EsxA membrane-permeabilizing activity plays a key role in mycobacterial cytosolic translocation and virulence: effects of single-residue mutations at glutamine 5

Abstract

EsxA is required for virulence of Mycobacterium tuberculosis (Mtb) and plays an essential role in phagosome rupture and translocation to the cytosol of macrophages. Recent biochemical studies have demonstrated that EsxA is a membrane-permeabilizing protein. However, evidence that link EsxA membrane-permeabilizing activity to Mtb cytosolic translocation and virulence is lacking. Here we found that mutations at glutamine 5 (Q5) could up or down regulate EsxA membrane-permeabilizing activity. The mutation Q5K significantly diminished the membrane-permeabilizing activity, while Q5V enhanced the activity. By taking advantage of the single-residue mutations, we tested the effects of EsxA membrane-permeabilizing activity on mycobacterial virulence and cytosolic translocation using the esxA/esxB knockout strains of Mycobacterium marinum (Mm) and Mtb. Compared to wild type (WT), the Q5K mutant exhibited significantly attenuated virulence, evidenced by intracellular survival and cytotoxicity in mouse macrophages as well as infection of zebra fish embryos. The attenuated virulence of the Q5K mutant was correlated to the impaired cytosolic translocation. On the contrary, the Q5V mutant had a significantly increased cytosolic translocation and showed an overall increased virulence. This study provides convincing evidence that EsxA contributes to mycobacterial virulence with its membrane-permeabilizing activity that is required for cytosolic translocation.

Nα-acetylation of EsxA is required for mycobacterial cytosolic translocation and virulence

“…For expression in E. coli: Using the previously reported plasmid pET22b-esxA-His6 as the template (20)(21)(22)29), the mutations T2A, T2Q, T2R, and T2S were introduced into the esxA gene by PCR using the primers listed in Table S1. All of the mutations were confirmed by DNA sequencing.…”

“…Protein expression was induced by adding 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) for 3-8 h at 37 °C. The cells were harvested and the proteins were purified as previously described (20)(21)(22)29).…”

“…Raw 264.7 macrophages were plated in a 24-well plate with a density of 5 x10 5 /well for infection on the following day. The Mm strains were prepared with a single cell preparation protocol as previously described (22,37). RAW264.7 cells were infected with various Mm strains at a multiplicity of infection (MOI) of 10 for 1 h. The macrophages were washed 3 times with PBS to remove free mycobacteria and incubated for another 3 h. The macrophages were stained using Calcein-AM and ethidium homodimer (Life Sciences) for 30 min, enabling visualization under a fluorescence microscope for green cells (live) and red cells (dead).…”

“…Mycobacterial cytosolic translocation was measured by CCF-4 FRET assay as previously described (17,22,38). Briefly, RAW264.7 cells were plated in a 6-well plate at a density of 2.5 x 10 6 cells/well.…”

A novel deep learning scheme for morphology-based classification of mycobacterial infection in unstained macrophages

“…Since Mm shares similarities with Mtb, in genomes, virulence factors and modes of infection in macrophages, it is widely accepted as a safer surrogate model for Mtb in laboratory research. Here, we use a Mm strain harboring a pMSP12:mCherry plasmid (Addgene, USA), which expresses a mCherry protein so that the bacteria emit cherry fluorescence [26]. Thus, the cherry fluorescence serves as an infection marker to train CNN and also to validate the classification results.…”