A novel fold for the factor H-binding protein BbCRASP-1 of Borrelia burgdorferi.

Borrelia burgdorferi, a spirochete transmitted to human hosts during feeding of infected Ixodes ticks, is the causative agent of Lyme disease. Serum-resistant B. burgdorferi strains cause a chronic, multisystemic form of the disease and bind complement factor H (FH) and FH-like protein 1 (FHL-1) on the spirochete surface. Here we report the atomic structure for the key FHL-1- and FH-binding protein BbCRASP-1 and reveal a homodimer that presents a novel target for drug design.

Identification and characterization of the factor H and FHL-1 binding complement regulator-acquiring surface protein 1 of the Lyme disease spirochete Borrelia spielmanii sp. nov.

Borrelia spielmanii, one of the etiological agents of Lyme disease found in Europe, evades host complement-mediated killing by recruitment of the immune regulators factor H and FHL-1 from human serum. Serum-resistant and intermediate serum-resistant isolates express up to 3 distinct complement regulator-acquiring surface proteins (CRASPs) that bind factor H and/or FHL-1. The present study describes identification and functional characterization of BsCRASP-1 as the dominant factor H and FHL-1 binding protein of B. spielmanii. BsCRASP-1 is a 27.7kDa outer surface lipoprotein, which after processing has a predicted mass of 24.9kDa. BsCRASP-1 is encoded by a single copy gene, cspA, that maps to a linear plasmid of approximately 55kb. Ligand affinity blot techniques revealed that both native and recombinant BsCRASP-1 from different isolates can strongly bind FHL-1, but only weakly factor H. Deletion mutants of recombinant BsCRASP-1 were generated and a high-affinity binding site for factor H and FHL-1 was mapped to its carboxy-terminal 10-amino-acid residue domain. Similarly, the dominant binding site of factor H and FHL-1 was localized to short consensus repeats (SCRs) 5-7. Factor H and FHL-1 maintained cofactor activity for factor I-mediated C3b inactivation when bound to full-length BsCRASP-1 but not to a deletion mutant lacking the carboxy-terminal 10-amino-acid residue domain. In conclusion, BsCRASP-1 binds the host immune regulators factor H and FHL-1, and is suggested to represent a key molecule of B. spielmanii for complement resistance. Thus, BsCRASP-1 most likely contributes to persistence of B. spielmanii and to pathogenesis of Lyme disease.

Mutational analyses of the BbCRASP-1 protein of Borrelia burgdorferi identify residues relevant for the architecture and binding of host complement regulators FHL-1 and factor H.

Borrelia burgdorferi exploits multiple strategies to evade host immune responses. One central immune escape mechanism is the inactivation of the host complement attack by acquisition host complement regulators FHL-1 and factor H via complement regulator-acquiring surface proteins (BbCRASPs). The BbCRASP-1 protein is the first bacterial factor H/FHL-1-binding protein for which the atomic structure has been solved. Previously, 3 regions including the C terminus were identified as putative contact sites for the two complement regulators by the pepspot analysis. Based on the crystallographic structure an in vitro mutagenesis approach was conducted to identify amino acid residues which are relevant for FHL-1 and factor H binding by exchanging single or multiple residues in region 1 and the C-terminally located region 3. Single changes at 4 positions in region 1 either reduced (Lys136, Lys141, Glu147) or completely eliminated (Leu146) binding of both complement regulators. Substitutions clustered within the C-terminal region decreased (Glu234, Lys238, Tyr239, Lys241, Asp244, Thr245) or abolished binding (Lys240, Asp242, Leu246) of both complement regulators. Mapping the mutations onto the atomic structure of BbCRASP-1 reveals that, in contrast to earlier assumption, the C-terminal mutations act indirectly on FHL-1 and factor H binding, whilst the region 1 mutations map the site of direct complement regulator interaction. The elucidation of BbCRASP-1 structure - function may allow development of novel therapeutic strategies against Lyme disease.

Influence of dTMP on the phenotypic appearance and intracellular persistence of Staphylococcus aureus.

Thymidine-dependent small-colony variants (SCVs) of Staphylococcus aureus are frequently associated with persistent and recurrent infections in cystic fibrosis patients. The phenotypic appearance of S. aureus SCVs or normal-colony variants (NCVs) is postulated to be affected by the intracellular amount of dTMP. This hypothesis was proven by metabolic pathway assays revealing altered intracellular dTMP concentrations, followed by investigation of the associated phenotype. Inhibition of the staphylococcal thymidylate synthase, which generated intracellular dTMP from dUMP, using 5-fluorouracil and co-trimoxazole resulted in an SCV phenotype. Inhibition of a nucleoside transporter, which provided the bacterial cell with extracellular thymidine, caused growth inhibition of SCVs. In turn, reversion of SCVs to NCVs was achieved by supplying extracellular dTMP. High-performance liquid chromatography additionally confirmed the intracellular lack of dTMP in SCVs, in contrast to NCVs. Moreover, the dTMP concentration is postulated to influence the intracellular persistence of S. aureus. Cell culture experiments with cystic fibrosis cells revealed that clinical and co-trimoxazole-induced SCVs with a diminished amount of dTMP showed significantly better intracellular persistence than NCVs. In conclusion, these results show that the dTMP concentration plays a key role in both the phenotypic appearance and the intracellular persistence of S. aureus.

Systematic analysis highlights the key role of TLR2/NF-kappaB/MAP kinase signaling for IL-8 induction by macrophage-like THP-1 cells under influence of Borrelia burgdorferi lysates.

Lyme borreliosis is a spirochetal infection caused by the Borrelia burgdorferi sensu lato complex that can proceed towards an inflammatory joint manifestation known as Lyme arthritis. Production of chemokines orchestrating neutrophil infiltration is supposed to be key to early arthritic pathogenesis. Using PMA-differentiated macrophage-like THP-1 (mTHP-1) cells we identified by antibody array methodology or mRNA analysis IL-8, GRO-alpha, NAP-2, and SDF-1alpha as being among those chemokines that are upregulated by bacterial lysates obtained from B. burgdorferi. Based on these observations, we set out to characterize in detail mechanisms mediating IL-8 release in this cellular model. TLR2 blocking antibodies, analysis of p65 translocation, and electromobility-shift analysis revealed activation of the TLR2/NF-kappaB axis by B. burgdorferi. The functional importance of this pathway was substantiated by suppression of IL-8 after inhibition of IkappaB kinase. Notably, MAP kinases, specifically the MEK1/2-ERK1/2 pathway, were essential for IL-8 secretion. Those data were confirmed by using freshly isolated adherent peripheral blood mononuclear cells. On the contrary, B. burgdorferi-induced IL-8 in mTHP-1 was unlikely related to flagellin, alpha3beta1-integrin signaling, lipopolysaccharide, bacterial DNA, NOD1/NOD2 agonists, or to intermediate production of IL-1beta and TNF-alpha. Induction of IL-8 by B. burgdorferi was not due to amplification of constitutive AP-1 DNA-binding activity detectable in mTHP-1 cells. Data presented herein validate that TLR2, particularly on mTHP-1 cells, holds a central position in mediating IL-8 secretion associated with extracellular B. burgdorferi and beyond that suggest inhibition of IkappaB kinase and MEK1/2 kinases as promising pharmacological strategies aiming at IL-8 in early Lyme arthritis.

The thymidine-dependent small-colony-variant phenotype is associated with hypermutability and antibiotic resistance in clinical Staphylococcus aureus isolates.

Thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus can be isolated from the airway secretions of patients suffering from cystic fibrosis (CF) and are implicated in persistent and treatment-resistant infections. These characteristics, as well as the variety of mutations in the thymidylate synthase-encoding thyA gene which are responsible for thymidine dependency, suggest that these morphological variants are hypermutable. To prove this hypothesis, we analyzed the mutator phenotype of different S. aureus phenotypes, in particular CF-derived TD-SCVs, CF-derived isolates with a normal phenotype (NCVs), and non-CF NCVs. The comparative analysis revealed that the CF isolates had significantly higher mutation rates than the non-CF isolates. The TD-SCVs, in turn, harbored significantly more strong hypermutators (mutation rate > or = 10(-7)) than the CF and non-CF NCVs. In addition, antimicrobial resistance to non-beta-lactam antibiotics, including gentamicin, ciprofloxacin, erythromycin, fosfomycin, and rifampin, was significantly more prevalent in TD-SCVs than in CF and non-CF NCVs. Interestingly, macrolide resistance, which is usually mediated by mobile genetic elements, was conferred in half of the macrolide-resistant TD-SCVs by the point mutation A2058G or A2058T in the genes encoding the 23S rRNA. Sequence analysis of mutS and mutL, which are involved in DNA mismatch repair in gram-positive bacteria, revealed that in hypermutable CF isolates and especially in TD-SCVs, mutL was often truncated due to frameshift mutations. In conclusion, these data provide direct evidence that TD-SCVs are hypermutators. This hypermutability apparently favors the acquisition of antibiotic resistance and facilitates bacterial adaptation during long-term persistence.

Thymidine-dependent Staphylococcus aureus small-colony variants: human pathogens that are relevant not only in cases of cystic fibrosis lung disease.

We report the isolation of thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus from unusual infection sites of patients with chronic soft tissue infection, tympanitis, bronchitis, peritonitis, and septicemia. Furthermore, we provide evidence that the essential growth factor for TD-SCVs, i.e., thymidine, and its metabolite dTMP are present in various human specimens.

Release of latent ClyA cytolysin from Escherichia coli mediated by a bacteriophage-associated putative holin (BlyA) from Borrelia burgdorferi.

Introduction of the Borrelia burgdorferi blyAB locus into Escherichia coli has recently been reported to cause a hemolytic phenotype that is dependent on the E. coli clyA (hlyE, sheA) gene (a cytolysin gene present in many E. coli strains, including E. coli K-12, which is repressed under standard in vitro growth conditions). The blyA gene product has been suggested to be a prophage-encoded holin, but the processes triggered in E. coli by the expression of blyA and/or blyB, which lead to the hemolytic phenotype, remained unclear. Here we show that expression of blyA in E. coli causes damage to the E. coli cell envelope and a clyA-dependent hemolytic phenotype, regardless whether blyB is present or absent. The expression of blyB in E. coli, on the other hand, did not have obvious phenotypic effects. Transcriptional studies demonstrated that the clyA gene is not induced in E. coli cells expressing blyA. Furthermore, protein analyses suggested that the impairment of the E. coli cell envelope by BlyA is responsible for the emergence of the hemolytic activity as it allows latent intracellular ClyA protein, derived from basal-level expression of the clyA gene, to leak into the medium and to lyse erythrocytes. These findings are compatible with the presumption that BlyA functions as a membrane-active holin.

Borrelia burgdorferi complement regulator-acquiring surface proteins (BbCRASPs): Expression patterns during the mammal-tick infection cycle.

Host complement is widely distributed throughout mammalian body fluids and can be activated immediately as part of the first line of defense against invading pathogens. The agent of Lyme disease, Borrelia burgdorferi sensu lato (s.l.), is naturally resistant to that innate immune defense system of its hosts. One resistance mechanism appears to involve binding fluid-phase regulators of complement to distinct borrelial outer surface molecules known as CRASPs (complement regulator acquiring surface proteins). Using sensitive molecular biology techniques, expression patterns of all three classes of genes encoding the CRASPs of B. burgdorferi sensu stricto (BbCRASPs) have been analyzed throughout the natural tick-mammal infection cycle. Each class shows a different expression profile in vivo and the results are summarized herein. Studies on the expression of B. burgdorferi genes using animal models of infection have advanced our knowledge on the ability of the causative agent to circumvent innate immune defenses, the contributions of CRASPs to spirochete infectivity, and the pathogenesis of Lyme disease.

Antimicrobial susceptibility of Borrelia burgdorferi sensu lato: what we know, what we don't know, and what we need to know.

Human Lyme borreliosis is a multisystem disorder that can progress in stages and is transmitted by ticks of the Ixodes ricinus complex infected with the spirochete Borrelia burgdorferi sensu lato. Today, Lyme borreliosis is regarded as the most important human tickborne illness in the northern hemisphere. Soon after the causative agent was correctly identified and successfully isolated in 1982, antibiotic treatment was shown to be effective and since then a variety of in vitro and in vivo studies have been performed to further characterize the activity of antimicrobial agents against B. burgdorferi s.l. Although many antimicrobial agents have been tested for their in vitro activity against borreliae, the full spectrum of antibiotic susceptibility in B. burgdorferi s.l. has not been defined for many compounds. Moreover, our current understanding of possible antimicrobial resistance mechanisms in B. burgdorferi s.l. is limited and is largely founded on in vitro experiments on relatively few borrelial isolates. This review will summarize what is and what is not known about antimicrobial resistance in B. burgdorferi s.l. and will discuss open questions that continue to fuel the current debate on treatment-resistant Lyme borreliosis.

Binding of human complement regulators FHL-1 and factor H to CRASP-1 orthologs of Borrelia burgdorferi.

The complement regulator-acquiring surface protein (CRASP)-1 is a member of the paralogous gene family gbb54 and the dominant FHL-1 and factor H binding protein of Borrelia burgdorferi sensu stricto (s.s.). It was shown recently that expression of BbCRASP-1 directly correlates with serum resistance of B. burgdorferi s.s. isolates. In the present study we have elucidated the putative potential of other members of the gbb54 paralogous family, including orthologs ZSA66, ZSA69, ZSA70, ZSA71, ZSA72 and ZSA73 of the European B. burgdorferi s.s. strain ZS7, to bind human FHL-1 and factor H. In spite of their overall similarity in protein sequence, between 47% and 67%, and the fact that the C-terminal region of ZSA69 shows 70% similarity with BbCRASP-1, none of the orthologous proteins was able to bind human FHL-1 and/or factor H. BbCRASP-1 is the only member of the paralogous gene family gbb54 that binds to human complement regulators, supporting the notion that BbCRASP-1 plays a critical role in evasion of complement by B. burgdorferi s.s. and thus may be helpful in the development of novel therapeutic strategies against Lyme borreliosis.

Host association of Borrelia burgdorferi sensu lato--the key role of host complement.

Borrelia burgdorferi sensu lato (s.l.), the tick-borne agent of Lyme borreliosis, is a bacterial species complex comprising 11 genospecies. Here, we discuss whether the delineation of genospecies is ecologically relevant. We provide evidence that B. burgdorferi s.l. is structured ecologically into distinct clusters that are host specific. An immunological model for niche adaptation is proposed that suggests the operation of complement-mediated selection in the midgut of the feeding tick. We conclude that vertebrate hosts rather than tick species are the key to Lyme borreliosis spirochaete diversity.

Borreliacidal activity of early Lyme disease sera against complement-resistant Borrelia afzelii FEM1 wild-type and an OspC-lacking FEM1 variant.

Sera obtained from 14 Lyme borreliosis patients at early stages (stages I and II) of the disease were examined for their borreliacidal properties against Borrelia afzelii isolate FEM1 by use of a growth inhibition assay. Five of 14 immune sera exhibited borreliacidal activity against isolate FEM1. Heat-inactivated immune sera failed to kill the spirochaetes. Immunoblotting experiments with outer-membrane preparations showed that OspC and 11 additional proteins of 14.0, 16.0, 17.7, 19.3, 21.7, 27.5, 32.7, 40.7, 48.9, 51.3 and 53.6 kDa were recognised by borreliacidal immune sera. To analyse the borreliacidal properties of anti-OspC antibodies, two sera (EM4 and EM5), which beside antibodies against a 51.3-kDa protein contained exclusively anti-OspC antibodies, were further investigated by comparative analysis with a FEM1 wild-type and a FEM1 variant lacking OspC in a growth inhibition assay. Only FEM1 wild-type and not variant FEM1OspC(-) was killed by immune sera EM4 and EM5. Complement-dependent killing of FEM1 wild-type was mediated by formation of the terminal complement complex that was found to be attached directly to the outer membrane as confirmed by immuno-electron microscopy. No complement deposition was observed on the surface of variant FEM1OspC(-) after incubation with immune sera EM4 and EM5, thereby suggesting that only anti-OspC antibodies in these two immune sera were responsible for borreliacidal activity. These results provide direct evidence that anti-OspC antibodies, once developed during the immune response, are of critical importance for efficient killing of borreliae in the early phase of infection.

New real-time PCR-based method for in vitro susceptibility testing of Anaplasma phagocytophilum against antimicrobial agents.

Up to now, only a few isolates of Anaplasma phagocytophilum have been tested for their susceptibility against a small number of antimicrobial agents. In addition, as with other fastidious or intracellular bacteria, the test methods are laborious and neither minimal inhibitory concentration (MIC) definitions, nor the test conditions and the inocula are standardised to date. A new 16S-rDNA-based real-time PCR assay has been developed and used under standardised conditions to analyse the activity of seven antimicrobial agents against two A. phagocytophilum isolates. After 72 h incubation, MICs were determined by software-assisted calculation of bacterial growth in samples and controls from semi-quantitative PCR results. In our study, the rank order of potency on a mg/l basis for the antimicrobial agents with enhanced in vitro activity against A. phagocytophilum was moxifloxacin (MIC: < or = 0.03 mg/l) > doxycycline (MIC: < or = 0.125 mg/l) > ciprofloxacin (MIC: 0.125 mg/l). Gentamicin, ampicillin, azithromycin and cethromycin showed no activity against the isolates tested in this investigation. Our new 16S-rDNA-PCR-based microdilution test system was shown to be sensitive, reproducible and reliable. The assay is capable of testing larger numbers of isolates and antimicrobial agents under standardised and very precise test conditions and may therefore offer a competent technical solution of the difficulties known to be associated with in vitro testing of other bacterial pathogens that grow intracellularly, such as chlamydia or rickettsia.

Complement regulator-acquiring surface proteins of Borrelia burgdorferi: a new protein family involved in complement resistance.

The innate immune system, particularly the host complement system, plays an important role in the elimination of invading pathogens. Borrelia burgdorferi, like other human pathogens, has developed strategies to prevent complement-mediated bacteriolysis. It is now well established that Borrelia differ in their complement resistance. In general terms, B. afzelii isolates are mainly resistant to complement-mediated bacteriolysis, whereas the majority of B. burgdorferi s.s. isolates display an intermediate complement-resistant phenotype. Most of the B. garinii isolates, in contrast, are efficiently killed by complement and, therefore, are classified as complement-sensitive. Complement resistance of B. afzelii and B. burgdorferi s.s. isolates correlates directly with the acquisition of the fluid-phase human complement regulators FHL-1/reconectin and factor H. To date, five distinct complement regulator-acquiring surface proteins (CRASPs) have been identified in B. afzelii and B. burgdorferi s.s. isolates. The individual CRASPs can be differentiated according to their size and their binding properties to FHL-1/reconectin and factor H. The domains that interact with CRASPs are localized at the C-terminal ends of these complement regulators. Thus, CRASPs represent a family of functional proteins involved in complement resistance of Borrelia. Furthermore, an alterable pattern of gene expression was observed for three CRASPs of B. afzelii: BaCRASP-1, BaCRASP-2, and BaCRASP-5 are up-regulated at 37 degrees C and down-regulated at 20 degrees C. The continued characterization of CRASPs at the molecular level is expected to identify new virulence factors and potential vaccine candidates.

Development and laboratory evaluation of a new recombinant ELISA for the serodiagnosis of Lyme disease.

OBJECTIVE: The use of recombinant proteins for serologic testing represents a modern approach for the improved laboratory diagnosis of Lyme disease (LD). The aim of the present study was to develop and evaluate a new recombinant ELISA (RE) for the detection of specific IgG and IgM antibodies against Borrelia burgdorferi. MATERIALS AND METHODS: The RE (Biotest AG, Dreieich, Germany) uses mixtures of recombinant p100, OspC, p18 of Borrelia afzelii and a fusion protein of recombinant internal fragments of the flagellum protein (p41) of Borrelia garinii strain PBi and Borrelia afzelii strain PKo. Serologic testing was performed on a commercially available ELISA processor without pre-absorption of sera. The sensitivity of the RE was determined by testing 226 sera obtained from patients suffering from Lyme disease (stage I: n = 148, stage II: n = 35, stage III: n = 43). Specificity of the RE was evaluated in 1107 sera from healthy blood donors and 275 sera from patients with other infectious diseases or autoimmune illnesses (leptospirosis: n = 53, syphilis: n = 70, toxoplasmosis: n = 60, herpes simplex virus: n = 30, HIV: n = 30; rheumatoid-factor positive: n = 32). In addition, 394 routine samples were prospectively tested in comparison with a well established whole-cell lysate extract ELISA (ENZYGNOST Borreliosis, DadeBehring, Germany) for relative sensitivity and specificity. RESULTS: Overall specificity, determined in 1107 healthy blood donors and 275 sera from patients with other diseases, was 94% for IgG and 91% for IgM. The overall sensitivities in 226 sera obtained from patients suffering from different stages of LD were 67-95%. Moreover, as revealed by prospective testing of 394 routine samples, the relative sensitivity of the RE in comparison with an established whole-cell lysate extract ELISA in the detection of seropositive samples was 81.1% for IgG and 86.5% for IgM with a relative specificity of 98.5% and 93% respectively. The ELISAs showed an overall agreement of 97% for IgG and 92.4% for IgM test results. CONCLUSION: The RE proved to be a reliable and specific screening test in the routine serodiagnosis of LD. In addition, the RE is easy to perform and requires no pre-absorption.

Quality of Lyme disease serology. Lessons from the German Proficiency Testing Program 1999-2001. A preliminary report.

OBJECTIVE: External quality control surveys are an important tool in regulating the quality of infection serology in general and of borreliosis serology in particular. We report on the results of a Lyme disease proficiency testing program which is regularly organised twice a year by our institutions in close cooperation with the Institute of Standardisation in the Medical Laboratory (INSTAND). METHODS: From 1999 to 2001, between 226 and 337 microbiological laboratories participated in each of the four surveys that have been held so far. In addition, between 23 and 30 laboratories from 13 other European countries also participated in each trial. In each survey two serum samples which had been unambiguously characterised by six reference laboratories to contain or not to contain antibodies against the Lyme disease spirochete were distributed in order to determine the accuracy of the diagnostic methods used in participating laboratories. The laboratories also reported interpretative statements of whether or not the test constellation suggested a possible borrelial infection and if an early or late phase of the specific antibody response was suspected. RESULTS: Test results were found to be in part highly variable and clearly correlated with the manufacturers and the applied test methodology. It was also clear that IgM tests were more difficult to handle than were IgG tests. ELISA-testing was more reproducible and proved to be more sensitive and specific than IFA and IHA testing. Quantification of test results and reporting of specific immunoblot bands also showed high variability. Moreover, for some assays a high number of false positive and false negative test results were reported by the participants. CONCLUSION: In view of our results further standardisation of Lyme disease serology is not just desirable but is urgently needed. Moreover, stronger criteria for the validation of available test kits must be applied.

Improved detection of blood stream pathogens by real-time PCR in severe sepsis.

OBJECTIVE: Evaluation of the technical and diagnostic feasibility of commercial multiplex real-time polymerase chain reaction (PCR) for detection of blood stream infections in a cohort of intensive care unit (ICU) patients with severe sepsis, performed in addition to conventional blood cultures. DESIGN: Dual-center cohort study. SETTING: Surgical ICU of two university hospitals. PATIENTS AND PARTICIPANTS: One hundred eight critically ill patients fulfilling the American College of Chest Physicians/Society of Critical Care Medicine (ACCP/SCCM) severe sepsis criteria were included. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: PCR results obtained in 453 blood samples from 108 patients were compared with corresponding blood culture results. PCR resulted in a twofold higher positivity rate when compared with conventional blood culture (BC) testing (114 versus 58 positive samples). In 40 out of 58 PCR positive assays the results of the corresponding blood cultures were identical to microorganisms detected by PCR. In 18 samples PCR and BC yielded discrepant results. Compared with conventional blood culture the sensitivity and specificity of PCR was 0.69 and 0.81, respectively. Further evaluation of PCR results against a constructed gold standard including conventional microbiological test results from other significant patient specimen (such as bronchio-alveolar lavage fluid, urine, swabs) and additionally generated clinical and laboratory information yielded sensitivity of 0.83 and specificity of 0.93. CONCLUSIONS: Our cohort study demonstrates improved pathogen detection using PCR findings in addition to conventional blood culture testing. PCR testing provides increased sensitivity of blood stream infection. Studies addressing utility including therapeutic decision-making, outcome, and cost-benefit following diagnostic application of PCR tests are needed to further assess its value in the clinical setting.

Linezolid resistance in Staphylococcus aureus: gene dosage effect, stability, fitness costs, and cross-resistances.

Linezolid resistance in Staphylococcus aureus is typically associated with mutations in the 23S rRNA gene. Here we show that the accumulation of a single point mutation, G2576T, in the different copies of this gene causes stepwise increases in resistance, impairment of the biological fitness, and cross-resistance to quinupristin-dalfopristin and chloramphenicol.

Deciphering the ligand-binding sites in the Borrelia burgdorferi complement regulator-acquiring surface protein 2 required for interactions with the human immune regulators factor H and factor H-like protein 1.

Borrelia burgdorferi, the etiologic agent of Lyme disease, employs sophisticated means to evade killing by its mammalian hosts. One important immune escape mechanism is the inhibition of complement activation mediated by interactions of the host-derived immune regulators factor H (CFH) and factor H-like protein 1 (CFHL1) with borrelial complement regulator-acquiring surface proteins (BbCRASPs). BbCRASP-2 is a distinctive CFH- and CFHL1-binding protein that is produced by serum-resistant B. burgdorferi strains. Here we show that binding of CFH by BbCRASP-2 is due to electrostatic as well as hydrophobic forces. In addition, 14 individual amino acid residues of BbCRASP-2 were identified as being involved in CFH and CFHL1 binding. Alanine substitutions of most of those residues significantly inhibited binding of CFH and/or CFHL1 by recombinant BbCRASP-2 proteins. To conclusively define the effects of BbCRASP-2 residue substitutions on serum sensitivity in the bacterial context, a serum-sensitive Borrelia garinii strain was transformed with plasmids that directed production of either wild-type or mutated BbCRASP-2 proteins. Critical amino acid residues within BbCRASP-2 were identified, with bacteria producing distinct mutant proteins being unable to bind either CFH or CFHL1, showing high levels of complement components C3, C6, and C5b-9 deposited on their surfaces and being highly sensitive to killing by normal serum. Collectively, we mapped a structurally sensitive CFH/CFHL1 binding site within borrelial BbCRASP-2 and identified single amino acid residues potentially involved in the interaction with both complement regulators.