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International audience; A wide spectrum of pathological conditions may result from the interaction of Aspergillus fumigatus and the immune system of its human host. Allergic bronchopulmonary aspergillosis is one of the most severe A.fumigatus-related diseases due to possible evolution toward pleuropulmonary fibrosis and respiratory failure. Allergic bronchopulmonary aspergillosis occurs almost exclusively in cystic fibrosis or asthmatic patients. An estimated 8%-10% of patients with cystic fibrosis experience this condition. The diagnosis of allergic bronchopulmonary aspergillosis relies on criteria first established in 1977. Progress in the understanding of host-pathogen interactions in A.fumigatus and patients with cystic fibrosis and the ongoing validation of novel laboratory tools concur to update and improve the diagnosis of allergic bronchopulmonary aspergillosis.

International audience; The intracellular bacterium Coxiella burnetii is responsible for Q fever, an infectious disease that increases the risk of abortion, preterm labor, and stillbirth in pregnant women. It has been shown that C. burnetii replicates in BeWo trophoblast cell line and inhibits the activation and maturation of decidual dendritic cells. Although tissue macrophages are known to be targeted by C. burnetii, no studies have investigated the interplay between placental macrophages and C. burnetii. Here, CD14 + macrophages from 46 full-term placentas were isolated by positive selection. They consisted of a mixed population of maternal and fetal origin as shown by genotype analysis. We showed that C. burnetii organisms infected placental macrophages after 4 h. When these infected macrophages were incubated for an additional 9-day culture, they completely eliminated organisms as shown by quantitative PCR. The ability of placental macrophages to form multinucleated giant cells was not affected by C. burnetii infection. The transcriptional immune response of placental macrophages to C. burnetii was investigated using quantitative real-time RT-PCR on 8 inflammatory and 10 immunoregulatory genes. C. burnetii clearly induced an inflammatory profile. Interestingly, the production by placental macrophages of interferon-γ, a cytokine known to be involved in efficient immune responses, was dramatically increased in response to C. burnetii. In addition, a clear correlation between interferon-γ production and C. burnetii elimination was found, suggesting that macrophages from full-term placentas eliminate C. burnetii under the control of an autocrine production of interferon-γ.

For 40 years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used in proteomics and biochemistry. It has been demonstrated in the last decade that MALDI-TOF MS can be used routinely to identify and classify numerous bacterial species or subspecies. We applied MALDI-TOF MS directly to intact mammalian cells, and we found that this method is valuable to identify human circulating cells and cells involved in the immune response including macrophages. We then stimulated human macrophages with cytokines, bacterial products, and a variety of bacteria. We found that MALDI-TOF MS discriminated unstimulated and stimulated macrophages and also detected multifaceted activation of macrophages. We conclude that whole-cell MALDI-TOF MS is an accurate method to identify various cell types and to detect subtle modifications in cell activity and therefore it can be beneficial in clinical practices for a rapid patient classification based on their immune profile.

International audience; Q fever is an infectious disease due to Coxiella burnetii. Following a primary-infection, C. burnetii may persist in some patients, leading to endocarditis and vascular infections. Mast cells (MCs), known for their role in allergic diseases, innate immunity and cardiac function, are produced by bone marrow, circulate as progenitors in the bloodstream and reach tissues for their maturation and activation. The latter may be estimated by measuring serum tryptase levels. We wondered if MC progenitors and tryptase were affected in Q fever. We showed a decrease in MC progenitor count in Q fever patients whereas serum tryptase levels were increased. Taken together, our results show alterations of MC numbers and activity in Q fever patients, suggesting that MC are involved in Q fever pathophysiology.

Mast cells (MCs) are found in tissues that are in close contact with external environment, such as skin, lungs, or intestinal mucosa but also in the placenta during pregnancy. If their role in mediating allergic conditions is established, several studies now highlight their importance during infection with extracellular pathogens. This study showed a new and effective antimicrobial mechanism of MCs against Coxiella burnetii, an intracellular bacterium whose infection during pregnancy is associated with abortion, preterm labor, and stillbirth. The data reveal that in response to C. burnetii, MCs release extracellular actin filaments that contain antimicrobial agents and are capable to trap and kill bacteria. We show that this mechanism is dependent on the cooperation of two membrane receptors, CD36 and Toll-like receptor 4, and may occur in the placenta during pregnancy by using ex vivo placental MCs. Overall, this study reports an unexpected role for MCs during infection with intracellular bacteria and suggests that MC response to C. burnetii infection is a protective defense mechanism during pregnancy., Mast cells (MCs) are critical mediators of inflammation; however, their microbicidal activity against invading pathogens remains largely unknown. Here, we describe a nonpreviously reported antibacterial mechanism used by MCs against Coxiella burnetii, the agent of Q fever. We show that C. burnetii interaction with MCs does not result in bacterial uptake but rather induces the formation of extracellular actin filaments named cytonemes. MC cytonemes express cathelicidin and neutrophil elastase and mediate the capture and destruction of entrapped bacteria. We provide evidence that MC cytoneme formation and microbicidal activity are dependent on the cooperation of the scavenger receptor CD36 and Toll-like receptor 4. Taken together, our results suggest that MCs use an extracellular sophisticated mechanism of defense to eliminate intracellular pathogens, such as C. burnetii, before their entry into host cells.

Background & Aims Infection with Tropheryma whipplei has a range of effects—some patients can be chronic carriers without developing any symptoms, whereas others can develop systemic Whipple disease, characterized by a lack a protective inflammatory immune response. Alterations in HLA-G function have been associated with several diseases. We investigated the role of HLA-G during T whipplei infection. Methods Sera, total RNA, and genomic DNA were collected from peripheral blood from 22 patients with classic Whipple’s disease, 19 patients with localized T whipplei infections, and 21 asymptomatic carriers. Levels of soluble HLA-G in sera were measured by enzyme-linked immuosorbent assay, and expressions of HLA-G and its isoforms were monitored by real-time polymerase chain reaction. HLA-G alleles were identified and compared with a population of voluntary bone marrow donors. Additionally, monocytes from healthy subjects were stimulated with T whipplei, and HLA-G expression was monitored by real-time polymerase chain reaction and flow cytometry. Bacterial replication was assessed by polymerase chain reaction in the presence of HLA-G or inhibitor of tumor necrosis factor (TNF) (etanercept). Results HLA-G mRNAs and levels of soluble HLA-G were significantly increased in sera from patients with chronic T whipplei infection compared with sera from asymptomatic carriers and control individuals. No specific HLA-G haplotypes were associated with disease or T whipplei infection. However, T whipplei infection of monocytes induced expression of HLA-G, which was associated with reduced secretion of TNF compared with noninfected monocytes. A neutralizing antibody against HLA-G increased TNF secretion by monocytes in response to T whipplei, and a TNF inhibitor promoted bacteria replication. Conclusions Levels of HLA-G are increased in sera from patients with T whipplei tissue infections, associated with reduced production of TNF by monocytes. This might promote bacteria colonization in patients.

International audience; We report 7 patients with interstitial lung disease seen at computed tomographic scan review. Coxiella burnetii infection was diagnosed in situ in 1 lung biopsy specimen. Q fever may be a cofactor of interstitial lung disease, especially in endemic areas.

International audience; Pulmonary and systemic antifungal immunity influences quality of life and survival of people with cystic fibrosis. Aspergillus fumigatus (Af) induces specific IgG and IgE. Mast cells respond to IgE, IgG and direct interactions with Af. Mast cells are the source of the protease tryptase. We aimed at evaluating serum baseline tryptase as a potential biomarker of the Af-host interaction in cystic fibrosis patients. Serum baseline tryptase, IgE and IgG directed to Af extract and Af molecular allergens were measured in 76 cystic fibrosis patients. The main findings were (i) lower levels of serum baseline tryptase in patients displaying specific IgE to Af (p < 0.0001) and (ii) an association between tryptase levels and IgE or IgG responses to Af and ribotoxin (Asp f 1). These findings suggest that serum baseline tryptase is influenced by Af-host interactions and thus might be a marker for mast cell regulation and pulmonary immune defenses. (C) 2018 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

International audience; Objectives: Chronic Q fever is a persistent infection with the intracellular Gram-negative bacterium Coxiella burnetii, which can lead to complications of infected aneurysms. Matrix metalloproteinases (MMPs) cleave extracellular matrix and are involved in infections as well as aneurysms. We aimed to study the role of MMPs in the pathogenesis of chronic Q fever. Methods: We investigated gene expression of MMPs through microarray analysis and MMP production with ELISA in C. burnetii-stimulated peripheral blood mononuclear cells (PBMCs) of patients with chronic Q fever and healthy controls. Twenty single nucleotide polymorphisms (SNPs) of MMP and tissue inhibitor of MMP genes were genotyped in 139 patients with chronic Q fever and 220 controls with similar cardiovascular co-morbidity. Additionally, circulating MMPs levels in patients with chronic Q fever were compared with those in cardiovascular controls with and without a history of past Q fever. Results: In healthy controls, the MMP pathway involving four genes (MMP1, MMP7, MMP10, MMP19) was significantly up-regulated in C. burnetii-stimulated but not in Escherichia coli lipopolysaccharide -stimulated PBMCs. Coxiella burnetii induced MMP-1 and MMP-9 production in PBMCs of healthy individuals (both p < 0.001), individuals with past Q fever (p < 0.05, p < 0.01, respectively) and of patients with chronic Q fever (both p < 0.001). SNPs in MMP7 (rs11568810) (p < 0.05) and MMP9 (rs17576) (p < 0.05) were more common in patients with chronic Q fever. Circulating MMP-7 serum levels were higher in patients with chronic Q fever (median 33.5 ng/mL, interquartile range 22.3-45.7 ng/mL) than controls (20.6 ng/mL, 15.9-33.8 ng/mL). Conclusion: Coxiella burnetii-induced MMP production may contribute to the development of chronic Q fever. (C) 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

SUMMARYCoxiella burnetiiis the agent of Q fever, or “query fever,” a zoonosis first described in Australia in 1937. Since this first description, knowledge about this pathogen and its associated infections has increased dramatically. We review here all the progress made over the last 20 years on this topic.C. burnetiiis classically a strict intracellular, Gram-negative bacterium. However, a major step in the characterization of this pathogen was achieved by the establishment of its axenic culture.C. burnetiiinfects a wide range of animals, from arthropods to humans. The genetic determinants of virulence are now better known, thanks to the achievement of determining the genome sequences of several strains of this species and comparative genomic analyses. Q fever can be found worldwide, but the epidemiological features of this disease vary according to the geographic area considered, including situations where it is endemic or hyperendemic, and the occurrence of large epidemic outbreaks. In recent years, a major breakthrough in the understanding of the natural history of human infection withC. burnetiiwas the breaking of the old dichotomy between “acute” and “chronic” Q fever. The clinical presentation ofC. burnetiiinfection depends on both the virulence of the infectingC. burnetiistrain and specific risks factors in the infected patient. Moreover, no persistent infection can exist without a focus of infection. This paradigm change should allow better diagnosis and management of primary infection and long-term complications in patients withC. burnetiiinfection.

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 18F–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. Electronic supplementary material The online version of this article (doi:10.1186/s12879-017-2656-6) contains supplementary material, which is available to authorized users.

International audience; Planarians, which are non-parasitic flatworms, are highly resistant to bacterial infections. To better understand the mechanisms underlying this resistance, we investigated the role of the circadian machinery in the anti-bacterial response of the freshwater planarian Schmidtea mediterranea. We identified Smed-Tim from S. mediterranea as a homolog of the mammalian clock gene Tim. We showed via RNA interference that Smed-Tim is required for the antimicrobial activities of Schmidtea mediterranea against Staphylococcus aureus infection during the light/dark cycle. Indeed, S. aureus infection leads to the expression of Smed-Tim, which in turn promotes Smed-Traf6 and Smed-morn2, but not Smed-p38 MAPK expression, 2 master regulators of planarian anti-microbial responses.

International audience; Brucella is a Gram-negative facultative intracellular bacterium responsible for a chronic disease known as brucellosis, the most widespread re-emerging zoonosis worldwide. Establishment of a Th1-mediated immune response characterized by the production of IL-12 and IFN gamma is essential to control the disease. Leukotrienes derived from arachidonic acid (AA) metabolism are known to negatively regulate a protective Th1 immune response against bacterial infections. Here, using genomics approaches we demonstrate that Brucella abortus strongly stimulates the prostaglandin (PG) pathway in dendritic cells (DC). We also show an induction of AA production by infected cells. This correlates with the expression of Ptgs2, a gene encoding the downstream cyclooxygenase-2 (COX-2) enzyme in infected DC. By comparing different infection routes (oral, intradermal, intranasal and conjunctival), we identified the intradermal inoculation route as the more potent in inducing Ptgs2 expression but also in inducing a local inflammatory response in the draining cervical lymph nodes (CLN). NS-398, a specific inhibitor of COX-2 enzymatic activity decreased B. melitensis burden in the CLN after intradermal infection. This effect was accompanied by a decrease of II10 and a concomitant increase of Ifng expression. Altogether, these results suggest that Brucella has evolved to take advantage of the PG pathway in the harsh environment of the CLN in order to persist and subvert immune responses. This work also proposes that novel strategies to control brucellosis may include the use of COX-2 inhibitors.

International audience; 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

Venous thromboses have been associated with tuberculosis, but the relationship with circulating anticoagulant has not been studied yet. In a cohort of 48 patients with tuberculosis, 22.9% of them presented with venous thromboses significantly associated with dose dependent level of antiphosphophatidyl-ethanolamine antibodies. Keywords: Tuberculosis, Deep vein thrombosis, Pulmonary embolism, Antiphospholipds antibodies, Mycobacterium tuberculosis, Anti-phosphatidylethanolamine antibodies

SummaryVariations in lipopolysaccharide (LPS), a bacterial outer membrane component, determine virulence of the obligate intracellular bacterium Coxiella burnetii, but the underlying mechanisms are unknown. We find that while avirulent C. burnetii LPS (avLPS) stimulates host p38α-MAPK signaling required for proper trafficking of bacteria containing compartments to lysosomes for destruction, pathogenic C. burnetii LPS (vLPS) does not. The defect in vLPS and pathogenic C. burnetii targeting to degradative compartments involves an antagonistic engagement of TLR4 by vLPS, lack of p38α-MAPK-driven phosphorylation, and block in recruitment of the homotypic fusion and protein-sorting complex component Vps41 to vLPS-containing vesicles. An upstream activator of p38α-MAPK or phosphomimetic mutant Vps41-S796E expression overrides the inhibition, allowing vLPS and pathogenic C. burnetii targeting to phagolysosomes. Thus, p38α-MAPK and its crosstalk with Vps41 play a central role in trafficking bacteria to phagolysosomes. Pathogenic C. burnetii has evolved LPS variations to evade this host response and thrive intracellularly.

Pathogenic rickettsiae of the spotted fever group (SFG) induce skin lesions called "eschars" at the sites of arthropod bites. We recently described a guinea pig model based on eschar formation to predict the eventual pathogenicity of new Rickettsia species, and in this study, we used the model to study cross-protection among SFG Rickettsia species and subspecies. We showed that the intradermal inoculation of guinea pigs with Rickettsia conorii subsp. conorii significantly reduced, but not completely prevented, the number of eschars after sub-challenges with the same pathogen (P=0.0004). The same effect was also observed for other subspecies of the R. conorii complex. Additionally, the bacterial load in the eschars was significantly lower in immunized animals than in naïve animals. No protection was observed when sub-challenges were performed with other Rickettsia species, such as R. africae, R. sibirica subsp. mongolitimonae, R. aeschlimannii and R. massiliae. Our data suggest that patients may experience several episodes of infection with related or with the same species of Rickettsia. Moreover, the absence of cross-protection between Rickettsia species may explain the co-existence of two or more tick-borne rickettsioses in the same geographic areas.

International audience; Bacteria can induce human lymphomas, whereas lymphoproliferative disorders have been described in patients with Q fever. We observed a lymphoma in a patient with Q fever that prompted us to investigate the association between the 2 diseases. We screened 1468 consecutive patients of the 2004 to 2014 French National Referral Center for Q fever database. The standardized incidence ratios (SIRs) of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) were calculated comparatively to the 2012 Francim Registry. The presence of Coxiella burnetii was tested using immunofluorescence and fluorescence in situ hybridization using a specific 16S ribosomal RNA probe and genomic DNA probe. Seven patients (0.48%) presented mature B-cell lymphoma consisting of 6 DLBCL and 1 FL. An excess risk of DLBCL and FL was found in Q fever patients compared with the general population (SIR [95% confidence interval], 25.4 [11.4-56.4] and 6.7 [0.9-47.9], respectively). C burnetii was detected in CD68(+) macrophages within both lymphoma and lymphadenitis tissues but localization in CD123(+) plasmacytoid dendritic cells (pDCs) was found only in lymphoma tissues. Q fever patients with persistent focalized infection were found more at risk of lymphoma (hazard ratio, 9.35 [1.10-79.4]). Interleukin-10 (IL10) overproduction (P = .0003) was found in patients developing lymphoma. These results suggest that C burnetii should be added to the list of bacteria that promote human B-cell non-Hodgkin lymphoma, possibly by the infection of pDCs and IL10 overproduction. Screening for early lymphoma diagnosis should be considered in the management of patients with Q fever, especially those with persistent focalized infections.

Whole-cell MALDI-TOF MS is routinely used to identify bacterial species in clinical samples. This technique has also proven to allow identification of intact mammalian cells, including macrophages. Here, we wondered whether this approach enabled the assessment human macrophages plasticity. The whole-cell MALDI-TOF spectra of macrophages stimulated with IFN-γ and IL-4, two inducers of M1 and M2 macrophage polarisation, consisted of peaks ranging from 2 to 12 kDa. The spectra of unstimulated and stimulated macrophages were clearly different. The fingerprints induced by the M1 agonists, IFN-γ, TNF, LPS and LPS+IFN-γ, and the M2 agonists, IL-4, TGF-β1 and IL-10, were specific and readily identifiable. Thus, whole-cell MALDI-TOF MS was able to characterise M1 and M2 macrophage subtypes. In addition, the fingerprints induced by extracellular (group B Streptococcus, Staphylococcus aureus) or intracellular (BCG, Orientia tsutsugamushi, Coxiella burnetii) bacteria were bacterium-specific. The whole-cell MALDI-TOF MS fingerprints therefore revealed the multifaceted activation of human macrophages. This approach opened a new avenue of studies to assess the immune response in the clinical setting, by monitoring the various activation patterns of immune cells in pathological conditions.

Background. The outcome of Q fever, an infectious disease caused by Coxiella burnetii, is associated with granuloma formation. Granulomas are present in patients with resolutive Q fever but are lacking in patients with chronic Q fever. Methods. Study of granuloma formation requires invasive approaches. Here, we took advantage of a recently described method that enables in vitro generation of human granulomas specific for C. burnetii. Results. Circulating mononuclear cells progressively accumulated around beads coated with C. burnetii extracts, and complete granulomas were generated in 8 days. Granuloma cells consisted of macrophages, lymphocytes, and, to a lesser extent, epithelioid cells and multinucleated giant cells. Early events that govern granuloma formation were studied using live-imaging microscopy. Monocytes migrated toward C. burnetii‐coated beads independently of the presence of T lymphocytes and then recruited T lymphocytes. About 90% of patients with chronic Q fever failed to form granulomas. This deficiency was associated with defective migration of monocytes toward coated beads. Conclusions. Monocytes were involved in the early stages of granuloma formation and recruited T lymphocytes to complete granuloma formation. This article describes a direct relationship between defective granuloma formation and defective migration of monocytes. Q fever is a zoonosis caused by Coxiella burnetii, an obligate intracellular bacterium that inhabits myeloid cells. The natural history of the disease reveals that the primary infection is symptomatic in a minority of exposed individuals. The acute form of Q fever usually results in cure and is associated with protective immune responses and granuloma formation. Q fever may become chronic in patients with valvular damage, in immunocompromised patients with lymphomas, and in pregnant women. In contrast to acute Q fever, chronic Q fever is characterized by defective cell-mediated immunity and granuloma formation [1, 2], suggesting that the formation of granulomas plays a major role in the

Intracellular pathogens have developed different strategies to survive within host cells. For example, these pathogens might interfere with the biogenesis of phagolysosomes, thereby forming replicative vacuoles. Although the complex mechanisms used by pathogens to hijack the biogenesis of phagolysosomes have been elucidated in naive leukocytes, the role of leukocyte activation in this process has not yet been investigated. Leukocytes are known to be activated by cytokines, and several reports have demonstrated that several cytokines modulate the endocytic pathway and thereby, affect phagosome biogenesis. These observations provide molecular evidence that endocytosis can be regulated by the immune environment. In this review, we highlight the effect of leukocyte activation by cytokines on the endocytic pathway and on phagosome biogenesis. We briefly describe the mechanism of phagolysosome formation before focusing on the strategies used by two bacterial pathogens, Coxiella burnetii and Mycobacterium tuberculosis, to hijack phagolysosome biogenesis. Finally, we emphasize the effect of leukocyte activation on the endocytic pathway and on phagolysosome formation, which has not been highlighted to date.

Rickettsia prowazekii, the agent of epidemic typhus, is an obligate intracellular bacterium that is transmitted to human beings by the body louse. Several strains that differ considerably in virulence are recognized, but the genetic basis for these variations has remained unknown since the initial description of the avirulent vaccine strain nearly 70 yr ago. We use a recently developed murine model of epidemic typhus and transcriptomic, proteomic, and genetic techniques to identify the factors associated with virulence. We identified four phenotypes ofR. prowazekiithat differed in virulence, associated with the up-regulation of antiapoptotic genes or the interferon I pathway in the host cells. Transcriptional and proteomic analyses ofR. prowazekiisurface protein expression and protein methylation varied with virulence. By sequencing a virulent strain and using comparative genomics, we found hotspots of mutations in homopolymeric tracts of poly(A) and poly(T) in eight genes in an avirulent strain that split and inactivated these genes. These includedrecO, putative methyltransferase, and exported protein. Passage of the avirulent Madrid E strain in cells or in experimental animals was associated with a cascade of gene reactivations, beginning withrecO, that restored the virulent phenotype. An area of genomic plasticity appears to determine virulence inR. prowazekiiand represents an example of adaptive mutation for this pathogen.

Converging studies show that M1 and M2 macrophages are functionally polarized in response to host mediators. Gene expression profiling of macrophages reveals that various bacteria induce the transcriptional activity of a common host response that includes genes belonging to the M1 program. The microbicidal machinery of M1 macrophages allows them to participate to the clearing of acute infections. However, excessive or prolonged M1 polarization can lead to tissue injury and contribute to pathogenesis. The so-called M2 macrophages play a critical role in the resolution of inflammation by producing anti-inflammatory mediators. These M2 macrophages cover a continuum of cells with different phenotypic and functional properties. Different bacterial pathogens escape from clearing by manipulating functions of M1 macrophages. It has recently been demonstrated that specific M2 programs induced in macrophages by bacterial pathogens are associated with the chronic evolution of infectious diseases.

Coxiella burnetii is an intracellular bacterial pathogen that causes Q fever, with symptoms ranging from self-limited febrile illness, pneumonia, hepatitis, meningitis, encephalitis, pericarditis, myocarditis, endocarditis, to osteomyelitis. This chapter provides an update on C. burnetii classification, epidemiology, identification, genetics, immunity, treatment and prevention.

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 Escherichia coli LPS or with the 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 activating macrophages by the disruption of the TLR-2 and TLR-4 association.

Q fever is an infectious disease caused byCoxiella burnetii, an obligate intracellular bacterium that replicates in macrophages. As cell-mediated immune response to microbial pathogens requires signals mediated by T-cell receptors and costimulatory molecules such as CD28, we wondered if CD28 is involved in protection againstC. burnetiiinfection. CD28-deficient (CD28−/−) mice were inoculated withC. burnetiiby intraperitoneal and intravenous routes. With both wild-type and CD28−/−mice,C. burnetiiorganisms were detected exclusively in spleen and liver. The antibody response againstC. burnetiiwas impaired in CD28−/−animals, but, surprisingly, the lack of CD28 decreasedC. burnetiiburden in the infected tissues, whatever the manner of inoculation of bacteria. The CD28 deficiency had no effect on either granuloma formation, which reflects cell-mediated immunity againstC. burnetii, or the production of gamma interferon and tumor necrosis factor, two cytokines known to be involved in granuloma formation. On the other hand, the production of interleukin-10 (IL-10) by peritoneal macrophages was highly impaired in CD28−/−mice. The results suggest that CD28 initiates a signal that favorsC. burnetiireplication through the modulation of the IL-10 pathway.

Background. Bartonella quintana, the etiological agent of bacillary angiomatosis (BA), causes endothelial cell proliferation. Erythromycin has dramatic effects on BA, and the effects are largely unexplained by the compound's bacteriostatic properties. Our aim here was to evaluate the possibility that erythromycin alters angiogenesis. Methods. The effect of erythromycin on B. quintana-induced endothelial cell proliferation was studied using a wild-type strain and an erythromycin-resistant B. quintana mutant. The latter was generated by serial subcultures on blood agar plates. Results. We show that erythromycin significantly inhibits the proliferation of dermal microvascular endothelial cells induced either by wild-type B. quintana or by our erythromycin-resistant mutant. Doxycycline and gentamycin failed to exert such an effect. Finally, we found that the resistant strain harbored a 27-bp insertion in the highly conserved region of the gene encoding the ribosomal protein L4; this insertion may explain the existence of the resistance to erythromycin. Conclusion. The data presented here indicate that erythromycin profoundly down-modulates endothelial cell proliferation irrespective of its bacteriostatic effects and suggest that this may be a key component of the efficacy of the compound in the treatment of patients with BA.

Whipple’s disease (WD) is a rare systemic disease caused by Tropheryma whipplei. We showed that T. whipplei was eliminated by human monocytes but replicated in monocyte-derived macrophages (Mφ) by inducing an original activation program. Two different host molecules were found to be key elements for this specific pattern. Thioredoxin, through its overexpression in infected monocytes, was involved in bacterial killing because adding thioredoxin to infected Mφ inhibited bacterial replication. IL-16, which was up-regulated in Mφ, enabled T. whipplei to replicate in monocytes and increased bacterial replication in Mφ. In addition, anti-IL-16 Abs abolished T. whipplei replication in Mφ. IL-16 down-modulated the expression of thioredoxin and up-regulated that of IL-16 and proapoptotic genes. In patients with WD, T. whipplei replication was higher than in healthy subjects and was related to high levels of circulating IL-16. Both events were corrected in patients who successfully responded to antibiotics treatment. This role of IL-16 was not reported previously and gives an insight into the understanding of WD pathophysiology.

Parachlamydia acanthamoebae is an obligate intracellular bacterium that naturally infects free-living amoebae. It is a potential agent of pneumonia that resists destruction by human macrophages. However, the strategy used by this Chlamydia-like organism in order to resist to macrophage destruction is unknown. We analysed the intracellular trafficking of P. acanthamoebae within monocyte-derived macrophages. Infected cells were immunolabelled for the bacteria and for various intracellular compartments by using specific antibodies. We analysed the bacteria colocalization with the different subcellular compartments by using epifluorescence and confocal microscopy. Bacterial replication took place 4-6 h post infection within acidic vacuoles. At that time, P. acanthamoebae colocalized with Lamp-1, a membrane marker of late endosomal and lysosomal compartments. A transient accumulation of EEA1 15 min post infection, and of rab7 and the mannose 6-phosphate receptor 30 min post infection confirmed that P. acanthamoebae traffic through the endocytic pathway. The acquisition of Lamp-1 was not different after infection with living and heat-inactivated bacteria. However, 24.5% and 79.5% of living and heat-inactivated P. acanthamoebae, respectively, colocalized with the vacuolar proton ATPase. Moreover, P. acanthamoebae did not colocalized with cathepsin D, a lysosomal hydrolase, suggesting that P. acanthamoebae interferes with maturation of its vacuole. Thus, P. acanthamoebae survives to destruction by human macrophages probably by controlling the vacuole biogenesis.

Q fever is caused by Coxiella burnetii, a bacterium that survives in monocytes/macrophages by resisting their natural microbicidal activity. Because the link between bacterial killing and phagosome maturation has yet to be demonstrated, we evaluated responses in monocytes from both immunologically naive control subjects and patients with various manifestations of Q fever. Monocytes from patients with chronic Q fever in evolution, who do not control the infection, exhibited defective phagosome maturation and impaired C. burnetii killing. Both responses were stimulated in patients recovering from Q fever. Phagosome maturation and C. burnetii killing were significantly correlated. Defective phagosome maturation and impaired C. burnetii killing were induced by adding interleukin (IL)-10 to monocytes from convalescent patients and were restored by IL-10 neutralization in chronic Q fever in evolution. We show that phagosome maturation and microbial killing are linked in Q fever and that IL-10 regulates both features of microbicidal activity.

Parachlamydia acanthamoebais an obligately intracellular bacterium that naturally infects free-living amoebae. It is a potential human pathogen and may survive in human macrophages. We studiedP. acanthamoebaentry into, and multiplication within, human monocyte-derived macrophages. After 8 h of incubation, 80% of macrophages were infected with a mean of 3.8P. acanthamoebaorganisms per cell. Electron microscopy demonstrated that parachlamydiae were in an intracellular vacuole. After infection with living organisms, the number of parachlamydiae per macrophage increased 4 times from day 0 to day 4, whereas heat-inactivated parachlamydiae were eliminated during the same period. Quantitative PCR confirmed thatP. acanthamoebareplicates within macrophages. Transcriptional activity ofP. acanthamoebawas detected by reverse transcription-PCR targeting the gene encoding ADP-ATP translocase (tlc).P. acanthamoebaexerted a cytopathic effect on macrophages. When macrophages were infected with living bacteria, their number decreased significantly from day 0 to day 4 due to apoptosis, as shown by annexin-V binding and electron microscopy. This study shows thatP. acanthamoebaenters and multiplies within human macrophages before inducing their apoptosis.

Whipple's disease may be diagnosed by periodic acid-Schiff (PAS) staining, electron microscopy, or polymerase chain reaction of intestinal biopsy specimens. The aim of this study was to evaluate the diagnostic value of immunohistochemistry and the quantification of infected cells in intestinal Whipple's disease. A total of 29 duodenal biopsy specimens from 15 patients with untreated and treated Whipple's disease were examined and compared with biopsy specimens from control patients with normal intestinal mucosa or various pathologic processes. Percentages of staining surfaces with PAS stain and antibodies directed against CD68, a macrophage marker, or the Whipple bacillus, Tropheryma whipplei, were studied quantitatively using a computerized system of image analysis. Positive detection of T. whipplei was obtained using immunohistochemistry in all 15 patients with Whipple's disease. No bacteria were detected in any of the negative controls. The use of quantitative image analysis showed a massive intestinal macrophagic infiltration before (20.3%) and after (13.4%) antibiotic therapy completion as compared with controls (2.1%). The 2 detection methods for T. whipplei, PAS stain and immunohistochemistry, were quantitatively similar before therapy (19.9% versus 17.5%), but the immunodetection-based surface area was significantly lower than the PAS staining surface area after therapy (2.8% versus 7.9%). Our findings indicate that immunohistochemistry is highly specific and sensitive and is applicable as a diagnostic method on intestinal tissue specimens to detect T. whipplei during active infection or in retrospective studies.

Phagocytosis is a highly localized event requiring the formation of spatially and temporally restricted signals. Numerous microorganisms have taken advantage of this property to invade host cells. Coxiella burnetii, the agent of Q fever, is an obligate intracellular bacterium that has developed a survival strategy in macrophages based on subversion of receptor-mediated phagocytosis. The uptake of C. burnetii is mediated by αvβ3 integrin and is restricted by impaired cross-talk of αvβ3 integrin and complement receptor 3 (CR3) (CD11b/CD18). In this study, we showed that CR3 molecules remained outside the pseudopodal extensions induced by C. burnetii in THP-1 monocytes, although αvβ3 integrin was present in the pseudopods. Chemoattractants such as RANTES restored CR3 localization to the front of pseudopodal extensions and increased C. burnetii phagocytosis, demonstrating that the localization of CR3 is critical for bacterial uptake. In addition, monocyte activation due to the expression of HIV-1 Nef protein also restored CR3-mediated phagocytosis of C. burnetii by allowing CR3 redistribution toward bacterial-induced pseudopods. The redistribution of CR3 and increased C. burnetii phagocytosis in THP-1 cells stimulated by RANTES or expressing Nef were associated with the inhibition of intracellular replication of C. burnetii. Hence, the localization of CR3 is critical for bacterial phagocytosis and also for the control of bacterial replication. This study describes a nonpreviously reported strategy of phagocytosis subversion by intracellular pathogens based on altered localization of monocyte receptors.

The prevalence of Q fever is higher in men than in women. Because the expression of circadian clock genes differs in male and female mice infected with Coxiella burnetii, we hypothesized that circadian genes are differently modulated in men and women with Q fever. The expression of the Per2 gene was significantly (P = .01) increased in males with acute Q fever compared with healthy volunteers. No significant difference was observed in females. We showed for the first time that gender altered the expression of a circadian gene, Per2, in an infectious disease.

Q fever is a disease caused by Coxiella burnetii, an obligate intracellular bacterium. Acute Q fever is spontaneously resolutive and is characterized by an efficient immune response. In contrast, chronic Q fever is characterized by dysregulated immune response, as demonstrated by the failure of C. burnetii to induce lymphoproliferation and the lack of granulomas. Recently, it has been demonstrated that when co-expressed in heterologous mammalian cell lines, the ligands of Numb proteins X1 and X2 (LNX1 and LNX2) regulate the level of the T-cell co-receptor CD8, which plays an essential role in T-cell-mediated immune response. We decided to investigate the expression of LNX1 and LNX2 genes in patients with acute or chronic Q fever. Interestingly, we found a high level of LNX1 and LNX2 mRNAs in endocarditis, the principal manifestation of chronic Q fever, but not in acute Q fever. Our data suggest that LNXs may be used as complementary biomarkers to follow the prognosis of chronic Q fever.

Chronic Q fever, which principally manifests as endocarditis, is characterized by Coxiella burnetii persistence and an impaired cell-mediated immune response. The long-term persistence of pathogens has been associated with the expansion of regulatory T cells (Tregs), the CD4+T-cell subset that is characterized by the expression of CD25 and Foxp3. We investigated the presence of Tregs in patients with acute Q fever (n = 17), known to exhibit an efficient immune response, patients with Q fever endocarditis (n = 54) and controls (n = 27) by flow cytometry. The proportion of CD3+, CD4+ and CD8+T cells was similar in controls and patients with Q fever. The percentage of CD4+T cells that expressed CD25 was similar in controls and patients with Q fever. The population of CD4+T cells that expressed both CD25 and Foxp3 was significantly (P

Q fever is a disease caused by Coxiella burnetii, an obligate intracellular bacterium. Acute Q fever is characterized by efficient immune response, whereas chronic Q fever is characterized by dysregulated immune response as demonstrated by the lack of granulomas, the failure of C. burnetii to induce lymphoproliferation, and interferon-γ production. The mitogen-activated protein kinase (MAPK) signaling pathway plays crucial roles in innate immune responses and control of bacterial infections. However, its role in Q fever has not been addressed. First, we investigated the activation of MAPKs p38, c-jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 in murine macrophages stimulated with C. burnetii. Coxiella burnetii NM phase I (virulent) and NM phase II (avirulent) induced the activation of JNK and ERK1/2. Avirulent C. burnetii activate p38, whereas C. burnetii did not induce the phosphorylation of p38. Second, the level of p38 activation was studied in Q fever patients. We found that p38 was activated in monocyte-derived macrophages from healthy donors and patients with acute Q fever in response to a potent agonist such as lipopolysaccharide. Interestingly, p38 was not activated in patients with active chronic Q fever and was activated in patients with cured chronic Q fever. These results suggest that the determination of p38 activation may serve as a tool for measuring Q fever activity.

Brucella is a Gram-negative bacterium responsible for brucellosis, a worldwide re-emerging zoonosis. Brucella has been shown to infect and replicate within Granulocyte macrophage colony-stimulating factor (GMCSF) in vitro grown bone marrow-derived dendritic cells (BMDC). In this cell model, Brucella can efficiently control BMDC maturation. However, it has been shown that Brucella infection in vivo induces spleen dendritic cells (DC) migration and maturation. As DCs form a complex network composed by several subpopulations, differences observed may be due to different interactions between Brucella and DC subsets. Here, we compare Brucella interaction with several in vitro BMDC models. The present study shows that Brucella is capable of replicating in all the BMDC models tested with a high infection rate at early time points in GMCSF-IL15 DCs and Flt3l DCs. GMCSF-IL15 DCs and Flt3l DCs are more activated than the other studied DC models and consequently intracellular bacteria are not efficiently targeted to the ER replicative niche. Interestingly, GMCSF-DC and GMCSF-Flt3l DC response to infection is comparable. However, the key difference between these 2 models concerns IL10 secretion by GMCSF DCs observed at 48 h post-infection. IL10 secretion can explain the weak secretion of IL12p70 and TNFα in the GMCSF-DC model and the low level of maturation observed when compared to GMCSF-IL15 DCs and Flt3l DCs. These models provide good tools to understand how Brucella induce DC maturation in vivo and may lead to new therapeutic design using DCs as cellular vaccines capable of enhancing immune response against pathogens.

The subversion of microbicidal functions of macrophages by intracellular pathogens is critical for their survival and pathogenicity. The replication of Coxiella burnetii, the agent of Q fever, in acidic phagolysosomes of nonphagocytic cells has been considered as a paradigm of intracellular life of bacteria. We show in this study that C. burnetii survival in THP-1 monocytes was not related to phagosomal pH because bacterial vacuoles were acidic independently of C. burnetii virulence. In contrast, virulent C. burnetii escapes killing in resting THP-1 cells by preventing phagosome maturation. Indeed, C. burnetii vacuoles did not fuse with lysosomes because they were devoid of cathepsin D, and did not accumulate lysosomal trackers; the acquisition of markers of late endosomes and late endosomes-early lysosomes was conserved. In contrast, avirulent variants of C. burnetii were eliminated by monocytes and their vacuoles accumulated late endosomal and lysosomal markers. The fate of virulent C. burnetii in THP-1 monocytes depends on cell activation. Monocyte activation by IFN-γ restored C. burnetii killing and phagosome maturation as assessed by colocalization of C. burnetii with active cathepsin D. In addition, when IFN-γ was added before cell infection, it was able to stimulate C. burnetii killing but it also induced vacuolar alkalinization. These findings suggest that IFN-γ mediates C. burnetii killing via two distinct mechanisms, phagosome maturation, and phagosome alkalinization. Thus, the tuning of vacuole biogenesis is likely a key part of C. burnetii survival and the pathophysiology of Q fever.

Tropheryma whippleiwas established as the agent of Whipple's disease in 2000, but the mechanisms by which it survives within host cells are still unknown. We show here thatT. whippleisurvives within HeLa cells by controlling the biogenesis of its phagosome. Indeed,T. whippleicolocalized with lysosome-associated membrane protein 1, a membrane marker of late endosomal and lysosomal compartments, but not with cathepsin D, a lysosomal hydrolase. This defect in phagosome maturation is specific to live organisms, since heat-killed bacilli colocalized with cathepsin D. In addition,T. whippleisurvived within HeLa cells by adapting to acidic pH. The vacuoles containingT. whippleiwere acidic (pH 4.7 ± 0.3) and acquired vacuolar ATPase, responsible for the acidic pH of late phagosomes. The treatment of HeLa cells with pH-neutralizing reagents, such as ammonium chloride,N-ethylmaleimide, bafilomycin A1, and chloroquine, increased the intravacuolar pH and promoted the killing ofT. whipplei. The ability ofT. whippleito survive in an acidic environment and to interfere with phagosome-lysosome fusion is likely critical for its prolonged persistence in host cells during the course of Whipple's disease. Our results suggest that manipulating the intravacuolar pH may provide a new approach for the treatment of Whipple's disease.

Programmed death ligand-1 (PD-L1) is a co-signaling molecule that regulates T-cell responses in vivo. Its role in bacterial infections, including Q fever, a zoonosis due to Coxiella burnetii infection, is not well understood. We showed by flow cytometry that PD-L1 membrane expression was specifically increased in T-cells from patients with acute Q fever, not from patients with Q fever endocarditis, suggesting that PD-L1 plays a role in the early phases of C. burnetii infection. To assess this hypothesis, we studied the role of PD-L1 in C. burnetii-infected mice. C. burnetii infection resulted in PD-L1 up-regulation in splenocytes. Anti-PD-L1 antibodies injected into the mice did not affect the total number of splenic T-cells but increased the relative number of CD4+ T-cells compared with CD8+ T-cells. Additionally, anti-PD-L1 antibodies significantly increased the number of splenic CD4+ and CD8+ T cells that expressed low membrane CD62L levels. Our results indicate that the increased expression of PD-L1 by T-cells is associated with a decreased number of memory T-cells during C. burnetii infection, opening new perspectives in the understanding of Q fever pathophysiology.

International audience; Dugesia japonica planarian flatworms are naturally exposed to various microbes but typically survive this challenge. We show that planarians eliminate bacteria pathogenic to Homo sapiens, Caenorhabditis elegans, and/or Drosophila melanogaster and thus represent a model to identify innate resistance mechanisms. Whole-transcriptome analysis coupled with RNAi screening of worms infected with Staphylococcus aureus or Legionella pneumophila identified 18 resistance genes with nine human orthologs, of which we examined the function of MORN2. Human MORN2 facilitates phagocytosis-mediated restriction of Mycobacterium tuberculosis, L. pneumophila, and S. aureus in macrophages. MORN2 promotes the recruitment of LC3, an autophagy protein also involved in phagocytosis, to M. tuberculosis-containing phagosomes and subsequent maturation to degradative phagolysosomes. MORN2-driven trafficking of M. tuberculosis to single-membrane, LC3-positive compartments requires autophagy-related proteins Atg5 and Beclin-1, but not Ulk-1 and Atg13, highlighting the importance of MORN2 in LC3-associated phagocytosis. These findings underscore the value of studying planarian defenses to identify immune factors.

Dendritic cells (DCs) orchestrate host defenses against microorganisms. In infectious diseases due to intracellular bacteria, the inefficiency of the immune system to eradicate microorganisms has been attributed to the hijacking of DC functions. In this study, we selected intracellular bacterial pathogens with distinct lifestyles and explored the responses of monocyte-derived DCs (moDCs). Using lipopolysaccharide as a control, we found that Orientia tsutsugamushi, the causative agent of scrub typhus that survives in the cytosol of target cells, induced moDC maturation, as assessed by decreased endocytosis activity, the ability to induce lymphocyte proliferation and the membrane expression of phenotypic markers. In contrast, Coxiella burnetii, the agent of Q fever, and Brucella abortus, the agent of brucellosis, both of which reside in vacuolar compartments, only partly induced the maturation of moDCs, as demonstrated by a phenotypic analysis. To analyze the mechanisms used by C. burnetii and B. abortus to alter moDC activation, we performed microarray and found that C. burnetii and B. abortus induced a specific signature consisting of TLR4, TLR3, STAT1 and interferon response genes. These genes were down-modulated in response to C. burnetii and B. abortus but up-modulated in moDCs activated by lipopolysaccharide and O. tsutsugamushi. This transcriptional alteration was associated with the defective interferon-β production. This study demonstrates that intracellular bacteria specifically affect moDC responses and emphasizes how C. burnetii and B. abortus interfere with moDC activation and the antimicrobial immune response. We believe that comparing infection by several bacterial species may be useful for defining new pathways and biomarkers and for developing new treatment strategies.

Expressing mspA porin gene from Mycobacterium smegmatis in Mycobacterium tuberculosis attenuated this pathogen. Intracellular growth of the transformants into free-living amoeba and murine and human macrophages decreased. Furthermore, transformants decreased the microbicidal program of human monocyte-derived macrophages. BALB/c mice inoculated with transformants exhibited higher weights, lower histological lesions and lower M. tuberculosis inoculum in the liver, spleen and lungs than control mice challenged with wild-type M. tuberculosis. Preliminary evaluation indicated that mice inoculated with this transformant showed higher weights and lower numbers of lung nodules and tissular mycobacteria than control mice when challenged with wild-type M. tuberculosis. Similar to the paradoxical “unbirthday” gift coined by Lewis Carroll in Alice's Adventures in Wonderland, adding mspA gene reduced the virulence of M. tuberculosis and yielded a protective effect. Lost of non-virulence genes is a mechanism for virulence in mycobacteria. Engineering non-virulence genes in M. tuberculosis may yield strains with decreased virulence and increased immunogenicity. © 2014 Published by Elsevier Ltd.

Tropheryma whipplei, the agent of Whipple's disease, inhibits phago-lysosome biogenesis to create a suitable niche for its survival and replication in macrophages. To understand the mechanism by which it subverts phagosome maturation, we used biochemical and cell biological approaches to purify and characterise the intracellular compartment where Tropheryma whipplei resides using mouse bone-marrow-derived macrophages. We showed that in addition to Lamp-1, the Tropheryma whipplei phagosome is positive for Rab5 and Rab7, two GTPases required for the early to late phagosome transition. Unlike other pathogens, inhibition of PI(3)P production was not the mechanism for Rab5 stabilisation at the phagosome. Overexpression of the inactive, GDP-bound form of Rab5 bypassed the pathogen-induced blockade of phago-lysosome biogenesis. This suggests that Tropheryma whipplei blocks the switch from Rab5 to Rab7 by acting on the Rab5 GTPase cycle. A bio-informatic analysis of the Tropheryma whipplei genome revealed a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) homologous with the GAPDH of Listeria monocytogenes, and this may be the bacterial protein responsible for blocking Rab5 activity. To our knowledge, Tropheryma whipplei is the first pathogen described to induce a "chimeric" phagosome stably expressing both Rab5 and Rab7, suggesting a novel and specific mechanism for subverting phagosome maturation.

Coxiella burnetii, the agent of Q fever, is an obligate intracellular microorganism that grows in monocytes/macrophages. The internalization of virulent organisms by monocytes is lower than that of avirulent variants and is associated with actin cytoskeleton reorganization. We studied the activation of protein tyrosine kinases (PTKs) byC. burnetiiin THP-1 monocytes. Virulent organisms induced early PTK activation and the tyrosine phosphorylation of several endogenous substrates, including Hck and Lyn, two Src-related kinases. PTK activation reflectsC. burnetiivirulence since avirulent variants were unable to stimulate PTK. We also investigated the role of PTK activation inC. burnetii-stimulated F-actin reorganization. Tyrosine-phosphorylated proteins were colocalized with F-actin inside cell protrusions induced byC. burnetii, and PTK activity was increased in Triton X-100-insoluble fractions. In addition, lavendustin A, a PTK inhibitor, and PP1, a Src kinase inhibitor, preventedC. burnetii-induced cell protrusions and F-actin reorganization. We finally assessed the role of PTK activation in bacterial phagocytosis. Pretreatment of THP-1 cells with lavendustin A and PP1 upregulated the uptake of virulentC. burnetiibut had no effect on the phagocytosis of avirulent organisms. Thus, it is likely that PTK activation byC. burnetiinegatively regulates bacterial uptake by interfering with cytoskeleton organization.