Françoise Guinet

Frequent ectopic recombination of virulence factor genes in telomeric chromosome clusters of P. falciparum.

Persistent and recurrent infections by Plasmodium falciparum malaria parasites result from the ability of the parasite to undergo antigenic variation and evade host immune attack. P. falciparum parasites generate high levels of variability in gene families that comprise virulence determinants of cytoadherence and antigenic variation, such as the var genes. These genes encode the major variable parasite protein (PfEMP-1), and are expressed in a mutually exclusive manner at the surface of the erythrocyte infected by P. falciparum. Here we identify a mechanism by which var gene sequences undergo recombination at frequencies much higher than those expected from homologous crossover events alone. These recombination events occur between subtelomeric regions of heterologous chromosomes, which associate in clusters near the nuclear periphery in asexual blood-stage parasites or in bouquet-like configurations near one pole of the elongated nuclei in sexual parasite forms. We propose that the alignment of var genes in heterologous chromosomes facilitates gene conversion and promotes the diversity of antigenic and adhesive phenotypes. The association of virulence factors with a specific nuclear subcompartment may also have implications for variation during mitotic recombination in asexual blood stages.

Detection of Yersinia pestis in Sputum by Real-Time PCR

ABSTRACT A 5′ nuclease PCR assay for detection of the Yersinia pestis plasminogen activator (pla) gene in human respiratory specimens with simulated Y. pestis infection was developed. An internal positive control was added to the reaction mixture in order to detect the presence of PCR inhibitors that are often found in biological samples. The assay was 100% specific for Y. pestis. In the absence of inhibitors, a sensitivity of 102 CFU/ml of respiratory fluid was obtained. When inhibitors were present, detection of Y. pestis DNA required a longer sample treatment time and an initial concentration of bacteria of at least 104 CFU/ml. The tests total turnaround time was less than 5 h. The assay described here is well suited to the rapid diagnosis of pneumonic plague, the form of plague most likely to result from a bioterrorist attack.

Defective Innate Cell Response and Lymph Node Infiltration Specify Yersinia pestis Infection

Since its recent emergence from the enteropathogen Yersinia pseudotuberculosis, Y. pestis, the plague agent, has acquired an intradermal (id) route of entry and an extreme virulence. To identify pathophysiological events associated with the Y. pestis high degree of pathogenicity, we compared disease progression and evolution in mice after id inoculation of the two Yersinia species. Mortality studies showed that the id portal was not in itself sufficient to provide Y. pseudotuberculosis with the high virulence power of its descendant. Surprisingly, Y. pseudotuberculosis multiplied even more efficiently than Y. pestis in the dermis, and generated comparable histological lesions. Likewise, Y. pseudotuberculosis translocated to the draining lymph node (DLN) and similar numbers of the two bacterial species were found at 24 h post infection (pi) in this organ. However, on day 2 pi, bacterial loads were higher in Y. pestis-infected than in Y. pseudotuberculosis-infected DLNs. Clustering and multiple correspondence analyses showed that the DLN pathologies induced by the two species were statistically significantly different and identified the most discriminating elementary lesions. Y. pseudotuberculosis infection was accompanied by abscess-type polymorphonuclear cell infiltrates containing the infection, while Y. pestis-infected DLNs exhibited an altered tissue density and a vascular congestion, and were typified by an invasion of the tissue by free floating bacteria. Therefore, Y. pestis exceptional virulence is not due to its recently acquired portal of entry into the host, but is associated with a distinct ability to massively infiltrate the DLN, without inducing in this organ an organized polymorphonuclear cell reaction. These results shed light on pathophysiological processes that draw the line between a virulent and a hypervirulent pathogen.

Accurate quantitation of Leishmania infection in cultured cells by flow cytometry.

BACKGROUND Leishmaniases are major parasitic diseases caused by protozoans that are obligate intracellular parasites during the mammalian phase of their life cycle. Quantitation of experimental mammalian cell infections is usually performed by time-consuming microscopic examination. In this report a flow cytometry (FCM)-based assay suitable for studying in vitro infections by L.amazonensis is presented. METHODS Intense fluorescence staining of the amastigote forms with a stage- and species-specific monoclonal antibody was obtained after permeabilization of both the host-cell cytoplasmic membrane and the parasitophorous vacuole membrane by saponin treatment. RESULTS Upon flow cytometry (FCM) analysis, parasitized cells separated sharply from the auto-fluorescence of the mammalian host cells, giving the assay a high degree of sensitivity and specificity. Ninety to 98% of cells in the more fluorescent population harbored parasites visible by phase-contrast and UV-light microscopy, while no parasites were observed in more than 95% of the cells in the population with background fluorescence. Comparisons of the FCM results with those from microscope counting and analysis of various dilutions of parasitized cells confirmed the reliability of the method. CONCLUSIONS The FCM assay provided rapid quantitation of Leishmania infection either in mouse macrophages, the natural host cell in murine leishmaniasis, or in Chinese hamster ovary (CHO) cells, a non-macrophage cell line proposed as an in vitro model for studying host-parasite interactions. The protocol described here should be adaptable to studies involving other parasites residing in nucleated cells.

NKT cells-containing inflammatory lesions induced by Yersinia pseudotuberculosis glycolipids

Valpha14-expressing NKT (invNKT) cells are a population of non-conventional T lymphocytes (TL) that bridge mammalian innate and adaptive immunity. Their role in infectious diseases and inflammatory processes is still largely ununderstood. A previous report has shown that an acute granulomatous-like reaction can be elicited by sub-cutaneous injection of Mycobacterium tuberculosis glycolipids in mice, and that recruitment of invNKT cells at the injection site is instrumental in this process. Here, we describe the mouse response to enterobacterium Yersinia pseudotuberculosis glycolipids extracts during the first week post injection. The cellular reaction is an acute inflammatory infiltrate where TL are abundant from early times on. InvNKT cells are present in the lesions, detectable as early as day 1 post injection. They compose all of the Valpha14-expressing TL, although conventional T cells expressing non-Valpha14 alpha-chains can be detected. The reaction is strictly dependent on ester-linked fatty acids as mild alkaline treatment of the extract prior to injection results in the absence of analysable lesions. Thus, glycolipids from Yersinia induce inflammatory lesions comparable to those induced by mycobacteria glycolipids, in spite of the totally different cell wall composition in the two genera. Moreover, the present findings show that invNKT cell response is not unique to mycobacterial glycolipids.

Abdominal Aortic Aneurysm Infected by Yersinia pseudotuberculosis

ABSTRACT Infected aneurysms caused by Yersinia are very uncommon and are principally due to Yersinia enterocolitica. We describe the first case of an infected aneurysm caused by Yersinia pseudotuberculosis in an elderly patient with a history of atherosclerotic cardiovascular disease.

Impact on the Host of the Yersinia pestis-specific Virulence Set and the Contribution of the Pla Surface Protease

From the closely related enteropathogen Yersinia pseudotuberculosis, the bubonic plague agent Yersinia pestis has recently evolved an exceptionally high virulence potential and the ability to infect via the intradermal route. However, Y. pseudotuberculosis inoculated at the same dose and the same site as Yersinia pestis remains less virulent, showing that the two species harbor distinct virulence sets. In particular, Y. pseudotuberculosis does not appear to be able to induce the formation of a typical bubo, characterized by extensive tissue destruction and bacterial invasive behavior, but rather of an organized and controlled lymph node abscess. Among the limited number of genetic differences between Y. pestis and Y. pseudotuberculosis is the pla gene, borne by the Y. pestis-specific pPla plasmid. Pla is a major virulence factor for bubonic plague and it has been shown in in vitro tests to be a multifunctional protein with proteolytic, adhesive, and invasive properties. The Y. pestis-specific character of Pla and its various in vitro functions led us to hypothesize that it may be involved in the appearance of the bubo-specific features. A model to investigate this hypothesis is presented as well as preliminary results indicating that Pla is important for the constitution of high bacterial burden in the draining lymph node and for some, but not all, of the plague histological lesions.

Comparative Approaches to Identify Host Factors Specifically Targeted by Yersinia pestis During the Infectious Process

The genus Yersinia includes three species pathogenic for humans and animals: Y. enterocolitica, Y. pseudotuberculosis and Y. pestis. The two former species behave like true enteropathogens, i.e. they cause mild intestinal symptoms and are transmitted by the fecal-oral route. In contrast, Y. pestis is the etiologic agent of plague, a highly severe and often fatal disease, which is transmitted by flea bites (bubonic plague) or aerosols (pneumonic plague). The plague bacillus is one of the most pathogenic microorganisms of the bacterial kingdom, but the mechanisms it specifically uses to kill its host so efficiently remain largely unknown. Despite drastically different clinical and epidemiological features between enteropathogenic Yersinia and the plague agent, it appears that Y. pestis is a clone recently emerged from Y. pseudotuberculosis (less than 20,000 years ago). Genome comparison indicates that the two species are genetically highly similar. This close genetic relationship is used to carry out comparative pathophysiological studies between Y. pestis and its recent ancestor Y. pseudotuberculosis, in an attempt to identify the host factors and/or cell lineages specifically targeted by the plague bacillus during the infectious process. Another comparative approach based on the identification of genetic and physiological differences between mouse strains that are either resistant or susceptible to plague is another mean to identify host responses specifically diverted by the plague bacillus.