A Genome-Wide Screen in Macrophages Defines Host Genes Regulating the Uptake of Mycobacterium abscessus.

The interactions between a host cell and a pathogen can dictate disease outcomes and are important targets for host-directed therapies. Mycobacterium abscessus (Mab) is a highly antibiotic resistant, rapidly growing nontuberculous mycobacterium that infects patients with chronic lung diseases. Mab can infect host immune cells, such as macrophages, which contribute to its pathogenesis. However, our understanding of initial host-Mab interactions remains unclear. Here, we developed a functional genetic approach to define these host-Mab interactions by coupling a Mab fluorescent reporter with a genome-wide knockout library in murine macrophages. We used this approach to conduct a forward genetic screen to define host genes that contribute to the uptake of Mab by macrophages. We identified known regulators of phagocytosis, such as the integrin ITGB2, and uncovered a key requirement for glycosaminoglycan (sGAG) synthesis for macrophages to efficiently take up Mab. CRISPR-Cas9 targeting of three key sGAG biosynthesis regulators, Ugdh, B3gat3, and B4galt7 resulted in reduced uptake of both smooth and rough Mab variants by macrophages. Mechanistic studies suggest that sGAGs function upstream of pathogen engulfment and are required for the uptake of Mab, but not Escherichia coli or latex beads. Further investigation found that the loss of sGAGs reduced the surface expression, but not the mRNA expression, of key integrins, suggesting an important role for sGAGs in modulating surface receptor availability. Together, these studies globally define and characterize important regulators of macrophage-Mab interactions and are a first step to understanding host genes that contribute to Mab pathogenesis and disease. IMPORTANCE Pathogen interactions with immune cells like macrophages contribute to pathogenesis, yet the mechanisms underlying these interactions remain largely undefined. For emerging respiratory pathogens, like Mycobacterium abscessus, understanding these host-pathogen interactions is important to fully understand disease progression. Given that M. abscessus is broadly recalcitrant to antibiotic treatments, new therapeutic approaches are needed. Here, we leveraged a genome-wide knockout library in murine macrophages to globally define host genes required for M. abscessus uptake. We identified new macrophage uptake regulators during M. abscessus infection, including a subset of integrins and the glycosaminoglycan synthesis (sGAG) pathway. While ionic characteristics of sGAGs are known to drive pathogen-cell interactions, we discovered a previously unrecognized requirement for sGAGs to maintain robust surface expression of key uptake receptors. Thus, we developed a flexible forward-genetic pipeline to define important interactions during M. abscessus infection and more broadly identified a new mechanism by which sGAGs control pathogen uptake.

A genetic selection for Mycobacterium smegmatis mutants tolerant to killing by sodium citrate defines a combined role for cation homeostasis and osmotic stress in cell death.

Mycobacteria can colonize environments where the availability of metal ions is limited. Biological or inorganic chelators play an important role in limiting metal availability, and we developed a model to examine Mycobacterium smegmatis survival in the presence of the chelator sodium citrate. We observed that instead of restricting M. smegmatis growth, concentrated sodium citrate killed M. smegmatis. RNAseq analysis during sodium citrate treatment revealed transcriptional signatures of metal starvation and hyperosmotic stress. Notably, metal starvation and hyperosmotic stress, individually, do not kill M. smegmatis under these conditions. A forward genetic transposon selection was conducted to examine why sodium citrate was lethal, and several sodium-citrate-tolerant mutants were isolated. Based on the identity of three tolerant mutants, mgtE, treZ, and fadD6, we propose a dual stress model of killing by sodium citrate, where sodium citrate chelate metals from the cell envelope and then osmotic stress in combination with a weakened cell envelope causes cell lysis. This sodium citrate tolerance screen identified mutants in several other genes with no known function, with most conserved in the pathogen M. tuberculosis. Therefore, this model will serve as a basis to define their functions, potentially in maintaining cell wall integrity, cation homeostasis, or osmotolerance. IMPORTANCE Bacteria require mechanisms to adapt to environments with differing metal availability. When Mycobacterium smegmatis is treated with high concentrations of the metal chelator sodium citrate, the bacteria are killed. To define the mechanisms underlying killing by sodium citrate, we conducted a genetic selection and observed tolerance to killing in mutants of the mgtE magnesium transporter. Further characterization studies support a model where killing by sodium citrate is driven by a weakened cell wall and osmotic stress, that in combination cause cell lysis.

GarD-ing the pathogen-containing vacuole from destruction.

The human pathogen Chlamydia trachomatis evades killing by IFNγ-activated mechanisms, yet how this occurs remains unclear. In this issue of Cell Host & Microbe, Walsh et al. identify an IFNγ-dependent antimicrobial mechanism mediated by the host ubiquitin ligase RNF213 that is evaded by the Chlamydia effector GarD.