Ebook: Molecular Biology of Spirochetes

This book is a compilation of presentations at a NATO Advanced Research Workshop on the “Molecular Biology of Spirochetes” held at the Czech Republic National Institute of Public Health, Prague, Czech Republic, December 5–8, 2005. This meeting was supported by the NATO Programme for Security Through Science, the United States National Institutes of Health, Office of Rare Diseases, the United States Institute of Allergy and Infectious Diseases, the Czech Republic National Institute of Public Health, Czech Republic and New York Medical College, Valhalla, New York, U.S.A. It was organized to foster the exchange of experience among scientists from NATO countries in North America, Western and Eastern Europe.

This type of encounter is valuable because diseases produced by spirochetes, including Lyme borreliosis, syphilis and leptospirosis, are on the rise worldwide, and because the biology of their causative organisms, their epidemiology, and clinical presentation display important variations in different geographical areas. For example B. burgdorferi sensu lato produces approximately 20,000 cases of Lyme borreliosis a year in the United States and 60,000 cases in Europe, but B. burgdorferi sensu stricto, B. afzelii and B. garinii are transmitted by different vectors and have different reservoirs and clinical presentations in these different geographic areas. Awareness and better understanding of these variations by researchers in the field is thus highly relevant to improvements in their prevention and treatment, and critical for improvement of human health.

The meeting was organized with oral presentations by major speakers and poster sessions by students and postdoctoral fellows from Eastern Europe. This volume includes not only the presentations of the major speakers but also several additional presentations by investigators who were invited but were unable to attend. For many reasons (including meeting organization and funding limitations), this volume does not intend to represent a comprehensive coverage of all aspects of spirochete biology. It rather focuses on a series of state of the art presentations of the research taking place in the laboratories of the contributors. As such, we hope that it may be useful as an introduction to those individuals entering in the burgeoning field of spirochete research. We would also like to believe that the meeting and this book will serve as a stimulus for researchers in the field to widen collaborations and exchanges between investigators in the different geographical areas where spirochetal diseases are common since these interactions can only be of benefit to the field.

Finally, we would like to thank the participants who risked the cold weather to attend the meeting, the authors who despite their inability to attend were willing to submit chapters to this book, the funding institutions mentioned above, and in particular, Drs. Phil Baker, Patti Rosa, Tom Schwan, Frank Gherardini, Ms. Marylin E. Kunzweiler (United States National Institute of Allergy and Infectious Diseases), Dr. Jorge Benach (Stony Brook University), Ms. Mary C. Demory (United States National Institutes of Health), Dr. Marina Cinco (University of Trieste), Dr. Michael Norgard (University of Texas Southwestern Medical Center) and Dr. Ira Schwartz (New York Medical College), whose efforts were critical to our securing some of the funds that supported the meeting, and TestLine, Qiagen, BioConsult, Roche, Biowestern, Generi BioTech and BagMed who provided additional support for this meeting. We would also like to thank Ms. Leonor Delgado for editorial assistance and Ms. Harriett V. Harrison for her outstanding and continuing assistance in organizing the meeting and in preparing the manuscripts that compose this book.

Felipe C. Cabello, New York Medical College, Valhalla, New York, USA

Dagmar Hulinska, Czech Republic National Institute of Public Health, Prague, Czech Republic

Henry P. Godfrey, New York Medical College, Valhalla, New York, USA

July, 2006

In all of the major human spirochetal infections, the fundamental pathogenetic event underlying the most serious complications of these diseases is documented or presumed hematogenous dissemination of the spirochete from the site of inoculation to distant sites. Lyme borreliosis is attractive for study of spirochetal dissemination for a variety of reasons including: the availability of a large patient base, the ability to identify patients early during the course of infection, and the ability to culture Lyme borrelia readily in vitro. Tick feeding per se does not directly lead to blood stream invasion by Lyme borrelia. The genotype of the strain of Borrelia burgdorferi introduced by the tick, however, does appear to be an important determinant of dissemination in humans and other mammals. Recent evidence suggests that host factors affect both the development of infection and subsequent dissemination of this spirochete as well. In one United States study, independent risk factors for hematogenous dissemination of B. burgdorferi included having a first episode of Lyme borreliosis and being more than 55 years of age. In another United States study, infection due to the least invasive genotype of B. burgdorferi was associated with carriage of the HLA class II allele DRB1^*0101. All of the major human spirochetal infections are also characterized by persistence of the spirochete in mammalian hosts, and depending on the specific spirochete and host involved, this phenomenon may be closely linked to the pathogenesis of important clinical manifestations and to communicability to uninfected hosts. In conclusion, spirochetemia and persistence are common and important pathogenetic features of the major human spirochetal infections.

Transposon mutagenesis is a powerful technique for generating random mutations. Previous studies had shown that the mariner transposon Himar1 could be utilized to introduce mutations in high-passage, noninfectious strains of Lyme disease Borrelia; however, transposon mutagenesis has not been reported in low-passage, infectious strains. In this pilot study, Himar1 transposon mutants were generated by transforming the infectious, highly transformable B. burgdorferi B31 clone 5A18 NP1 with the transposon vector pMarGent. The data provide 'proof in concept' that random transposon mutagenesis can be utilized for the global analysis of genes involved in the pathogenesis of Lyme disease Borrelia.

Borrelia burgdorferi, the causative agent of Lyme disease, is the most prevalent arthropod-borne infectious agent in the United States and, along with infections attributed to other related Borrelia spp. worldwide, contributes to a significant amount of morbidity. Much of what is known regarding pathogenic mechanisms of B. burgdorferi has been limited to biochemical analyses due to the genetic intractability of this spirochetal pathogen. Recently, advances by a number of investigators have made it possible to isolate and genetically complement isogenic mutants in infectious B. burgdorferi as a first step in determining the role of specific genes in Lyme borreliosis. The approach described here involves a customized transposon that contains an antibiotic resistance marker expressed from a strong borrelial promoter, which is used to mutagenize a borrelial gene target in vitro. The resulting transposon mutagenized candidates are then mapped and sequenced. The desired constructs, containing the inactivated gene, are transformed into a low passage but non-infectious B. burgdorferi strain lacking the 25 kb linear plasmid (lp25) and putative transformants selected with the appropriate antibiotic. The lp25 deficient strain used here is more amenable to transformation due to the absence of an lp25-specific restriction/modification system. Using this genetic background, several borrelial genes purported to be involved in B. burgdorferi pathogenesis have been genetically inactivated. The loss of infectivity associated with the lp25 deficient background can be restored back to wild-type levels by transformation with the borrelial shuttle vector pBBE22, which encodes the lp25 bbe22 locus. The bbe22 (pncA) gene of this strain encodes a nicotinamidase enzyme required for de novo biosynthesis of NAD only during infection. Thus, transformation with pBBE22 serves two purposes, as it: (i) restores infectivity in the parent background, and (ii) asks whether the resulting mutant exhibits an infectivity deficient phenotype relative to the parent background using the well-established mouse model of infection. Subsequent genetic complementation with an intact version of the targeted gene in pBBE22 is then carried out to ensure that the mutant phenotype observed can be attributed to the inactivated gene. The strategy presented here could be applied to the genetic analysis of other borrelial genes to determine their significance in the pathogenesis of Lyme borreliosis.

Motility and chemotaxis are important in the disease process for many species of bacteria. When infected ticks feed, B. burgdorferi moves from the midgut to the salivary gland, and when uninfected ticks feed on infected animals, the spirochete migrates from the animal into the tick. Motility and chemotaxis are likely to be involved in this traveling of B. burgdorferi between the two hosts. Compared with the well-studied Escherichia coli, motility and chemotaxis in B. burgdorferi is quite complex: it has 2 cheA, 3 cheY, 2 cheB, 2 cheR and 2 fliG homologues. Instead of the E. coli phosphatase CheZ, B. burgdorferi has a CheY-P phosphatase called CheX. Furthermore, B. burgdorferi has to coordinate two bundles of periplasmic flagella, extending from opposite ends of the cell, to move toward attractants and away from repellents. B. burgdorferi exhibits different motility modes, consisting of runs, flexes, and reverses, which depend on movement of the bundles of periplasmic flagella. Analysis of the B. burgdorferi genome sequence revealed that it has no homologues of the sigma-28 or anti-sigma factor genes responsible for cascade control of motility gene regulation found in E. coli or Salmonella enterica serovar Typhimurium. Rather, studies indicate that motility genes in B. burgdorferi are regulated by sigma-70 promoters. To determine the role(s) of individual motility genes, we deployed targeted mutagenesis technology and analyzed the resulting cell phenotypes based on the flagellar structure, cell swimming behavior, and effect of specific gene inactivation on other genes and on chemotaxis. Novel functions of many of these genes of the Lyme disease organism are summarized.

The genus Leptospira belongs to the order Spirochaetales and is composed of both saprophytic and pathogenic members, such as Leptospira biflexa and L. interrogans, respectively. A major factor contributing to our ignorance of spirochetal biology is the lack of methods for genetic analysis of these organisms. We have developed a system for transposon mutagenesis of L. biflexa using a mariner transposon, Himar1. This mutagenesis approach yields a randomly distributed set of insertion mutations throughout the genome, which can be screened for specific phenotypes. An analysis of transposon mutants has allowed the identification of genes required for diverse biological activities such as amino acid biosynthesis and metal transport. The development of numerous genetic tools for saprophytic species of Leptospira enables the use of these bacteria such as L. biflexa as a model bacterium We therefore sequenced the genome of the saprophyte L. biflexa consisting of a 3.6-megabase large chromosome and a 278-kilobase small chromosome. Comparative genomics, in combination with gene inactivation, give us significant functional information on iron homeostasis in Leptospira.

Antibiotic resistance is not a clinical problem with Lyme disease, but it has been extensively employed to genetically dissect the causative agent Borrelia burgdorferi. The first selectable marker was a coumermycin A1-resistant gyrB allele, which encodes a subunit of DNA gyrase, a target of several antibiotics. The utility of coumermycin A1 resistance has been compromised by technical and genetic barriers; resistance to other antibiotics has replaced the gyrB marker. Fluoroquinolones are another class of antibiotics that target DNA gyrase, as well as its homolog topoisomerase IV. Fluoroquinolone resistance in B. burgdorferi maps to parC, which encodes a subunit of topoisomerase IV, suggesting that this enzyme is the primary target of fluoroquinolones in Borrelia. A fluoroquinolone-resistant parC allele has been fashioned into a counter-selectable marker, a genetic tool used to select for the loss of DNA. One of the second-generation selectable markers is a heterologous aadA gene that confers resistance to spectinomycin and streptomycin, which target the small subunit of the ribosome. Selection with spectinomycin failed due to a high frequency of mutants in the population. These had mutations in 16S rRNA and were able to compete with wild type in vitro. This lack of a significant fitness cost for the mutant may contribute to the spread of antibiotic resistance.

Electroporation of Treponema denticola allowed for construction of monospecific mutants in this oral spirochete. The subsequent construction of shuttle plasmids enabled the expression of heterologous genes in these organisms. More recently, a comparable expression system has been developed in T. phagedenis.

We have determined draft whole genome sequences for B. burgdorferi isolates N40 and JD1 and the plasmids from isolate 297. The comparison of these draft sequences sheds considerable light on a number of aspects of their biology including horizontal exchange of genetic information, plasmid diversity, lipoprotein variability, and plasmid partitioning. We find that plasmids cp9, cp26, the cp32s, and lp54 are quite constant in gene organization among the four strains, but the remaining linear plasmids are quite variable in organization. We analyze the replication, partitioning, and compatibility gene cluster on the plasmids from these four strains and propose to use them in a universal B. burgdorferi plasmid nomenclature system.

Treponemes include the agents of syphilis, yaws, and other diseases, but as a group are fastidious organisms that often can only be grown in animals. This has hampered molecular analysis, but much progress is being made since genome sequences for these bacteria have been completed. The current state of the art involves comparative genomics, microarrays, whole genome clone sets, and other tools to unravel the biology of these pathogens.

The presence of two chromosomes makes Leptospira unusual amongst its closest relatives in the bacterial world. The Leptospira genome is in a state of flux, as indicated by the presence of many chromosomal rearrangements that alter genetic organization between individual serovars. It is therefore somewhat remarkable that at least two Leptospira loci (LPS biosynthetic genes and the S10-spc-alpha ribosomal protein operon) form large, extended operons that are among the longest bacterial operons reported to date. Insertion sequences (IS) that are distributed throughout Leptospira genomes contribute to the formation of rearrangements. These elements can transpose and disrupt the integrity of genes, or alternatively, can activate cryptic genes by providing promoter activity to genomic sequences downstream of the insertion site. Bioinformatics and experimental functional analyses were used to characterize the L. interrogans genomes and thus gain insight into this organism's biology. Quantitative analysis of the L. interrogans serovars Lai and Copenhageni genomes showed that these bacteria are proficient in environmental sensing and response, and in nutrient transport. These data support epidemiological evidence that L. interrogans is transmitted primarily by passage through environmental sources. Few pseudogenes were detected in either strain, suggesting that there is sufficient selective pressure to maintain a highly functional genome. However, several genes were identified that are complete in one strain but have frameshifts in the other that may affect phenotype. Further differences in phenotype may also result from gene acquisition, and we found several large, serovar-specific gene clusters. Analysis of an ECF locus from L. interrogans serovar Pomona is used to show how RT-PCR and expression vectors can be used to localize promoters in L. interrogans. Antisera produced against recombinant fusion proteins were used to detect invasion of lung, liver, and kidney during experimental infection of hamsters with serovar Pomona. These data are consistent with some of the clinical manifestations of severe leptospirosis and help to illustrate how genomic analysis can aid in the understanding of these pathogenic bacteria.

Leptospirosis, an emerging infectious disease, is a worldwide zoonosis of human and veterinary concern. Caused by pathogenic spirochaetes of the genus Leptospira, the disease presents greater incidence in tropical and subtropical regions. Humans can be infected by exposure to chronically infected animals and their environment. The genome sequence of Leptospira interrogans serovar Copenhageni was recently reported. It contains a broad array of genes encoding for regulatory system, signal transduction, and methyl-accepting chemotaxis proteins, conforming to the organism's ability to respond to diverse environmental stimuli. A large number of exported lipoproteins and transmembrane outer membrane proteins were identified that may be involved in leptospiral pathogenesis and protective immunity. Comparative analysis with the Leptospira interrogans serovar Lai genome revealed that, despite genetic similarity, there are structural differences, including a large chromosomal inversion. The leptospiral genome sequence, combined with bioinformatics tools, offered a unique opportunity to search for immune targets to be used for vaccine or diagnostic kit development. Out of a hundred recombinant proteins tested, sixteen were recognized by antibodies present in sera from patients diagnosed with leptospirosis and might be useful for these purposes. The most important results obtained within genome sequences, comparative genomics, and outer membrane genome-derived protein expressed in E. coli are reviewed here.

Lyme disease, the most commonly reported arthropod-borne disease in the United States, is caused by infection with the spirochete, Borrelia burgdorferi. The acute stage of the infection has a varied presentation, ranging from mild, localized disease (characterized by a skin lesion) to highly symptomatic, disseminated disease. We hypothesize that this is due, in part, to B. burgdorferi genotypic variation. By combining PCR amplification of the 16S-23S rDNA spacer with restriction fragment length polymorphism (RFLP) analysis, several genotypes were identified among B. burgdorferi clinical isolates obtained from either skin or blood of early Lyme disease patients. Hematogenous dissemination in humans is associated with a distinct genotype and disease severity and spirochete burden was also associated with this same genotype in a murine model of Lyme disease. A genomic approach was undertaken to elucidate the differences in genome content and/or gene expression that may result in disease variability. Comparative transcriptional profiling of two clinical isolates with distinct genotypes (invasive and attenuated) was performed using whole genome arrays. A total of 78 ORFs had significantly different expression levels in the two isolates. Nearly 25% of the differentially expressed genes are predicted to be localized on the cell surface, implying that these two isolates have considerably different cell surface properties. Comparative genome hybridization demonstrated that genotypic variation largely results from differences in plasmid content and/or sequence and revealed several plasmid-encoded candidate genes that are uniquely absent in attenuated strains. A number of genes identified in these investigations are currently under further study by genetic analysis to substantiate a possible role in virulence.

Thirty years after it was introduced, whole DNA/DNA hybridization (WDDH) is still considered as the gold standard for bacterial taxonomy. However, WDDH has serious limitations, as it requires large volumes of cultures, is a highly fastidious and time consuming procedure, lacks of reproducibility, and requires pairwise comparisons between unknown genome and genomes from previously described species. These limits have hampered progress in Borrelia taxonomy. With the development of sequencing, it is inviting to propose MLSA as an alternative to WDDH. We chose fragments from seven loci: rrs (16S rRNA), flaB, groEL, hbb, recA, ospA, and the internal transcribed spacer rrf-rrl (ITS). We sequenced these loci for 2–4 representative Borrelia burgdorferi sensu lato strains from each known species present on both the European and North American continents. Phylogenetic trees were built from sequences of each locus, confirming that lateral transfer was a rare event, and from the concatenated sequence of the seven loci from each strain. Genetic distances were calculated from concatenated sequences and compared to those obtained by WDDH (both published and unpublished). The two methods were strictly correlated. A precise cutoff of MLSA-based genetic distances (0.021) allowed us to delineate new species more safely and accurately than the usual 70% DNA relatedness cutoff. We have confirmed in this way a recently described sixth species in Europe, B. spielmanii, whose reservoirs are dormice. Three new nonpathogenic Borrelia species are delineated in California, genomospecies 1, 2, and 3. Finally, the phylogenetic tree obtained from the concatenated sequences from 42 isolates (from both Europe and North America) shows 12 clusters, each corresponding to a species. These clusters form two main groups. One group comprises only European species, whereas the other group comprises B. burgdorferi sensu stricto isolates from Europe and the USA, including both Borrelia spp. “border line” to B. burgdorferi sensu stricto. This clustering confirms our previous hypothesis of the American origin of B. burgdorferi sensu stricto and closely related isolates that have been imported recently into Europe from the USA.

Diversity of Lyme disease spirochetes and their protein encoding genes play a major role in pathogenesis and diagnosis of Lyme borreliosis. In a survey of 72 CSF isolates from Germany, we found 15 (20.5%) Borrelia afzelii, 39 (53.4%) B. garinii, and 18 (24.7%) B. burgdorferi sensu stricto ; among 160 skin isolates, B. afzelii was present in 107 (66.8%), B. garinii in 39 (24.3%), B. burgdorferi sensu stricto in 10 (6.3%), and the new species B. spielmanii in 4 (2.5%). A small number of isolates from synovial fluids (n=6) revealed heterogeneity of the causative strains (2 B. burgdorferi sensu stricto , 2 B. afzelii, and 2 B. garinii). Among 507 PCR-positive ticks from Southern Germany, 22% harboured B. burgdorferi sensu stricto , 25% B. afzelii, 34% B. garinii, 16% B. valaisiana, and 6% B. spielmanii. Sequence identities among major immunodominant proteins (DbpA, VlsE, OspC, OspA, BmpA, p83/100, p58, and flagellin) from the three main human pathogenic species increase from 40–44% to 96–97%. Comparison of dbpA sequences with ospC sequences from a panel of 59 strains revealed all kinds of cross-connections indicating processes of lateral gene transfer. The extent of sequence identities among the dbpA genes decreased from the DNA (67%) to the AA level (44%) about 23%, and ospC sequence identities differed about 10%, an indication that both proteins, but especially DbpA, play a role in immune escape. A combination of four DbpAs (including two different B. garinii DbpAs) was crucial for sensitive serodiagnosis. The use of primarily in vivo-expressed recombinant proteins like OspC, DbpA, and VlsE significantly improved antibody detection. VlsE and DbpA are the most sensitive antigens for IgG antibody detection. OspC is not only the immunodominant protein of the early (IgM) response in the infected host, but it is also crucial for host infectivity. It is controversial to assume that OspC is necessary for borrelial invasion of the tick salivary glands. We found that an OspC-positive B. afzelii wild type strain disseminated from the midgut to the salivary glands, while an OspC-negative mutant was only present in the tick midguts. Colonization of salivary glands by the mutant was restored by complementation of this strain with a plasmid construct that constitutively expresses the wild type ospC. Thus Borrelia strains might also differ in their potential to disseminate from the gut to the ticks' salivary glands.

Relapsing fever Borrelia challenge microbiological typing as, unlike other microbes, they possess segmented genomes maintaining essential genes on large linear plasmids. Antigenic variation further complicates typing. Intragenic spacer (IGS, between 16S-23S genes), provides resolution among Lyme-associated and some relapsing fever spirochaetes. When applied to East African relapsing fever borreliae, two and four types were found respectively among Borrelia recurrentis and B. duttonii. However, IGS typing was unable to discriminate between the tick- and louse-borne forms of disease, raising the question as to whether these are indeed separate species. To address this question, further genes were sequenced to produce a multi-locus approach to resolve whether these are either a single or different species. Various housekeeping genes were selected from data deposited for B. hermsii (limited sequence information exists for either B. recurrentis or B. duttonii). Of selected targets, sufficient data was produced only from glpQ. Further genes analysed included flaB, rrs rDNA, and P66 outer membrane protein. Sequence comparison of multiple genes was undertaken, but restricted through the limited number of available isolates of these notoriously fastidious organisms that until recently were considered non-cultivable. Whereas the IGS typing was applied to a range of clinical isolates, patient blood samples and arthropod vectors, other genes were sequenced only from cultivable strains, potentially introducing a bias to the results. Our data highlights the remarkable similarity between these Borrelia with only minor differences at the nucleotide level. Collectively, this suggests a common ancestral lineage for these spirochaetes, with the limited differences revealed at the nucleotide level from these cultivable strains being able to divide both “species” into separate clades; however, it must be stressed that these differences ranged from 2 to 10 nucleotides depending on the gene used. It is more likely that these are clades of the same species, which have accumulated adaptive changes through time and pressures of different vector transmission. In contrast, the IGS sequence, being non-coding, is not under such selection and in consequence probably reflects changes accumulated over time alone, but without the constraints of producing functional gene products. Full genomic sequence analysis should reveal further insights into the taxonomic relationship between these microbes and elucidate the molecular basis of arthropod competence and pathogenicity among these spirochaetes.

The Borrelia culture collection at the Vector Laboratory of the Gamaleya Research Institute of Epidemiology and Microbiology, Moscow dates from 1983. Today, this collection consists of almost 1,200 primary isolates from different sources from various geographic regions. A PCR–RFLP analysis of isolates from Russia and neighboring countries has shown that they include primarily genospecies of B. garinii and B. afzelii, which are widespread and epidemiologically significant, along with B. burgdorferi sensu stricto, B. valaisiana, B. lusitaniae, and B. spielmanii (group A14S). Considerable genetic heterogeneity of B. afzelii has been revealed by comparing the sequences of the rrf (5S)-rrl (23S) ribosomal intergenic spacer in 139 primary isolates obtained from ixodid ticks and from several species of small mammals. All these isolates were previously identified as B. afzelii by PCR–RFLP analysis of amplicons of the 5S–23S rRNA spacer region. Two genomic subgroups have now been identified. The majority of the isolates have a high degree of homology to the most widespread genomic subgroup of B. afzelii, strain VS461 (98.8–99.6%). The remainder have a high degree of homology to the other genomic subgroup B. afzelii, strain NT28 (98.4–99.6%). Several genovariants have been identified within each subgroup with the degree of nucleotide homology within most of them reaching 100%. Seven of ten genovariants belong to subgroup VS461, three genovariants belong to subgroup NT28. Seven allelovariants have been detected by comparing the sequences of a p66 gene fragment (246–377 bp) in 45 B. afzelii isolates. Three of seven B. afzelii VS461 genovariants are apparently common in Eurasian natural foci among different species of vectors and reservoir hosts, with one genovariant probably circulating mainly in foci where I. persulcatus is the main vector of Borrelia infection. The remaining four genovariants of this subgroup appear to be associated with I. ricinus ticks and circulate mainly in Europe. The distribution of the three genovariants belonging to B. afzelii NT28 subgroup needs further study. Nucleotide sequences of approximately 100 isolates of different Borrelia genospecies have been deposited in GenBank.

Spirochaetes within the Leptospiraceae family, encompassing the genera Leptospira, Leptonema and Turneria, are known to display substantial phenotypic and genetic variation resulting in their capacity to colonize diverse natural habitats worldwide. There are many lines of evidence to suggest that these microorganisms belong to at least three ecotypes. The first type consists of free-living nonparasitic leptospires inhabiting water, including marine and soil environments (Leptospira biflexa sensu lato). Not infrequently, saprophytic leptospires contaminate nutrition media, their occasional alleged isolation from mammalian hosts or humans resulting not only in misdiagnosis, but also in taxonomic confusion. The second ecological type consists ofparasitic leptospires showing pathogenic potential for humans and various animal species as well as host and tissue specificity of a wide spectrum (L. interrogans sensu lato). The third group comprises leptospires with life styles intermediate between independent and symbiotic (also within L. interrogans sensu lato). The latter (for example, L. kirshneri serovar grippotyphosa) display features of so-called sapronotic agents: high survival and competitive potential in environmental ecosystems, widespread mammalian host range and pronounced antigenic and genetic heterogeneity. This paper addresses ecological and genetic diversity within the Leptospiraceae family as relevant to epidemiological and taxonomic issues.

Lyme disease has continued to spread in the Czech Republic, and there have been increasing numbers of cases in central and eastern Bohemia and Moravia. We have shown that Borrelia burgdorferi sensu lato causes infection by migration through connective tissues of the skin, heart, liver, and synovium without inflammation and by adhesion to host cells. Twenty-three isolates of B. burgdorferi sensu lato originally cultured from cerebrospinal fluid, blood, and tissue samples were characterized phenotypically by immunosorbent electron microscopy and Western blots and genotypically by sequence analysis of the outer surface (Osp) A and C gene fragments. Comparison of genotyping of borreliae performed directly in patients and in Ixodes ricinus ticks collected in the same endemic regions with typing of cultures revealed that a large proportion of directly typed samples had more heterogenic Borrelia types or combined sequence variants. In addition, B. garinii OspA types 6 and 5 were in coinfection with B. valaisiana and rarely with B. burgdorferi, but OspA subtype 4 was not found in the mixure. More frequent and heterogenic direct proof of B. burgdorferi sensu lato in CSF and blood versus culture reflects either different behavior and exigencies of different genospecies or intergenospecies exchange caused by human and animal migration.

Arthropod-borne bacteria can cause a variety of human infectious diseases and have a high diversity of geographic distributions, animal reservoirs, arthropod vectors, and pathogenic properties. Human-biting, questing adult Ixodes persulcatus and Dermacentor reticulatus ticks, as well as mosquitoes and midges from Western Siberia, Russia, were tested for infection with Borrelia, Bartonella, Rickettsia, Anaplasma/Ehrlichia, and Babesia using nested PCR assays with subsequent sequencing. I. persulcatus ticks were found to be infected with Borrelia spp. (39.5 ± 4.5%), Bartonella spp. (37.6 ± 4.3%), Rickettsia tarasevichiae (90.0 ± 4.8%), Anaplasma phagocytophilum (2.4 ± 1.4%), and Ehrlichia muris (8.8 ± 2.5%), whereas D. reticulatus ticks contained DNA of Borrelia spp. (3.6 ± 2.0%), Bartonella spp. (21.4 ± 4.5%), R. tarasevichiae (3.2 ± 3.2%), Rickettsia sp. RpA4 (51.6 ± 9.1%), and Babesia canis canis (3.6 ± 2.0%). Borrelia garinii, B. afzelii, and their mixed infections were observed among I. persulcatus, whereas B. garinii and B. spielmanii DNA were present in samples from D. reticulatus. Surprisingly, only two human pathogens—Bartonella henselae and B. quintana—were found in ixodid ticks in Siberia, despite long-term sample collection and phylogenetic analysis of all known Bartonella species. Moreover, currently both B. henselae and B. quintana, but none of other tick-borne infectious agents studied, were found in mosquitos of the genus Aedes. Bartonella DNA was detected in 1.9 ± 2.1% Aedes cantans mosquitoes but not in samples isolated from mosquitoes of other species including Ae. punctor, Ae. cinereus, and Ae. communis, or in Byssodon maculata midges. Ba. canis canis was the only subspecies found in D. reticulatus, but no Babesia species were observed in I. persulcatus. Thus, both bacterial infection rates and loads for I. persulcatus exceeded those for D. reticulatus and Aedes spp. Considering the large number of ticks implicated in the transmission of bacteria, human exposure to these infectious agents may be more substantial than is currently believed. From 369 patients with clinical manifestations of generalized infection following tick bites, Borrelia DNA was found in 43 plasma samples, Bartonella DNA in 73 blood cell samples, and R. sibirica DNA in a single sample. Molecular typing indicated the prevalence of infection with B. garinii and B. quintana. Neither Ehrlichia/Anaplasma nor Babesia was found in human specimens in the Novosibirsk region.

The development of genetic systems for B. burgdorferi has allowed identification of several regulatory and structural genes involved in the ability of this bacterium to colonize and disseminate in its arthropod and mammalian hosts. Progress has been slow as a result of the complex biology of this pathogen, and its genetic distance from Gram-positive and Gram-negative bacteria has impeded easy adaptation to B. burgdorferi of genetic tools developed for other bacteria. We have repeatedly encountered these issues in our attempts to use molecular genetic tools to analyze the role of the members of the paralogous bmp gene family in the biology and virulence of B. burgdorferi. For example, an attempt to characterize the role of bmpC in virulence using a bmpC deletion mutant of an infectious B. burgdorferi 297 strain and an extrachromosomally complemented derivative of this mutant was only partially successful. While deletion of bmpC did not decrease infectivity in mice, it significantly decreased B. burgdorferi DNA in joints and arthritis. Extrachromosomal complementation of this null mutation reduced infectivity and did not restore tissue pathogenicity. To overcome these problems, we have adapted the Tet system of controlled gene expression developed for other bacteria and eukaryotic cells to manipulate gene expression in B. burgdorferi. In a two plasmid Tet system, where one plasmid contains the TetR repressor fused to the flaB promoter and the other plasmid contains a hybrid borrelial promoter with a tet operator (Ptetl-1), expression of the gene fused to the Ptetl-1 promoter (truncated BmpA, green fluorescent protein) is regulated by anhydrotetracycline in a concentration dependent manner over a wide range of concentrations. Because the two plasmid Tet system has some inherent instability manifested by escape from TetR repression and potential plasmid loss requiring continuous antibiotic selection, we constructed a B. burgdorferi strain containing the flaB/tetR fusion inserted in a nonessential gene (luxS) and designed a new hybrid promoter containing two instead of one tet operators (Ptetl-2). The functionality of this Tet system was demonstrated by manipulating expression of the B. burgdorferi bmpA gene. These findings strongly suggest that the Tet system can be used in B. burgdorferi to manipulate gene expression and open the door to manipulating gene expression by antisense RNA technology and gene fusions that will permit isolation of conditional lethal mutants in B. burgdorferi.