Modeling pulmonary alveolar lining fluid to identify genes required  for survival of Mycobacterium tuberculosis during transmission
Mycobacterium tuberculosis (Mtb) must withstand physical and chemical stresses during its airborne transmission from one person to another, including during the desiccation of aerosols that are small enough to reach pulmonary alveoli in a new host. There, Mtb encounters pulmonary alveolar lining fluid (ALF) before it is engulfed by macrophages. To study the genetic requirements for these interactions in vitro, we formulated a model alveolar lining fluid (MALF) that mimics the composition of ALF with respect to metabolites, lipids, surfactants, iimmunoglobulins, hydrolases and other proteins, as inferred from the composition of human bronchoalveolar lavage fluid (BALF). In three independent experiments, we compared the transcriptome of log-phase Mtb in MALF to the transcriptome of Mtb in BALF reconstituted (rcBALF) after saline lavage of lungs of three different healthy adults. The variation among replicates in MALF and the variation among replicates in rcBALF were each greater than the variation in genes regulated similarly between MALF and rcBALF. Log-phase Mtb survived quantitatively in MALF and rcBALF for at least 24 hours . However, Mtb that had passed through three earlier stages of transmission, including desiccation of microdroplets in air, began to succumb sometime between 3-6 hours in MALF, which was enough time for Mtb to be phagocytized by alveolar macrophage-like cells cultured at the air-liquid interface with pulmonary epithelial cells. Screening of a genome-wide CRISPRi library of Mtb identified 35 genes as uniquely required by Mtb to survive the transition from microdroplet desiccation into rehydration in MALF, rather than in the three earlier stages of modeled transmission. Most of these genes are non-essential under conventional laboratory conditions and ten have unknown functions. Thirteen of the 35 genes were additionally required for Mtb to survive in the air-liquid interface culture.