Phosphoproteomics reveals new insights into the role of PknG during the persistence of pathogenic mycobacteria in host macrophages

Abstract

Pathogenic mycobacteria, such as Mycobacterium tuberculosis, modulate the host immune system to evade clearance and promote long-term persistence, resulting in disease progression or latent infection. Understanding the mechanisms pathogenic mycobacteria use to escape elimination by the host immune system is critical to better understanding the molecular mechanisms of mycobacterial infection. Protein kinase G (PknG) in pathogenic mycobacteria has been shown to play an important role in avoiding clearance by macrophages through blocking phagosome-lysosome fusion; however, the exact mechanism is not completely understood. Here, to further investigate the role of mycobacterial PknG during early events of macrophage infection, RAW 264.7 macrophage cell lines were infected with M. bovis BCG wild-type and PknG knock-out mutant strains. After proteolysis, phosphopeptides were enriched via TiO2 columns and subjected to LC-MS/MS to identify differentially phosphorylated peptides between the wild-type and PknG mutant infected macrophages. A total of 1401 phosphosites on 914 unique proteins were identified. Following phosphoproteome normalisation and differential expression analysis, a total of 149 phosphosites were differentially phosphorylated in the wild-type infected RAW 264.7 macrophages versus the PknG knock-out mutant. A subset of 95 phosphosites was differentially up-regulated in the presence of PknG. Functional analysis of our data revealed that PknG kinase activity reprograms normal macrophage function through interfering with host cytoskeletal organisation, spliceosomal machinery, translational initiation, and programmed cell death. Differentially phosphorylated proteins in this study serve as a foundation for further validation and PknG host substrate assignment.ImportanceTuberculosis (TB) remains one of the leading causes of death from infection worldwide, due to the ability of Mycobacterium tuberculosis (Mtb) to survive and replicate within the host, establishing reservoirs of live bacteria that promote persistence and recurrence of disease. Understanding the mechanisms that Mtb uses to evade the host immune system is thus a major goal in the TB field. Protein kinase G is thought to play an important role in Mtb avoiding clearance by the host through disruption of macrophage function, but the underlying molecular mechanisms of this are not well understood. Here, our new phosphoproteomic data reveals that mycobacterial PknG substantially reprograms normal macrophage function through extensive PknG-mediated post-translational control of critical host cellular processes. These novel findings therefore considerably increase our knowledge of mycobacterial pathogenicity, including specific host cellular pathways that might be re-activatable through host-directed therapy, thereby restoring macrophage ability to eliminate Mtb.

Strain-Specific Behavior of Mycobacterium tuberculosis in Interruption of Autophagy Pathway in Human Alveolar Type II Epithelial A549 Cells

“…The increased expression of pknG gene is another factor that may reduce autophagy in our studied strains. A previous study suggests that PknG enhances mycobacterial virulence and proliferation within the host by mitigating the autophagy-mediated clearance through the phosphorylation of P62/ SQSTM1 [35] . Changes in the expression of these two genes (P62/SQSTM1 and pknG) in the Beijing and CAS strains can be effective in reducing autophagy.…”

Proteomic Approaches to Unravel Mechanisms of Antibiotic Resistance and Immune Evasion of Bacterial Pathogens

“…Ultra-high pressure reversed phase liquid chromatography (uHPLC, often with C18 beads as the stationary phase) utilizing a water—acetonitrile gradient as the mobile phase system is a common choice for the chromatographic system. Most biofluids, from urine ( 34 ) to blood ( 35 ) to CSF ( 36 ) to bacterial biofilms ( 37 ), cell samples ( 38 ) or even soil ( 39 ) and waste-water samples ( 40 ) can be processed along similar principles: The sample is homogenized and the protein extracted, digested, desalted, and analyzed by the appropriate MS system, typically with inline (i.e., with the fractionated sample being ionized for analysis continuously as eluted) chromatographic separation and often with some enrichment step or pre-fractionation where applicable or needed ( Figure 1B ). However, there remain many deviations from this general rule with direct sampling by MALDI-MS2 for added tissue-level spatial information being one that differs in nearly every aspect listed before, and one being used ever increasingly.…”

“…Examples of the use of proteomic methodologies in disentangling host-pathogen interactions include studies focussed on the proteomics of the pathogen cell-wall as the major interface ( 86 , 87 ), analyses of intracellular host proteomes during infection ( 38 ) and analyses of secreted pathogen proteins, among others. Using combinations such as protein cross-linking with MS and computational modeling or affinity purification strategies with SWATH-MS, all integrated in the system biology frame are useful to unravel the networks formed during these host-pathogen interactions too.…”