Use of in vivo Expression Technology for the Identification of Putative Host Adaptation Factors of the Lyme Disease Spirochete

Abstract

The causative agent of Lyme disease, Borrelia burgdorferi, is an obligate parasite that requires either a tick vector or a mammalian host for survival. Identification of the bacterial genes that are specifically expressed during infection of the mammalian host could provide targets for novel therapeutics and vaccines. In vivo expression technology (IVET) is a reporter-based promoter trap system that utilizes selectable markers to identify promoters of bacterial host-specific genes. Using previously characterized genes for in vivo and in vitro selection, this study utilized an IVET system that allows for selection of B. burgdorferi sequences that act as active promoters only during murine infection. This promoter trap system was able to successfully distinguish active promoter sequences both in vivo and in vitro from control sequences and a library of cloned B. burgdorferi genomic fragments. However, a bottleneck effect during the experimental mouse infection limited the utility for genome-wide promoter screening. Overall, IVET was demonstrated as a tool for the identification of in vivo-induced promoter elements of B. burgdorferi, and the observed infection bottleneck apparent using a polyclonal infection pool provides insight into the dynamics of experimental infection with B. burgdorferi.

DNA Methylation by Restriction Modification Systems Affects the Global Transcriptome Profile in Borrelia burgdorferi

“…The MTase genes bbe02 and bbq67 were first identified due to the high transformability of strains lacking these loci (24). As a consequence, many laboratories utilize MTase-deficient strains, including the BbΔe02/q67 Ϫ strain used in this study (5A18-NP1), as surrogates for wild-type B. burgdorferi because of their ease of genetic manipulation (27)(28)(29)(30)(31)(32)(33). The findings reported here suggest that these strains may not be appropriate model organisms for precise studies of gene regulatory networks in B. burgdorferi, particularly when interpreting conclusions for strains with differential MTase enzyme profiles.…”

“…It is not currently known whether the MTases associated with these RM systems can also function as gene expression regulators. The complements of identified MTases differ between commonly studied B. burgdorferi isolates (25), and mutant strains lacking endogenous MTases are often used as surrogates for "wild-type" strains in laboratory studies because of their increased transformation efficiency and ease of genetic manipulation (27)(28)(29)(30)(31)(32)(33). As such, a more comprehensive characterization of the DNA methylation systems in B. burgdorferi and their effects on gene regulation will aid in the interpretation of these studies and could prove crucial for a more thorough understanding of B. burgdorferi biology.…”

Genetic Manipulation of Borrelia

“…This concept is the basis of in vivo expression technology (IVET), a powerful and versatile method for genome-wide identification of promoters that are active in a host environment, by complementation of an auxotrophic mutation through gene fusion (Mahan et al, 1993). IVET has been adapted for B. burgdorferi using the pncA gene (Ellis et al, 2013;Casselli and Bankhead, 2015), which encodes a nicotinamidase activity that is required during infection of a mammalian host (Purser et al, 2003). The pncA promoter trap provides strong selection for in vivo-active promoters, as only those clones from the IVET library that express pncA during infection will survive in the mouse.…”

“…Population bottlenecks during infection have been experimentally demonstrated using both Tn-Seq and genetic bar-coding approaches (Troy et al, 2013;Rego et al, 2014). A significant population bottleneck was found to occur at the site of infection, due in part to early innate immune responses (Troy et al, 2013;Casselli and Bankhead, 2015). Population bottlenecks were also detected during persistent infection of mice, as well as during spirochete acquisition and transmission by ticks (Rego et al, 2014;Phelan et al, 2019).…”

Analysis of In Vivo Transcriptome of Intracellular Bacterial Pathogen Salmonella enterica serovar Typhmurium Isolated from Mouse Spleen

“…Many of these adaptive processes are regulated at the transcriptional level [ 8 ]. In recent years, many studies have confirmed that many bacteria that induce the high expression of genes in vivo are pathogenic [ 9 , 10 , 11 , 12 , 13 ]. Type III secretion systems (T3SSs) are central to the interactions of many pathogenic bacteria with their hosts [ 14 ].…”