Investigating Syphilis Pathogenesis Through Genetic Engineering of Treponema pallidum

ABSTRACT In several high-income nations, including the United States, infectious syphilis has been resurgent for over two decades now, while syphilis is still endemic in low- and middle-income countries. Syphilis is therefore still a public global health concern, particularly in consideration that it can lead to neurological

sequelae such as dementia and stroke-like syndrome, as well as cardiovascular manifestations potentially leading to death. Furthermore, every year, about half a million pregnancies are adversely affected by congenital transmission of the pathogen. The partial success of recent syphilis control campaigns promoted

by the CDC and WHO clearly highlights the necessity of devising novel ways to control this serious infection. Improving our understanding of syphilis pathogenesis and the virulence factors that allow the syphilis agent, Treponema pallidum subsp. pallidum (T. pallidum) to establish infection and persist in the host despite a

robust immune response might be the key to new control strategies. However, two significant obstacles have hindered our ability to unravel the complexities of syphilis pathogenesis since the first T. pallidum strain was isolated in 1912. These barriers were the inability to propagate T. pallidum in vitro and, consequently, to genetically manipulate this difficult pathogen. In 2018,

the “in vitro propagation” barrier was overcome by the discovery that T. pallidum could be propagated in a cell culture-based system. In 2021, for the first time, we overcame the “genetic manipulation” barrier and derived a T. pallidum knock-out (KO) isolate, in which a functional kanamycin resistance (kanR) cassette

was used to successfully replace the non-essential T. pallidum tprA (tp0009) locus through homologous recombination after transforming the syphilis agent with a suicide vector. This newly found ability to genetically alter T. pallidum will allow us to pinpoint more clearly the role of putative virulence factors of this pathogen during infection. Here, by using an array of newly generated T.

pallidum KO strains lacking critical components of the pathogen`s antigenic variation system, we propose to study the contribution of antigenic variation to T. pallidum ability for immune evasion and persistence during infection. If successful, these studies will provide our research community with an array of T. pallidum mutants

to be used in comparative studies. Furthermore, our results will help settle ongoing controversies surrounding the function of the T. pallidum TprK virulence factors that originated over the last two decades due to working with a difficult pathogen that, until now, could not be genetically engineered.