Functional testing of putative oligopeptide permease (Opp) proteins of Borrelia burgdorferi: a complementation model in opp(-) Escherichia coli

Studies of the protein function of Borrelia burgdorferi have been limited by a lack of tools for manipulating borrelial DNA. We devised a system to study the function of a B. burgdorferi oligopeptide permease (Opp) orthologue by complementation with Escherichia coli Opp proteins. The Opp system of E. coli has been extensively studied and has well defined substrate specificities. The system is of interest in B. burgdorferi because analysis of its genome has revealed little identifiable machinery for synthesis or transport of amino acids and only a single intact peptide transporter operon. As such, peptide uptake may play a major role in nutrition for the organism. Substrate specificity for ABC peptide transporters in other organisms is determined by their substrate binding protein. The B. burgdorferi Opp operon differs from the E. coli Opp operon in that it has three separate substrate binding proteins, OppA-1, -2 and -3. In addition, B. burgdorferi has two OppA orthologues, OppA-4 and -5, encoded on separate plasmids. The substrate binding proteins interact with integral membrane proteins, OppB and OppC, to transport peptides into the cell. The process is driven by two ATP binding proteins, OppD and OppF. Using opp-deleted E. coli mutants, we transformed cells with B. burgdorferi oppA-1, -2, -4 or -5 and E. coli oppBCDF. All of the B. burgdorferi OppA proteins are able to complement E. coli OppBCDF to form a functional Opp transport system capable of transporting peptides for nutritional use. Although there is overlap in substrate specificities, the substrate specificities for B. burgdorferi OppAs are not identical to that of E. coli OppA. Transport of toxic peptides by B. burgdorferi grown in nutrient-rich medium parallels borrelial OppA substrate specificity in the complementation system. Use of this complementation system will pave the way for more detailed studies of B. burgdorferi peptide transport than currently available tools for manipulating borrelial DNA will allow.     

Host-specific functional compartmentalization within the oligopeptide transporter during the Borrelia burgdorferi enzootic cycle

Borrelia burgdorferi must acquire all of its amino acids (AAs) from its arthropod vector and vertebrate host. Previously, we determined that peptide uptake via the oligopeptide (Opp) ABC transporter is essential for spirochete viability in vitro and during infection. Our prior study also suggested that B. burgdorferi employs temporal regulation in concert with structural variation of oligopeptide-binding proteins (OppAs) to meet its AA requirements in each biological niche. Herein, we evaluated the contributions to the B. burgdorferi enzootic cycle of three of the spirochete’s five OppAs (OppA1, OppA2, and OppA5). An oppA1 transposon (tn) mutant lysed in the hyperosmolar environment of the feeding tick, suggesting that OppA1 imports amino acids required for osmoprotection. The oppA2tn mutant displayed a profound defect in hematogenous dissemination in mice, yet persisted within skin while inducing only a minimal antibody response. These results, along with slightly decreased growth of the oppA2tn mutant within DMCs, suggest that OppA2 serves a minor nutritive role, while its dissemination defect points to an as yet uncharacterized signaling function. Previously, we identified a role for OppA5 in spirochete persistence within the mammalian host. We now show that the oppA5tn mutant displayed no defect during the tick phase of the cycle and could be tick-transmitted to naïve mice. Instead of working in tandem, however, OppA2 and OppA5 appear to function in a hierarchical manner; the ability of OppA5 to promote persistence relies upon the ability of OppA2 to facilitate dissemination. Structural homology models demonstrated variations within the binding pockets of OppA1, 2, and 5 indicative of different peptide repertoires. Rather than being redundant, B. burgdorferi’s multiplicity of Opp binding proteins enables host-specific functional compartmentalization during the spirochete lifecycle.     

Sequence of the complete osp operon encoding two major outer membrane proteins of a European Borrelia burgdorferi isolate (B29)

The nucleotide sequence of the operon encoding the major outer surface proteins, OspA and OspB, of a European isolate of Borrelia burgdorferi (strain B29) was determined and compared to the osp operon of the American strain, B31. An amino acid (aa) identity of 80.7% was found when comparing the OspA of B29 with that of B31, whereas the aa sequence of the OspB of B29 reveals only 61.3% identity with the OspB of B31. Thus, strains B31 and B29 can be regarded as representatives of different B. burgdorferi groups.     

Cross-antigenicity between the major surface proteins (ospA and ospB) and other proteins of Borrelia burgdorferi

Two of the major surface Ag of Borrelia burgdorferi, the 31-kDa OspA and 34-kDa OspB proteins, are encoded by a 49-kb plasmid. In this study, mAb and monospecific polyclonal antibodies were used to define cross-antigenicity of the OspA and OspB protein to each other and to other lower molecular mass proteins by Western blot analysis. Two mAb studied, 105.5 and 184.1, were directed predominantly against the 31-kDa OspA protein. However, each also reacted with other minor bands, though with different specificities. Using V8 protease digestion and cleavage by cyanogen bromide, we demonstrated that each mAb reacted to the 31-kDa protein differently. Monospecific polyclonal rabbit and human antibodies directed against the 34-, 31-, 22-, and 20-kDa proteins were eluted from blots and used to further corroborate the cross-reactivity among these Ag. Rabbit antibodies to the 31- and 22-kDa Ag gave remarkably similar peptide maps after V8 protease digestion of the 31-kDa OspA protein, as did mAb 184.1, suggesting that this mAb recognized an immunodominant epitope common to the 22- and 31-kDa proteins. It seems likely therefore that the humoral immune response to Borrelia surface Ag may be due to a limited number of cross-reactive epitopes on distinct, but related, gene products.     

Molecular characterization of a flagellar/chemotaxis operon in the spirochete Borrelia burgdorferi

A chemotaxis gene cluster from Borrelia burgdorferi, the spirochete that causes Lyme disease, was cloned, sequenced, and analyzed. This cluster contained three chemotaxis gene homologs (cheA, cheW and cheY) and an open reading frame we identified as cheX. Although the major functional domains for B. burgdorferi CheW and CheY were well conserved, the size of cheW was significantly different from the homolog of other bacteria. Phylogenetic analysis of CheY indicated that B. burgdorferi constitutes a distinct branch with Treponema pallidum and is closely associated with Archea and Gram-positive bacteria. RT-PCR analysis indicated that the chemotaxis genes and the upstream flagellar gene flaA constitute an operon. Western blot analysis using antibody to Escherichia coli CheA resulted in two reactive proteins in the cell lysates of B. burgdorferi that is consistent with two cheA homologs being present in this organism. The results taken together suggest both similarities and differences in the chemotaxis apparatus of B. burgdorferi compared to those of other bacteria.     

Regulation of peptide transport in Escherichia coli: induction of the trp-linked operon encoding the oligopeptide permease.

Growth of Escherichia coli in medium containing leucine results in increased entry of exogenously supplied tripeptides into the bacterial cell. This leucine-mediated elevation of peptide transport required expression of the trp-linked opp operon and was accompanied by increased sensitivity to toxic tripeptides, by an enhanced capacity to utilize nutritional peptides, and by an increase in both the velocity and apparent steady-state level of L-[U-14C]alanyl-L-alanyl-L-alanine accumulation for E. coli grown in leucine-containing medium relative to these parameters of peptide transport measured with bacteria grown in media lacking leucine. Direct measurement of opp operon expression by pulse-labeling experiments demonstrated that growth of E. coli in the presence of leucine resulted in increased synthesis of the oppA-encoded periplasmic binding protein.     

Borrelia burgdorferi in ticks of the Canton Tessin (Switzerland)

In the northern Alps it is hard to find an Ixodes ricinus population which does not harbour Borrelia burgdorferi. The infection rates range from 5 to 34% for adults and nymphs to 3.1% for larvae. This study shows that the infection rates on the southern side of the Alps are similar, at 25% for adults, 36.2% for nymphs and 3.2% for larvae. With respect to tick species other than I. ricinus, we did not succeed in finding any spirochetes in Dermacentor marginatus, Haemaphysalis punctata, Ixodes hexagonus, Rhipicephalus sanguineus.     

Shaping the Borrelia burgdorferi genome: crystal structure and binding properties of the DNA-bending protein Hbb

The genome of the Lyme disease-causing spirochete Borrelia burgdorferi encodes only a single polypeptide from the integration host factor (IHF)/HU or 'DNABII' family of nucleoid-associated proteins - Hbb. DNABII proteins induce large bends in DNA and serve as architectural factors in a variety of prokaryotic cellular processes. We have solved the crystal structure of an Hbb-DNA complex in which the DNA is bent by over 180 degrees . We find that like IHF, Hbb relies exclusively on indirect readout to recognize its cognate site. Additional binding studies show that the sequence preferences of Hbb are related to, yet distinct from those of IHF. Defining these binding characteristics may help to uncover additional roles for Hbb in Borrelia DNA metabolism as well as further our understanding of the mechanism of indirect readout.     

Transmission of Lyme disease spirochetes (Borrelia burgdorferi)

The field and laboratory evidence incriminating nymphalIxodes dammini as the main vectors ofBorrelia burgdorferi is substantial. Furthermore, other members of theIxodes (Ixodes) ricinus complex, includingI. ricinus, I. persulcatus, I. pacificus, andI. scapularis, are competent vectors of the Lyme disease spirochete. Although ticks in other genera are also naturally infected withB. burgdorferi, experimental evidence suggests thatAmblyomma andDermacentor ticks are inefficient vectors of these spirochetes. Current research on the kinetics ofB. burgdorferi growth within ticks demonstrates that Lyme disease spirochetes are dramatically influenced by physiological events during the tick's life-cycle.     

Purification and properties of the plasmid maintenance proteins from the Borrelia burgdorferi linear plasmid lp17

The Lyme disease spirochete Borrelia burgdorferi carries more plasmids than any other bacterium, many of which are linear with covalently closed hairpin ends. These plasmids have also been referred to as mini-chromosomes and essential genetic elements and are integral components of its segmented genome. We have investigated two plasmid maintenance proteins, BBD14 (the replication initiator) and BBD21 (a presumptive ParA orthologue), encoded by the linear plasmid lp17; these proteins are representatives of paralogous families 62 and 32, respectively. We have purified recombinant 6-his-BBD21 and shown it possesses an ATPase activity. 6-his-BBD14 initially could not be overexpressed in Escherichia coli by itself. It was only effectively overproduced in recombinant form through coexpression with other B. burgdorferi proteins and codon optimization. Although the mechanism for increased production through coexpression is not clear, this method holds promise for expression and purification of other B. burgdorferi proteins, a number of which have remained recalcitrant to purification from E. coli. Finally, we present evidence for the physical interaction of BBD14 and BBD21, a feature suggesting that BBD21 and the paralogous family 32 proteins are more likely involved in DNA replication than functioning as simple ParA orthologues as previously surmised based upon sequence homology. Such a role would not preclude a function in plasmid partitioning through interaction with the replication initiator.     

Evidence of Dbps (decorin binding proteins) among European strains of Borrelia burgdorferi sensu lato and in the immune response of LB patient sera

Decorin binding proteins DbpA and DbpB act as Borrelia burgdorferi (B. burgdorferi) adhesins to decorin, and are able to elicit a persistent antibody response in the mouse; accordingly DbpA protein would seem to be promising in immunoprofilaxis of Lyme borreliosis (LB). This study examines the distribution of Dbp epitopes in European strains of B. burgdorferi, of different genospecies and the presence of antibodies to Dbps in human sera from patients suffering from early and late LB, as revealed by immunoblotting. Different levels of expression of Dbp epitopes were found both among and within genospecies; data from human sera indicate that Dbps are expressed during infection though not as strongly as in the mouse infection.     

Identification of a large motility operon in Borrelia burgdorferi by semi-random PCR chromosome walking

Motility has been implicated in the invasive process of Borrelia burgdorferi (Bb), the etiologic agent of Lyme disease. To identify Bb motility related genes, we used a method termed `semi-random PCR chromosome walking' (SRPCW) to walk through a large motility gene cluster. The major advantage of this approach over other PCR walking methods is that it employs a secondary PCR amplification of cloned fragments which can be readily sequenced and analyzed. Starting with a primer specific to flgE, we identified and sequenced 14 open reading frames (ORFs) spanning 11 kb downstream of the flgE gene. The genes identified include flbD, motA, motB, fliL, fliM, fliN, fliZ, fliP, fliQ, fliR, flhB, flhA, flhF and flbE. Twelve of the deduced proteins shared extensive homology with flagellar proteins from other bacteria. The gene products and order of genes within this cluster are most similar to those of Treponema pallidum (Tp) and Bacillus subtilis (Bs). One of the unique genes identified, flbD, demonstrated homology to an ORF from the same operon of Tp. Another ORF, flbE, showed similarity to genes from both Tp and Bs. RT-PCR and primer extension analysis revealed that this gene cluster is transcribed as a single unit indicating that it is part of a large motility operon spanning more than 21 kb. Antisera to Escherichia coli and Salmonella typhimurium FliN, FliM, FlhB and FlhA reacted with proteins of the predicted molecular weights in cell lysates of Bb. The results suggest that the flagellar system is highly conserved in evolution and thus underscore the importance of motility in bacterial survival and pathogenesis.     

Immunodominant proteins of Borrelia burgdorferi,the etiological agent of lyme borreliosis

Lyme borreliosis, a multisystem disorder involving the skin, the nervous system, the heart, the joints and many other organs, is a worldwide infectious disease which is transmitted by ticks of the Ixodes complex. Most frequently diagnosis is accomplished by detection of antibodies because the Borrelia are difficult to cultivate. Present serodiagnostic methods, however, are impaired by low sensitivity and unspecific reactions. The selection of immunodominant antigens with low cross-reactivity to other bacteria should improve antibody detection. Borrelia burgdorferi proteins have been analysed for cross-reactivity with immune sera from unrelated bacteria, and sera from patients with different stages of the disease. Suitable antigens for improving serodiagnosis have been detected and are reported here. In view of the immunological heterogeneity of Borrelia proteins, sensitivity of antibody detection may possibly be increased by using recombinant antigens derived from different strains. Immunization with recombinant OspA (a flagellum-associated protein) from a North American isolate protected mice from the challenge with three North American isolates. However, for development of an effective vaccine (especially in Europe), the heterogeneity of OspA has to be considered.This paper was presented at the IUMS Symposium on New Developments in Diagnosis and Control of Infectious Diseases held in conjunction with the Eighth International Congress of Virology, Berlin, Germany, 24–31 August 1990.     

Analysis of the bmp gene family in Borrelia burgdorferi sensu lato

BmpA, BmpB, BmpC, and BmpD are homologous Borrelia burgdorferi lipoproteins of unknown functions, encoded by the bmp genes of paralogous chromosomal gene family 36. At least some of the Bmp proteins are immunogens in infected vertebrate hosts. The genetic organization of the bmp region has been characterized for a variety of B. burgdorferi sensu lato strains by Southern hybridization, PCR amplification, and DNA sequencing. All four bmp genes were present in the same relative order in all B. burgdorferi sensu lato low- and high-passage-number isolates. While there were no differences in the relative orders of the bmp genes in these species, variations in DNA sequence in the bmpD-bmpC and bmpC-bmpA intergenic regions were significantly more common than in the corresponding 3' bmpD and bmpC coding regions. The genetic structure of the chromosomal region containing the bmp genes thus appears to be well conserved across different species of B. burgdorferi, but variations in DNA fine structure that prevent PCR primer annealing may occur in this region and make Southern hybridization much more reliable than PCR for detection of the presence of these genes. Our results also suggest that bmp gene products may be used as reagents in the preparation of vaccines and diagnostic assays to protect against and diagnose Lyme disease produced by B. burgdorferi sensu lato.