Molecular Genetic Basis of the Infectious Cycle of Borrelia burgdorferi

National Institute Of Allergy And Infectious Diseases

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Abstract

Lyme disease is the most common tick-borne illness in the United States and Europe. It is caused by Borrelia burgdorferi, a bacterial pathogen that is maintained in nature in a zoonotic cycle between various species of small mammals and an ixodid tick vector. A hallmark of the Lyme disease spirochete is its unusual, segmented genome, comprising a linear chromosome and approximately 20 linear and circular plasmids. An increasing body of data demonstrates that plasmid-encoded functions are critical for successful adaptation of B. burgdorferi to the different environments that the spirochete encounters during its natural infectious cycle. We have developed genetic tools to investigate basic aspects of the unusual genomic organization, cellular structure and metabolism of B. burgdorferi. We have extended this investigation to an in vivo setting with an experimental system that closely mimics the natural arthropod vector/rodent host infectious cycle. Through an understanding of the basic molecular biology of the organism, we hope to gain insight into the infectious strategy utilized by this significant vector-borne pathogen and thereby facilitate efforts to prevent, diagnose and treat Lyme disease. The alternative sigma factor RpoS plays a central role in a critical adaptive response of the Lyme disease spirochete that is induced during tick feeding and prepares the spirochete for infection of the vertebrate host. Previous work from our lab identified the plasmid-encoded BBD18 protein as a negative regulator of RpoS, but inactivation of the bbd18 gene in wild-type spirochetes was never achieved. In FY2022, we utilized a conditional bbd18 mutant, genome-wide transcriptomic, metabolomic, and protein profiling, and the first engineered displacement of all cp32 prophage plasmids, to investigate the lethal phenotype that accompanies BBD18 depletion and unmodulated production of RpoS in infectious B. burgdorferi. Additionally, we conducted in vivo studies to identify the precise stage and location during the mouse-tick infectious cycle at which spirochetes require BBD18 for survival. As a result of these experiments, we provide the first report of a link between transducing phage and the RpoS-dependent adaptive response that Lyme disease spirochete undergo during tick feeding. We infer that the multipartite structure of the Borrelia genome facilitates phage-mediated genetic exchange and reassortment, and thereby fosters the level of diversity that is required for spirochete maintenance in the natural enzootic cycle. Senior post-doctoral IRTA Fellow Dr. Jenny Wachter played a lead role in this exciting study and prepared a manuscript describing these results that received positive reviews and is currently in revision at Nature Communications (1). In a study culminating in FY2022, we investigated the contribution of a previously uncharacterized gene on linear plasmid lp54 during the infectious cycle of B. burgdorferi. Linear plasmid lp54 is universally conserved among the Lyme disease spirochetes and exceptionally stable during in vitro propagation, suggesting that lp54 is not only essential for the spirochete's survival throughout the infectious cycle, but also contributes to efficient growth in vitro. However, the functions of most lp54-encoded proteins remain unknown. BBA30, the gene on lp54 that was the focus of our study, is regulated by RpoS and present in all Borrelia, but without any identified homologs outside the genus. Modeling the structure of the highly conserved amino acid sequence of BBA30 identified a Helix-Turn-Helix nucleic acid binding motif near the amino terminus of the protein, suggesting a possible regulatory function. Interestingly, an adjacent gene adjacent on lp54 is homologous to prophage DNA packaging genes and has paralogs on the cp32 prophage plasmids of Borrelia. Surprisingly, despite these features, we demonstrated that BBA30 is not required by the Lyme disease spirochete at any stage of the experimental mouse/tick infectious cycle. We suggest that key but undefined features of the enzootic cycle of Borrelia underlie strict conservation of lp54 and bba30 in nature. Former postdoctoral Visiting Fellow Dr. Bharti Bhatia initiated and played a primary role in this study, which was recently published in Infection and Immunity (2). In FY2022, as part of a longstanding collaboration with Drs. Constantin Takacs and Christine Jacobs-Wagner at Stanford University, we assisted in an investigation of the pathways involved in stable inheritance of the highly segmented genome of the Lyme disease spirochete. This seminal study identified and visualized the polyploid genome of exponentially growing spirochetes in the tick vector and in culture, with reiterated copies of the linear chromosome and each plasmid uniformly positioned along the length of the bacterium. The unique pattern and spatial organization of the Borrelia genome are achieved with both canonical and novel DNA binding proteins. These findings provide novel insights into a hallmark feature of this important human pathogen and have universal implications for mechanisms of genome inheritance in other bacteria. This work is described in a manuscript that received positive reviews and is currently in revision for publication at Nature Communications (3). In FY 2022, as part of an ongoing collaboration with Dr. Patrick Secor at the University of Montana, Missoula, we assisted in their investigation of phage resistance in the opportunistic human pathogen Pseudomonas aeruginosa. Many P. aeruginosa clinical isolates carry integrated filamentous prophage genomes that precipitate cell lysis if super-infected by another bacteriophage. In this study, described in a recent publication in mBio (4), the mechanism of phage exclusion was shown to entail a small, highly conserved phage protein that inhibited assembly of the type IV pili, which is an important virulence factor in addition to the phage cell surface receptor of P. aeruginosa. In FY2022, we assisted Drs. Melina Garcia Guizzo, Jose Ribeiro and Lucas Tirloni in an investigation of the interactions between the endosymbiont Rickettsia buchneri, its tick host Ixodes scapularis, and the Lyme disease agent B. burgdorferi. The preliminary findings of this study are quite interesting and additional experiments are ongoing. In FY2022 we also assisted Drs. Joel Vega-Rodriguez and Lucas Tirloni in an investigation of the contributions of a tick salivary gland apyrase during tick feeding and acquisition of B. burgdorferi from an infected murine host. The results of the initial experiment were somewhat inconclusive, potentially due to technical issues. 1. Wachter, J., Cheff, B., Hillman, C., Carracoi, V., Dorward, D.W., Martens, C., Barbian, K., Olano, L.R., Kinnersly, M., Secor, P.R., and Rosa, P.A. Coupled induction of prophage and virulence factors during tick transmission of the Lyme disease spirochete. in revision, Nature Communications, August 2022. 2. Bhatia, B., Hillman, C., Stewart, P., Rosa, P. Probing the role of bba30, a highly conserved gene of the Lyme disease spirochete, throughout the mouse-tick infectious cycle. Infect. Immun. 89:e00333-21, doi: 10.1128/IAI.00333-21, 2021. 3. Takacs, C.N., Wachter, J., Xiang, Y., Karaboja, X., Ren, Z., Scott, M., Irnov, I., Jannetty, N., Rosa, P.A., Wang, X., Jacobs-Wagner, C. Polyploidy, regular patterning of genome copies, and unusual control of DNA partitioning in the Lyme disease spirochete. in revision, Nature Communications, August 2022. 4. Schmidt, A., Fitzpatrick, A., Schwartzkopf, C., Faith, D., Jennings, L., Coluccio, A., Hunt, D., Michaels, L., Hargil, A., Chen, Q., Bollyky, P., Dorward, D., Wachter, J., Rosa, P., Maxwell, K., Secor, P. A filamentous bacteriophage protein inhibits type IV pili to prevent superinfection of Pseudomomas aeruginosa. mBio e02441-21,