By Lusanda Kunene
Imagine a world without tuberculosis (TB). While that vision remains out of reach, scientists around the world are working towards making it a reality. Currently, TB is one of the leading infectious causes of death worldwide. In 2023 alone, 10.8 million people were infected, and 1.25 million people died from the disease. South Africa is among the top countries that have a high TB burden accounting for two-thirds of the global epidemic.
To help combat this disease, researchers are working to understand how Mycobacterium tuberculosis (M.tb), the pathogen that causes TB, evades the immune system. Uncovering the mechanisms that allow the bacterium to survive opens the door to discovering new targets for more effective treatments for the disease.
One of the survival strategies of M.tb involves secreting proteins called kinases and phosphatases during infection, which interfere with host signalling pathways. One of the most important of these proteins is Protein Kinase G (PknG). This kinase enables M.tb to establish a niche inside macrophages, allowing progression of the infection.
Under normal physiological conditions, when a pathogen enters the body, the immune system is activated. Macrophages recognize and engulf the invading pathogen, enclosing it within a phagosome. The phagosome then fuses with a lysosome, which contains digestive enzymes that break down the pathogen, preventing the infection from progressing. However, M.tb uses PknG to evade this defence mechanism and survive. The exact molecular mechanism by which PknG enables M.tb to establish a niche within macrophages remains unclear. (Figure 1 is a graphic depicting engulfment of M.tb).
To investigate the mechanism behind how PknG establishes this niche, a study was conducted using phosphoproteomics to analyse changes in protein phosphorylation to identify the host signalling pathways altered by PknG.
Methodology
For the study, RAW 264.7 macrophages were infected with either Mycobacterium bovis BCG wild-type or PknG knockout mutant strains. The infected macrophages were lysed and proteins extracted, followed by a TiO2-based phosphopeptide enrichment to capture phosphorylated proteins. The enriched proteins were then analysed using liquid chromatography mass spectrometry to identify changes in protein phosphorylation. Then functional analysis using KEGG pathways and Gene Ontology terms was done to determine which biological processes and cellular pathways were altered in the presence of PknG. Uninfected macrophages were used as the control.
Key findings
The study found that both the M.tb wild type and PknG mutant strains were internalized by the macrophages. However, comparing the survival between the two strains, the wild type strain proliferated significantly while the mutant strain exhibited impaired growth with a decline in CFU.
Figure 2: Graph illustrating the colony forming unit (CFU) of the bacteria at different time intervals post infection
Through functional analysis, they found that the host biological processes and signalling pathways that PknG perturbs are primarily involved in cytoskeletal organization, phagosome maturation, and programmed cell death (apoptosis). By disrupting these pathways, PknG reprograms normal macrophage function. As a result, the phagosome does not mature and fuse with the lysosome, preventing the bacterium from being degraded by lysosomal enzymes. At the same time, the macrophage’s normal programmed cell death response is impaired, creating an intracellular environment that allows M. tb to survive and the infection to progress.
Figure 3: KEGG pathways altered by changes in protein abundance and phosphorylation in the presence of PknG
Figure 4: Bar chart ranking of the biological processes enriched by the differentially abundant proteins across the bacterial strains
Conclusion
Although there is still much to learn about how M.tb evades the immune system, this study provides a major step towards understanding one of TB’s greatest survival strategies, utilizing PknG. Researchers are revealing vulnerabilities that could be targeted for future therapies. A world without TB may not yet be here, but every discovery brings that vision a little closer.
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