Role of Plasmid Encoded Genes in the Pathopsyiology of Lyme Disease

Borrelia burgdorferi (Bb), the tick-borne agent of Lyme disease, is a unique spirochete with limited metabolic capabilities and intimately dependent on host-derived nutrients to alter its gene expression profiles in response to conditions that are highly divergent during its tick and mammalian phases of infection. An array of zinc-dependent transcriptional and post-transcriptional regulators of gene expression have been identified while the intricacies of regulatory changes mediated by zinc as a co-factor during different stages of its infectious life cycle are yet to be fully understood. Bioinformatic and whole genome transcriptional analysis revealed the presence of Borrelia zinc-finger domain containing Protein (BziP) whose role in the patho-physiology of Bb is unknown. My hypothesis is that BziP is linked to housekeeping sigma factor σ70 and is thus essential for Bb’s survival during the tick and mammalian phases of infection. In order to test this hypothesis, constitutive expression of bziP in Bb was achieved by expressing bziP under the control of PflgB-a strong constitutive borrelial promoter using a borrelial shuttle vector pTM61 which also expressed green fluorescent protein to facilitate rapid isolation of transformants. Constitutive expression of bziP results in an increase in the levels of major regulators of gene expression such as BosR and RpoS with a concomitant increase in the levels of several lipoproteins such as Outer surface protein C (OspC) and Decorin Binding Proteins A/B (DbpA/B) critical for transmission, dissemination and survival of Bb in mammalian hosts. BziP contains two conserved CXXC motifs shown to be critical for zinc binding and both these motifs are known to play critical roles in the functions of BziP homologs present in other bacteria. Site-specific changes replacing the cysteine residues of two CXXC (C223SxxC226S and C198SxxC201S) motifs with serines and a histidine at position 202 with alanine (H202A) decreased the levels of BosR, RpoS and OspC. The C198SxxC201S motif and the H202A also show a decrease in expression in BziP itself, indicating that these two site-directed changes destabilize BziP. Phenotypic analysis of the morphology of mutants expressing either the wild type or site directed mutants revealed that spirochetes with three site specific changes have a more elongated rod-shape when compared to the wildtype. Taken together, the constitutive expression of BziP increases the expression of many essential mammalian virulence-related genes and the elongated rod-shaped spirochetes of site specifically altered strains suggest the possibility of BziP playing a key role in motility/morphology of Bb. Based on these phenotypic observations it is likely that BziP could play a critical role in the transmission and infectivity in the mammalian host as both motility/morphology is essential for Bb infection. RNA Seq analysis and infectivity phenotype of these mutants in the C3H/HeN mouse model of Lyme disease will further aid in our understanding of the role of BziP in patho-physiology of Bb.