Utilizing Two Borrelia bavariensis Isolates Naturally Lacking the PFam54 Gene Array To Elucidate the Roles of PFam54-Encoded Proteins

Abstract

Lyme borreliosis is the most common vector-borne disease in the Northern hemisphere, caused by spirochetes belonging to the Borrelia burgdorferi sensu lato ( Bb sl) species complex which are transmitted by ixodid ticks. Bb sl species produce a family of proteins on the linear plasmid 54 (PFam54), some of which confer the functions of cell adhesion and inactivation of complement, the first line of host defense. However, the impact of PFam54 in promoting Bb sl pathogenesis remains unclear because of the hurdles to simultaneously knock out all PFam54 proteins in a spirochete. Here, we describe two Borrelia bavariensis ( Bbav ) strains, PBN and PNi, isolated from patients naturally lacking PFam54 but maintaining the rest of the genome with greater than 95% identity to the reference Bbav strain PBi. We found that PBN and PNi less efficiently survive in human serum than PBi. Such defects were restored by introducing two Bbav PFam54 recombinant proteins, BGA66 and BGA71, confirming the role of these proteins in providing complement evasion of Bbav . Further, we found that all three strains remain detectable in various murine tissues 21 days post subcutaneous infection, supporting the non-essential role of Bbav PFam54 in promoting spirochete persistence. This study identified and utilized isolates deficient in PFam54 to associate the defects with the absence of these proteins, building the foundation to further study the role of each PFam54 protein in contributing to Bb sl pathogenesis. Importance To establish infections, Lyme borreliae utilize various means to overcome the host’s immune system. Proteins encoded by the PFam54 gene array play a role for spirochete survival in vitro and in vivo . Moreover, this gene array has been described in all currently available Lyme borreliae genomes. By investigating the first two Borrelia bavariensis isolates naturally lacking the entire PFam54 gene array, we showed that both patient isolates display an increased susceptibility to human serum, which can be rescued in the presence of two PFam54 recombinant proteins. However, both isolates remain infectious to mice after intradermal inoculation suggesting the non-essential role of PFam54 during long-term but may differ slightly in the colonization of specific tissues. Furthermore, these isolates show high genomic similarity to type-strain PBi (>95%) and could be used in future studies investigating the role of each PFam54 protein in Lyme borreliosis pathogenesis.

Unprecedented genetic variability of PFam54 paralogs among Eurasian Lyme borreliosis‐causing spirochetes

“…This includes the loss of genes encoding for proteins known to interact with the complement system of vertebrates, namely BGA66 and BGA71 of B. bavariensis , PKO2068 of B. afzelii , and ZQA68 of B. garinii (Hammerschmidt et al., 2016 ; Hart et al., 2021 ; Kraiczy, 2016 ). This is supported by recent work which showed that the loss of the whole PFam54 gene array in two B. bavariensis isolates (PBN, PNi) impacted susceptibility to human complement but did not influence infection of mice further highlighting the probable, functional redundancy in the overall Borrelia genome and especially in relation to the studied species (Rollins et al., 2022 ).…”

“…shown that short-read data was able to reconstruct the full absence of the PFam54 gene array in B. bavariensis isolates PBN and PNi with confirmation through long-read sequencing (Rollins et al, 2022).…”

“…impacted susceptibility to human complement but did not influence infection of mice further highlighting the probable, functional redundancy in the overall Borrelia genome and especially in relation to the studied species (Rollins et al, 2022).…”

“…This could explain the absence of some genes in our analysis and also the presence of many, singular architecture types potentially due to lower resolution in specific samples. Nevertheless, short‐reads have been shown to adequately reconstruct the lp54 where the PFam54 gene array is located (Margos et al., 2017 ) and previous work has shown that short‐read data was able to reconstruct the full absence of the PFam54 gene array in B. bavariensis isolates PBN and PNi with confirmation through long‐read sequencing (Rollins et al., 2022 ). Taken together, this suggests short‐read sequencing should be adequate to reconstruct the PFam54 gene array but future analysis should include long‐read sequencing data to determine any bias that could arise due to sequencing methodology.…”

“…One well‐studied protein belonging to the paralogous protein family PFam54 Clade IV, CspA ( bba68 ), is capable of binding the fluid‐phase complement regulatory proteins Factor H and Factor H‐like protein 1 (FHL‐1) (Hart et al., 2018 ; Kraiczy, 2016 ). Members of the PFam54 are encoded by genes predominantly arranged in a multi‐gene array located at the 5´‐terminal end of the linear plasmid 54 (lp54) in the majority of Bb sl isolates studied (Casjens et al., 2012 ; Hart et al., 2018 ; Rollins et al., 2022 ; Schwartz et al., 2021 ; Wywial et al., 2009 ). The PFam54 gene array can be separated into five major Clades where Clades I, II, III, and V share one‐to‐one orthology among the Bb sl species studied to date (Wywial et al., 2009 ).…”