Unit: Molecular and Medical Bacteriology
Director: Guy BARANTON
The possibility of inactivating genes in Leptospira allows to envisage a study of their functions. The participation of Toll-like receptors in innate immune responses generated by leptospires and its LPS is under study. In Borrelia, the ospC gene analysis, when compared to genus phylogeny, allows to differentiate three distinct bacterial populations: invasive, cutaneous and non infectious for vertebrates. In Yersinia, analysis of chromosomal determinants and their comparison among different pathogenic species opens a new field of investigation to understand the exceptional pathogenicity of Y. pestis.
"Toll-like receptors" (TLRs) and leptospiral lipopolysaccharide (LPS) (C. Werts, E. Fournié-Amazouz, M-A. Nahori and I. Saint Girons).
Toll-like receptors (TLRs) are essential components of innate immunity that are able to sense conserved microbial patterns (so called Pathogen Associated Molecular Patterns or PAMPs) like LPS, lipoproteins, peptidoglycan, bacterial DNA. We showed that TLR2, instead of TLR4 used by LPS from Gram negative bacteria, was required for cell activation by leptospiral LPS (Werts et al., 2001). The peculiar structure of leptospiral lipidA has been deciphered by the group of C.R.H. Raetz (Duke, Durham, USA) (Que et al., in preparation). We are now studying the relationships between the structure of leptospiral lipidA and its biological functions. Preliminary results indicate that despite a lack of endotoxic properties, leptospiral lipidA is able to activate mammalian cells but differs from whole LPS in the TLR use. These results may help to understand some of the features of leptospirosis. Furthermore we are now interested in other PAMPs, as heat shock proteins that have recently been associated with inflammation for Helicobacter pylori.
Characterization of amino acids and heme biosynthesis pathways in Leptospira spp (M. Picardeau, R. Guégan, H. Bauby and I. Saint Girons).
Unlike the spirochetes Borrelia burgdorferi and Treponema pallidum, Leptospira spp. contain genes encoding the enzymes for amino acids and heme biosynthesis. Recently, the development of new genetic tools has facilitated the construction of targeted mutants in saprophytic Leptospira spp. In a previous study, we have shown that disruption of the L. meyeri trpE gene resulted in tryptophan auxotrophy. Since methionine usually controls a large number of cellular processes, we also studied its biosynthesis in Leptospira spp. Our mutational and sequence analysis shows that the methionine biosynthesis of Leptospira is far more intertwined than suspected and includes two distinct pathways: the enterobacterial type transsulfuration pathway and the sulfhydrylation pathway.
Heme is a tetrapyrrole derivative commonly used as a prosthetic group in proteins such as cytochromes, catalases and peroxidases. Sequence analysis of the L. interrogans genome shows that all heme biosynthetic genes, except hemD which is not present in the entire genome, are clustered in a 15-kb region of the CII secondary chromosome. The L. interrogans hemC gene (encoding porphobilinogen deaminase) was able to restore uroporphyrinogen III synthase activity in an Escherichia coli ΔhemD mutant, suggesting that the L. interrogans hemC gene encodes a bifunctional enzyme. The last step of heme biosynthesis is catalyzed by ferrochelatase (encoded by the hemH gene). We constructed a L. biflexa hemH mutant. Mutants containing an inactivated hemH gene were recovered only when exogenous hemin was present. The results indicate that heme is an essential growth factor for Leptospira, and that these spirochetes are capable of both de novo synthesis or uptake of heme. This may have implications in a better understanding of the pathogenesis of Leptospira.
Chromosomal toxin-antitoxin locus of Leptospira interrogans (M. Picardeau, S. Ren and I. Saint Girons)
Toxin-antitoxin systems encoded by bacterial plasmids and chromosomes typically consist of a toxin that inhibits growth of the host cell and a specific antitoxin. In this report, the chpK gene from the chromosomal toxin-antitoxin locus of the spirochete Leptospira interrogans was studied in both prokaryotic and eukaryotic systems. Cloning of the spirochetal chpK gene into a mycobacterial expressing vector lead to dramatic reductions of transformation efficiency in both Mycobacterium smegmatis and M. bovis BCG, suggesting that L. interrogans ChpK is highly toxic for mycobacteria. Presence of the L. interrogans chpK gene was also found to inhibit cell growth of the yeast Saccharomyces cerevisiae. These results show that ChpK possesses a broad activity against both prokaryotes and eukaryotes, suggesting that the cellular target of the toxin is conserved in these organisms.
CNR and WHO Collaborating Center
D. Postic and G. Baranton in collaboration with Institut de Veille Sanitaire (InVS): Leptospirosis and heatwave.
The heatwave which spread over continental France from June to September 2003 lead to increased (both frequency and duration) contacts with fresh waters. The previous experience of such heatwaves was not helpful. In 1947 and 1949, the number of leptospirosis cases was about twice the mean number recorded in this period. In contrast in 1976, the number of cases decreased as compared to the seventies mean. It was therefore difficult to anticipate. However as soon as August, it was obvious that the increase was moderate and restricted to three regions: Aquitaine (mainly Dordogne), Champagne-Ardennes (mainly Aube) and Poitou-Charentes. This trend was confirmed in September (59 cases instead of 40-50 usually) in spite of a drastic increase of serology demands. This increase of demands was due to messages of alert published in the press which lead to inadequate behaviour (serology asked for asymptomatic swimmers ). In October and November, the number of cases became inferior to the usual mean. In conclusion, people increased drastically their contacts with fresh waters which were in contrast rather less contaminated than previous years. A possible explanation was that heavy rains associated with storms did not occur during this summer (excepted in South West areas). These rains usually drain leptospires in rivers leading to water contamination.
The CNR and InVS are in contact to document and evaluate alert systems to face extreme meteorological phenomenons such as heatwaves, floodings which are supposed to occur more frequently with global climate changes.
Consult also: http://www.pasteur.fr/recherche/Leptospira/LeptospiraF.html
Borrelia in Morocco (M. Sarih, F. Jouda, L. Gern and D. Postic).
In the frame of an European project (associating 7 partners and coordinated by D. Postic) whose aim is to evaluate the Lyme disease risk among mediterranean countries (in fact in North Africa). After Tunisia, Morocco is studied. As in Tunisia, ticks infestation rates are high (48%) and the prominent species is a non pathogenic Borrelia: B. lusitaniae. However, 3 B. garinii and 3 B. burgdorferi sensu stricto strains have been detected. B. burgdorferi had not been identified in Africa up to now.
ospC and pathogenicity of Borrelia (V. Lagal, D. Postic, E. Ruzic-Sabljic and G. Baranton).
SSCP-PCR studies of the ospC gene from 80 Borrelia burdorferi sensu lato isolates of human origin have been performed. Most of studied isolates came from Slovenia where Lyme disease is both frequent and severe. The study showed that most genotypes indeed correlate with invasive phenotypes. The ospC gene encodes an outer-membrane protein and therefore may be considered as a pathogenicity marker of a given strain. When correlated with phylogenetic trees previously drawn with either VNTRs or AP-PCR, the ospC type shows that each phenotype (either invasive or cutaneous or non infectious) is associated with specific clones. Additionally, it seems that non infectious clones are in ancestral position.
CNR Borrelia (D. Postic, E. Ferquel and C. Pérez-Eid)
Lyme disease, the most common vector born disease in Europe as in North America, is our main research activity. We focus our work on two goals: the vector and the transmitted bacteria and human incidence of the disease in France.
In Europe, I. ricinus, the vector of Lyme disease, transmits the three species of the B. burgdorferi complex responsible for human cases. Each of the three species of Borrelia seems to be associated with specific clinical manifestations depending on the tropism of the Borrelia species for a specific organ. It is therefore of interest to know the respective geographical distribution of each Borrelia species. Such a study needs to be done in the vector and not in humans since (1) the identification of the Borrelia species is easier in the vector; (2) few biological specimens are available in humans; (3) the organ which is most likely to allow the isolation of the Borrelia is not known in human and finally (4) it is very difficult to conclude on the geographical origin of a Borrelia isolated in humans. In 2002, we collected ticks on the field in Brittany, the most occidental area of France and Europe, and in the centre of France (Department of Allier). Collects are done using a method allowing statistic analysis of the results according to a methodology previously adjusted by our team, to evaluate their density. The infectious status of ticks for Borrelia was evaluated by using PCR technique.
The incidence of Lyme disease in France is presently unknown, the results from older studies varying from 16.4 to 40 cases for 100 000 inhabitants. Our goal is to obtain more accurate data in order to be able to better know the incidence and to detect possible differences in the incidence and in the symptoms of the disease in different geographical areas. For this purpose, we are performing a retrospective study based on serological analysis requests from the Pasteur Cerba laboratory (one of the two major medical analysis laboratories in France) and a prospective study in 2002, with a net of physicians (from private practice and hospital) working as generalists or in different specialities, and biologists.
YERSINIA (Head: E. Carniel)
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 completed recently. A comparative genomics approach is now undertaken;
- 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 2003 dealt with:
Presence of the high pathogenicity island in Salmonella enterica subspecies III and VI (Contributor: E. Carniel - Collaborators: T.A. Oelschlaeger, D. Zhang, S. Schubert, W. Rabsch, H. Karch and J. Hacker).
The genus Salmonella is divided into eight groups but the majority of Salmonella isolates pathogenic for warm blood animals, including humans, are members of group I. The screening of 74 Salmonella strains for the presence of the High-Pathogenicity Island (HPI), initially described in Yersinia, revealed that Salmonella groups IIIa, IIIb, and VI did harbor the island. Most HPI positive isolates produced yersiniabactin under iron limiting conditions and were positive for the high molecular weight proteins HMWP1 and HMWP2. However, only strains of group VI had an HPI identical to that of Yersinia, including the presence of an integrase gene downstream of ybtS and an insertion site into an asn tRNA gene. In contrast, strains of groups IIIa and IIIb harbored several genes (int, ybtS, ybtP-ybtA intergenic region, irp1) containing insertions or deletions. Their HPI was not inserted into an asn tRNA gene but downstream of ych. The HPI is thus an ubiquitous island, present in a wide variety of members of the family of Enterobacteriaceae.
Analysis of the relationship between bacterial virulence and nucleotide metabolism (Contributors: V. Chenal-Francisque, J. Foulon and E. Carniel - Collaborators: H. Munier-Lehmann, M. Ionescu, P. Christova and O. Bârzu).
Nucleoside monophosphate kinases (NMPKs) are essential catalysts for bacterial growth and multiplication. These enzymes display high primary sequence identities among members of the family Enterobacteriaceae. Although Y. pesti belongs to this family, its thymidylate kinase (TMPKyp) was previously shown to exhibit biochemical properties significantly different from those of its Escherichia coli counterpart. We thus studied and characterized another NMPK, the adenylate kinase (AK) of Y. pestis (AKyp). As for TMPKyp, AKyp displayed a lower thermodynamic stability than other studied AKs. Two mutations in AK (S129F and P87S), previously shown to induce a thermosensitive growth defect in E. coli, were introduced into AKyp. The recombinant variants had a lower stability than wild-type AKyp and a higher susceptibility to proteolytic digestion. When the P87S substitution was introduced into the chromosomal adk gene of Y. pestis, growth of the mutant strain was altered at the non-permissive temperature of 37°C. In virulence testings, while less than 50 CFU of wild-type Y. pestis killed 100% of the mice upon subcutaneous infection, bacterial loads as high as 1.5 x 104 CFU of the adk mutant were unable to kill any animal.
Detection of Yersinia pestis in sputum by Real-Time PCR (Contributor: F. Guinet - Collaborators: C. Loïez, S. Herwegh, F. Wallet, S. Armand and R.J. Courcol).
A 5' nuclease PCR assay for detection of the Y. pestis plasminogen activator 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 test total turnaround time was less than 5 h. This assay is thus well suited for the rapid diagnosis of pneumonic plague, the form of plague most likely to result from a bioterrorist attack.
Development and validation of a dipstick for the rapid diagnosis of Y. pestis (Contributors: J. Foulon and E. Carniel - Collaborators: S. Chanteau, L. Rahalison, L. Ralafiarisoa, M. Ratsitorahina and L. Ratsifasoamanana).
Delayed diagnosis of plague is one of the major causes of human death and the spread of the disease since it limits access to effective treatments and implementation of control measures. In endemic foci, the availability of a simple and specific diagnostic test that local health workers could carry out in a few minutes on biological samples from suspected patients or dead rodents was a key priority in the fight against this disease. We developed this type of rapid diagnostic test for plague, based on F1 antigen detection and immunochromatography. The test can be performed in less than 15 min. It has a threshold of 0.5 ng/ml and a shelf life of 21 days at 60°C. Its sensitivity and specificity are 100%. It detected 41.6% and 31% more positive clinical specimens than did bacteriological methods and ELISA, respectively. Once distributed to health workers at 26 pilot field sites in Madagascar, the agreement rate between tests performed in the field and at the Central Laboratory was 89.8%. This test will be of key importance for the control of plague in endemic countries.
Use of O-antigen gene cluster specific PCRs for the identification and O-genotyping of Yersinia pseudotuberculosis and Yersinia pestis (Contributor: E. Carniel - Collaborators: T. Bogdanovich, H. Fukushima and M. Skurnik).
Y. pestis is a very recently evolved clone of Y. pseudotuberculosis serotype O:1b. This close relationship causes potential difficulties in DNA-based diagnostic methods. Analysis of the O-antigen gene clusters in these two organisms identified two regions that were used to specifically identify Y. pestis-Y. pseudotuberculosis as a group, or Y. pestis alone. Both PCR assays were found to be 100% specific when tested on a large collection of Yersinia species and other Enterobacteriaceae. Furthermore, advantage was taken of the different setups of the O-antigen gene clusters of the 21 known Y. pseudotuberculosis serotypes to develop a multiplex PCR assay to replace the conventional serotyping method of Y. pseudotuberculosis by O-genotyping. The multiplex PCR assay contained nine sets of specific PCRs in a single tube and when used on Y. pseudotuberculosis reference strains, allowed the distinction of 14 individual serotypes and two duplex serotypes (O:4a-O:8 and O:12-O:13). Serotype O:7, O:9, and O:10 strains required additional PCRs for O-genotyping. Once applied to Y. pseudotuberculosis strains of various origins, a very good correlation between classical serotypes and O-genotypes was observed, although some discrepancies were found. O-genotyping also proved useful to correct misidentification of some strains and to type Y. pseudotuberculosis isolates that had lost the expression of the O-antigen. The PCR-based O-genotyping can easily be applied in conventional laboratories, without the need for tedious preparation of a large set of specific antisera.
Development of a rapid and simple method for inactivating chromosomal genes in Yersinia (Contributors: A. Derbise, B. Lesic, D. Dacheux and E. Carniel - Collaborator: J.M. Ghigo).
The methodology commonly used in Yersinia to generate knockout mutants by chromosomal gene replacement requires several subcloning steps into a suicide delivery vector, followed by electrotransformation into E. coli and subsequent transfer back into Yersinia by electroporation or conjugation. Such a methodology represents a major limitation for large-scale mutagenesis in post-genomic studies. To circumvent this fastidious reverse genetics approach, we developed a chromosomal gene inactivation method that does not require any cloning step. The technique is based on the expression of the phage red operon encoded by plasmid pKOBEG which promotes recombination between the chromosomal region of interest and a PCR-generated resistance marker flanked by regions of homology with the target DNA. The construction of a pKOBEG-sacB vector and the use of a three-steps PCR procedure to generate 500 bp flanking regions of homology allowed us to obtain efficient allelic exchanges of genes in both Y. pestis and Y. pseudotuberculosis.
National Reference Laboratory and WHO Collaborating Center for Yersinia (L. Martin, A. Leclercq, F. Guinet and E. Carniel)
See the Yersinia National Reference Center web site: http://www.pasteur.fr/sante/clre/cadrecnr/yersinia-index.html
Keywords: Spirochetes, Leptospira, Borrelia, TLR, lipid A, genetics, allelic exchange, epidemiology, phylogeny, VNTR, ospC, Yersinia, genomics, virulence