiae, accumulated at juxtanuclear quality-control compartments (JUNQs) and insoluble protein deposits (IPODs), characteristic of aggregative or misfolded proteins. AnkA, which was not ubiquitinated, accumulated exclusively at the IPOD. CaeA, but not AnkA or the other effectors, caused oxidative damage in yeast. We discuss that CaeA and AnkA behavior in yeast may rather reflect misfolding than recognition of conserved targets in the heterologous system. In contrast, CBU0077 accumulated at vacuolar membranes and abnormal ER extensions, suggesting that it interferes with vesicular traffic, whereas AnkB associated with the yeast nucleolus. Both effectors shared common localization features in HeLa and yeast cells. Our results support the idea that C. burnetii T4BSS effectors manipulate multiple host cell targets, which can be conserved in higher and lower eukaryotic cells. However, the behavior of CaeA and AnkA prompt us to conclude that heterologous protein aggregation and proteostatic stress can be a limitation to be considered when using the yeast model to assess the function of bacterial effectors.

Coxiella burnetii, the causative agent of Q fever, is recognized by TLR2. TLR10 can act as an inhibitory receptor on TLR2-derived immune responses. Therefore, we investigated the role of TLR10 on C. burnetii-induced cytokine production and assessed whether genetic polymorphisms in TLR10 influences the development of chronic Q fever. HEK293 cells, transfected with TLR2, TLR10 or TLR2/TLR10, and human peripheral blood mononuclear cells (PBMCs) in the presence of anti-TLR10, were stimulated with C. burnetii. In both assays, the absence of TLR10 resulted in increased cytokine responses after C. burnetii stimulation. In addition, the effect of single nucleotide polymorphisms (SNPs) in TLR10 was examined in healthy volunteers whose PBMCs were stimulated with C. burnetii Nine Mile or the Dutch outbreak isolate C. burnetii 3262. Individuals bearing SNPs in TLR10 displayed increased cytokine production upon C. burnetii 3262 stimulation. Furthermore, 139 chronic Q fever patients and 220 controls were genotyped for TLR10 N241H, I775V and I369L. None of these polymorphisms were associated with increased susceptibility to chronic Q fever. In conclusion, TLR10 has an inhibitory effect on in vitro cytokine production by C. burnetii, but the presence of TLR10 polymorphisms does not lead to an increased risk of developing chronic Q fever., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Coxiella burnetii is a highly infectious bacterium that promotes its own replication in macrophages by inhibiting several host cell responses. Here, we show that C. burnetii inhibits caspase-1 activation in primary mouse macrophages. By using co-infection experiments, we determine that the infection of macrophages with C. burnetii inhibits the caspase-11-mediated non-canonical activation of the NLRP3 inflammasome induced by subsequent infection with Escherichia coli or Legionella pneumophila. Genetic screening using flagellin mutants of L. pneumophila as a surrogate host, reveals a novel C. burnetii gene (IcaA) involved in the inhibition of caspase activation. Expression of IcaA in L. pneumophila inhibited the caspase-11 activation in macrophages. Moreover, icaA(-) mutants of C. burnetii failed to suppress the caspase-11-mediated inflammasome activation induced by L. pneumophila. Our data reveal IcaA as a novel C. burnetii effector protein that is secreted by the Dot/Icm type IV secretion system and interferes with the caspase-11-induced, non-canonical activation of the inflammasome.

Genes involved in human immune response are well recognized to influence the clinical course of infection. The association of host genetics with susceptibility to and severity of clinical symptoms in acute Q fever was investigated. Single nucleotide polymorphisms (SNPs) in the IFNG (rs2430561/rs1861493), STAT1 (rs1914408), and VDR (rs2228570) genes were determined in 85 patients from the 2007 Dutch acute Q fever outbreak, and a symptom score was calculated. IFNG rs1861493 showed a significant association with the symptom score; IFNG rs2430561 showed a similar trend. These SNPs were then used to reproduce results in a 2009 outbreak population (n = 123). The median symptom score differed significantly in both populations: 2 versus 7. The significant association of IFNG rs1861493 with symptom score in the first population was not reproduced in the second population. We hypothesize that individuals in the second outbreak were exposed to a higher Coxiella burnetii dose compared to the first, which overruled the protection conferred by the A-allele of IFNG rs1861493 in the first population.

To survive in macrophages, Coxiella burnetii hijacks the activation pathway of macrophages. Recently, we have demonstrated that C. burnetii, via its lipopolysaccharide (LPS), avoids the activation of p38α-MAPK through an antagonistic engagement of Toll-like receptor (TLR)-4. We investigated the fine-tuned mechanism leading to the absence of activation of the p38α-MAPK despite TLR-4 engagement. In macrophages challenged with LPS from the avirulent variants of C. burnetii, TLR-4 and TLR-2 co-immunoprecipitated. This association was absent in cells challenged by the LPS of pathogenic C. burnetii. The disruption makes TLRs unable to signal during the recognition of the LPS of pathogenic C. burnetii. The disruption of TLR-2 and TLR-4 was induced by the re-organization of the macrophage cytoskeleton by C. burnetii LPS. Interestingly, blocking the actin cytoskeleton re-organization relieved the disruption of the association TLR-2/TLR-4 by pathogenic C. burnetii and rescued the p38α-MAPK activation by C. burnetii. We elucidated an unexpected mechanism allowing pathogenic C. burnetii to avoid macrophage activation by the disruption of the TLR-2 and TLR-4 association.

The formation of granulomas is associated with the resolution of Q fever, a zoonosis due to Coxiella burnetii; however the molecular mechanisms of granuloma formation remain poorly understood. We generated human granulomas with peripheral blood mononuclear cells (PBMCs) and beads coated with C. burnetii, using BCG extracts as controls. A microarray analysis showed dramatic changes in gene expression in granuloma cells of which more than 50% were commonly modulated genes in response to C. burnetii and BCG. They included M1-related genes and genes related to chemotaxis. The inhibition of the chemokines, CCL2 and CCL5, directly interfered with granuloma formation. C. burnetii granulomas also expressed a specific transcriptional profile that was essentially enriched in genes associated with type I interferon response. Our results showed that granuloma formation is associated with a core of transcriptional response based on inflammatory genes. The specific granulomatous response to C. burnetii is characterized by the activation of type 1 interferon pathway.

Coxiella burnetii, the causative agent of Q fever, is a human intracellular pathogen that utilizes the Icm/Dot type IVB secretion system to translocate effector proteins into host cells. To identify novel C. burnetii effectors, we applied a machine-learning approach to predict C. burnetii effectors, and examination of 20 such proteins resulted in the identification of 13 novel effectors. To determine whether these effectors, as well as several previously identified effectors, modulate conserved eukaryotic pathways, they were expressed in Saccharomyces cerevisiae. The effects on yeast growth were examined under regular growth conditions and in the presence of caffeine, a known modulator of the yeast cell wall integrity (CWI) mitogen-activated protein (MAP) kinase pathway. In the presence of caffeine, expression of the effectors CBU0885 and CBU1676 caused an enhanced inhibition of yeast growth, and the growth inhibition of CBU0388 was suppressed. Furthermore, analysis of synthetic lethality effects and examination of the activity of the CWI MAP kinase transcription factor Rlm1 indicated that CBU0388 enhances the activation of this MAP kinase pathway in yeast, while CBU0885 and CBU1676 abolish this activation. Additionally, coexpression of CBU1676 and CBU0388 resulted in mutual suppression of their inhibition of yeast growth. These results strongly indicate that these three effectors modulate the CWI MAP kinase pathway in yeast. Moreover, both CBU1676 and CBU0885 were found to contain a conserved haloacid dehalogenase (HAD) domain, which was found to be required for their activity. Collectively, our results demonstrate that MAP kinase pathways are most likely targeted by C. burnetii Icm/Dot effectors., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

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&#95;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.

The acute sickness response to infection is a conserved set of changes in physiology and behaviour, featuring fever, fatigue, musculo-skeletal pain, disturbed mood, and cognitive difficulties. The manifestations differ somewhat between individuals, including those infected with pathogens which do not have genetic variability--suggesting host determinants. Principal components analysis (PCA) was applied to acute phase, self-report symptom data from subjects in the Dubbo Infection Outcomes Study (n=296) to empirically derive indices of fatigue, pain, neurocognitive difficulties, and mood disturbance, as well as overall illness severity. Associations were sought with functional single nucleotide polymorphisms (SNPs) in the cytokine genes, interleukin (IL)-6, tumour necrosis factor (TNF)-α, interferon (IFN)-γ, and IL-10. The summed individual symptom indices correlated with overall severity and also with functional status. The relative contribution of individual symptom domains to the overall illness was stable over time within subjects, but varied between subjects with the same infection. The T allele of the IFN-γ +874 T/A SNP was associated with increased fatigue (p=0.0003; OR: 3.3). The C allele of the IL-10 -592 C/A SNP exerted a protective effect on neurocognitive difficulties (p=0.017; OR: 0.52); while the A allele for the IL-10 -592 SNP was associated with increased mood disturbance (p=0.044; OR: 1.83), as was the G allele of the IL-6 -174 G/C SNP (p=0.051; OR: 1.83). The acute sickness response has discrete symptom domains including fatigue, which have unique genetic associations. These data provide novel insights into the pathophysiology of fatigue states., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Coxiella burnetii infection is frequently unrecognized or misdiagnosed, as symptoms generally mimic an influenza-like illness. However, the disease (Q fever) may result in chronic infection, usually manifesting as potentially fatal endocarditis. The development of a chronic fatigue-like sequela may also occur. Infected ruminants are the major reservoir for infection in humans, primarily through exposure to birth products or aerosols that transmit the bacterium over wide regions. A vaccine against C. burnetii infection has been in use in Australia for abattoir and agricultural workers for many years. The possibility of adverse reactions in those with previous exposure to the agent has prevented its use elsewhere. Subunit vaccines, utilizing chemical extraction of components thought to cause adverse reactions, are in development, but none are yet licensed. Others have sought to combine immunogenic peptides with or without selected lipopolysaccharide components to produce a vaccine without the possibility of adverse reactions. Selected immunogenic proteins have been shown to induce both humoral and cellular immune responses. Although current diagnosis of infection relies on serological testing, the presentation of specific antibody occurs 7-15 days following the onset of symptoms, delaying treatment that may result in prolonged morbidity. PCR detection of DNA to specific C. burnetii antigens in the blood is possible early in infection, but PCR may become negative when PII IgG antibodies appear. PCR is useful for early diagnosis when Q fever is suspected, as in large epidemics, and shortens the delay in the identification of Q fever endocarditis. Others have combined PCR with ELISA or other methods to increase the ability to detect infection at any stage. The search for new diagnostic reagents and vaccines has utilized new methods for discovery of immunoreactive proteins. DNA analysis of the heterogeneity of C. burnetii isolates has led to a greater understanding of the diversity of isolates and a means to determine whether there is a correlation between strain and disease severity. 2-D SDS PAGE of immunogenic proteins reactive with human or animal infection sera and mass spectrometric analysis of specific secreted or outer membrane proteins have identified candidate antigens. Microarrays have allowed the analysis of peptide libraries of open reading frames to evaluate the immunogenicity of complete genomes.

Recent proteomic studies of C. burnetii, the etiological agent of Q fever, have brought a deeper insight into the pathogen's physiology and offered new possibilities in investigations of inter- or intra-species relatedness. The data generated from these studies in conjunction with the current genomic sequence databases may reveal additional identities for conserved and unique C. burnetii biomarkers and aid in creating algorithms and/or databases that could develop into diagnostic and detection tools for the pathogen. Moreover, wide scale screening and further characterization of potential C. burnetii protein antigens along with a comprehensive evaluation of the humoral immune response will be of fundamental importance towards research and development of a safe and efficacious vaccine as well as improved serodiagnostic tests for rapid and sensitive detection of the Q fever pathogen. Given these advances, proteomics may make marked contributions to the improvement of human health protection against C. burnetii in the coming years.

Acute Q fever is commonly resolved without an antibiotic regimen, but a primary infection may develop into a chronic infection in a minority of cases. Coxiella burnetii, the causative agent of Q fever, is known to infect macrophages both in vitro and in vivo. It has been observed that the intracellular survival of C. burnetii requires the subversion of the microbicidal properties of macrophages. Adaptive immunity is also essential to cure C. burnetii infection, as demonstrated by clinical studies and animal models. Indeed, the control of infection in patients with primary Q fever involves a systemic cell-mediated immune response and granuloma formation with an essential role for interferon-γ in the protection against C. burnetii. In contrast, chronic Q fever is characterized by defective cell-mediated immunity with the defective formation of granulomas and over-production of interleukin-10, an immunoregulatory cytokine. Finally, epidemiological data demonstrate that age and gender are risk factors for Q fever. The analysis of gene expression programs in mice reveals the importance of sex-related genes in C. burnetii infection because only 14% of the modulated genes are sex-independent, while the remaining 86% are differentially expressed in males and females. These results open a new field to understand how host metabolism controls C. burnetii infection in humans.

Lipids are essential components of both eukaryotic and prokaryotic cells, serving diverse functions including energy metabolism and membrane structure. Intracellular vacuolar pathogens such as Coxiella burnetii require lipids for both normal bacterial functions as well as formation of the acidic, phagolysosomal-like parasitophorous vacuole (PV) surrounding the bacteria. As an intracellular pathogen, C. burnetii can acquire lipid through both de novo bacterial synthesis and subversion of host cell pools. The C. burnetii genome encodes enzymes required for de novo synthesis of fatty acids and phospholipids. The high percentage of branched fatty acids suggests C. burnetii modifies these molecules to generate a bacterial cell envelope that can resist the harsh environment of the PV, such as the acidic pH. In addition to fatty acids and their derivatives, C. burnetii requires isoprenoids, particularly sterols as the PV membrane is cholesterol-rich. With the exception of two eukaryote-like sterol reductases, C. burnetii does not have the capability to generate cholesterol, suggesting sterols are actively diverted from the host cell. While C. burnetii utilizes host cell lipids for membrane biogenesis and possibly energy, bacterial manipulation of host cell lipid signaling pathways may support establishment of the intracellular niche. For example, effectors secreted by the C. burnetii Type IV secretion system may either directly or indirectly modify host cell lipids. Further understanding of the lipid biosynthetic capabilities of C. burnetii, along with C. burnetii's manipulation of host cell lipids, will provide insight into the host-pathogen relationship.

Coxiella burnetii, the causative agent of Q fever, is the only known intracellular pathogenic bacterium that replicates within the acidic, degradative confines of a eukaryotic phagolysosome. It appears that the bacterium nullifies or thwarts the toxic elements of this vacuole and evades both innate and adaptive immune responses. C. burnetii is ubiquitous in its geographic distribution with the disease prevalence more widespread than previously considered. Recent molecular and cell biological advances, along with improved instrumentation, have provided unique insight into the host-parasite interrelationship and revealed previously unknown virulence strategies of C. burnetii. An example is completion of the genome sequences of several strains of C. burnetii that has improved our understanding of pathogenic mechanisms used by the organism to cause disease in mammalian hosts.

C. burnetii is a Gram-negative intracellular Y-proteobacteria that causes the zoonotic disease Q fever. Q fever can manifest as an acute or chronic illness. Different typing methods have been previously developed to classify C. burnetii isolates to explore its pathogenicity. Here, we report a comprehensive genomotyping method based on the presence or absence of genes using microarrays. The genomotyping method was then tested in 52 isolates obtained from different geographic areas, different hosts and patients with different clinical manifestations. The analysis revealed the presence of 10 genomotypes organized into 3 groups, with a topology congruent with that obtained through multi-spacer typing. We also found that only 4 genomotypes were specifically associated with acute Q fever, whereas all of the genomotypes could be associated to chronic human infection. Serendipitously, the genomotyping results revealed that all hard tick isolates, including the Nine Mile strain, belong to the same genomotype.

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.

Background: Q fever, a zoonosis due to Coxiella burnetii infection, exhibits sexual dimorphism; men are affected more frequently and severely than women for a given exposure. Here we explore whether the severity of C. burnetii infection in mice is related to differences in male and female gene expression profiles., Methodology/principal Findings: Mice were infected with C. burnetii for 24 hours, and gene expression was measured in liver cells using microarrays. Multiclass analysis identified 2,777 probes for which expression was specifically modulated by C. burnetti infection. Only 14% of the modulated genes were sex-independent, and the remaining 86% were differentially expressed in males and females. Castration of males and females showed that sex hormones were responsible for more than 60% of the observed gene modulation, and this reduction was most pronounced in males. Using functional annotation of modulated genes, we identified four clusters enriched in males that were related to cell-cell adhesion, signal transduction, defensins and cytokine/Jak-Stat pathways. Up-regulation of the IL-10 and Stat-3 genes may account for the high susceptibility of men with Q fever to C. burnetii infection and autoantibody production. Two clusters were identified in females, including the circadian rhythm pathway, which consists of positive (Clock, Arntl) and negative (Per) limbs of a feedback loop. We found that Clock and Arntl were down-modulated whereas Per was up-regulated; these changes may be associated with efficient bacterial elimination in females but not in males, in which an exacerbated host response would be prominent., Conclusion: This large-scale study revealed for the first time that circadian rhythm plays a major role in the anti-infectious response of mice, and it provides a new basis for elucidating the role of sexual dimorphism in human infections.

Background: The authors have previously reported genomic subtypes of chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) based on expression of 88 human genes., Aim: To attempt to reproduce these findings, determine the specificity of this signature to CFS/ME, and test for associations between CFS/ME subtype and infection., Methods: Expression levels of 88 human genes were determined in blood of 62 new patients with idiopathic CFS/ME (according to Fukuda criteria), six patients with Q-fever-associated CFS/ME from the Birmingham Q-fever outbreak (according to Fukuda criteria), 14 patients with endogenous depression (according to DSM-IV criteria) and 29 normal blood donors., Results: In patients with CFS/ME, differential expression was confirmed for all 88 genes. Q-CFS/ME had similar patterns of gene expression to idiopathic CFS/ME. Gene expression in patients with endogenous depression was similar to that in the normal controls, except for upregulation of five genes (APP, CREBBP, GNAS, PDCD2 and PDCD6). Clustering of combined gene data in CFS/ME patients for this and the authors' previous study (117 CFS/ME patients) revealed genomic subtypes with distinct differences in SF36 scores, clinical phenotypes, severity and geographical distribution. Antibody testing for Epstein-Barr virus, enterovirus, Coxiella burnetii and parvovirus B19 revealed evidence of subtype-specific relationships for Epstein-Barr virus and enterovirus, the two most common infectious triggers of CFS/ME., Conclusions: This study confirms the involvement of these genes in CFS/ME.

Coxiella burnetii is an obligate intracellular bacterium that generates large vacuoles in which this pathogen replicates and survives. We have previously demonstrated that C. burnetii interacts with the autophagic pathway as a strategy for its survival and replication. Coxiella displays an anti-apoptotic activity to maintain host cell viability, leading to a persistent infection. Our recent study reveals that Beclin 1 is recruited to the Coxiella-membrane vacuole favoring its development and bacterial replication. In contrast, the anti-apoptotic protein Bcl-2 alters the normal development of the Coxiella-replicative compartment. In addition, our results indicate that C. burnetii infection modulates autophagy and apoptotic pathways via Beclin 1-Bcl-2 interplay to establish a successful infection in the host cell. Of note, this pathogen-host cell model has allowed uncovering a novel function of Beclin 1 as a regulator of the anti-apoptotic activity of Bcl-2. We have also established that a proper interplay between Beclin 1 and Bcl-2 is required for both autophagy and apoptosis modulation.

Coxiella burnetii is a macrophage-tropic, Gram-negative organism, which causes acute Q fever infection in humans. This zoonotic infection causes illness ranging from asymptomatic seroconversion to severe and protracted disease featuring hepatitis and pneumonia. Interactions between C. burnetii lipopolysaccharide (LPS) and host Toll-like receptors (TLR)-2 and -4 have been implicated in pathogen recognition, phagocytosis and signaling responses. Nonconservative single nucleotide polymorphisms in the coding regions of TLR-2 (Arg677Trp and Arg753Gln) and TLR-4 (Asp299Gly) have been found to correlate with mycobacterial infections and Gram-negative sepsis respectively. Associations between the TLR-2 and -4 polymorphisms, illness characteristics and immune response parameters were examined in subjects with acute Q fever (n=85) and comparison subjects with viral infections (n=162). No correlation was demonstrated between these polymorphisms and susceptibility to Q fever, illness severity or illness course.

Q fever (infection with Coxiella burnetii) has been uncommon in Australia's Northern Territory, with no reported cases until 2002. Since then, twelve cases of Q fever have been reported, representing a much lower notification rate than in surrounding Australian states. Three cases were identified in Central Australia during 2006, prompting this review of clinical and epidemiological features of all notified Northern Territory cases. Three patients required Intensive Care admission, 1 died, 5 had moderately severe illness, 2 were treated as outpatients and 2 were excluded as unlikely Q fever cases on clinical grounds. Hospital stays were long (median length of stay 9.5 days), and diagnosis and definitive therapy were generally delayed. Although macrolides and quinolones have some reported efficacy against C. burnetii, 2 patients experienced prolonged fever (5 and 9 days respectively) despite azithromycin, and the fatality occurred in a patient treated with multiple antibiotics including ciprofloxacin. Four patients were Aboriginal, 3 were tested for HTLV-1 and 2 were positive. The patient who died was diabetic. None of acute and chronic manifestations of Q fever is required in the Northern Territory. Early institution of doxycyline in suspected cases is recommended, and more rapid diagnostic methods including polymerase chain reaction testing should be considered. Host risk factors for chronicity, which may be of particular importance in Indigenous patients, merit attention. Given the lack of occupational exposure in these cases, there seems little reason to change the current Northern Territory policy of opting out of the National Q Fever Vaccination Program. Recognised alternative exposures, such as non-occupational livestock and domestic animal contact, require consideration as local Q fever sources.