Wunder, E. A. | Search bioRxiv

bioRxiv2025

DMEM and EMEM are suitable surrogate media to mimic host environment and expand leptospiral pathogenesis studies using in vitro tools

Pathogenic Leptospira species can survive and thrive in a wide range of environments. Distinct environments expose the bacteria to different temperatures, osmolarities, and amounts and sources of nutrition. However, leptospires are mostly cultured, in a laboratory setting under in vitro conditions that do not reflect natural environments. This constraint on laboratory cultures limits the applicability of in vitro studies to the understanding of even simple pathogenic processes. Here we report, investigate, and identify a medium and conditions that mimic the host environment during leptospirosis infection, expanding the available in vitro tools to evaluate leptospiral pathogenesis. We quantified genome-wide gene expression of pathogenic Leptospira interrogans cultured in different in vitro media compositions (EMJH, DMEM, EMEM, and HAN). Using EMJH as standard, we compared gene expression in these compositions to genome-wide gene expression gathered in a host environment: whole blood (WB) of hamsters after infection with pathogenic leptospires. Leptospires cultured in DMEM and EMEM media shared 40% and 47% of all differentially expressed genes (DEGs) of leptospires present within WB (FDR<0.01), while leptospires cultured in HAN media only shared 20% of DEGs with those from WB. Furthermore, gene and pathway expression of leptospires cultured on DMEM and EMEM media exhibited a better correlation with leptospires grown in WB, including promoting expression of a similar leptospiral lipid A profile to the one identified directly in host tissues. Taken together, these results indicate that commercial cell-culture media EMEM or DMEM are better surrogates for in vivo pathogenic studies than EMJH or HAN media in Leptospira. These alternative culture conditions, using media that are a standard supply worldwide, provide a reproducible and cost-effective approach that can accelerate research investigation and reduce the number of animal infections necessary for basic research of leptospirosis.

microbiology↗

bioRxiv2023

Lysinoalanine crosslinking is a conserved post-translational modification in the spirochete flagellar hook

Spirochete bacteria cause Lyme disease, leptospirosis, syphilis and several other human illnesses. Unlike other bacteria, spirochete flagella are enclosed within the periplasmic space where the filaments distort and push the cell body by action of the flagellar motors. We previously demonstrated that the oral pathogen Treponema denticola (Td) catalyzes the formation of covalent lysinoalanine (Lal) crosslinks between conserved cysteine and lysine residues of the FlgE protein that composes the flagellar hook. Although not necessary for hook assembly, Lal is required for motility of Td, presumably due to the stabilizing effect of the crosslink. Herein, we extend these findings to other, representative spirochete species across the phylum. We confirm the presence of Lal crosslinked peptides in recombinant and in vivo-derived samples from Treponema spp., Borreliella spp., Brachyspira spp., and Leptospira spp.. Like with Td, a mutant strain of the Lyme disease pathogen Borreliella burgdorferi unable to form the crosslink has impaired motility. FlgE from Leptospira spp. does not conserve the Lal-forming cysteine residue which is instead substituted by serine. Nevertheless, Leptospira interrogans also forms Lal, with several different Lal isoforms being detected between Ser-179 and Lys-145, Lys-148, and Lys-166, thereby highlighting species or order-specific differences within the phylum. Our data reveals that the Lal crosslink is a conserved and necessary post-translational modification across the spirochete phylum and may thus represent an effective target for spirochete-specific antimicrobials. Significance StatementThe phylum Spirochaetota contains bacterial pathogens responsible for a variety of diseases, including Lyme disease, syphilis, periodontal disease, and leptospirosis. Motility of these pathogens is a major virulence factor that contributes to infectivity and host colonization. The oral pathogen Treponema denticola produces a post-translational modification (PTM) in the form of a lysinoalanine (Lal) crosslink between neighboring subunits of the flagellar hook protein FlgE. Herein, we demonstrate that representative spirochetes species across the phylum all form Lal in their flagellar hooks. T. denticola and B. burgdorferi cells incapable of forming the crosslink are non-motile, thereby establishing the general role of the Lal PTM in the unusual type of flagellar motility evolved by spirochetes.

microbiology↗