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The possibility of inactivating genes in Leptospira allows for the first time to envisage a study of their functions. For Borrelia, the observation that the potential invasivity of a strain is conferred by a single gene opens also a new field of investigation. In Yersinia, the study of chromosomal determinants and their comparison among the various pathogenic species has paved a new way to a better understanding of the mechanisms underlying the exceptional pathogenicity potential of Y. pestis.
One of the research pole of the unit is the study of spirochetes with a fundamental approach: genetics and interaction of spirochetes with their host and an applied approach: taxonomy, phylogeny and diagnostic. The other research pole concerns Yersinia species whose genome structure and degree of stability are investigated. Some virulence genes clustered on an unstable pathogenicity island are studied in these species. Molecular epidemiology is also a factor of interest for both spirochetes and Yersinia.
Spirochetes, agents of leptospirosis (anthropozoonosis), Lyme borreliosis (transmitted by ticks) and other spirochetosis are pathogenic bacteria which grow slowly. Demonstration of diversity of American and European strains of Borrelia burgdorferi, in concert with diverse symptomatology (dermatologic, neurologic and articular) has led to individualisation of new species
Penicillin binding proteins. (A. Brenot, D. Trott, I.Saint Girons, R. Zuerner).
Most eubacteria possess proteins, which bind covalently penicillin ("PBP").These PBP are anchored into the cytoplasmic membrane and participate to the terminal steps of peptidoglycan assembly, thus to the maintenance of cellular morphology. The b-lactams act as substrate analogs for transpeptidation and bind covalently active sites of PBPs. 6 penicillin binding proteins in L. interrogans have been shown to exist by a functional assay. The ponA and pbpB genes had been isolated. PonA and pbpB encode PBP1 and PBP3 respectively. The L. interrogans PBP1 andPBP3 proteins were synthesized in E. coli and were modified with ampicillin using a digoxygenin-ampicillin conjugate. These data show that both genes encode functional penicillin-binding proteins.
Restriction-modification enzymes in leptospires. (Brenot, A. Werts, C., Ottone, C, Sertour, Charon, N., Postic, D., Baranton, G, and Saint Girons I.).
Thanks to the results of titration of a leptobacteriophage, we showed evidence for a restriction-modification system in Leptospira. These results indicate the importance of the knowledge of the presence of restriction and modification enzymes for the choice of the receptor strain when assaying for genetic transfer.
Reverse genetics for saprophytic Leptospira: a non motile mutant. (M. Picardeau, A. Brenot, I. Saint Girons).
An essential tool for understanding the biology of bacteria is the ability to inactivate any gene at will. Targeted gene replacement is routine in many bacteria but has proved difficult for Leptospira. The availability of a L. biflexa-E. coli shuttle vector has solved many questions, in particular the functionality of a kanamycin resistance marker from a Gram-positive bacterium. However, allelic exchange required a further step, namely the enhancement of homologous recombination. We choose flaB (encoding flagellin) as a target gene and obtained a flaB: kanR mutant which is non motile, with an altered morphology and absence of a visible endoflagellum. These results demonstrate that FlaB is implicated in flagellum assembly and also show the feasibility of reverse genetics in saprophytic Leptospira. Two other genes, recA and metY have been inactivated, indicating that reverse genetics is now a bona fide tool for Leptospira. Experiments are under way to extend these results to pathogenic Leptospira.
Leptospira at the era of genomics: Identification of a toxin/antitoxin locus. (M. Picardeau, Shuangxi Ren (China), I. Saint Girons).
The total sequence of the two chromosomes (4500+350 kb) from Leptospira interrogans strain lai had been performed by the Chinese National Human Genome Center at Shanghai (China). An early access to 100 kb of the sequence has allowed us to show the existence of a toxin-antitoxin locus with homologies to the pem/chp locus. The physiological role of this chromosomal toxin-antitoxin locus which is found in Gram-positive, Gram-negative, Archae and spirochetes bacteria is not clear. It had been suggested that it is part of an answer to environmental stimuli like the nutritional stress.
Toll-like receptors (TLRS) and Leptospiral LPS. (Werts,C., R. I. Tapping, J. C. Mathison, T-H Chuang, V. Kravchenko, I. Saint Girons, D. Haake, P.J. Godowski, F. Hayashi, A. Ozinsky, D. Underhill, C.J. Kirschning, H. Wagner, A. Aderem, P.S. Tobias, and R.J. Ulevitch).
Bacterial pathogens activate the host innate immune system via receptors, called "Toll-like" (TLRs) which recognize conserved microbial components, such as lipopolysacharide (LPS), bacterial DNA or peptidoglycan. We show that LPS from Leptospira activates macrophages via CD14 and TLR2. In contrast to LPS from Gram-negative bacteria, TLR4 is not involved in cellular responses initiated by Leptospiral LPS. These data provide a new basis for understanding the innate immune response during Leptospira infection. LPS from Leptospira constitutes a useful tool to study the cellular responses initiated by the TLRs.
National Reference Center (NRC) and WHO/FAO Collaborating Center (WHO/FAO CC) for Leptospires. (E. Fournié, N. Sertour, E. Bellenger, P. Bourhy, A. Duvaquier, D. Postic, G. Baranton).
See (after March 2002) the Leptospira National reference center:
Borrelia are, in Europe as in United States or in Asia responsible for Lyme disease. Informations are available on:
Predominance of Borrelia lusitaniae in Tunisia. (H. Younsi, D. Postic, G. Baranton).
Initiated by Tunisian colleagues, a study of Ixodes ricinus ticks infected by Borrelia has been performed. A high prevalence of infection (over 30% in both adults and nymphs) is reported in ticks (n = 490). Unexpectedly, both by PCR (60 out of 61 amplicons) and culture (15 out of 16 isolates), B. lusitaniae, a non pathogenic species rare in Europe, has been identified. However, one of the isolates has been identified as B. garinii, a species pathogenic for human. This represents the first bacteriological confirmation of the existence of the agent responsible for Lyme disease on the African continent.
Lateral transferts of small DNA fragments of Borrelia burgdorferi leading to a mosaïc ospC gene: hypothesis of a Gene Transfert Agent. (D. E. Dykhuizen, G. Baranton).
In collaboration with Dan Dykhuizen (Stony Brook, USA), we have shown that the ospC gene exhibits an unusual diversity due to a rare evolutional way: diversifying selection. It allows to develop a true "repertoire" of antigenic motifs. This repertoire is a way for the bacterium to escape the protective anti OspC antibodies elicited by potential hosts previously infected with a Borrelia strain. Since no restriction-modification system is encoded by the Borrelia genome, none of the usual genetic transfers is suitable to explain those mosaic ospC genes. An hypothesis is that a Borrelia plasmid should play a Gene Transfert Agent (GTA) role. GTAs are phages with such small heads that they cannot harbour the phage genome but instead small DNA fragments of bacterial origin: altruist phages.
ospC gene sequence allows to predict the evolution of Lyme disease in human. (G. Theodore, D. Dykhuizen, D. Postic, G. Baranton).
Also in collaboration with Dan Dykhuizen and his team, we showed that all Borrelia strains isolated from so called "invasive" (i.e. other than from early Erythema migrans) forms of Lyme disease belonged to a small subset of ospC gene clusters of sequences. Indeed only ten out of one hundred clusters (204 sequences in databanks) comprise the 36 sequences of all isolates from invasive Lyme disease due to any of the 3 pathogenic Borrelia species. Therefore, 36 other sequences from either ticks or Erythema migrans which also comprise these 10 clusters represent potentially invasive strains. Concerning most of the ticks isolates, even though belonging to human pathogenic Borrelia species, we hypothesize they are not infectious for human.
LEGIONELLA AND FRANCISELLA
The Legionella genus comprises several species which live in natural or artficial (water supplies, climatisation towers) aquatic environments. Its medical importance is linked to one of these species: L. pneumophila which is associated to a pneumonary syndrom in immunocompromised human. It is known as Legionnaires disease.
Francisella tularensis, responsible for a zoonosis, is a very small bacterium infecting rodents and Leporids. It is the only bacterium able to pass through the intact skin. This caracteristic along with the 30% letality associated to the North American strains (subsp. tularensis) make them suitable as a biological weapon.
Laboratory of Legionella and Francisella. (C. Tram).
- Legionella. 66 waters samples have been analysed up to now (November the 30th)). The 66 corresponding isolates have been identified by both PCR (mip gene) and monoclonal antibodies.
- Francisella tularensis subsp. holartica. In 2001, 6 strains isolated from human tularemia (including 2 exceptional pulmonary clinical presentations) were confirmed and identified. 83 sera were also submitted to both WesternBlot and/or PCR. This year, the Maisons Alfort Lab whose staff has been trained in the Unit could apply our techniques to the veterinary strains.
The genus Yersinia is composed of 3 species pathogenic for humans: the enteropathogens Y. pseudotuberculosis and Y. enterocolitica, and the plague agent Y. pestis.
The main fields of activity of the Yersinia laboratory are:
- The characterization of a pathogenicity island whose presence confers to the host bacterium the ability to cause systemic infections in humans and to be lethal in mice.
- The molecular bases for the exceptional pathogenicity of Y. pestis. Sequencing of the genome of Y. pseudotuberculosis, a bacterium genetically almost identical to Y. pestis but of much lower pathogenicity, has been recently completed. A comparative genomics approach will now be undertaken, along with a comparative transcriptome analysis.
- The relations between Y. pestis and its insect vector, the flea.
- The physiopathology of Yersinia infection.
- The evolution of Y. pestis since its recent emergence from Y. pseudotuberculosis.
- The resistance of pathogenic Yersinia to antibiotics and the evaluation of new treatments.
- Public health (French Reference Center and WHO Collaborating Center for Yersinia).
The works published in 2001 dealt with:
1. Transferable plasmid-mediated resistance to streptomycin in a clinical isolate of Yersinia pestis. (Guiyoule A., G. Gerbaud, C. Buchrieser, M. Galimand, L. Rahalison, S. Chanteau, P. Courvalin and E. Carniel).
Plasmid-mediated high-level resistance to multiple antibiotics was reported in a clinical isolate of Yersinia pestis in Madagascar in 1997. We described a second Y. pestis strain with high-level resistance to streptomycin, isolated from a human case of bubonic plague in Madagascar. The resistance determinants were carried by a self-transferable plasmid that could conjugate at high frequencies to other Y. pestis isolates. The plasmid and the host bacterium were different from those previously associated with multiple-drug resistance, indicating that acquisition of resistance plasmids is occurring in this bacterial species. Emergence of resistance to streptomycin in Y. pestis represents a critical public health problem since this antibiotic is used as the first-line treatment against plague in many countries.
2. Silencing and reactivation of urease in Y. pestis. (Sebanne F., A. Devalckenaere, J. Foulon, E. Carniel and M. Simonet).
Yersinia pestis is a naturally non-ureolytic microorganism, while all other Yersinia species display a potent urease activity. We demonstrated that Y. pestis harbors a complete urease locus composed of three structural (ureABC) and four accessory (ureEFGD) genes. Absence of ureolytic activity is due to the presence of one additional G residue in a poly(G) stretch, which introduces a premature stop codon in ureD. The presence of the same additional G in eight other Y. pestis isolates indicates that this mutation is species specific. Spontaneous excision of the extra G occurs at a frequency of 10-4 to 10-5 and restores a ureolytic phenotype to Y. pestis. The virulence of two independent ureolytic clones of Y. pestis injected either intravenously, subcutaneously, or intragastrically did not differ from that of the parental strain in the mouse infection model. Coinfection experiments with an equal number of ureolytic and non-ureolytic bacteria did not evidence any difference in the ability of the two variants to multiply in vivo and to cause a lethal infection. Altogether our results demonstrate that variation of one extra G residue in ureD determines the ureolytic activity of Y. pestis but does not affect its virulence for mice or its ability to multiply and disseminate.
3. Identification and characterization of the hemophore-dependent heme acquisition system of Yersinia pestis. (Rossi M. S., J. D. Fetherston, S. Létoffé, E. Carniel, R. D. Perry and J.-M. Ghigo).
Yersinia pestis possesses a heme-protein acquisition system (Hmu) that allows it to utilize heme and heme-protein complexes as sole sources of iron. Analysis of the Y. pestis CO92 genomic sequence revealed a second heme-protein acquisition gene cluster sharing homology with the hemophore-dependent heme-acquisition system (Has system) of S. marcescens. This locus consisted of hasRyp receptor gene, hasAyp hemophore gene and genes encoding components of the HasAyp dedicated ABC-transporter factor (hasDEyp) as well as a TonB-homologue (hasByp). Using a reconstituted secretion system in E. coli, we showed that HasAyp is a secreted heme-binding protein and that expression of HasAyp is iron-regulated in E. coli. The use of a transcriptional reporter fusion showed that the hasRADEB promoter is Fur-regulated and has increased activity at 37°C. No contribution of the Has system in heme utilization was observed in either E. coli or Y. pestis under the conditions we tested. Previously it was shown that a deletion of the Hmu system had no effect on the virulence of Y. pestis in a mouse model of bubonic plague. An Hmu- Has- double mutant also retained full virulence in this model of infection.
4. Analysis of the NKT cells-containing inflammatory lesions induced by Yersinia pseudotuberculosis glycolipids. (Guinet F., C. Ronet, M. Mempel, M. Huerre, E. Carniel, and G. Gachelin).
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. The mouse response to enterobacterium Yersinia pseudotuberculosis glycolipids extracts during the first week post injection was investigated. 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 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 mycobacterial glycolipids, in spite of the totally different cell wall composition in the two genera.
5. National Reference Laboratory and WHO Collaborating Center for Yersinia (L. Martin, F. Guinet, E. Carniel).
See the Yersinia National Reference Center web site
6. Sequencing of the Y. pseudotuberculosis genome. (V. Chenal, D. Dacheux and E. Carniel. E. Garcia, P. Chain).
Comparison of the genomes of Y. pestis (recently sequenced) and Y. pseudotuberculosis (a bacterium genetically closely related to Y. pestis but of much lower virulence) might be a highly useful tool for identifying genes responsible for the extraordinary pathogenicity of Y. pestis. Sequencing of the genome of Y. pseudotuberculosis has been undertaken and has been recently completed at the Lawrence Livermore National Laboratory (US), in collaboration with our laboratory. The comparative genomics phase has now started.
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