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1.
Article in English | MEDLINE | ID: mdl-31024862

ABSTRACT

Despite their high degree of genomic similarity, different spotted fever group (SFG) Rickettsia are often associated with very different clinical presentations. For example, Rickettsia conorii causes Mediterranean spotted fever, a life-threatening disease for humans, whereas Rickettsia montanensis is associated with limited or no pathogenicity to humans. However, the molecular basis responsible for the different pathogenicity attributes are still not understood. Although killing microbes is a critical function of macrophages, the ability to survive and/or proliferate within phagocytic cells seems to be a phenotypic feature of several intracellular pathogens. We have previously shown that R. conorii and R. montanensis exhibit different intracellular fates within macrophage-like cells. By evaluating early macrophage responses upon insult with each of these rickettsial species, herein we demonstrate that infection with R. conorii results in a profound reprogramming of host gene expression profiles. Transcriptional programs generated upon infection with this pathogenic bacteria point toward a sophisticated ability to evade innate immune signals, by modulating the expression of several anti-inflammatory molecules. Moreover, R. conorii induce the expression of several pro-survival genes, which may result in the ability to prolong host cell survival, thus protecting its replicative niche. Remarkably, R. conorii-infection promoted a robust modulation of different transcription factors, suggesting that an early manipulation of the host gene expression machinery may be key to R. conorii proliferation in THP-1 macrophages. This work provides new insights into the early molecular processes hijacked by a pathogenic SFG Rickettsia to establish a replicative niche in macrophages, opening several avenues of research in host-rickettsiae interactions.


Subject(s)
Gene Expression Regulation , Host-Pathogen Interactions , Macrophages/immunology , Macrophages/microbiology , Rickettsia conorii/growth & development , Rickettsia/growth & development , Gene Expression Profiling , Humans , Immune Evasion , Microbial Viability , Rickettsia/immunology , Rickettsia conorii/immunology , THP-1 Cells
3.
Lab Chip ; 16(9): 1625-35, 2016 04 26.
Article in English | MEDLINE | ID: mdl-27025227

ABSTRACT

We present a microfluidic immunoassay platform based on the use of linear microretroreflectors embedded in a transparent polymer layer as an optical sensing surface, and micron-sized magnetic particles as light-blocking labels. Retroreflectors return light directly to its source and are highly detectable using inexpensive optics. The analyte is immuno-magnetically pre-concentrated from a sample and then captured on an antibody-modified microfluidic substrate comprised of embedded microretroreflectors, thereby blocking reflected light. Fluidic force discrimination is used to increase specificity of the assay, following which a difference imaging algorithm that can see single 3 µm magnetic particles without optical calibration is used to detect and quantify signal intensity from each sub-array of retroreflectors. We demonstrate the utility of embedded microretroreflectors as a new sensing modality through a proof-of-concept immunoassay for a small, obligate intracellular bacterial pathogen, Rickettsia conorii, the causative agent of Mediterranean Spotted Fever. The combination of large sensing area, optimized surface chemistry and microfluidic protocols, automated image capture and analysis, and high sensitivity of the difference imaging results in a sensitive immunoassay with a limit of detection of roughly 4000 R. conorii per mL.


Subject(s)
Immunoassay/instrumentation , Lab-On-A-Chip Devices , Rickettsia conorii/isolation & purification , Animals , Antibodies, Immobilized/metabolism , Automation, Laboratory , Cells, Immobilized , Computer-Aided Design , Equipment Design , Image Processing, Computer-Assisted , Immunoassay/methods , Immunomagnetic Separation , Limit of Detection , Magnetic Phenomena , Microscopy , Microscopy, Electron, Scanning , Microspheres , Microtechnology/methods , Polymethyl Methacrylate/chemistry , Proof of Concept Study , Reproducibility of Results , Rickettsia conorii/growth & development , Rickettsia conorii/immunology , Surface Properties
5.
Infect Immun ; 79(9): 3733-43, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21690236

ABSTRACT

Infection of the endothelial cell lining of blood vessels with Rickettsia conorii, the causative agent of Mediterranean spotted fever, results in endothelial activation. We investigated the effects of R. conorii infection on the status of the Janus kinase (JAK)-signal transducer and activator of transcription protein (STAT) signaling pathway in human microvascular endothelial cells (HMECs), the most relevant host cell type, in light of rickettsial tropism for microvascular endothelium in vivo. R. conorii infection induced phosphorylation of STAT1 on tyrosine 701 and serine 727 at 24, 48, and 72 h postinfection in HMECs. Employing transcription profile analysis and neutralizing antibodies, we further determined that beta interferon (IFN-ß) production and secretion are critical for STAT1 activation. Secreted IFN-ß further amplified its own expression via a positive-feedback mechanism, while expression of transcription factors interferon regulatory factor 7 (IRF7) and IRF9, implicated in the IFN-ß-STAT1 feedback loop, was also induced. Metabolic activity of rickettsiae was essential for the IFN-ß-mediated response(s) because tetracycline treatment inhibited R. conorii replication, IFN-ß expression, and STAT1 phosphorylation. Inclusion of IFN-ß-neutralizing antibody during infection resulted in significantly enhanced R. conorii replication, whereas addition of exogenous IFN-ß had the opposite inhibitory effect. Finally, small interfering RNA-mediated knockdown further confirmed a protective role for STAT1 against intracellular R. conorii replication. In concert, these findings implicate an important role for IFN-ß-mediated STAT1 activation in innate immune responses of vascular endothelium to R. conorii infection.


Subject(s)
Blood Vessels/microbiology , Endothelial Cells/microbiology , Interferon-beta/metabolism , Microvessels/microbiology , Rickettsia conorii/growth & development , Rickettsia conorii/metabolism , STAT1 Transcription Factor/metabolism , Antibodies, Monoclonal , Cell Line , Humans , Interferon Regulatory Factor-7/biosynthesis , Interferon-Stimulated Gene Factor 3, gamma Subunit/biosynthesis , Interferon-beta/biosynthesis , Interferon-beta/immunology , Janus Kinases/metabolism , Phosphorylation , RNA Interference , RNA, Small Interfering , STAT1 Transcription Factor/biosynthesis , Signal Transduction , Tetracycline/pharmacology
7.
Int J Antimicrob Agents ; 32(4): 344-8, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18703317

ABSTRACT

Recent data suggest that statins may have a beneficial effect during sepsis. In this study, we tested the effect of lovastatin and pravastatin on the cellular culture of Rickettsia conorii using a quantitative plaque assay model associated with an original image analysis algorithm. Statins added at the time of infection did not modify plaque formation, whereas pre-incubation with statins for 48h resulted in a significant 30-68% plaque reduction, depending on the tested compounds and doses. These preliminary findings raise the hypothesis that statins may prevent or moderate rickettsial disease in exposed people.


Subject(s)
Anticholesteremic Agents/pharmacology , Lovastatin/pharmacology , Pravastatin/pharmacology , Rickettsia conorii/drug effects , Algorithms , Animals , Culture Media , Image Processing, Computer-Assisted , L Cells , Mice , Microbial Sensitivity Tests/methods , Rickettsia conorii/growth & development
8.
Res Microbiol ; 156(2): 211-8, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15748986

ABSTRACT

Rickettsia conorii is an obligate intracellular bacterium transmitted to humans by Rhipicephalus sanguineus ticks. The success of this microorganism at surviving in nature implicates the ability to efficiently adapt to different environments, including the arthropod vector and the mammalian host. Numerous bacterial species possess a highly evolved system for stress adaptation. This so-called stringent response is mediated by guanosine 3',5'-bispyrophosphate and guanosine 3'-diphosphate 5'-triphosphate which are under spoT control in some Gram-negative bacteria. Interestingly, annotation of the R. conorii genome evidenced 5 spoT paralogs. We hypothesized that these spoT genes play a role in adaptation to environmental changes specifically encountered by rickettsiae during their different life cycles. Transcription of the spoT paralogs was examined by RT-PCR from infected Vero cells maintained in rich or deficient culture media, from infected C6/36 insect cells cultured at various temperatures and from infected ticks. Our results demonstrated that the 5 spoT genes can be transcribed. SpoT1 (RC0374) is only transcribed upon stringent response. Transcription of spoT3 (RC0888) was never observed in arthropod cells or ticks, but was specific to R. conorii RNA extracted from infected Vero cells. These results indicate that rickettsial spoT paralogs are independently transcribed, depending on the different infected hosts and the adaptive capacity of the pathogen. Bioinformatics analysis of these possibly encoded proteins is also reported.


Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Heat-Shock Response , Rickettsia conorii/physiology , Transcription, Genetic , Animals , Bacterial Proteins/genetics , Cell Line , Chlorocebus aethiops , Gene Expression Profiling , Guanosine Tetraphosphate/genetics , Guanosine Tetraphosphate/metabolism , Rhipicephalus sanguineus/microbiology , Rickettsia conorii/genetics , Rickettsia conorii/growth & development , Rickettsia conorii/pathogenicity , Temperature , Vero Cells
9.
Ann N Y Acad Sci ; 1063: 215-21, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16481517

ABSTRACT

To analyze the host dependency of rickettsial growth, NIAS-AeAl-2 insect cells (AeAl2) derived from mosquito were first used in this study. It was demonstrated that typhus group rickettsiae (TGR) grew well in AeAl2 cells, but spotted fever group rickettsiae (SFGR) failed. To elucidate the inhibitory process of the growth of SFGR in AeAl2 cells, the adherence and invasion were first analyzed. SFGR possessed abilities to adhere to and invade AeAl2 cells as well as TGR in contrast to their inability of the growth in the cells. Morphologically, generation of microvilli could not be observed on AeAl2 cells inoculated with either group of rickettsiae. On the contrary, Vero cells inoculated with rickettsiae generated a great number of microvilli that adhered to rickettsiae and engulfed them into the cells. The roles of rickettsial major outer membrane protein A and B (rOmpA and rOmpB) were later investigated using E. coli expressing either rOmpA or rOmpB on their surface. Bacteria expressing either one of the major outer membrane proteins of rickettsiae as well as bacteria not expressing these proteins showed adherence to and invasion of AeAl2 cells. Thus, it is yet to be elucidated whether these major outer membrane proteins have any roles in these steps.


Subject(s)
Aedes/cytology , Aedes/microbiology , Rickettsia/growth & development , Animals , Bacterial Adhesion/immunology , Cell Culture Techniques , Cell Line , Chlorocebus aethiops , Rickettsia/pathogenicity , Rickettsia/ultrastructure , Rickettsia conorii/growth & development , Rickettsia conorii/pathogenicity , Rickettsia prowazekii/growth & development , Rickettsia prowazekii/pathogenicity , Rickettsia typhi/growth & development , Rickettsia typhi/pathogenicity , Rickettsia typhi/ultrastructure , Vero Cells
10.
Virchows Arch ; 446(1): 21-7, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15480764

ABSTRACT

Fractalkine (CX3CL1) is a chemokine expressed mainly by endothelial cells, which are the major cellular targets of rickettsiae. We used immunohistochemistry to investigate the normal expression of CX3CL1 in mice and the kinetics of expression of this chemokine throughout the course of lethal and sublethal rickettsial infections in a mouse model of spotted-fever group rickettsioses. The peak of expression of fractalkine on day 3 of infection coincided with the time of infiltration of macrophages into infected tissues and preceded the peak of rickettsial content in tissues.


Subject(s)
Boutonneuse Fever/immunology , Chemokines, CX3C/analysis , Membrane Proteins/analysis , Animals , Chemokine CX3CL1 , Chemokines, CX3C/physiology , Immunohistochemistry , Macrophages/pathology , Membrane Proteins/physiology , Mice , Mice, Inbred C3H , Rickettsia conorii/growth & development
11.
J Clin Microbiol ; 42(12): 5757-66, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15583310

ABSTRACT

By use of the nearly perfectly colinear genomes of Rickettsia conorii and Rickettsia prowazekii, we compared the usefulness of three types of sequences for typing of R. conorii isolates: (i) 5 variable coding genes comprising the 16S ribosomal DNA, gltA, ompB, and sca4 (gene D) genes, which are present in both genomes, and the ompA gene, which is degraded in R. prowazekii; (ii) 28 genes degraded in R. conorii but intact in R. prowazekii, including 23 split and 5 remnant genes; and (iii) 27 conserved and 25 variable intergenic spacers. The 4 conserved and 23 split genes as well as the 27 conserved intergenic spacers each had identical sequences in 34 human and 5 tick isolates of R. conorii. Analysis of the ompA sequences identified three genotypes of R. conorii. The variable intergenic spacers were significantly more variable than conserved genes, split genes, remnant genes, and conserved spacers (P < 10(-2) in all cases). Four of the variable intergenic spacers (dksA-xerC, mppA-purC, rpmE-tRNA(fMet), and tRNA(Gly)-tRNA(Tyr)) had highly variable sequences; when they were combined for typing, multispacer typing (MST) identified 27 different genotypes in the 39 R. conorii isolates. Two batches from the same R. conorii strain, Malish (Seven), with different culture passage histories were found to exhibit the same MST type. MST was more discriminatory for strain genotyping than multiple gene sequencing (P < 10(-2)). Phylogenetic analysis based on MST sequences was concordant with the geographic origins of R. conorii isolates. Our study supports the usefulness of MST for strain genotyping. This tool may be useful for tracing a strain and identifying its source during outbreaks, including those resulting from bioterrorism.


Subject(s)
Bacterial Typing Techniques , DNA, Ribosomal Spacer/analysis , Rickettsia conorii/classification , Sequence Analysis, DNA , Animals , Bacterial Proteins/genetics , Base Sequence , Boutonneuse Fever/microbiology , DNA, Bacterial/analysis , Genetic Variation , Genome, Bacterial , Genotype , Humans , Molecular Sequence Data , RNA, Ribosomal, 16S/genetics , Rickettsia Infections/microbiology , Rickettsia conorii/genetics , Rickettsia conorii/growth & development , Rickettsia prowazekii/classification , Rickettsia prowazekii/genetics
12.
Infect Immun ; 72(6): 3524-30, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15155660

ABSTRACT

A recent study demonstrated that polyclonal antibodies to Rickettsia conorii and monoclonal antibodies to outer membrane proteins A (OmpA) and B (OmpB) provided effective, Fc-dependent, passive immunity, even in severe combined immunodeficient mice with an established infection. In order to determine the mechanism of protection, mouse endothelial and macrophage-like cell lines were infected with R. conorii that had been exposed to polyclonal antibodies, monoclonal antibodies to OmpA or OmpB, Fab fragments of the polyclonal antibodies, or normal serum or that were left untreated. At 0 h, Fc-dependent antibody enhancement of R. conorii adherence to endothelial and macrophage-like cell lines was inhibited by the presence of normal serum, suggesting Fc receptor-mediated adherence of opsonized rickettsiae. At 3 h, the opsonized rickettsiae had been internalized. After 72 h, inhibited survival of rickettsiae exposed to polyclonal antibodies or monoclonal antibodies to OmpA or OmpB was evident compared with growth of untreated and normal serum-treated and polyclonal Fab antibody-treated R. conorii. Polyclonal antibodies and an anti-OmpB monoclonal antibody inhibited the escape of R. conorii from the phagosome, resulting in intraphagolysosomal rickettsial death. At 48 h of infection, rickettsicidal activity of macrophages by opsonized rickettsiae was inhibited by NG-monomethyl-L-arginine, superoxide dismutase, mannitol, or supplemental L-tryptophan, and endothelial rickettsicidal activity against opsonized rickettsiae was inhibited by NG-monomethyl-L-arginine, superoxide dismutase, catalase, or supplemental L-tryptophan. Thus, Fc-dependent antibodies protected against R. conorii infection of endothelium and macrophages by opsonization that inhibited phagosomal escape and resulted in phagolysosomal killing mediated by nitric oxide, reactive oxygen intermediates, and L-tryptophan starvation.


Subject(s)
Antibodies, Bacterial/pharmacology , Antibodies, Monoclonal/pharmacology , Endothelial Cells/microbiology , Macrophages/microbiology , Rickettsia Infections/prevention & control , Rickettsia conorii/pathogenicity , Animals , Antibodies, Bacterial/immunology , Antibodies, Monoclonal/immunology , Bacterial Adhesion , Bacterial Outer Membrane Proteins/immunology , Cell Line , Chlorocebus aethiops , Cytosol/microbiology , Endothelial Cells/immunology , Macrophages/immunology , Mice , Opsonin Proteins/metabolism , Phagosomes/microbiology , Receptors, Fc/metabolism , Rickettsia Infections/immunology , Rickettsia conorii/drug effects , Rickettsia conorii/growth & development , Rickettsia conorii/physiology , Vero Cells
13.
J Clin Microbiol ; 41(7): 3317-9, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12843081

ABSTRACT

Three different spotted-fever group rickettsiae-Rickettsia conorii, R. massiliae, and R. rhipicephali-were detected and identified by PCR-restriction fragment length polymorphism analysis in Rhipicephalus ticks collected from domestic animals in the Fokida region of Greece, where a high seroprevalence of antibodies to R. conorii was previously demonstrated. The infection rate of ticks was 1.6%. Moreover, R. conorii was isolated from one Rhipicephalus sanguineus tick.


Subject(s)
Polymerase Chain Reaction/methods , Polymorphism, Restriction Fragment Length , Rickettsia conorii , Rickettsia conorii/classification , Rickettsia conorii/isolation & purification , Ticks/microbiology , Animals , Animals, Domestic/parasitology , Bacterial Typing Techniques , Chlorocebus aethiops , DNA, Bacterial/analysis , Greece , Rickettsia conorii/genetics , Rickettsia conorii/growth & development , Tick Infestations/parasitology , Ticks/classification , Vero Cells
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