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. Genetic manipulation of B. burgdorferi is currently feasible but inefficient, requiring microgram quantities of DNA while yielding only a few transformants. This severely limits the application of effective genetic screens to the Lyme disease spirochete. Endogenous plasmid-encoded restriction/modification (R/M) systems constitute part of the barrier to stable introduction of foreign DNA in B. burgdorferi. We previously identified the DNA sequence motifs recognized by R/M systems of the widely used B. burgdorferi type strain B31, which facilitated the design of shuttle vectors and selectable markers that lack these R/M sites. In FY2021, as part of an ongoing collaboration with Dr. Craig Martens of the Research Technologies Branch (RTB) at RML, we completed this analysis and identified the R/M motifs of additional B. burgdorferi strains and prototypic B. garinii and B. afzelii strains that are agents of Lyme borreliosis in Europe, and equally important targets of genetic investigation. In FY2021, in collaboration with Dr. Craig Martens, we also completed comparative RNAseq analyses of the transcriptomes of Borrelia strains that differ in R/M gene content. In contrast to a recent report from another lab, we found no evidence for epigenetic regulation of gene expression by methylation in B. burgdorferi, whereas we did identify a non-random distribution of R/M sites in plasmid-encoded genes encoding immunodominant outer surface proteins. This finding suggests a potential role for R/M systems in recombination-driven antigenic variation in Borrelia, which is critical for immune evasion by the spirochete during host infection. Dr. Jenny Wachter, a senior postdoctoral fellow in MGS, took a lead role in the R/M project and was first and/corresponding author of the manuscript describing these findings, which was published in 2021 (1). In FY2021, as part of an ongoing collaboration with Drs. Constantin Takacs and Christine Jacobs-Wagner at Stanford University, we assisted with the development of a CRISPR interference platform for selective downregulation of gene expression in B. burgdorferi (2). This CRISPRi genetic approach represents an easy and efficient complement to traditional homologous recombination-based methods of gene inactivation, particularly as a tool for the investigation of essential genes. In FY2021, Dr. Rosa and Dr. Wachter also collaborated with the Jacobs-Wagner lab to extend their investigation of the replication and segregation of the highly segmented Borrelia genome to the experimental mouse/tick infectious cycle, thereby validating results in an in vivo setting. During tick feeding, environmental signals upregulate the alternative sigma factor RpoS in spirochetes colonizing the tick midgut, which drives a global transcriptional response that is critical for spirochete transmission and survival in the mammalian host. Surprisingly, we and others have found that engineered over-expression of RpoS is lethal for the spirochete. We previously identified an essential plasmid-encoded negative regulator of RpoS, termed BBD18, that could not be inactivated in wild-type B. burgdorferi. In FY2021, we continued to investigate the genetic mechanism by which BBD18 modulates RpoS activity and how/where BBD18 functions during the mouse-tick infectious cycle. We manipulated bbd18 gene expression with an inducible promoter and observed spirochete lysis when BBD18 was depleted, phenotypically similar to engineered over-expression of rpoS. Reduction in BBD18 resulted in increased gene expression and copy number of the cp32 plasmids, which encode transducing prophage whose contribution to the infectious cycle is currently undefined. In collaboration with Dr. Dave Dorward of RTB, electron microscopic analyses revealed that loss of BBD18 correlated with the appearance of phage particles in the supernatant of lysing cells and phage-like elements within intact spirochetes. Furthermore, culture supernatants contained nuclease-resistant DNA of the same size as cp32 plasmids. In FY2021, in an ongoing collaboration with Dr. Patrick Secor at the University of Montana, we continued a physical characterization of phage particles in BBD18-depleted cells. In FY2021, in collaboration with Dr. Craig Martens of RTB, we also conducted RNA-seq analyses to probe transcriptional changes in B. burgdorferi that immediately follow de-induction of BBD18 and precede cell lysis. In FY2021 we utilized a molecular genetic approach to sequentially displace cp32 plasmids from wild type and BBD18-inducible spirochetes to assess the contribution of cp32 prophage to cell lysis when BBD18 is depleted. Finally, we investigated the contribution of BBD18 during the infectious cycle and found that bbd18 gene expression decreases during tick feeding, accompanying transmission to the host, but that BBD18 is required for spirochete survival following the bloodmeal. We hypothesize that transducing phage are a natural component of the RpoS-dependent host-adaptive response, thereby facilitating horizontal gene transfer between spirochetes in infected ticks prior to transmission, and that BBD18 modulates RpoS activity to circumvent uncontrolled activation of lytic prophage. Dr. Jenny Wachter played a lead role in the BBD18 studies and is preparing a manuscript describing these exciting results. (1) Wachter J, Martens C, Barbian K, Rego ROM, Rosa P (2021). Epigenomic Landscape of Lyme Disease Spirochetes Reveals Novel Motifs. mBio 12, e0128821. https://doi.org/10.1128/mBio.01288-21 (2) Takacs CN, Scott M, Chang Y, Kloos ZA, Irnov I, Rosa PA, Liu J, Jacobs-Wagner C (2020). A CRISPR interference platform for selective downregulation of gene expression in Borrelia burgdorferi. Appl Environ Microbiol 87, e02519-20. https://doi.org/10.1128/AEM.02519-20