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ABSTRACT Borrelia burgdorferi is an extreme amino acid (AA) auxotroph whose genome encodes few free AA transporters and an elaborate oligopeptide transport system ( B. burgdorferi Opp [ Bb Opp]). Bb Opp consists of five oligopeptide-binding proteins (OBPs), two heterodimeric permeases, and a heterodimeric nucleotide-binding domain (NBD). Homology modeling based on the crystal structure of liganded Bb OppA4 revealed that each OBP likely binds a distinct range of peptides. Transcriptional analyses demonstrated that the OBPs are differentially and independently regulated whereas the permeases and NBDs are constitutively expressed. A conditional NBD mutant failed to divide in the absence of inducer and replicated in an IPTG (isopropyl-β- d -thiogalactopyranoside) concentration-dependent manner. NBD mutants grown without IPTG exhibited an elongated morphotype lacking division septa, often with flattening at the cell center due to the absence of flagellar filaments. Following cultivation in dialysis membrane chambers, NBD mutants recovered from rats not receiving IPTG also displayed an elongated morphotype. The NBD mutant was avirulent by needle inoculation, but infectivity was partially restored by oral administration of IPTG to infected mice. We conclude that peptides are a major source of AAs for B. burgdorferi both in vitro and in vivo and that peptide uptake is essential for regulation of morphogenesis, cell division, and virulence. IMPORTANCE Borrelia burgdorferi , the causative agent of Lyme disease, is an extreme amino acid (AA) auxotroph with a limited repertoire of annotated single-AA transporters. A major issue is how the spirochete meets its AA requirements as it transits between its arthropod vector and mammalian reservoir. While previous studies have confirmed that the B. burgdorferi oligopeptide transport ( opp ) system is capable of importing peptides, the importance of the system for viability and pathogenesis has not been established. Here, we evaluated the opp system structurally and transcriptionally to elucidate its ability to import a wide range of peptides during the spirochete’s enzootic cycle. Additionally, using a novel mutagenesis strategy to abrogate opp transporter function, we demonstrated that peptide uptake is essential for bacterial viability, morphogenesis, and infectivity. Our studies revealed a novel link between borrelial physiology and virulence and suggest that peptide uptake serves an intracellular signaling function regulating morphogenesis and division. 

ABSTRACT In recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) of Treponema pallidum subspecies pallidum , the syphilis spirochete, and identifying its surface-exposed β-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of β-barrel-encoding regions of tprC , tprD , and bamA in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we used T. pallidum 0548 ( tp0548 ) genotyping and tp0558 sequences to assign strains to the Nichols or SS14 clades. We found that (i) β-barrels in clinical strains could be grouped according to allelic variants in T. pallidum subsp. pallidum reference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored β-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. On the basis of structural models, nonconservative amino acid substitutions in predicted transmembrane β-strands of T. pallidum repeat C (TprC) and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures drive T. pallidum subsp. pallidum OMP diversity and that genetic exchange contributes to the evolutionary biology of T. pallidum subsp. pallidum . They also set the stage for topology-based analysis of antibody responses to OMPs and help frame strategies for syphilis vaccine development. IMPORTANCE Despite recent progress characterizing outer membrane proteins (OMPs) of Treponema pallidum , little is known about how their surface-exposed, β-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the β-barrel-encoding regions of three OMP loci, tprC , tprD , and bamA , in T. pallidum subsp. pallidum isolates from a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within β-barrel domains occurs predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function were also noted. Our findings suggest that selection pressures exerted within human populations drive T. pallidum subsp. pallidum OMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance of T. pallidum subsp. pallidum and frame strategies for vaccine development based upon conserved OMP extracellular loops.