Interdependence of environmental factors influencing reciprocal patterns of gene expression in virulent Borrelia burgdorferi.

The paradigm for differential antigen expression in Borrelia burgdorferi, the agent of Lyme disease, is the reciprocal expression of its outer surface (lipo)proteins (Osp) A and C; as B. burgdorferi transitions from its arthropod vector into mammalian tissue, ospC is upregulated, and ospA is downregulated. In the current study, using B. burgdorferi cultivated under varying conditions in BSK-H medium, we found that a decrease in pH, in conjunction with increases in temperature (e.g. 34 degrees C or 37 degrees C) and cell density, acted interdependently for the reciprocal expression of ospC and ospA. The lower pH (6.8), which induced the reciprocal expression of ospC and ospA in BSK-H medium, correlated with a drop in pH from 7.4 to 6.8 of tick midgut contents during tick feeding. In addition to ospC and ospA, other genes were found to be regulated in reciprocal fashion. Such genes were either ospC-like (e.g. ospF, mlp-8 and rpoS) (group I) or ospA-like (lp6.6 and p22) (group II); changes in expression occurred at the mRNA level. That the expression of rpoS, encoding a putative stress-related alternative sigma factor (sigma(s)), was ospC-like suggested that the expression of some of the group I genes may be controlled through sigma(s). The combined results prompt a model that allows for predicting the regulation of other B. burgdorferi genes that may be involved in spirochaete transmission, virulence or mammalian host immune responses.

Decorin-binding protein A (DbpA) of Borrelia burgdorferi is not protective when immunized mice are challenged via tick infestation and correlates with the lack of DbpA expression by B. burgdorferi in ticks.

Previous studies showed that decorin-binding protein A (DbpA) of Borrelia burgdorferi was a protective immunogen in the murine model of Lyme borreliosis when mice were challenged (needle inoculated) intradermally with in vitro-cultivated spirochetes. In the present study, DbpA-immunized C3H/HeJ mice were not protected from infection when infested with Ixodes scapularis nymphs harboring virulent B. burgdorferi 297. This lack of protection correlated with the failure to detect DbpA on B. burgdorferi in ticks, suggesting that DbpA is not available as a target for bactericidal antibodies in serum when B. burgdorferi-infected ticks take their blood meal from an immunized host. The failure of DbpA immunization to protect tick-challenged mice contradicts the results of earlier needle inoculation vaccination experiments and suggests that DbpA may not be suitable as a Lyme disease vaccine.

Identification, characterization, and expression of three new members of the Borrelia burgdorferi Mlp (2.9) lipoprotein gene family.

We previously reported on the existence of a family of lipoprotein genes, designated 2.9 lipoprotein genes, encoded in at least seven versions on the circular (supercoiled) cp32 and cp18 plasmids of Borrelia burgdorferi 297. A distinguishing feature of the 2.9 lipoproteins were highly similar signal sequences but variable mature polypeptides that segregated into two antigenic classes. Further screenings of B. burgdorferi 297 genomic libraries led to the identification of three additional 2.9 lipoprotein genes, renamed herein mlp, for multicopy lipoprotein genes. Computer analyses and immunoblotting revealed that Mlp-9 segregated with the antigenic class I lipoproteins, whereas Mlp-8 and Mlp-10 were members of class II. Northern blotting showed that all three of the mlp genes were expressed when B. burgdorferi was cultivated in vitro at 34 degrees C, although mlp-9 and mlp-10 transcripts were expressed at very low levels. Additional combined immunoblotting and comparative reverse transcription-PCR analyses performed on borreliae cultivated in vitro at 23, 34, or 37 degrees C indicated that although Mlp-8 was substantially more abundant than Mlp-9 or Mlp-10, all three of the mlp genes were upregulated during B. burgdorferi replication at 37 degrees C. Expression of the same three lipoproteins was further enhanced upon growth of the spirochetes within dialysis membrane chambers (DMCs) implanted intraperitoneally in rats (i.e., spirochetes in a mammalian host-adapted state), suggesting that temperature alone did not account for maximal upregulation of the mlp genes. That certain mlp genes are likely expressed during the growth of B. burgdorferi in mammalian tissues was supported by findings of antibodies against all three Mlp lipoproteins in mice after challenge with Ixodes scapularis nymphs harboring B. burgdorferi 297. The combined data suggest that as opposed to being differentially expressed in any reciprocal fashion (e.g., OspA/OspC), at least three mlp genes are simultaneously upregulated by temperature (37 degrees C) and some other mammalian host factor(s). The findings have importance not only for understanding alternative modes of differential antigen expression by B. burgdorferi but also for assessing whether one or more of the Mlp lipoproteins represent new candidate vaccinogens for Lyme disease.

Physicochemical evidence that Treponema pallidum TroA is a zinc-containing metalloprotein that lacks porin-like structure.

Although TroA (Tromp1) was initially reported to be a Treponema pallidum outer membrane protein with porin-like properties, subsequent studies have suggested that it actually is a periplasmic substrate-binding protein involved in the transport of metals across the treponemal cytoplasmic membrane. Here we conducted additional physicochemical studies to address the divergent viewpoints concerning this protein. Triton X-114 phase partitioning of recombinant TroA constructs with or without a signal sequence corroborated our prior contention that the native protein's amphiphilic behavior is due to its uncleaved leader peptide. Whereas typical porins are trimers with extensive beta-barrel structure, size exclusion chromatography and circular dichroism spectroscopy revealed that TroA was a monomer and predominantly alpha-helical. Neutron activation, atomic absorption spectroscopy, and anomalous X-ray scattering all demonstrated that TroA binds zinc in a 1:1 molar stoichiometric ratio. TroA does not appear to possess structural features consistent with those of bacterial porins.

Decorin-binding protein of Borrelia burgdorferi is encoded within a two-gene operon and is protective in the murine model of Lyme borreliosis.

Isolated outer membranes of Borrelia burgdorferi were used in immunoblotting experiments with sera from immune mice to identify new putative Lyme disease vaccine candidates. One immunoreactive polypeptide migrated on polyacrylamide gels just proximal to outer surface protein C and comigrated with [3H]palmitate-labeled polypeptides. A degenerate oligonucleotide primer based upon internal amino acid sequence information was used to detect the corresponding gene within a B. burgdorferi total genomic library. The relevant open reading frame (ORF) encoded a polypeptide comprised of a 24-amino-acid putative signal peptide terminated by LLISC, a probable consensus sequence for lipoprotein modification, and a mature protein of 163 amino acids. Immunoblots of a recombinant fusion protein corresponding to this ORF supported the idea that the encoded protein was a previously reported decorin-binding protein (DBP) of B. burgdorferi N40 (B. P. Guo, S. J. Norris, L. C. Rosenberg, and M. Höök, Infect. Immun. 63:3467-3472, 1995). However, further DNA sequencing revealed the presence of a second ORF, designated ORF-1, whose termination codon was 119 bp upstream of the dbp gene. ORF-1 also encoded a putative lipoprotein with a mature length of 167 amino acids. Northern blots, Southern blots, and primer extension analyses indicated that ORF-1 and dbp comprised a two-gene operon located on the 49-kb linear plasmid. Both proteins, which were 40% identical and 56% similar, partitioned into Triton X-114 detergent extracts of B. burgdorferi isolated outer membranes. Mice infected with B. burgdorferi produced high titers of antibodies against the ORF-1-encoded protein and DBP during both early and later stages of chronic infection. Both DBP and the ORF-1-encoded protein were sensitive to proteinase K treatment of intact borreliae, suggesting that they were surface exposed. In active immunization experiments, 78% of mice immunized with recombinant DBP were immune to challenge. While it is not clear whether the two lipoproteins encoded by the ORF-1-dbp operon have analogous decorin-binding functions in vivo, the combined studies implicate DBP as a new candidate for a human Lyme disease vaccine.

The MtrR repressor binds the DNA sequence between the mtrR and mtrC genes of Neisseria gonorrhoeae.

Gonococcal resistance to antimicrobial hydrophobic agents (HAs) is due to energy-dependent removal of HAs from the bacterial cell by the MtrCDE membrane-associated efflux pump. The mtrR (multiple transferrable resistance Regulator) gene encodes a putative transcriptional repressor protein (MtrR) believed to be responsible for regulation of mtrCDE gene expression. Gel mobility shift and DNase I footprint assays that used a maltose-binding protein (MBP)-MtrR fusion protein demonstrated that the MtrR repressor is capable of specifically binding the DNA sequence between the mtrR and mtrC genes. This binding site was localized to a 26-nucleotide stretch that includes the promoter utilized for mtrCDE transcription and, on the complementary strand, a 22-nucleotide stretch that contains the -35 region of the mtrR promoter. A single transition mutation (A-->G) within the MtrR-binding site decreased the affinity of the target DNA for MtrR and enhanced gonococcal resistance to HAs when introduced into HA-susceptible strain FA19 by transformation. Since this mutation enhanced expression of the mtrCDE gene complex but decreased expression of the mtrR gene, the data are consistent with the notion that MtrR acts as a transcriptional repressor of the mtrCDE efflux pump protein genes.

Evidence for a methyl-accepting chemotaxis protein gene (mcp1) that encodes a putative sensory transducer in virulent Treponema pallidum.

The clinical and histopathological manifestations of syphilis and the invasive behavior of Treponema pallidum in tissue culture systems reflect the propensity for treponemes to migrate through skin, hematogenously disseminate, and invade targeted tissues. Treponemal motility is believed to be essential to this process and thereby an important facet of syphilis pathogenesis. By analogy with other bacterial pathogens, it is plausible that treponemal motility and tissue invasion are modulated by sensory transduction events associated with chemotactic responses. Recent studies have demonstrated the existence in T. pallidum of accessory molecules typically associated with sensory transduction events involving methyl-accepting chemotaxis proteins (MCPs). Intrinsic radiolabeling of T. pallidum in vitro with L-[methyl-3H] methionine revealed one methylated treponemal polypeptide with an apparent molecular mass of 64 kDa. A degenerate oligonucleotide probe corresponding to a highly conserved C-terminal domain within Bacillus subtilis and Escherichia coli MCPs was used in Southern blotting of T. pallidum DNA to identify and subsequently clone a putative T. pallidum MCP gene (mcp1). Computer analyses predicted a near-consensus promoter upstream of mcp1, and primer extension analysis employing T. pallidum RNA revealed a transcriptional initiation site. T. pallidum mcp1 encoded a 579-amino-acid (64.6-kDa) polypeptide which was highly homologous to at least 69 other known or putative sensory transducer proteins, with the highest degrees of homology existing between the C terminus of mcp1 and the C-terminal (signaling) domains of the other bacterial MCPs. Other salient features of Mcp1 included (i) six potential membrane-spanning domains at the N terminus, (ii) two predicted alpha-helical coiled coil regions containing at least three putative methylation sites, and (iii) homologies with two ligand-binding domains (LI-1 and LI-2) of the E. coli MCPs Trg and Tar. This study is the first to provide both metabolic and genetic evidence for an MCP sensory transducer in T. pallidum. The combined findings prompt key questions regarding the relationship(s) among sensory transduction, regulation of endoflagellar rotation, and chemotactic responses (in particular, the role of glucose) during virulence expression by T. pallidum.

Molecular characterization of a 6.6-kilodalton Borrelia burgdorferi outer membrane-associated lipoprotein (lp6.6) which appears to be downregulated during mammalian infection.

Isolated outer membranes of Borrelia burgdorferi 297 were utilized to obtain partial amino acid sequence information for a low-molecular-weight, outer membrane-associated polypeptide. Degenerate oligonucleotide primers based upon this information were used to amplify a 100-bp probe for detection of the corresponding full-length gene within a B. burgdorferi total genomic library. The relevant open reading frame (ORF) encoded a polypeptide comprised of a 17-amino-acid putative signal peptide terminated by LFVAC, a probable consensus sequence for lipoprotein modification, and a mature protein of 51 amino acids (predicted molecular mass of 5.8 kDa). The DNA sequences of the corresponding ORFs in B. burgdorferi 297 and B31 were identical; the corresponding ORF in strain N40 differed by only one nucleotide. Assuming conventional processing and acylation, the molecular weight of the lipoprotein, designated lp6.6, is about 6,600. The lp6.6 gene, which was localized to the 49-kb linear plasmid of B. burgdorferi, subsequently was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase. Immunoblot analysis with monoclonal antibody 240.7 revealed that lp6.6 was identical to a low-molecular-weight, highly conserved B. burgdorferi lipoprotein reported previously (L. I. Katona, G. Beck, and G. S. Habicht, Infect. Immun. 60:4995-5003, 1992). Results of indirect immunofluorescence assays, growth inhibition assays, passive immunizations, and active immunizations indicated that this outer membrane-associated antigen is not surface exposed in B. burgdorferi. Particularly interesting was the finding that mice and rhesus monkeys chronically infected with B. burgdorferi failed to develop antibodies against this antigen. We propose that high-level expression of lp6.6 is associated with the arthropod phase of the spirochetal life cycle and that expression of the gene is downregulated during mammalian infection.

A mgl-like operon in Treponema pallidum, the syphilis spirochete.

A 38-kDa lipoprotein of Treponema pallidum subsp. pallidum (T. pallidum), the syphilis spirochete, previously was identified as a putative homolog of E. coli MglB [Becker et al. (1994) Infect. Immun. 62, 1381-1391]. In the present study, genome walking in regions adjacent to the T. pallidum 38-kDa lipoprotein gene has identified three contiguous genes (tp-mglB [formerly tpp38], tp-mglA, and tp-mglC) which appear to comprise a mgl-like operon in T. pallidum. A prominent transcript corresponding to tp-mglB, the first gene of the operon which encodes the carbohydrate receptor, is synthesized by T. pallidum along with lesser abundant transcript(s) corresponding to the entire T. pallidum mgl operon. An active promoter 135 bp upstream of tp-mglB is believed to direct mRNA synthesis for the operon. This is the first membrane protein-encoding operon of T. pallidum for which a putative function (glucose import) has been assigned. Furthermore, by analogy with E. coli MglB which interacts with the sensory transducer Trg to induce a chemotactic response, it is possible that T. pallidum also contains a homolog of E. coli Trg or other methyl-accepting chemotaxis proteins. The existence of a mgl operon in T. pallidum thus may have important implications with respect to T. pallidum survival, tissue dissemination, and sensory transduction during virulence expression.

Transcriptional control of the mtr efflux system of Neisseria gonorrhoeae.

The capacity of Neisseria gonorrhoeae to resist structurally diverse hydrophobic agents (HAs) because of the mtr (multiple transferable resistance) efflux system was found to be regulated at the level of transcription by two distinct mechanisms. This was surmised because a deletion that removed > 90% of the coding sequence of the mtrR (multiple transferrable resistance regulator) gene or a single-base-pair deletion within a 13-bp inverted repeat sequence located in its promoter resulted in altered expression of the mtrC gene; mtrC encodes a 44-kDa membrane lipoprotein essential for the efflux of HAs. However, the single-base-pair deletion had the more significant impact on gene expression since it resulted in the loss of expression of mtrR and a threefold increase in the expression of mtrC. Hence, the mtr efflux system in gonococci is subject to both MtrR-dependent and MtrR-independent regulation, and the levels of mtrC mRNA correlate well with HA resistance levels in gonococci.

Importance of lipooligosaccharide structure in determining gonococcal resistance to hydrophobic antimicrobial agents resulting from the mtr efflux system.

Levels of gonococcal resistance to antimicrobial hydrophobic agents (HAs) are controlled by the mtr (multiple transferrable resistance) system, composed of the mtrRCDE genes. The mtrR gene encodes a transcriptional repressor that appears to regulate expression of the upstream and divergent mtrCDE operon. The mtrCDE genes encode membrane proteins analogous to the MexABOprK proteins of Pseudomonas aeruginosa that mediate export of structurally diverse antimicrobial agents. In this study we found that a single base pair deletion in a 13 bp inverted repeat sequence within the mtrR promoter resulted in increased resistance of gonococci to both crystal violet (CV) and erythromycin (ERY) as well as to the more lipophilic non-ionic detergent Triton X-100 (TX-100). However, this cross-resistance was contingent on the production of a full-length lipooligosaccharide (LOS) by the recipient strain used in transformation experiments. Introduction of this mutation (mtrR-171) into three chemically distinct deep-rough LOS mutants by transformation resulted in a fourfold increase in resistance to TX-100 compared with a 160-fold increase in an isogenic strain producing a full-length LOS. However, both wild-type and deep-rough LOS strains exhibited an eightfold increase in resistance to CV and ERY as a result of the mtrR-171 mutation. This suggests that gonococci have different LOS structural requirements for mtr-mediated resistance to HAs that differ in their lipophilic properties. Evidence is presented that gonococci exclude HAs by an energy-dependent efflux process mediated by the mtr system.

Missense mutations that alter the DNA-binding domain of the MtrR protein occur frequently in rectal isolates of Neisseria gonorrhoeae that are resistant to faecal lipids.

Resistance of Neisseria gonorrhoeae to structurally diverse hydrophobic agents (HAs) has been associated with missense or deletion mutations in the mtrR (multiple transferable resistance Regulator) gene of laboratory-derived strains but their prevalence in clinical isolates was heretofore unknown. Since faecal lipids provide strong selective pressure for the emergence of variants resistant to HAs (HAR), the nucleotide sequence of the mtrR gene from rectal isolates of N. gonorrhoeae, which displayed different levels of HAR, was determined. Compared to the mtrR gene possessed by the HA-sensitive strain FA19, each clinical isolate contained mutations in the coding and/or promoter regions of their mtrR gene. A missense mutation in codon 45 (Gly-45 to Asp) was the most common mutation found in the strains studied and impacted the structure of the helix-turn-helix domain of the MtrR protein thought to be important in DNA-binding activity. Two clinical isolates bearing a missense mutation in codon 45 also contained a single basepair deletion in a 13 bp inverted sequence positioned within the mtrR promoter region. Introduction of mtrR sequences amplified from the clinical isolates into strain FA19 revealed that acquisition of the single basepair deletion was correlated with high level HAR while mutations in the mtrR-coding region provided for an intermediate level of HAR.

Resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic agents is modulated by the mtrRCDE efflux system.

The mtr (multiple transferable resistance) system of Neisseria gonorrhoeae determines levels of gonococcal resistance to hydrophobic agents (HAs), including detergent-like fatty acids and bile salts that bathe certain mucosal surfaces. The genetic organization of the mtr system was determined and found to consist of the mtrR gene, which encodes a transcriptional regulator (MtrR), and three tandemly linked genes termed mtrCDE. The mtrCDE genes were organized in the same apparent transcriptional unit, upstream and divergent from the mtrR gene. The mtrCDE-encoded proteins of N. gonorrhoeae were analogous to a family of bacterial efflux/transport proteins, notably the MexABOprK proteins of Pseudomonas aeruginosa and the AcrAE and EnvCD proteins of Escherichia coli, that mediate resistance to drugs, dyes, and detergents. Inactivation of the mtrC gene resulted in loss of the MtrC lipoprotein and rendered gonococci hypersusceptible to structurally diverse HAs; this revealed the importance of the mtr system in determining HAR in gonococci. Further support for a role of the mtrCDE gene complex in determining levels of HAR in gonococci was evident when transformants bearing mutations in the mtrR gene were analysed. In this respect, missense and null mutations in the mtrR gene were found to result in increased levels of MtrC and HAR. However, high levels of MtrC and HAR, similar to those observed for clinical isolates, were associated with a single bp deletion in a 13 bp inverted repeat sequence that intervened the divergent mtrR and mtrC genes. We propose that the 13 bp inverted-repeat sequence represents a transcriptional control element that regulates expression of the mtrRCDE gene complex, thereby modulating levels of gonococcal susceptibility to HAs.