METABOLIC VIRULENCE DETERMINANTS AND RAPID MOLECULAR DIAGNOSTICS OF PATHOGENIC SPIROCHETES
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Borrelia burgdorferi and Leptospira interrogans are pathogenic spirochetes that elicit serious health threats, termed as Lyme disease and leptospirosis. Key areas of spirochete research involve a better understanding of their intriguing biology and infection, including identification of novel virulence factors and improvements in diagnostic and preventive methods. Notably, certain bacterial metabolic enzymes are surface-exposed, having evolved to acquire additional functions referred to as protein moonlighting that contributes in microbial virulence. Comparative genome analysis revealed that certain components of sugar metabolism pathways are either absent or seemingly inactive in pathogenic spirochetes, which were studied herein for their potential roles as metabolic virulence factors. Of nine borrelial enzymes investigated, only phosphomannose isomerase (PMI) was found to be surface-exposed and remained enzymatically active in the spirochete outer membrane. PMI is critical for mannose metabolism and facilitates the interconversion of fructose 6-phosphate and mannose-6-phosphate, although its occurrence in borrelial surface remains enigmatic. PMI may provide a critical function for B. burgdorferi viability as it is constitutively expressed and all attempts to create genetic mutants remained unsuccessful. Active immunization studies using recombinant PMI did not influence the outcome of infection within tick or murine hosts, although a significant reduction in bacterial levels within the joints of mice was recorded, suggesting its involvement in spirochete persistence in a tissue-specific manner. Despite substantial advancement, the development of more effective diagnostics for leptospirosis and Lyme disease still remains a critical need since human vaccines are unavailable. Antibiotic treatment can resolve these infections but is most effective when administered early during infection, prior to pathogen dissemination to distant organs. As diagnostic methods for spirochete infection still depends on ineffective and antiquated technologies, we sought to develop novel RNA-based assays for better detection of early spirochete infection. Results indicated that targeting specific regions of 16S and 23S ribosomal RNA targets provided the highest possible sensitivity and specificity of detection, which was far superior to current serological, microbiological or molecular methods used to detect presence of invading pathogens.