Leptospirosis is a re-emerging form of zoonosis that is caused by the spirochete pathogen Leptospira. Extracellular proteins play critical roles in the pathogenicity and survival of this pathogen in the host and environment. Extraction and analysis of extracellular proteins is a difficult task due to the abundance of enrichments like serum and bovine serum albumin in the culture medium, as is distinguishing them from the cellular proteins that may reach the analyte during extraction. In this study, extracellular proteins were separated as secretory proteins from the culture supernatant and surface proteins were separated during the washing of the cell pellet. The proteins identified were sorted based on the proportion of the cellular fractions and the extracellular fractions. The results showed the identification of 56 extracellular proteins, out of which 19 were exclusively extracellular. For those proteins, the difference in quantity with respect to their presence within the cell was found to be up to 1770-fold. Further, bioinformatics analysis elucidated characteristics and functions of the identified proteins. Orthologs of extracellular proteins in various Leptospira species were found to be closely related among different pathogenic forms. In addition to the identification of extracellular proteins, this study put forward a method for the extraction and identification of extracellular proteins.

Prokaryotes lack precise subcellular organelles but exhibit distinct regions such as cytoplasm, inner membrane, periplasm, and outer membrane, where most biochemical and physiological functions are organized. Thus, understanding the functional characteristics of proteins necessitates elucidating their subcellular localization. However, extracting subcellular proteins from gram-negative bacteria poses challenges due to their complex phospholipid bilayer. Although Triton X-114 has shown promise in outer membrane protein (OMP) extraction, a concise protocol remains elusive. This protocol demonstrates a step-by-step workflow for extracting subcellular proteins using the spirochete Leptospira as a model. This technique, featuring subcellular fractionation and phase separation, yields distinct fractions for cytoplasmic, outer, and inner membrane proteins. The detergent Triton X-114 is well-suited for phase separation due to its optimal cloud point temperature (~22 °C) and low critical micelle concentration (CMC), enabling efficient extraction and purification of native proteins with minimal denaturation. Notably, the analysis reveals the efficiency in discriminating proteins from the inner and outer membranes distinct from the cytoplasm.