Preparation of Mycobacterium tuberculosis Culture Filtrate to Understand TB Pathogenesis.

Mycobacterium tuberculosis (Mtb) secretes many proteins into the host to modulate various cellular processes, such as host signaling, immune response, apoptosis, etc., to enhance survival. While the function of a few secretory factors has been elucidated, many are yet to be characterized. Hence, investigating the Mtb secretory protein is an essential area of research. Mtb secretes proteins into the extracytoplasmic space with the help of three different secretion systems: general, twin-arginine, and specialized Type VII secretion system. One has to prepare the secretome to identify novel secretory molecules and functionally characterize the known factors. Here, we outline the method for preparing the secretome and sample preparation for mass spectrometry. Secretome preparation requires the utmost care to avoid contamination of cytosolic proteins, which can be monitored by probing with antibodies against well-characterized membrane, secretory, and cytosolic proteins. The culture filtrate thus prepared can be used for different purposes, which include investigating host immunomodulation properties.