Comparative and functional genomic analyses of iron transport and regulation in Leptospira spp.

The spirochetes of the Leptospira genus contain saprophytic and pathogenic members, the latter being responsible for leptospirosis. Despite the recent sequencing of the genome of the pathogen L. interrogans, the slow growth of these bacteria, their virulence in humans, and a lack of genetic tools make it difficult to work with these pathogens. In contrast, the development of numerous genetic tools for the saprophyte L. biflexa enables its use as a model bacterium. Leptospira spp. require iron for growth. In this work, we show that Leptospira spp. can acquire iron from different sources, including siderophores. A comparative genome analysis of iron uptake systems and their regulation in the saprophyte L. biflexa and the pathogen L. interrogans is presented in this study. Our data indicated that, for instance, L. biflexa and L. interrogans contain 8 and 12 genes, respectively, whose products share homology with proteins that have been shown to be TonB-dependent receptors. We show that some genes involved in iron uptake were differentially expressed in response to iron. In addition, we were able to disrupt several putative genes involved in iron acquisition systems or iron regulation in L. biflexa. Comparative genomics, in combination with gene inactivation, gives us significant functional information on iron homeostasis in Leptospira spp.

Killing effect and antitoxic activity of the Leptospira interrogans toxin-antitoxin system in Escherichia coli.

We report the first evidence of a chromosome-encoded toxin-antitoxin locus in spirochetes. This locus has been found in the pathogenic spirochete Leptospira interrogans and exhibits homologies with the pem/chp loci. The L. interrogans chp locus consists of two genes: chpK (for "killer protein") and its upstream partner chpI (for "inhibitory protein"). Expression of ChpK in Escherichia coli results in the inhibition of bacterial growth. The coexpression of ChpI neutralizes ChpK toxicity. By Southern blot analysis, chp homologs were found in all representative pathogenic strains of L. interrogans.

First evidence for a restriction-modification system in Leptospira sp.

The LE1 leptophage exhibited a host range restricted to the saprophytic Leptospira biflexa [Saint Girons et al., Res. Microbiol. 141 (1990) 1131-1133] and mainly to the Patoc 1 strain (hereafter called PFRA) kept in the Paris, France collection. Results of titration of LE1 lysates indicated the presence of a host-controlled modification and restriction system within PUSA (Patoc 1 strain maintained in the Morgantown, WV, USA collection) that was absent in PFRA. Because genomic DNA of PITAL (Patoc 1 strain maintained in Trieste, Italy) appeared smeared in pulsed field gel electrophoresis (PFGE), this strain is likely to contain nucleases that are activated upon DNA isolation. Moreover, comparative NotI digestions of PUSA and PFRA DNAs, as visualized by PFGE, indicated that PUSA belonged to a different serovar than PFRA. Finally, 16S ribosomal sequence analysis indicated that PUSA belonged to the saprophytic Leptospira meyeri species, while PITAL and PFRA appertained to L. biflexa. The evolutionary significance and the importance of the restriction and modification enzymes or non-specific nucleases within strains for genetic experiments are discussed.

First evidence for gene replacement in Leptospira spp. Inactivation of L. biflexa flaB results in non-motile mutants deficient in endoflagella.

Leptospira spp. offer many advantages as model bacteria for the study of spirochaetes. However, homologous recombination between introduced DNA and the corresponding chromosomal loci has never been demonstrated. A unique feature of spirochaetes is the presence of endoflagella between the outer membrane sheath and the cell cylinder. We chose the flaB flagellin gene, constituting the flagellar core, as a target for gene inactivation in the saprophyte Leptospira biflexa. The amino acid sequence of the FlaB protein of L. biflexa was most similar to those of spirochaetes Brachyspira hyodysenteriae (agent of swine dysentery), Leptospira interrogans (agent of leptospirosis) and Treponema pallidum (agent of syphilis). A suicide vector containing the L. biflexa flaB gene disrupted by a kanamycin marker was UV irradiated or alkali denatured before electroporation. This methodology allowed the selection of many kanamycin-resistant colonies resulting from single and double cross-over events at the flaB locus. The double recombinant mutants are non-motile, as visualized in both liquid and semi-solid media. In addition, a flaB mutant selected for further analysis was shown to be deficient in endoflagella by electron microscopy. However, most of the transformants had resulted from a single homologous recombination event, giving rise to the integration of the suicide vector. We evaluated the effect of the sacB and rpsL genes in L. biflexa as potential counterselectable markers for allelic exchange, and then used the rpsL system for the positive selection of flaB double recombinants in a streptomycin-resistant strain. Like the flaB mutant studied above, the Strr double cross-over mutant was non-motile and deficient in endoflagella. Our results demonstrate that FlaB is involved in flagella assembly and motility. They also show the feasibility of performing allelic replacement in Leptospira spp. by homologous recombination.

Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism.

Leptospira interrogans are zoonotic pathogens that have been linked to a recent increased incidence of morbidity and mortality in highly populated tropical urban centers. They are unique among invasive spirochetes in that they contain outer membrane lipopolysaccharide (LPS) as well as lipoproteins. Here we show that both these leptospiral outer membrane constituents activate macrophages through CD14 and the Toll-like receptor 2 (TLR2). Conversely, it seems that TLR4, a central component for recognition of Gram-negative LPS, is not involved in cellular responses to L. interrogans. We also show that for intact L. interrogans, it is LPS, not lipoprotein, that constitutes the predominant signaling component for macrophages through a TLR2 pathway. These data provide a basis for understanding the innate immune response caused by leptospirosis and demonstrate a new ligand specificity for TLR2.

Penicillin-binding proteins in Leptospira interrogans.

The Leptospira interrogans ponA and pbpB genes were isolated and characterized. ponA and pbpB encode the penicillin-binding proteins (PBPs) 1 and 3, respectively. There is little sequence variation between the PBP genes from two L. interrogans strains (serovar icterohaemorrhagiae strain Verdun and serovar pomona strain RZ11). The deduced L. interrogans PBP 1 and PBP 3 protein sequences from the two strains shared over 50% similarity to homologous proteins from Escherichia coli. It was demonstrated for strain Verdun that ponA and pbpB are transcribed individually from their own promoter. The ponA and pbpB genes from both strains are separated by 8 to 10 kb and oriented such that their transcription is convergent. The L. interrogans PBP 1 and PBP 3 proteins were synthesized in E. coli and were modified with ampicillin using a digoxigenin-ampicillin conjugate. These data show that both genes encode functional PBPs.

Localization of the Leptospira interrogans metF gene on the CII secondary chromosome.

An open reading frame of 885 nucleotides was identified as the Leptospira interrogans metF gene. The deduced amino acid sequence (294 amino acids) showed similarities with Escherichia coli methylene tetrahydrofolate reductase (MetF or MTHFR) (33% identity) and with the N-terminal part of human MTHFR (33% identity). The L. interrogans metF gene complements an E. coli metF mutant to prototrophy, suggesting the functionality of the folate branch converging to form methionine. In addition, the L. interrogans MetF was found to be thermolabile. The metF gene belonged to the CII secondary chromosome, in contrast to the previously isolated metY and metX genes, which have been localized to the CI chromosome of Leptospira sp.

A clonal subpopulation of Leptospira interrogans sensu stricto is the major cause of leptospirosis outbreaks in Brazil.

Leptospira is a highly diverse genus comprising many species and serogroups in Brazil as well as all over the world. However, a study by arbitrarily primed PCR of 44 leptospiral strains isolated from humans during three different outbreaks in Brazilian urban centers reveals that 43 of 44 isolates exhibit very similar fingerprints. Analysis of these isolates indicates that they belong to a clonal subpopulation of Leptospira interrogans sensu stricto.

Identification of Borrelia burgdorferi sensu lato species in Europe.

Characterisation at the species level of 142 Borrelia isolates obtained from ticks, humans and rodents in Western Europe was carried out and their geographical distribution was described. Borrelia garinii was the predominant species representing 44% of the isolates and B. afzelii and B. burgdorferi sensu stricto constituted 27% and 19% of isolates respectively. B. valaisiana, (formerly group VS116) constituted 10.5% of isolates. Some differences in the Borrelia species distribution were observed from one country to another, possibly linked to different sources of samples. In the human samples, which were mostly collected in Austria, B. afzelii was preferentially isolated from skin and B. garinii from CSF. B. afzelii was consistently isolated from rodents captured in Switzerland, but one isolate of B. garinii was obtained from a rodent in Austria. B. garinii was by far the most abundant species isolated from Ixodes ricinus ticks in all studied countries. B. valaisiana was isolated from I. ricinus ticks collected from vegetation and from I. ricinus engorged on birds.

Leptospira genomics.

The bacterial species Leptospira interrogans (sensu stricto) has a complex genome containing two circular chromosomal replicons. Comparative analysis of the larger chromosome reveals a fluid genetic organization with many large rearrangements differentiating two closely related strains. In the present study new genes were identified by partial sequence analysis of randomly cloned fragments of L. interrogans DNA. These genes were localized in regions of the genome by nucleic acid hybridization with DNA fragments separated by pulsed-field gel electrophoresis. The resulting genetic maps provide improved resolution for each strain and provide evidence for additional chromosomal rearrangements. Insertion elements may be involved in recombination events, as several are near regions of the chromosome that have undergone rearrangement.

Direct sulfhydrylation for methionine biosynthesis in Leptospira meyeri.

A gene library of the Leptospira meyeri serovar semaranga strain Veldrat S.173 DNA has been constructed in a mobilizable cosmid with inserts of up to 40 kb. It was demonstrated that a Leptospira DNA fragment carrying metY complemented Escherichia coli strains carrying mutations in metB. The latter gene encodes cystathionine gamma-synthase, an enzyme which catalyzes the second step of the methionine biosynthetic pathway. The metY gene is 1,304 bp long and encodes a 443-amino-acid protein with a molecular mass of 45 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced amino acid sequence of the Leptospira metY product has a high degree of similarity to those of O-acetylhomoserine sulfhydrylases from Aspergillus nidulans and Saccharomyces cerevisiae. A lower degree of sequence similarity was also found with bacterial cystathionine gamma-synthase. The L. meyeri metY gene was overexpressed under the control of the T7 promoter. MetY exhibits an O-acetylhomoserine sulfhydrylase activity. Genetic, enzymatic, and physiological studies reveal that the transsulfuration pathway via cystathionine does not exist in L. meyeri, in contrast to the situation found for fungi and some bacteria. Our results indicate, therefore, that the L. meyeri MetY enzyme is able to perform direct sulfhydrylation for methionine biosynthesis by using O-acetylhomoserine as a substrate.

Recognition of two new species of intestinal spirochetes: Serpulina intermedia sp. nov. and Serpulina murdochii sp. nov.

On the basis of DNA-DNA hybridization data, nine intestinal spirochete strains were grouped into five genospecies. Three of these genospecies were previously recognized Serpulina species, Serpulina hyodysenteriae (type strain, B78), Serpulina innocens (type strain, B256), and Serpulina pilosicoli (type strain, P43/6/78; previously "Anguillina coli"). The other two genospecies were found to be new Serpulina species, for which we propose the names Serpulina intermedia sp. nov. (with type strain PWS/A) and Serpulina murdochii sp. nov. (with type strain 56-150). S. intermedia and S. murdochii cells had a typical spirochete ultrastructure with 22 to 28 periplasmic flagella per cell. Various soluble sugars were growth substrates for S. intermedia and S. murdochii. During growth in basal heart infusion broth supplemented with fetal calf serum beneath an O2-N2 (1:99) atmosphere, cells of these new species consumed oxygen and glucose and produced H2, CO2, acetate, butyrate, and ethanol. The G + C content of the DNA of S. murdochii 56-150T was 27 mol%, and the G + C content of the DNA of S. intermedia PWS/AT was 25 mol%. In addition, a restriction fragment length polymorphism-PCR assay for the detection of intestinal spirochetes was developed. The assay was based on generation and restriction endonuclease analysis (with HinfI, TaqI, Sau3A, and MboII) of a 558-bp amplicon of ribosomal DNA (rDNA) encoding 16S rRNA. The PCR amplification was specific for Serpulina species and Brachyspira aalborgi. Four restriction digest patterns were found for the five Serpulina species. HinfI restriction differentiated S. murdochii and S. innocens from the other species. Sau3A and TaqI restrictions gave unique fragment patterns for S. murdochii and S. pilosicoli, respectively. S. hyodysenteriae and S. intermedia DNAs gave the same fragment pattern regardless of the enzyme tested. B. aalborgi was differentiated from the Serpulina species by MboII digestion of the 16S rDNA amplicon.

Homoserine O-acetyltransferase, involved in the Leptospira meyeri methionine biosynthetic pathway, is not feedback inhibited.

The Leptospira meyeri serovar semaranga metX gene was identified by complementation of an Escherichia coli metA mutant, i.e., devoid of homoserine O-succinyltransferase. However, the MetX protein exhibited a homoserine O-acetyltransferase activity in agreement with its similarity to homoserine O-acetyltransferases. Reverse transcription-PCR analysis demonstrated that metX is the second gene of an operon.

Borrelia burgdorferi uridine kinase: an enzyme of the pyrimidine salvage pathway for endogenous use of nucleotides.

The 621 bp udk gene encoding Borrelia burgdorferi potential uridine kinase, involved in the pyrimidine salvage pathway, was cloned and sequenced. The B burgdorferi protein has a molecular mass of 24 kDa in sodium dodecyl sulfate-polyacrylamide gel. The N-terminal sequence of the protein, Ala-Lys-Ile-Ile, is identical to that predicted but lacks N-terminal methionine. udk is located at around 15 kb from the left telomere and forms an operon with an upstream ORF. A likely hypothesis for the role of the pyrimidine salvage pathway is the sole use of endogenous nucleotides for Borrelia.

Molecular characterization of a large Borrelia burgdorferi motility operon which is initiated by a consensus sigma70 promoter.

A large motility operon, referred to as the flgB operon, was identified, characterized, and mapped at 310 to 320 kb on the linear chromosome of the spirochete Borrelia burgdorferi. This is the first report that a sigma70-like promoter rather than a sigma28-like promoter is involved in the transcription of a major motility operon in bacteria. From these results in conjunction with results from a previous study (Y. Ge and N. W. Charon, Gene, in press), we have identified 26 genes in this operon that are relevant to motility and flagellar synthesis. With few exceptions, the gene order and deduced gene products were most similar to those of other spirochetes and Bacillus subtilis. Primer extension analysis indicated that transcription initiated from a conserved sigma70-like promoter immediately upstream of flgB; this promoter mapped within the heat-shock-induced protease gene hslU. Reverse transcriptase PCR analysis indicated that a single transcript of 21 kb initiated at this promoter and extended through flgE and (with our previous results) onto the putative motility gene flbE. The flgB promoter element had strong activity in both Escherichia coli and Salmonella typhimurium. As expected, a mutant of S. typhimurium with an inactivated flagellum-specific sigma28 factor did not affect the function of this promoter. Western blot analysis indicated that B. burgdorferi recombinant FliG and FliI were antigenically similar to those of E. coli and other spirochetes. Although complementation of E. coli or S. typhimurium fliG or fliI mutants with the B. burgdorferi genes was unsuccessful, B. burgdorferi recombinant FliI completely inhibited flagellar synthesis and motility of wild-type E. coli and S. typhimurium. These results show that spirochete motility genes can influence flagellar synthesis in other species of bacteria. Finally, Western blot analysis with sera from infected humans and animals indicated a weak or nondetectable response to recombinant FliG and FliI. These results indicate that these antigens are not favorable candidate reagents to be used in the diagnosis of Lyme disease.

A cheA cheW operon in Borrelia burgdorferi, the agent of Lyme disease.

Borrelia burgdorferi sensu stricto homologues of cheA and cheW were cloned and characterized. A combination of strategies such as polymerase chain reaction (PCR) using degenerate primers, random-primed gene walking PCR and construction of a lamda library were used to identify the putative cheA gene. Sequence analysis of the DNA fragments obtained from the CT strain identified a 2,592-bp open reading frame (ORF) encoding an 864-amino-acid protein with significant similarity (53-64.6% identical residues) to the CheA of several genera of eubacteria. In particular, hallmarks of a histidine kinase family were found such as the location of the histidine autophosphorylation domain very close to the NH2 terminus and the nucleotide-binding site. A second ORF located immediately downstream from the putative borrelial cheA gene encoded a 195-amino-acid protein which displayed a high level of similarity to bacterial CheW. Using reverse transcription PCR, we demonstrated that cheA and cheW form an operon with an upstream, unidentified ORF. The cheA and cheW homologues were located at 722-737 kbp, 738-768 kbp and 743-824 kbp on the linear chromosomes of B. burgdorferi sensu stricto, B. garinii and B. afzelii, respectively. Identification of cheA and cheW is the first step toward elucidation of a possible role of chemotaxis in virulence of the Lyme disease borreliae.

Identification and characterization of Serpulina pilosicoli isolates recovered from the blood of critically ill patients.

The phenotypic and genetic characteristics of spirochetes isolated from the blood of one U.S. and six French patients with severe clinical disease or impaired immunity were examined. All spirochetes were anaerobic, weakly beta-hemolytic, positive for hippurate hydrolysis, and negative for beta-glucosidase activity. Cell lengths ranged from 4 to 8 microm, and each isolate had between 8 and 12 periplasmic flagella per cell. These features were consistent with the spirochetes' being Serpulina pilosicoli, the agent of intestinal spirochetosis. All isolates were positive in a PCR assay amplifying a portion of the S. pilosicoli 16S rRNA gene, and they all grouped with fecal isolates of S. pilosicoli in multilocus enzyme electrophoresis (MLEE). The blood isolates could be differentiated from each other by MLEE, although the U.S. and two French isolates were closely related. Apparently S. pilosicoli may translocate from the large intestine to establish spirochetemia. The clinical significance of this finding remains uncertain and requires further investigation.

FliH and fliI of Borrelia burgdorferi are similar to flagellar and virulence factor export proteins of other bacteria.

Two motility genes (fliH and fliI) of the Lyme disease spirochete Borrelia burgdorferi were cloned, physically mapped and sequenced, FliH and FliI showed extensive homology to the proteins involved in the export of flagellar components and to virulence factors found in both animal and plant bacterial pathogens. The results suggest that the flagellar apparatus and associated protein export pathway are well conserved in evolution.

First isolation and characterisation of Borrelia garinii, agent of Lyme borreliosis, from Irish ticks.

Nymphal Ixodes ricinus, the tick vector of Lyme borreliosis, were collected from the edges of paths in Muckross Demesne, Killarney National Park, Co. Kerry, Ireland. Examination of some of these nymphs by indirect immunofluorescence showed an infection prevalence of 12% with Borrelia burgdorferi sensu lato, the spirochaete agent of Lyme borreliosis. Gerbils (Meriones unguiculatus) were infected by infesting them with other nymphs from the same batch. Subsequently uninfected laboratory larvae were applied to the gerbils and the contents of the resulting infected engorged ticks were then placed in media and the spirochaetes cultured. The spirochaetes were identified as B. burgdorferi sensu lato by indirect immunofluorescence using monoclonal antibodies and they were further characterised by polymerase chain reaction and pulsed-field gel electrophoresis. Both of these latter techniques showed that spirochaetes in all samples belonged to the genomic species, Borrelia garinii.

IS1500, an IS3-like element from Leptospira interrogans.

Copies of an insertion-sequence (IS)-like element were isolated from two closely related serovars of Leptospira interrogans sensu stricto. Nucleotide sequence analysis of the 1236 bp element showed a characteristic IS structure with terminal imperfect inverted repeats (IRs) flanking a 1159 bp central region. This element was designated IS1500. Four open reading frames (orfA-orfD) were found in the central 'unique' region of IS1500. Similarities were detected between ORFA and ORFB and the putative transposases from members of the IS3 family of transposable elements. IS1500 or IS1500-like sequences were also detected in all other pathogenic leptospiral serovars, but not in the saprophytic species L. biflexa. Differences in IS1500 copy numbers in members of the same species suggest that this element can transpose. Physical mapping of IS1500 insertions in L. interrogans serovars icterohaemorrhagiae and pomona showed insertions were only on the large chromosomal replicon. The location of some IS1500 insertions coincides with regions of the genome that have undergone large rearrangements.