Investigating unknown yet essential mechanisms of gene regulation for tick-borne relapsing fever spirochetes.

Baylor College Of Medicine, Houston TX

Investigators

Abstract

Project Summary Relapsing fever (RF) Borrelia are vector-borne pathogens that causes significant morbidity and mortality in low- and middle-income countries, while we are also observing the emergence of the disease in populated regions of North America. Pathogenic spirochetes are transmitted by the human body louse and ixodid and argasid ticks. Since most species of RF Borrelia are transmitted by argasid ticks in the Ornithodoros genus, we developed this model. Unfortunately, we do not understand the molecular mechanisms driving RF spirochete transmission leading to an absence of preventative interventions that disrupt the pathogens’ life cycle. While RF- and Lyme disease-causing spirochetes are in the same family, their biology significantly differs indicating what is known for one system is not necessarily applicable to the other. One key difference is that populations of RF spirochetes persistently colonize Ornithodoros midguts and salivary glands. The salivary gland population is essential because those spirochetes are preadapted for entry into the vertebrate host and are transmitted within seconds of tick bite. Upon transmission, vector-specific genes are downregulated and the variable major protein (vmp) locus, which is responsible for antigenic variation and persistent mammalian infection, is upregulated. However, very little is known regarding which genes are expressed in the tick and vertebrate and the regulatory mechanisms controlling their expression. We are addressing this through advances in RF spirochete genomics. We utilized Oxford Nanopore Technology (ONT) to generate plasmid-resolved genomes needed for comprehensive transcriptional studies. Furthermore, since it is challenging to obtain enough spirochete RNA from the tick for transcriptional studies, we characterized salivary glands and found that they are highly oxidative. This allows us to grow the pathogen in in vitro conditions that better mimic the tick environment. We also identified essential RF Borrelia genes expressed in the tick (bta132 – bta136) and will build on the importance of vmp to delineate regulatory mechanisms. We hypothesize that RF spirochetes possess unique mechanisms to regulate genes differentially expressed during the tick-mammalian transmission cycle. In Aim 1, we will utilize the ONT platform for a comprehensive analysis of RF Borrelia genes and operons that are upregulated in the tick salivary glands versus murine blood and determine their transcriptional start sites. The objectives are: 1) perform long- read ONT-Cappable-seq using in vitro grown spirochetes; 2) analyze data and select candidates; and 3) validate findings in vivo. In Aim 2, we will investigate the regulatory mechanisms of important genes upregulated in the tick (bta132-bta136) and mammal (vmp locus) through the identification of promotor regions and DNA-binding proteins. The objectives are: 1) identify promoter sequences; and 2) identify DNA-binding proteins that interact with promoters of bta132 - bta136 and the vmp expression locus. Our findings will result in the identification of genes to target for the development of countermeasures against a significant yet neglected pathogen.