Deciphering the role of Leptospira trigger factor domains in proteostasis and their function as heat shock regulators...

Trigger Factor (TF) is an essential ribosome-associated molecular chaperone that plays a critical role in protein folding, stabilization, and proteostasis maintenance across various bacterial systems. The multifunctional roles of TF, particularly its involvement in virulence modulation, stress response, and adhesion, are well established across multiple organisms, including Leptospira interrogans. However, its precise function has yet to be fully explored. Transcriptomic studies of Leptospira have unequivocally demonstrated that the genes tig and clpP1 are co-transcribed, indicating a significant regulatory interplay with the Clp protease complex. To further elucidate this relationship, this study engineered three LinTF mutant variants, targeting the N-terminal (LinTFΔNTD) and C-terminal regions (LinTFΔCTD and LinTFΔpCTD). Functional assays confirmed that LinTFΔpCTD and LinTFΔCTD markedly enhanced LinClpP activity by 49% and 34%. No increase in LinClpP activity was observed with LinTFΔNTD. Transmission electron microscopy and immunofluorescence techniques decisively revealed that LinTF is localized on the spirochete surface, a conclusion reinforced by subcellular fractionation. While LinTF is predominantly cytoplasmic, a small membrane-associated fraction indicates surface-associated moonlighting function. Full-length LinTF complemented the E. coli ΔtigΔdnaK strain, fully restoring growth at 37 °C. Conversely, LinTFΔpCTD complementation permitted only moderate recovery, while neither LinTFΔCTD nor LinTFΔNTD exhibited any rescuing effects on the E. coli ΔtigΔdnaK strain. Moreover, LinTF significantly promoted the refolding of chemically denatured GAPDH, as evidenced by a fluorescence emission shift and dynamic light scattering. Overall, these findings decisively highlight the domain-specific contributions of LinTF to LinClp protease regulation and protein folding, reaffirming its critical role in maintaining proteostasis in L.interrogans.