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Borrelia burgdorferi, the agent of Lyme disease, is maintained in nature within an enzootic cycle involving a mammalian reservoir and an Ixodes sp. tick vector. The transmission, survival and pathogenic potential of B. burgdorferi depend on the bacterium's ability to modulate its transcriptome as it transits between vector and reservoir host. Herein, we employed an amplification-microarray approach to define the B. burgdorferi transcriptomes in fed larvae, fed nymphs and in mammalian host-adapted organisms cultivated in dialysis membrane chambers. The results show clearly that spirochetes exhibit unique expression profiles during each tick stage and during cultivation within the mammal; importantly, none of these profiles resembles that exhibited by in vitro grown organisms. Profound shifts in transcript levels were observed for genes encoding known or predicted lipoproteins as well as proteins involved in nutrient uptake, carbon utilization and lipid synthesis. Stage-specific expression patterns of chemotaxis-associated genes also were noted, suggesting that the composition and interactivities of the chemotaxis machinery components vary considerably in the feeding tick and mammal. The results as a whole make clear that environmental sensing by B. burgdorferi directly or indirectly drives an extensive and tightly integrated modulation of cell envelope constituents, chemotaxis/motility machinery, intermediary metabolism and cellular physiology. These findings provide the necessary transcriptional framework for delineating B. burgdorferi regulatory pathways throughout the enzootic cycle as well as defining the contribution(s) of individual genes to spirochete survival in nature and virulence in humans., (© 2014 John Wiley & Sons Ltd.)

P66 is a Borrelia burgdorferi surface protein with β₃ integrin binding and channel forming activities. In this study, the role of P66 in mammalian and tick infection was examined. B. burgdorferiΔp66 strains were not infectious in wild-type, TLR2⁻/⁻- or MyD88⁻/⁻-deficient mice. Strains with p66 restored to the chromosome restored near wild-type infectivity, while complementation with p66 on a shuttle vector did not restore infectivity. Δp66 mutants are cleared quickly from the site of inoculation, but analyses of cytokine expression and cellular infiltrates at the site of inoculation did not reveal a specific mechanism of clearance. The defect in these mutants cannot be attributed to nutrient limitation or an inability to adapt to the host environment in vivo as Δp66 bacteria were able to survive as well as wild type in dialysis membrane chambers in the rat peritoneum. Δp66 bacteria were able to survive in ticks through the larva to nymph moult, but were non-infectious in mice when delivered by tick bite. Independent lines of evidence do not support any increased susceptibility of the Δp66 strains to factors in mammalian blood. This study is the first to define a B. burgdorferi adhesin as essential for mammalian, but not tick infection., (© 2012 Blackwell Publishing Ltd.)

In a microarray analysis of the RpoS regulon in mammalian host-adapted Borrelia burgdorferi, bb0728 (cdr) was found to be dually transcribed by the sigma factors σ(70) and RpoS. The cdr gene encodes a coenzyme A disulphide reductase (CoADR) that reduces CoA-disulphides to CoA in an NADH-dependent manner. Based on the abundance of CoA in B. burgdorferi and the biochemistry of the enzyme, CoADR has been proposed to play a role in the spirochaete's response to reactive oxygen species. To better understand the physiologic function(s) of BbCoADR, we generated a B. burgdorferi mutant in which the cdr gene was disrupted. RT-PCR and 5'-RACE analysis revealed that cdr and bb0729 are co-transcribed from a single transcriptional start site upstream of the bb0729 coding sequence; a shuttle vector containing the bb0729-cdr operon and upstream promoter element was used to complement the cdr mutant. Although the mutant was no more sensitive to hydrogen peroxide than its parent, it did exhibit increased sensitivity to high concentrations of t-butyl-hydroperoxide, an oxidizing compound that damages spirochetal membranes. Characterization of the mutant during standard (15% oxygen, 6% CO(2)) and anaerobic (< 1% O(2) , 9-13% CO(2)) cultivation at 37°C revealed a growth defect under both conditions that was particularly striking during anaerobiosis. The mutant was avirulent by needle inoculation and showed decreased survival in feeding nymphs, but displayed no survival defect in unfed flat nymphs. Based on these results, we propose that BbCoADR is necessary to maintain optimal redox ratios for CoA/CoA-disulphide and NAD(+) /NADH during periods of rapid replication throughout the enzootic cycle, to support thiol-disulphide homeostasis, and to indirectly protect the spirochaete against peroxide-mediated membrane damage; one or more of these functions are essential for infection of the mammalian host by B. burgdorferi., (© 2011 Blackwell Publishing Ltd.)

Definitive identification of Treponema pallidum rare outer membrane proteins (OMPs) has long eluded researchers. TP0326, the sole protein in T. pallidum with sequence homology to a Gram-negative OMP, belongs to the BamA family of proteins essential for OM biogenesis. Structural modelling predicted that five polypeptide transport-associated (POTRA) domains comprise the N-terminus of TP0326, while the C-terminus forms an 18-stranded amphipathic β-barrel. Circular dichroism, heat modifiability by SDS-PAGE, Triton X-114 phase partitioning and liposome incorporation supported these topological predictions and confirmed that the β-barrel is responsible for the native protein's amphiphilicity. Expression analyses revealed that native TP0326 is expressed at low abundance, while a protease-surface accessibility assay confirmed surface exposure. Size-exclusion chromatography and blue native polyacrylamide gel electrophoresis revealed a modular Bam complex in T. pallidum larger than that of Escherichia coli. Non-orthologous ancillary factors and self-association of TP0326 via its β-barrel may both contribute to the Bam complex. T. pallidum-infected rabbits mount a vigorous antibody response to both POTRA and β-barrel portions of TP0326, whereas humans with secondary syphilis respond predominantly to POTRA. The syphilis spirochaete appears to have devised a stratagem for harnessing the Bam pathway while satisfying its need to limit surface antigenicity., (© 2011 Blackwell Publishing Ltd.)

Borrelia burgdorferi must adapt physiologically to two markedly different host milieus and efficiently transit between its mammalian host and arthropod vector during tick feeding. Differential production of lipoproteins is essential for spirochaetes to survive, multiply and migrate within both hosts. Outer-surface protein C (OspC), which is induced during the blood meal, is critical for transmission of Lyme disease spirochaetes by nymphal ticks. Its biological function is poorly understood, however, despite the fact that its crystal structure has been solved. Evidence has accumulated that OspC blocks clearance of spirochaetes following inoculation in skin, and it is thought to do so by facilitating evasion of innate immunity. The study by Liang and co-workers in this edition of Molecular Microbiology extends this work by showing that OspC prevents early elimination and promotes dissemination. Surprisingly, they also show that unrelated borrelial outer-surface lipoproteins can replace these functions in an ospC mutant. They propose that an abundance of lipoprotein(s) is needed to stabilize the borrelial outer membrane against innate defences. This provocative work clearly runs counter to prevailing orthodoxies of bacterial pathogenesis. It also points the way towards future studies that will clarify the 'partially specific' roles of this enigmatic molecule in Lyme disease pathogenesis.

Borrelia burgdorferi (Bb) adapts to its arthropod and mammalian hosts by altering its transcriptional and antigenic profiles in response to environmental signals associated with each of these milieus. In studies presented here, we provide evidence to suggest that mammalian host signals are important for modulating and maintaining both the positive and negative aspects of mammalian host adaptation mediated by the alternative sigma factor RpoS in Bb. Although considerable overlap was observed between genes induced by RpoS during growth within the mammalian host and following temperature-shift, comparative microarray analyses demonstrated unequivocally that RpoS-mediated repression requires mammalian host-specific signals. A substantial portion of the in vivo RpoS regulon was uniquely upregulated within dialysis membrane chambers, further underscoring the importance of host-derived environmental stimuli for differential gene expression in Bb. Expression profiling of genes within the RpoS regulon by quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed a level of complexity to RpoS-dependent gene regulation beyond that observed by microarray, including a broad range of expression levels and the presence of genes whose expression is only partially dependent on RpoS. Analysis of Bb-infected ticks by qRT-PCR established that expression of rpoS is induced during the nymphal blood meal but not within unfed nymphs or engorged larvae. Together, these data have led us to postulate that RpoS acts as a gatekeeper for the reciprocal regulation of genes involved in the establishment of infection within the mammalian host and the maintenance of spirochetes within the arthropod vector.

Members of the ospE/ospF/elp lipoprotein gene families of Borrelia burgdorferi, the Lyme disease agent, are transcriptionally upregulated in response to the influx of blood into the midgut of an infected tick. We recently have demonstrated that despite the high degree of similarity between the promoters of the ospF (P(ospF)) and ospE (P(ospE)) genes of B. burgdorferi strain 297, the differential expression of ospF is RpoS-dependent, while ospE is controlled by sigma(70). Herein we used wild-type and RpoS-deficient strains of B. burgdorferi and Escherichia coli to analyse transcriptional reporters consisting of a green fluorescent protein (gfp) gene fused to P(ospF), P(ospE), or two hybrid promoters in which the -10 regions of P(ospF) and P(ospE) were switched [P(ospF ) ((E - 10)) and P(ospE) ((F - 10)) respectively]. We found that the P(ospF)-10 region is both necessary and sufficient for RpoS-dependent recognition in B. burgdorferi, while sigma(70) specificity for P(ospE) is dependent on elements outside of the -10 region. In E. coli, sigma factor selectivity for these promoters was much more permissive, with expression of each being primarily due to sigma(70). Alignment of the sequences upstream of each of the ospE/ospF/elp genes from B. burgdorferi strains 297 and B31 revealed that two B31 ospF paralogues [erpK (BBM38) and erpL (BBO39)] have -10 regions virtually identical to that of P(ospF). Correspondingly, expression of gfp reporters based on the erpK and erpL promoters was RpoS-dependent. Thus, the sequence of the P(ospF)-10 region appears to serve as a motif for RpoS recognition, the first described for any B. burgdorferi promoter. Taken together, our data support the notion that B. burgdorferi utilizes sequence differences at the -10 region as one mechanism for maintaining the transcriptional integrity of RpoS-dependent and -independent genes activated at the onset of tick feeding.

Borrelia burgdorferi, a spirochaete that causes Lyme borreliosis, contains 21 linear and circular plasmids thought to be important for survival in mammals or ticks. Our results demonstrate that the gene BBE22 encoding a nicotinamidase is capable of replacing the requirement for the 25 kb linear plasmid lp25 during mammalian infection. Transformation of B. burgdorferi lacking lp25 with a shuttle vector containing the lp25 gene BBE22 (pBBE22) restored infectivity in mice to a level comparable to that of wild-type Borrelia. This complementation also restored the growth and host adaptation of lp25-B. burgdorferi in dialysis membrane chambers (DMCs) implanted in rats. A single Cys to Ala conversion at the putative active site of BBE22 abrogated the ability of pBBE22 to re-establish infectivity or growth in DMCs. Additional Salmonella typhimurium complementation studies and enzymatic analysis demonstrated that the BBE22 gene product has nicotinamidase activity and is most probably required for the biosynthesis of NAD. These results indicate that some plasmid-encoded products fulfil physiological functions required in the enzootic cycle of pathogenic Borrelia.

The 32kb circular plasmid (cp32) family of Borrelia burgdorferi has been the subject of intensive investigation because its members encode numerous differentially expressed lipoproteins. As many as nine different cp32s appear to be capable of stable replication within a single spirochaete. Here, we show that a construct (pCE310) containing a 4 kb fragment from the putative maintenance region of a B. burgdorferi CA-11.2A cp32 was capable of autonomous replication in both high-passage B. burgdorferi B31 and virulent B. burgdorferi 297. Deletion analysis revealed that only the member of paralogous family 57 and the adjacent non-coding segment were essential for replication. The PF32 ParA orthologue encoded by the pCE310 insert was almost identical to the PF32 orthologues encoded on the B31 and 297 cp32-3 plasmids. The finding that cp32-3 was selectively deleted in both B31 and 297 transformants carrying pCE310 demonstrated the importance of the PF32 protein for cp32 compatibility and confirmed the prediction that cp32 plasmids expressing identical PF32 paralogues are incompatible. A shuttle vector containing the CA-11.2A cp32 plasmid maintenance region was used to introduce green, yellow and cyan fluorescent protein reporters into B. burgdorferi. Flow cytometry revealed that the green fluorescent protein was well expressed by almost 90% of both avirulent and infectious transformants. In addition to enhancing our understanding of B. burgdorferi plasmid biology, our results further the development of genetic systems for dissecting pathogenic mechanisms in Lyme disease.