Lactate-responsive lysine lactylation in Campylobacter revealed by antibody-based detection of a conserved post-translational modification

Lysine lactylation (Kla) links lactate metabolism to protein regulation yet remains largely unexplored in bacteria. We used a Kla-specific rabbit polyclonal anti-{varepsilon}-N-L-lactyl-lysine antibody to detect for this modification in Campylobacter jejuni (11168H, 81-176) and Campylobacter coli (M8). Immunoblotting of whole-cell lysates resolved discrete, strain-modulated bands under basal conditions and after physiologically relevant L-lactate pulses (4 or 16 mM). A conserved 42-45 kDa species was detected across strains, including in the absence of added lactate. Sample-load titrations revealed additional low-intensity bands, consistent with substoichiometric occupancy typical of a regulatory post-translational modification (PTM). Because Campylobacter, a member of the Epsilonproteobacteria, relies on amino-acid-driven, microaerophilic metabolism, we propose that endogenous and host-derived lactate generates lactyl donors (e.g., lactyl-CoA or lactoyl-glutathione) that enable Kla. We hypothesise that a CobB-like sirtuin mediates reversibility. To our knowledge, these data provide the first antibody-based evidence of Kla in Campylobacter and establish a framework for site-resolved mapping and functional tests that connect redox state and carbon flux to protein function, stress responses, and host interactions. Significance StatementLactate, classically a metabolic end product, can modify lysine residues to regulate proteins, but this process is largely uncharted in bacteria. We examined Campylobacter jejuni and Campylobacter coli and detected lysine lactylation using a polyclonal antibody recognising{varepsilon} -N-L-lactyl-lysine. Immunoblotting of whole-cell lysates revealed discrete, strain-modulated signals under basal conditions and after exposure to physiologically relevant L-lactate. A recurring 42-45 kDa band was present across strains, including in the absence of added lactate, and additional faint bands emerged with greater sample load, consistent with selective, low-occupancy modification. Because Campylobacter relies on amino-acid-driven metabolism under microaerophilic conditions, we suggest that endogenous and host-derived lactate generates activated lactyl donors (e.g., lactyl-CoA or lactoyl-glutathione) that support lysine lactylation, with a CobB-like sirtuin providing reversibility. These findings provide, to our knowledge, the first antibody-based evidence for lysine lactylation in Campylobacter and establish a basis for mapping sites and testing how metabolic state influences protein function, stress responses, and interactions with the hosts.