Leptospira interrogans HtrA protease is a potent inducer of multifaceted cellular responses.

Leptospirosis, a bacterial zoonosis caused by select pathogenic spirochetes belonging to the genus Leptospira, can trigger a systemic inflammatory response syndrome due to a cytokine storm that can occur during infection. However, the molecular mechanisms underlying the extensive inflammatory responses during leptospiral infection remain poorly understood. In this study, we characterized a high temperature requirement protein A (HtrA) homologue encoded by the L. interrogans gene locus LIC12812, referred herein as LepHtrA. We used AlphaFold 3 to predict the structure of this protein. And we show that LepHtrA is located on the surface of L. interrogans, and its expression is upregulated by increased temperature. An in vitro enzyme assay using purified LepHtrA provides direct evidence of its serine protease activity. We demonstrate that LepHtrA induces a robust pro-inflammatory response in RAW264.7 cells, a mouse macrophage cell line, by promoting their differentiation into pro-inflammatory M1 macrophages. This differentiation leads to an increased production of pro-inflammatory cytokines and the generation of reactive oxygen species, ultimately inducing cellular apoptosis via modulation of Bcl-2/Bax expression. Collectively, our study suggests that LepHtrA is a temperature-induced and surface-exposed antigen that can contribute to cellular inflammation, stress, and apoptosis, potentially influencing the pathogenesis of leptospirosis.