The plasmid-encoded members of paralogous gene family 52 are dispensable to the enzootic cycle of Borrelia burgdorferi.

Lyme disease, the leading vector-borne disease in the United States and Europe, develops after infection with Borrelia burgdorferi sensu lato bacteria. Transmission of the spirochete from the tick vector to a vertebrate host requires global changes in gene expression that are controlled, in part, by the Rrp2/RpoN/RpoS alternative sigma factor cascade. Transcriptional studies defining the B. burgdorferi RpoS regulon have suggested that RpoS activates the transcription of paralogous family 52 (PFam52) genes. In strain B31, PFam52 genes (bbi42, bbk53, and bbq03) encode a set of conserved hypothetical proteins with >89% amino acid identity that are predicted to be surface-localized. Extensive homology among members of paralogous families complicates studies of protein contributions to pathogenicity as the potential for functional redundancy will obfuscate findings. Using a sequential mutagenesis approach, we generated clones expressing a single PFam52 paralog, as well as a strain deficient in all three. The single paralog expressing strains were used to confirm BBI42, BBK53, and BBQ03 surface localization and RpoS regulation. Surprisingly, the PFam52-deficient strain was able to infect mice and complete the enzootic cycle similar to the wild-type parental strain. Indeed, the presence of numerous pseudogenes that contain frameshifts or internal stop codons among the PFam52 genes suggests that they may be subjected to gene loss in B. burgdorferi's reduced genome. Alternatively, the lack of phenotype might reflect the limitations of the experimental mouse infection model.

Borrelia burgdorferi BB0346 is an Essential, Structurally Variant LolA Homolog that is Primarily Required for Homeostatic Localization of Periplasmic Lipoproteins.

In diderm bacteria, the Lol pathway canonically mediates the periplasmic transport of lipoproteins from the inner membrane (IM) to the outer membrane (OM) and therefore plays an essential role in bacterial envelope homeostasis. After extrusion of modified lipoproteins from the IM via the LolCDE complex, the periplasmic chaperone LolA carries lipoproteins through the periplasm and transfers them to the OM lipoprotein insertase LolB, itself a lipoprotein with a LolA-like fold. Yet, LolB homologs appear restricted to γ-proteobacteria and are missing from spirochetes like the tick-borne Lyme disease pathogen Borrelia burgdorferi, suggesting a different hand-off mechanism at the OM. Here, we solved the crystal structure of the B. burgdorferi LolA homolog BB0346 (LolABb) at 1.9 Å resolution. We identified multiple structural deviations in comparative analyses to other solved LolA structures, particularly a unique LolB-like protruding loop domain. LolABb failed to complement an Escherichia coli lolA knockout, even after codon optimization, signal I peptide adaptation, and a C-terminal chimerization which had allowed for complementation with an α-proteobacterial LolA. Analysis of a conditional B. burgdorferi lolA knockout strain indicated that LolABb was essential for growth. Intriguingly, protein localization assays indicated that initial depletion of LolABb led to an emerging mislocalization of both IM and periplasmic OM lipoproteins, but not surface lipoproteins. Together, these findings further support the presence of two separate primary secretion pathways for periplasmic and surface OM lipoproteins in B. burgdorferi and suggest that the distinct structural features of LolABb allow it to function in a unique LolB-deficient lipoprotein sorting system.

Development and validation of systems for genetic manipulation of the Old World tick-borne relapsing fever spirochete, Borrelia duttonii.

Relapsing fever (RF), a vector-borne disease caused by Borrelia spp., is characterized by recurring febrile episodes due to repeated bouts of bacteremia. RF spirochetes can be geographically and phylogenetically divided into two distinct groups; Old World RF Borrelia (found in Africa, Asia, and Europe) and New World RF Borrelia (found in the Americas). While RF is a rarely reported disease in the Americas, RF is prevalent in endemic parts of Africa. Despite phylogenetic differences between Old World and New World RF Borrelia and higher incidence of disease associated with Old World RF spirochete infection, genetic manipulation has only been described in New World RF bacteria. Herein, we report the generation of genetic tools for use in the Old World RF spirochete, Borrelia duttonii. We describe methods for transformation and establish shuttle vector- and integration-based approaches for genetic complementation, creating green fluorescent protein (gfp)-expressing B. duttonii strains as a proof of principle. Allelic exchange mutagenesis was also used to inactivate a homolog of the Borrelia burgdorferi p66 gene, which encodes an important virulence factor, in B. duttonii and demonstrate that this mutant was attenuated in a murine model of RF. Finally, the B. duttonii p66 mutant was complemented using shuttle vector- and cis integration-based approaches. As expected, complemented p66 mutant strains were fully infectious, confirming that P66 is required for optimal mammalian infection. The genetic tools and techniques reported herein represent an important advancement in the study of RF Borrelia that allows for future characterization of virulence determinants and colonization factors important for the enzootic cycle of Old World RF spirochetes.

Characterization of the arthropod associated lipoprotein (Alp) in the tick-mammalian transmission cycle of Borrelia turicatae.

Pathogenic species of Borrelia are etiological agents of tick-borne relapsing fever (TBRF). Most species of TBRF Borrelia are transmitted by argasid ticks, and persistent colonization of the salivary glands is vital for spirochete transmission. This is due to the fast-feeding dynamics of the vector. However, the molecular mechanisms leading to vector colonization by the spirochete and their transmission to the vertebrate host remain vague. Previous work in Borrelia hermsii identified the arthropod associated lipoprotein (Alp) as being produced by spirochetes colonizing tick salivary glands. Upon transmission to mice, alp expression was down-regulated and the protein was undetectable in B. hermsii infecting mouse blood. Furthermore, Alp has homologs in multiple TBRF Borrelia species including Borrelia turicatae, Borrelia duttonii, and Borrelia recurrentis. To further evaluate the role of Alp in tick colonization and transmission, the gene was deleted in B. turicatae and the mutant's phenotype was evaluated. Our findings indicate that Alp is dispensable for colonization of the tick salivary glands and for the establishment of infection in laboratory mice.

The Diadenylate Cyclase CdaA Is Critical for Borrelia turicatae Virulence and Physiology.

Relapsing fever (RF), caused by spirochetes of the genus Borrelia, is a globally distributed, vector-borne disease with high prevalence in developing countries. To date, signaling pathways required for infection and virulence of RF Borrelia spirochetes are unknown. Cyclic di-AMP (c-di-AMP), synthesized by diadenylate cyclases (DACs), is a second messenger predominantly found in Gram-positive organisms that is linked to virulence and essential physiological processes. Although Borrelia is Gram-negative, it encodes one DAC (CdaA), and its importance remains undefined. To investigate the contribution of c-di-AMP signaling in the RF bacterium Borrelia turicatae, a cdaA mutant was generated. The mutant was significantly attenuated during murine infection, and genetic complementation reversed this phenotype. Because c-di-AMP is essential for viability in many bacteria, whole-genome sequencing was performed on cdaA mutants, and single-nucleotide polymorphisms identified potential suppressor mutations. Additionally, conditional mutation of cdaA confirmed that CdaA is important for normal growth and physiology. Interestingly, mutation of cdaA did not affect expression of homologs of virulence regulators whose levels are impacted by c-di-AMP signaling in the Lyme disease bacterium Borrelia burgdorferi Finally, the cdaA mutant had a significant growth defect when grown with salts, at decreased osmolarity, and without pyruvate. While the salt treatment phenotype was not reversed by genetic complementation, possibly due to suppressor mutations, growth defects at decreased osmolarity and in media lacking pyruvate could be attributed directly to cdaA inactivation. Overall, these results indicate CdaA is critical for B. turicatae pathogenesis and link c-di-AMP to osmoregulation and central metabolism in RF spirochetes.

The BB0345 Hypothetical Protein of Borrelia burgdorferi Is Essential for Mammalian Infection.

During the natural enzootic life cycle of Borrelia burgdorferi (also known as Borreliella burgdorferi), the bacteria must sense conditions within the vertebrate and arthropod and appropriately regulate expression of genes necessary to persist within these distinct environments. bb0345 of B. burgdorferi encodes a hypothetical protein of unknown function that is predicted to contain an N-terminal helix-turn-helix (HTH) domain. Because HTH domains can mediate protein-DNA interactions, we hypothesized that BB0345 might represent a previously unidentified borrelial transcriptional regulator with the ability to regulate events critical for the B. burgdorferi enzootic cycle. To study the role of BB0345 within mammals, we generated a bb0345 mutant and assessed its virulence potential in immunocompetent mice. The bb0345 mutant was able to initiate localized infection and disseminate to distal tissues but was cleared from all sites by 14 days postinfection. In vitro growth curve analyses revealed that the bb0345 mutant grew similar to wild-type bacteria in standard Barbour-Stoenner-Kelley II (BSK-II) medium; however, the mutant was not able to grow in dilute BSK-II medium or dialysis membrane chambers (DMCs) implanted in rats. Proteinase K accessibility assays and whole-cell partitioning indicated that BB0345 was intracellular and partially membrane associated. Comparison of protein production profiles between the wild-type parent and the bb0345 mutant revealed no major differences, suggesting BB0345 may not be a global transcriptional regulator. Taken together, these data show that BB0345 is essential for B. burgdorferi survival in the mammalian host, potentially by aiding the spirochete with a physiological function that is required by the bacterium during infection.

The RpoS Gatekeeper in Borrelia burgdorferi: An Invariant Regulatory Scheme That Promotes Spirochete Persistence in Reservoir Hosts and Niche Diversity.

Maintenance of Borrelia burgdorferi within its enzootic cycle requires a complex regulatory pathway involving the alternative σ factors RpoN and RpoS and two ancillary trans-acting factors, BosR and Rrp2. Activation of this pathway occurs within ticks during the nymphal blood meal when RpoS, the effector σ factor, transcribes genes required for tick transmission and mammalian infection. RpoS also exerts a 'gatekeeper' function by repressing σ70-dependent tick phase genes (e.g., ospA, lp6.6). Herein, we undertook a broad examination of RpoS functionality throughout the enzootic cycle, beginning with modeling to confirm that this alternative σ factor is a 'genuine' RpoS homolog. Using a novel dual color reporter system, we established at the single spirochete level that ospA is expressed in nymphal midguts throughout transmission and is not downregulated until spirochetes have been transmitted to a naïve host. Although it is well established that rpoS/RpoS is expressed throughout infection, its requirement for persistent infection has not been demonstrated. Plasmid retention studies using a trans-complemented ΔrpoS mutant demonstrated that (i) RpoS is required for maximal fitness throughout the mammalian phase and (ii) RpoS represses tick phase genes until spirochetes are acquired by a naïve vector. By transposon mutant screening, we established that bba34/oppA5, the only OppA oligopeptide-binding protein controlled by RpoS, is a bona fide persistence gene. Lastly, comparison of the strain 297 and B31 RpoS DMC regulons identified two cohorts of RpoS-regulated genes. The first consists of highly conserved syntenic genes that are similarly regulated by RpoS in both strains and likely required for maintenance of B. burgdorferi sensu stricto strains in the wild. The second includes RpoS-regulated plasmid-encoded variable surface lipoproteins ospC, dbpA and members of the ospE/ospF/elp, mlp, revA, and Pfam54 paralogous gene families, all of which have evolved via inter- and intra-strain recombination. Thus, while the RpoN/RpoS pathway regulates a 'core' group of orthologous genes, diversity within RpoS regulons of different strains could be an important determinant of reservoir host range as well as spirochete virulence.

Assessing the Contribution of an HtrA Family Serine Protease During Borrelia turicatae Mammalian Infection.

Tick-borne relapsing fever (TBRF), characterized by recurring febrile episodes, is globally distributed and among the most common bacterial infections in some African countries. Despite the public health concern that this disease represents, little is known regarding the virulence determinants required by TBRF Borrelia during infection. Because the chromosomes of TBRF Borrelia show extensive colinearity with those of Lyme disease (LD) Borrelia, the exceptions represent unique genes encoding proteins that are potentially essential to the disparate enzootic cycles of these two groups of spirochetes. One such exception is a gene encoding an HtrA family protease, BtpA, that is present in TBRF Borrelia, but not in LD spirochetes. Previous work suggested that btpA orthologs may be important for resistance to stresses faced during mammalian infection. Herein, proteomic analyses of the TBRF spirochete, Borrelia turicatae, demonstrated that BtpA, as well as proteins encoded by adjacent genes in the B. turicatae genome, were produced in response to culture at mammalian body temperature, suggesting a role in mammalian infection. Further, transcriptional analyses revealed that btpA was expressed with the genes immediately upstream and downstream as part of an operon. To directly assess if btpA is involved in resistance to environmental stresses, btpA deletion mutants were generated. btpA mutants demonstrated no growth defect in response to heat shock, but were more sensitive to oxidative stress produced by t-butyl peroxide compared to wild-type B. turicatae. Finally, btpA mutants were fully infectious in a murine relapsing fever (RF) infection model. These results indicate that BtpA is either not required for mammalian infection, or that compensatory mechanisms exist in TBRF spirochetes to combat environmental stresses encountered during mammalian infection in the absence of BtpA.

Infection of Primary Human Alveolar Macrophages Alters Staphylococcus aureus Toxin Production and Activity.

Pulmonary pathogens encounter numerous insults, including phagocytic cells designed to degrade bacteria, while establishing infection in the human lung. Staphylococcus aureus is a versatile, opportunistic pathogen that can cause severe pneumonia, and methicillin-resistant isolates are of particular concern. Recent reports present conflicting data regarding the ability of S. aureus to survive and replicate within macrophages. However, due to use of multiple strains and macrophage sources, making comparisons between reports remains difficult. Here, we established a disease-relevant platform to study innate interactions between S. aureus and human lungs. Human precision-cut lung slices (hPCLS) were subjected to infection by S. aureus LAC (methicillin-resistant) or UAMS-1 (methicillin-sensitive) isolates. Additionally, primary human alveolar macrophages (hAMs) were infected with S. aureus, and antibacterial activity was assessed. Although both S. aureus isolates survived within hAM phagosomes, neither strain replicated efficiently in these cells. S. aureus was prevalent within the epithelial and interstitial regions of hPCLS, with limited numbers present in a subset of hAMs, suggesting that the pathogen may not target phagocytic cells for intracellular growth during natural pulmonary infection. S. aureus-infected hAMs mounted a robust inflammatory response that reflected natural human disease. S. aureus LAC was significantly more cytotoxic to hAMs than UAMS-1, potentially due to isolate-specific virulence factors. The bicomponent toxin Panton-Valentine leukocidin was not produced during intracellular infection, while alpha-hemolysin was produced but was not hemolytic, suggesting that hAMs alter toxin activity. Overall, this study defined a new disease-relevant infection platform to study S. aureus interaction with human lungs and to define virulence factors that incapacitate pulmonary cells.

DksA Controls the Response of the Lyme Disease Spirochete Borrelia burgdorferi to Starvation.

The pathogenic spirochete Borrelia burgdorferi senses and responds to changes in the environment, including changes in nutrient availability, throughout its enzootic cycle in Ixodes ticks and vertebrate hosts. This study examined the role of DnaK suppressor protein (DksA) in the transcriptional response of B. burgdorferi to starvation. Wild-type and dksA mutant B. burgdorferi strains were subjected to starvation by shifting cultures grown in rich complete medium, Barbour-Stoenner-Kelly II (BSK II) medium, to a defined mammalian tissue culture medium, RPMI 1640, for 6 h under microaerobic conditions (5% CO2, 3% O2). Microarray analyses of wild-type B. burgdorferi revealed that genes encoding flagellar components, ribosomal proteins, and DNA replication machinery were downregulated in response to starvation. DksA mediated transcriptomic responses to starvation in B. burgdorferi, as the dksA-deficient strain differentially expressed only 47 genes in response to starvation compared to the 500 genes differentially expressed in wild-type strains. Consistent with a role for DksA in the starvation response of B. burgdorferi, fewer CFU of dksA mutants were observed after prolonged starvation in RPMI 1640 medium than CFU of wild-type B. burgdorferi spirochetes. Transcriptomic analyses revealed a partial overlap between the DksA regulon and the regulon of RelBbu, the guanosine tetraphosphate and guanosine pentaphosphate [(p)ppGpp] synthetase that controls the stringent response; the DksA regulon also included many plasmid-borne genes. Additionally, the dksA mutant exhibited constitutively elevated (p)ppGpp levels compared to those of the wild-type strain, implying a regulatory relationship between DksA and (p)ppGpp. Together, these data indicate that DksA, along with (p)ppGpp, directs the stringent response to effect B. burgdorferi adaptation to its environment.IMPORTANCE The Lyme disease bacterium Borrelia burgdorferi survives diverse environmental challenges as it cycles between its tick vectors and various vertebrate hosts. B. burgdorferi must withstand prolonged periods of starvation while it resides in unfed Ixodes ticks. In this study, the regulatory protein DksA is shown to play a pivotal role controlling the transcriptional responses of B. burgdorferi to starvation. The results suggest that DksA gene regulatory activity impacts B. burgdorferi metabolism, virulence gene expression, and the ability of this bacterium to complete its natural life cycle.

Generation of Conditional Mutants in Borrelia burgdorferi.

Mutational studies aimed at characterizing the function(s) of bacterial genes required for growth or viability are constrained by the inability to generate deletion strains lacking the gene of interest. To circumvent this limitation, it is possible to generate conditional mutants in which a copy of the gene of interest is introduced into the bacteria to compensate for the loss of the native allele. Expression of the non-native copy of the target gene is typically under control of an inducible promoter, which allows for controllable and regulated production of the gene of interest. Conditional mutants are also broadly useful for phenotypic analyses of genes that require a tightly regulated and artificially inducible copy of the target gene. Herein, we describe the methods used to generate and confirm conditional mutant clones in Borrelia burgdorferi utilizing the Borrelia-adapted lac operator/repressor system.

BB0744 Affects Tissue Tropism and Spatial Distribution of Borrelia burgdorferi.

Borrelia burgdorferi, the etiologic agent of Lyme disease, produces a variety of proteins that promote survival and colonization in both the Ixodes species vector and various mammalian hosts. We initially identified BB0744 (also known as p83/100) by screening for B. burgdorferi strain B31 proteins that bind to α1ß1 integrin and hypothesized that, given the presence of a signal peptide, BB0744 may be a surface-exposed protein. In contrast to this expectation, localization studies suggested that BB0744 resides in the periplasm. Despite its subsurface location, we were interested in testing whether BB0744 is required for borrelial pathogenesis. To this end, a bb0744 deletion was isolated in a B. burgdorferi strain B31 infectious background, complemented, and queried for the role of BB0744 following experimental infection. A combination of bioluminescent imaging, cultivation of infected tissues, and quantitative PCR (qPCR) demonstrated that Δbb0744 mutant B. burgdorferi bacteria were attenuated in the ability to colonize heart tissue, as well as skin locations distal to the site of infection. Furthermore, qPCR indicated a significantly reduced spirochetal load in distal skin and joint tissue infected with Δbb0744 mutant B. burgdorferi. Complementation with bb0744 restored infectivity, indicating that the defect seen in Δbb0744 mutant B. burgdorferi was due to the loss of BB0744. Taken together, these results suggest that BB0744 is necessary for tissue tropism, particularly in heart tissue, alters the ability of B. burgdorferi to disseminate efficiently, or both. Additional studies are warranted to address the mechanism employed by BB0744 that alters the pathogenic potential of B. burgdorferi.

SaeRS-dependent inhibition of biofilm formation in Staphylococcus aureus Newman.

The SaeRS two-component regulatory system of Staphylococcus aureus is known to affect the expression of many genes. The SaeS protein is the histidine kinase responsible for phosphorylation of the response regulator SaeR. In S. aureus Newman, the sae system is constitutively expressed due to a point mutation in saeS, relative to other S. aureus strains, which results in substitution of proline for leucine at amino acid 18. Strain Newman is unable to form a robust biofilm and we report here that the biofilm-deficient phenotype is due to the saeSP allele. Replacement of the Newman saeSP with saeSL, or deletion of saeRS, resulted in a biofilm-proficient phenotype. Newman culture supernatants were observed to inhibit biofilm formation by other S. aureus strains, but did not affect biofilm formation by S. epidermidis. Culture supernatants of Newman saeSL or Newman ΔsaeRS had no significant effect on biofilm formation. The inhibitory factor was inactivated by incubation with proteinase K, but survived heating, indicating that the inhibitory protein is heat-stable. The inhibitory protein was found to affect the attachment step in biofilm formation, but had no effect on preformed biofilms. Replacement of saeSL with saeSP in the biofilm-proficient S. aureus USA300 FPR3757 resulted in the loss of biofilm formation. Culture supernatants of USA300 FPR3757 saeSP, did not inhibit biofilm formation by other staphylococci, suggesting that the inhibitory factor is produced but not secreted in the mutant strain. A number of biochemical methods were utilized to isolate the inhibitory protein. Although a number of candidate proteins were identified, none were found to be the actual inhibitor. In an effort to reduce the number of potential inhibitory genes, RNA-Seq analyses were done with wild-type strain Newman and the saeSL and ΔsaeRS mutants. RNA-Seq results indicated that sae regulates many genes that may affect biofilm formation by Newman.

BB0238, a presumed tetratricopeptide repeat-containing protein, is required during Borrelia burgdorferi mammalian infection.

The Lyme disease spirochete, Borrelia burgdorferi, occupies both a tick vector and mammalian host in nature. Considering the unique enzootic life cycle of B. burgdorferi, it is not surprising that a large proportion of its genome is composed of hypothetical proteins not found in other bacterial pathogens. bb0238 encodes a conserved hypothetical protein of unknown function that is predicted to contain a tetratricopeptide repeat (TPR) domain, a structural motif responsible for mediating protein-protein interactions. To evaluate the role of bb0238 during mammalian infection, a bb0238-deficient mutant was constructed. The bb0238 mutant was attenuated in mice infected via needle inoculation, and complementation of bb0238 expression restored infectivity to wild-type levels. bb0238 expression does not change in response to varying culture conditions, and thus, it appears to be constitutively expressed under in vitro conditions. bb0238 is expressed in murine tissues during infection, though there was no significant change in expression levels among different tissue types. Localization studies indicate that BB0238 is associated with the inner membrane of the spirochete and is therefore unlikely to promote interaction with host ligands during infection. B. burgdorferi clones containing point mutations in conserved residues of the putative TPR motif of BB0238 demonstrated attenuation in mice that was comparable to that in the bb0238 deletion mutant, suggesting that BB0238 may contain a functional TPR domain.

Identification of lysine residues in the Borrelia burgdorferi DbpA adhesin required for murine infection.

Decorin-binding protein A (DbpA) of Borrelia burgdorferi mediates bacterial adhesion to heparin and dermatan sulfate associated with decorin. Lysines K82, K163, and K170 of DbpA are known to be important for in vitro interaction with decorin, and the DbpA structure, initially solved by nuclear magnetic resonance (NMR) spectroscopy, suggests these lysine residues colocalize in a pocket near the C terminus of the protein. In the current study, we solved the structure of DbpA from B. burgdorferi strain 297 using X-ray crystallography and confirmed the existing NMR structural data. In vitro binding experiments confirmed that recombinant DbpA proteins with mutations in K82, K163, or K170 did not bind decorin, which was due to an inability to interact with dermatan sulfate. Most importantly, we determined that the in vitro binding defect observed upon mutation of K82, K163, or K170 in DbpA also led to a defect during infection. The infectivity of B. burgdorferi expressing individual dbpA lysine point mutants was assessed in mice challenged via needle inoculation. Murine infection studies showed that strains expressing dbpA with mutations in K82, K163, and K170 were significantly attenuated and could not be cultured from any tissue. Proper expression and cellular localization of the mutated DbpA proteins were examined, and NMR spectroscopy determined that the mutant DbpA proteins were structurally similar to wild-type DbpA. Taken together, these data showed that lysines K82, K163, and K170 potentiate the binding of DbpA to dermatan sulfate and that an interaction(s) mediated by these lysines is essential for B. burgdorferi murine infection.

Insights into the biology of Borrelia burgdorferi gained through the application of molecular genetics.

Borrelia burgdorferi, the vector-borne bacterium that causes Lyme disease, was first identified in 1982. It is known that much of the pathology associated with Lyme borreliosis is due to the spirochete's ability to infect, colonize, disseminate, and survive within the vertebrate host. Early studies aimed at defining the biological contributions of individual genes during infection and transmission were hindered by the lack of adequate tools and techniques for molecular genetic analysis of the spirochete. The development of genetic manipulation techniques, paired with elucidation and annotation of the B. burgdorferi genome sequence, has led to major advancements in our understanding of the virulence factors and the molecular events associated with Lyme disease. Since the dawn of this genetic era of Lyme research, genes required for vector or host adaptation have garnered significant attention and highlighted the central role that these components play in the enzootic cycle of this pathogen. This chapter covers the progress made in the Borrelia field since the application of mutagenesis techniques and how they have allowed researchers to begin ascribing roles to individual genes. Understanding the complex process of adaptation and survival as the spirochete cycles between the tick vector and vertebrate host will lead to the development of more effective diagnostic tools as well as identification of novel therapeutic and vaccine targets. In this chapter, the Borrelia genes are presented in the context of their general biological roles in global gene regulation, motility, cell processes, immune evasion, and colonization/dissemination.

Rrp2, a prokaryotic enhancer-like binding protein, is essential for viability of Borrelia burgdorferi.

The Lyme disease spirochete, Borrelia burgdorferi, exists in two diverse niches (i.e., an arthropod tick vector and mammalian host) during its enzootic life cycle. To effectively adapt to these unique environments, the bacterium alters the expression of numerous genes, including several major outer surface (lipo)proteins that are required for infection and transmission. An enhancer-binding protein (EBP), known as Rrp2, is one identified activator of the RpoN/RpoS alternative sigma factor cascade. Because initial efforts to generate an rrp2 deletion strain were unsuccessful, the role of Rrp2 in the activation of the RpoN/RpoS pathway was first defined using a strain of B. burgdorferi carrying an rrp2 point mutant that was defective in its ability to activate RpoN-dependent transcription. The fact that subsequent attempts to disrupt rrp2 have also been unsuccessful has led investigators to hypothesize that Rrp2 has other undefined functions which are essential for B. burgdorferi survival and independent of its EBP function. We used a lac-based inducible expression system to generate a conditional rrp2 mutant in virulent B. burgdorferi. In this strain, an isopropyl-ß-D-thiogalactopyranoside-inducible copy of the rrp2 gene is expressed in trans from a borrelial shuttle vector. We found that the chromosomal copy of rrp2 could be inactivated only when rrp2 was induced, and the maintenance of rrp2 expression was required for the growth of the mutants. In addition, the overexpression of rrp2 is detrimental to B. burgdorferi growth in a manner that is independent of the RpoN/RpoS pathway. These studies provide the first direct evidence that rrp2 is an essential gene in B. burgdorferi.

Invariant NKT cell development requires a full complement of functional CD3 zeta immunoreceptor tyrosine-based activation motifs.

Invariant NKT (iNKT) cells regulate early immune responses to infections, in part because of their rapid release of IFN-gamma and IL-4. iNKT cells are proposed to reduce the severity of Lyme disease following Borrelia burgdorferi infection. Unlike conventional T cells, iNKT cells express an invariant alphabeta TCR that recognizes lipids bound to the MHC class I-like molecule, CD1d. Furthermore, these cells are positively selected following TCR interactions with glycolipid/CD1d complexes expressed on CD4+CD8+ thymocytes. Whereas conventional T cell development can proceed with as few as 4/10 CD3 immunoreceptor tyrosine-based activation motifs (ITAMs), little is known about the ITAM requirements for iNKT cell selection and expansion. We analyzed iNKT cell development in CD3 zeta transgenic lines with various tyrosine-to-phenylalanine substitutions (YF) that eliminated the functions of the first (YF1,2), third (YF5,6), or all three (YF1-6) CD3 zeta ITAMs. iNKT cell numbers were significantly reduced in the thymus, spleen, and liver of all YF mice compared with wild type mice. The reduced numbers of iNKT cells resulted from significant reductions in the expression of the early growth response 2 and promyelocytic leukemia zinc finger transcription factors. In the mice with few to no iNKT cells, there was no difference in the severity of Lyme arthritis compared with wild type controls, following infections with the spirochete B. burgdorferi. These findings indicate that a full complement of functional CD3 zeta ITAMs is required for effective iNKT cell development.

Rrp2, a sigma54-dependent transcriptional activator of Borrelia burgdorferi, activates rpoS in an enhancer-independent manner.

Rrp2 is the sole sigma(54)-dependent transcriptional activator present in the Borrelia burgdorferi genome. We showed that recombinant Rrp2 binds to DNA in a sequence-nonspecific manner. During infection, Rrp2 activates sigma(54)-dependent rpoS expression without an apparent upstream enhancer element commonly associated with other sigma(54)-dependent transcriptional activators.

Transcriptional interplay among the regulators Rrp2, RpoN and RpoS in Borrelia burgdorferi.

The RpoN-RpoS alternative sigma factor pathway is essential for key adaptive responses by Borrelia burgdorferi, particularly those involved in the infection of a mammalian host. A putative response regulator, Rrp2, is ostensibly required for activation of the RpoN-dependent transcription of rpoS. However, questions remain regarding the extent to which the three major constituents of this pathway (Rrp2, RpoN and RpoS) act interdependently. To assess the functional interplay between Rrp2, RpoN and RpoS, we employed microarray analyses to compare gene expression levels in rrp2, rpoN and rpoS mutants of parental strain 297. We identified 98 genes that were similarly regulated by Rrp2, RpoN and RpoS, and an additional 47 genes were determined to be likely regulated by this pathway. The substantial overlap between genes regulated by RpoS and RpoN provides compelling evidence that these two alternative sigma factors form a congruous pathway and that RpoN regulates B. burgdorferi gene expression through RpoS. Although several known B. burgdorferi virulence determinants were regulated by the RpoN-RpoS pathway, a defined function has yet to be ascribed to most of the genes substantially regulated by Rrp2, RpoN and RpoS.