Yassina Bechah

Identification of Rickettsial Infections by Using Cutaneous Swab Specimens and PCR

To determine the usefulness of noninvasive cutaneous swab specimens for detecting rickettsiae, we tested skin eschars from 6 guinea pigs and from 9 humans. Specimens from eschars in guinea pigs were positive for rickettsiae as long as lesions were present. Optimal storage temperature for specimens was 4°C for 3 days.

Persistent Coxiella burnetii Infection in Mice Overexpressing IL-10: An Efficient Model for Chronic Q Fever Pathogenesis

Interleukin (IL)-10 increases host susceptibility to microorganisms and is involved in intracellular persistence of bacterial pathogens. IL-10 is associated with chronic Q fever, an infectious disease due to the intracellular bacterium Coxiella burnetii. Nevertheless, accurate animal models of chronic C. burnetii infection are lacking. Transgenic mice constitutively expressing IL-10 in macrophages were infected with C. burnetti by intraperitoneal and intratracheal routes and infection was analyzed through real-time PCR and antibody production. Transgenic mice exhibited sustained tissue infection and strong antibody response in contrast to wild-type mice; thus, bacterial persistence was IL-10-dependent as in chronic Q fever. The number of granulomas was low in spleen and liver of transgenic mice infected through the intraperitoneal route, as in patients with chronic Q fever. Macrophages from transgenic mice were unable to kill C. burnetii. C. burnetii–stimulated macrophages were characterized by non-microbicidal transcriptional program consisting of increased expression of arginase-1, mannose receptor, and Ym1/2, in contrast to wild-type macrophages in which expression of inducible NO synthase and inflammatory cytokines was increased. In vivo results emphasized macrophage data. In spleen and liver of transgenic mice infected with C. burnetii by the intraperitoneal route, the expression of arginase-1 was increased while microbicidal pathway consisting of IL-12p40, IL-23p19, and inducible NO synthase was depressed. The overexpression of IL-10 in macrophages prevents anti-infectious competence of host, including the ability to mount granulomatous response and microbicidal pathway in tissues. To our knowledge, this is the first efficient model for chronic Q fever pathogenesis.

Genomic, proteomic, and transcriptomic analysis of virulent and avirulent Rickettsia prowazekii reveals its adaptive mutation capabilities

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 of R. prowazekii that differed in virulence, associated with the up-regulation of antiapoptotic genes or the interferon I pathway in the host cells. Transcriptional and proteomic analyses of R. prowazekii surface 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 included recO, 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 with recO, that restored the virulent phenotype. An area of genomic plasticity appears to determine virulence in R. prowazekii and represents an example of adaptive mutation for this pathogen.

Transmission potential of Rickettsia felis infection by Anopheles gambiae mosquitoes

Significance Rickettsia felis is a ubiquitous, recently described human pathogen that has been identified as an important cause of unexplained fever in patients in sub-Saharan Africa. The epidemiology of R. felis infection, including its potential arthropod vectors, is poorly understood, however. The results of our experimental model of infection suggest that Anopheles gambiae mosquitoes, the primary malarial vectors in sub-Saharan Africa, have the potential to be vectors of R. felis infection. A growing number of recent reports have implicated Rickettsia felis as a human pathogen, paralleling the increasing detection of R. felis in arthropod hosts across the globe, primarily in fleas. Here Anopheles gambiae mosquitoes, the primary malarial vectors in sub-Saharan Africa, were fed with either blood meal infected with R. felis or infected cellular media administered in membrane feeding systems. In addition, a group of mosquitoes was fed on R. felis-infected BALB/c mice. The acquisition and persistence of R. felis in mosquitoes was demonstrated by quantitative PCR detection of the bacteria up to day 15 postinfection. R. felis was detected in mosquito feces up to day 14. Furthermore, R. felis was visualized by immunofluorescence in salivary glands, in and around the gut, and in the ovaries, although no vertical transmission was observed. R. felis was also found in the cotton used for sucrose feeding after the mosquitoes were fed infected blood. Natural bites from R. felis-infected An. gambiae were able to cause transient rickettsemias in mice, indicating that this mosquito species has the potential to be a vector of R. felis infection. This is particularly important given the recent report of high prevalence of R. felis infection in patients with “fever of unknown origin” in malaria-endemic areas.

Infection of Endothelial Cells with Virulent Rickettsia prowazekii Increases the Transmigration of Leukocytes

Rickettsia prowazekii, the etiologic agent of epidemic typhus, infects vascular endothelium, leading to vasculitis and tissue infiltration of leukocytes. Murine and human endothelial cells (ECs) were infected with R. prowazekii, including the virulent Breinl strain and the attenuated Madrid E strain. The transendothelial migration (TM) of murine and human peripheral blood mononuclear cells (PBMCs) across ECs infected with Breinl organisms was significantly increased compared with that for uninfected ECs or for ECs infected with attenuated organisms, demonstrating that increased TM was related to R. prowazekii virulence. Increased TM was associated with a specific inflammatory pattern. Indeed, only Breinl organisms induced the expression of transcripts for inflammatory cytokines and chemokines by ECs. Murine PBMCs that had transmigrated across ECs infected with Breinl organisms overexpressed inflammatory cytokines and chemokines as well as tissue factor, whereas interleukin-10 expression was down-regulated. The impact of R. prowazekii infection on the TM of PBMCs may play a prominent role in the development of lesions in epidemic typhus.

Rickettsial diseases: from Rickettsia-arthropod relationships to pathophysiology and animal models

Rickettsiae cause spotted fevers and typhus-related diseases in humans. Some of these diseases occur worldwide and are life-threatening, for example, epidemic typhus is still a major health problem despite the apparent efficiency of antibiotic treatment. In addition, Rickettsia prowazekii, the agent of epidemic typhus, and R. rickettsii, the agent of Rocky Mountain spotted fever, are microorganisms that could potentially be used as bioweapons to induce panic in the population. Rickettsiae are obligate intracellular bacteria in both vertebrate and invertebrate hosts, but rickettsial species differ in terms of association with arthropods, behavior of the vector to infection, pathophysiology and outcome of the disease. Understanding the pathogenic steps of rickettsioses is essential to develop protective strategies against these bacteriological threats. Unfortunately, the mechanisms involved in the pathogenesis of many rickettsioses are poorly characterized, and protective immunity is incompletely understood, in part because accurate animal models that mimic human diseases are lacking. In the past, murine models have been of limited value because infection of mice was without effect or resulted in erratic mortality. Recent studies have reported that rickettsial infection can be established in mice, depending on the genetic background of mice, the type of rickettsial species and the route of inoculation. These models may be useful for analyzing the pathogenesis of rickettsioses, especially epidemic typhus, evaluating new therapeutic molecules and vaccine candidates, and preventing future outbreaks.

Rickettsia prowazekii and Real-time Polymerase Chain Reaction

This highly standardized and adaptable assay could improve epidemic typhus surveillance.

Escherichia coli α-Hemolysin Counteracts the Anti-Virulence Innate Immune Response Triggered by the Rho GTPase Activating Toxin CNF1 during Bacteremia

The detection of the activities of pathogen-encoded virulence factors by the innate immune system has emerged as a new paradigm of pathogen recognition. Much remains to be determined with regard to the molecular and cellular components contributing to this defense mechanism in mammals and importance during infection. Here, we reveal the central role of the IL-1β signaling axis and Gr1+ cells in controlling the Escherichia coli burden in the blood in response to the sensing of the Rho GTPase-activating toxin CNF1. Consistently, this innate immune response is abrogated in caspase-1/11-impaired mice or following the treatment of infected mice with an IL-1β antagonist. In vitro experiments further revealed the synergistic effects of CNF1 and LPS in promoting the maturation/secretion of IL-1β and establishing the roles of Rac, ASC and caspase-1 in this pathway. Furthermore, we found that the α-hemolysin toxin inhibits IL-1β secretion without affecting the recruitment of Gr1+ cells. Here, we report the first example of anti-virulence-triggered immunity counteracted by a pore-forming toxin during bacteremia.

MALDI-TOF MS as an innovative tool for detection of Plasmodium parasites in Anopheles mosquitoes

BackgroundMalaria is still a major public health issue worldwide, and one of the best approaches to fight the disease remains vector control. The current methods for mosquito identification include morphological methods that are generally time-consuming and require expertise, and molecular methods that require laboratory facilities with relatively expensive running costs. Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) technology, routinely used for bacterial identification, has recently emerged in the field of entomology. The aim of the present study was to assess whether MALDI-TOF MS could successfully distinguish Anopheles stephensi mosquitoes according to their Plasmodium infection status.MethodsC57BL/6 mice experimentally infected with Plasmodium berghei were exposed to An. stephensi bites. For the determination of An. stephensi infection status, mosquito cephalothoraxes were dissected and submitted to mass spectrometry analyses and DNA amplification for molecular analysis. Spectra were grouped according to mosquitoes’ infection status and spectra quality was validated based on intensity and reproducibility within each group. The in-lab MALDI-TOF MS arthropod reference spectra database, upgraded with representative spectra from both groups (infected/non-infected), was subsequently queried blindly with cephalothorax spectra from specimens of both groups.ResultsThe MALDI TOF MS profiles generated from protein extracts prepared from the cephalothorax of An. stephensi allowed distinction between infected and uninfected mosquitoes. Correct classification was obtained in blind test analysis for (79/80) 98.75% of all mosquitoes tested. Only one of 80 specimens, an infected mosquito, was misclassified in the blind test analysis.ConclusionsMatrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry appears to be a promising, rapid and reliable tool for the epidemiological surveillance of Anopheles vectors, including their identification and their infection status.

Prediction of rickettsial skin eschars in humans using an experimental guinea pig model.

Until now, when a new Rickettsia species was isolated in a tick, it was not possible to predict whether it was a human pathogen or if it would cause a skin eschar at the infection site. Guinea pigs are injected intradermally with 25 different Rickettsia species or subspecies: 16 induced an eschar, 5 induced inflammatory lesions and 4 have no effect. We observed that the occurrence of skin eschars in this model was significantly correlated (P <0.05) with observations of skin eschars in humans (14/16). The most common histological finding was mononuclear cell infiltration. Polymorphonuclear cell infiltration was observed for Rickettsia australis, Rickettsia japonica, and, as in humans, Rickettsia africae. The treatment of guinea pigs with corticosteroids prevents the apparition of eschar following Rickettsia bellii inoculation. Virulent, but not avirulent, Rickettsia prowazekii induced transient inflammatory lesions that were associated with dermal vasculitis, as is Rickettsia typhi. Therefore, the intradermal injection of Rickettsia in guinea pigs appears to be a relevant model for the prediction of the development of escharotic lesions following Rickettsia infection in humans. We speculate that skin eschar is the reflect of a local control avoiding extreme virulence.