To identify genes essential for survival in a macrophage: In order to survive in the host and eventually cause disease, the bacteria need to survive in the hostile environment of the macrophage. The macrophage is a professional phagocytic cell with multiple pathways of killing opsonized bacteria. Many of these pathways involve the fusion of the phagosome with vacuoles containing effector molecules ("phagosome maturation into a lysosome"), such as oxygen and nitrogen radicals, acid, antibacterial peptides, etc. Bacterial killing is also accompanied by presentation of bacterial antigens to other immune cells (enhancing immune response), and mechanisms favoring autophagy rather than cell death by necrosis (which does not promote further immunity). Pathogens that reside in macrophages have developed several mechanisms to overcome these killing mechanisms, either surviving in the macrophage for long periods of time, or bringing about the death of the macrophage by necrosis, thus liberating many new bacteria to the environment and precluding an effective immune response. These pathogens include Listeria, Salmonella, Toxoplasma, Leishmania as well as all the pathogenic mycobacteria, including MAP. Killing-evasion mechanisms differ between pathogens, but mycobacteria like Mtb use mechanisms such as interference with intra-cellular signaling (thus interrupting phagosome maturation) by secreting acid phosphatases (SapM and PtpA), resistance to acid, resistance to nitric oxide and turnover of damaged proteins in the proteasome. MAP may use some of these mechanisms, and may have unique mechanisms. The interference with these mechanisms may damage the bacteria’s ability to survive in macrophages, and to avoid an immune attack. Also, mutants defective in some of these mechanisms were shown to have increased immunogenicity in BCG and Mtb. The identification of the equivalent mechanisms in MAP is therefore imperative for the creation of attenuated vaccine strains.

To identify the genes responsible for evading killing by macrophages, we will infect macrophages (mouse cell lines, as well as fresh, bovine mononuclear-cell derived macrophages) by our library of mutants. The spectrum of mutations in the library will be known (by deep sequencing, as described previously). Briefly, after infection of macrophages and allowing the bacteria to multiply for several days (depending on the exact protocol), the eukaryotic cell is lysed and the intracellular bacteria are collected. These bacteria will undergo the same deep-sequencing procedure, and the surviving mutants will be identified. Triplicate infections (to both mouse macrophages and bovine ones) will undergo deep sequencing together with the control mutants library (as described above) to produce about 7.5 million reads for each sample, such that at least 5 million reads per sample are expected to be left after quality filtering. The determination of the conditional gene essentiality will be carried by running the software ESSENTIALS that was previously described. This time, the normalized counts for each gene will be compared between the infections and the control conditions, and significant genes (absent in infections, but exist in control) will be determined using EdgeR. Those mutants present in the pool of mutants used to infect the cells (control), but absent from the pool extracted from it after macrophage passage, represent mutants in which a gene essential (or at least highly important) for survival in macrophages was inactivated. These genes will be identified, their structure compared across several mycobacteria, and their potential to be exploited for development of an attenuated mutant vaccine will be assessed.