Insights into the inter-ring plasticity of caseinolytic proteases from the X-ray structure of Mycobacterium tuberculosis ClpP1.

Mycobacterium tuberculosis caseinolytic protease ClpP1 (Mt ClpP1) is a self-compartmentalized protease consisting of two heptameric rings stacked on top of each other, thus enclosing a catalytic chamber. Within the chamber, which can be reached through two axial pores, each of the 14 identical monomers possesses a serine protease active site. The unfolding and translocation of substrates into the chamber are mediated by associated hexameric ATPases covering the axial pores. Three crystal structures of Mt ClpP1, determined by molecular replacement, are presented in this study. Two of the models were refined to a resolution of 2.6 A and the third to 3.0 A. It was found that disorder in the handle domain affects the formation and configuration of the tetradecamer and results in condensed structures with larger equatorial pores when compared with ClpPs from other species. Additionally, this disorder accompanies conformational changes of the residues in the catalytic triad. The models also reveal structural differences within the N-terminal hairpin-loop domain, which possibly reflect the significant differences in amino-acid sequence between Mt ClpP1 and other ClpP homologues in this region.

A single-run, real-time PCR for detection and identification of Borrelia burgdorferi sensu lato species, based on the hbb gene sequence.

Lyme borreliosis is the most important vector-borne disease caused by spirochetes within the Borrelia burgdorferi sensu lato (B. burgdorferi sl) complex. There is strong evidence that different species of this group of genetically diverse spirochetes are involved in distinct clinical manifestations of the disease. In order to differentiate species within this bacterial complex, we developed a real-time-PCR protocol, which targets the hbb gene. We designed a fluorescein-labeled probe specific of a region of this gene harboring a polymorphism linked to species. An internally Red640 labeled primer allowed a fluorescence resonance energy transfer to occur. The sensitivity of this method was in the range of 10 bacteria per assay. After amplification, a melting curve was generated for genotyping. Analysis of these melting curves clearly allowed the distinction between the main European species of B. burgdorferi sl. One hundred seventy tick extracts were analysed by this hbb-based method and in parallel by amplification of the 5S-23S intergenic spacer and RFLP analyses. There was a good correlation between these two methods. We conclude that this hbb-based real-time-PCR is suitable for epidemiological studies on field-collected ticks, although rare mutations in the genomic sequence spanned by the probe could lead to misidentification.

Borrelia burgdorferi sensu stricto invasiveness is correlated with OspC-plasminogen affinity.

Borrelia burgdorferi sensu lato, the causative agent of Lyme borreliosis, is transmitted through tick bite. Lyme borreliosis evolves in two stages: a primary red skin lesion called erythema migrans; later on, invasive bacteria disseminate to distant sites inducing secondary manifestations (neuropathies, arthritis, carditis, late skin disorders). It has been previously suggested that the ospC gene could be associated with invasiveness in humans depending on its sequence. Here, we confirm the pattern of invasiveness, according to B. burgdorferi sensu stricto (B. b. ss) ospC group, using the mouse as an experimental host of B. b. ss. As it has been shown that the host plasminogen activation system is used by B. burgdorferi to disseminate throughout the host, we studied the interaction of plasminogen with OspC proteins from invasive and non-invasive groups of B. b. ss. Using two methods, ELISA and surface plasmon resonance, we demonstrate that indeed OspC is a plasminogen-binding protein. Moreover, significant differences in binding affinity for plasminogen are correlated with different invasiveness patterns in mice. These results suggest that the correlation between ospC polymorphism and Borrelia invasiveness in humans is linked, at least in part, to differences in OspC affinity for plasminogen.

A rapid and quantitative method for the detection of Leptospira species in human leptospirosis.

Prompt laboratory diagnosis of leptospirosis infection facilitates patient management and initiation of therapy. A cost effective real-time PCR assay using SYBR Green I was developed for detection of pathogenic leptospires in serum specimens. Specific PCR products were obtained only with DNA of pathogenic Leptospira genomospecies. LightCycler PCR ability to distinguish between species was possible using melting curves, providing an approach for identification with a specific Tm assigned to a single species or set of species. Assay sensitivity was approximately 50 leptospires/ml, corresponding to one to two genome copies in a PCR mixture. Fifty-one patients who had clinical symptoms consistent with leptospirosis were tested both with a previously described rrs amplification and our real-time assay. Our LFB1 real-time assay confirmed the diagnosis for 25 patients (49%, 25/51) and revealed an estimated density of 8.0x10(1)-3.9x10(4) leptospires/ml of blood. The total assay time for 12 clinical samples from sample to data analysis was less than 3 h. These data illustrate the potential of our LFB1 real-time assay for the rapid detection of leptospires in serum samples and their subsequent quantification in a single run.