Protein Detection Analysis Provides New Insights into Tuberculosis
Protein Detection Techniques have been used as a tool to unravel the puzzle of Tuberculosis virulence
March 03, 2022
It may come as a surprise to learn that Tuberculosis (TB), a disease many people might have thought to have been more or less eradicated, is in fact still the 13th leading cause of death worldwide and amongst infectious diseases was second only to COVID-19 as the most deadly infection of 2020. Even in the US and EU, multidrug-resistant TB (MDR-TB) is still considered a public health crisis. Given the continued global threat of TB, the United Nations has established one of its Health Targets for 2030 as the ending the TB epidemic, and research continues apace to understand the causative agent of TB, the bacterium Mycobacterium tuberculosis, and to identify new mechanisms to fight the disease, and prevent its spread.
New research sheds light on an old enemy
As part of the global effort to eradicate TB, a new study has answered the question of how bacterial novel virulence factors affect TB infectivity. 
The study, carried out at the Pharmacology and Structural Biology Institute in Toulouse, France, was focused on a newly discovered gene shown to be linked to the initiation of TB infection in the lungs of mice.
How M. tuberculosisis able to bypass respiratory clearance mechanisms and seed itself into the lungs of an infected person is still largely unknown. The Toulouse research group used a combination of whole-genome screening, cell culture, and protein detection to first identify bacterial genes important for lung seeding and replication of TB, and secondly, to determine the gene’s precise function
Virulence protein detection and elucidation
The researchers identified genes essential to lung tissue seeding and infection initiation by screening a whole-genome library of M. tuberculosis mutants for strains that show significant impairment of these functions. The scientists were then able to match the impaired bacterial strains to specific genetic mutations. Using this method, they identified several genes that had already been determined as important for virulence, as well as a handful of novel candidate genes of unknown function.
One of these genes, rv0180c, was particularly intriguing because directed disruption of the gene impairs both M. tuberculosis replication and initiation of TB infection in the lungs of mice. This gene was ultimately shown by the Toulouse team to play an important role inM. tuberculosisassault on host macrophages.
Macrophages are the first cellular target of M. tuberculosis during infection and the primary cell type for the growth and persistence of TB during infection. The Toulouse research group used human monocyte-derived macrophages to model TB infection in vitro and were able to determine that the gene is critical to the successful invasion of macrophages.Host cells infected by M. tuberculosisin which the rv0180c gene was disrupted carried 33% fewer copies of the bacteria than host cells infected with the wild type.
Protein Detection using transfer membranes
The next step was to determine exactly how the rv0180c gene exerts its effects. Gene sequencing pointed toward rv0180c coding for an integral membrane protein. The scientists knew that bacterial entry into host cells is usually accomplished through binding with host macrophage phagocytic receptors, in this case, the CR3 receptor. Blocking the CR3 binding site erased the difference between wild-type bacteria containing an intact gene, and one containing a disrupted rv0180c gene. However, the length of the protein encoded by the newly discovered gene made it unlikely that the protein could be interacting directly with macrophage receptors associated with phagocytosis. This observation made it more likely that rv0180c was instead coding for a protein that affects the functionality of the bacterial cell envelope itself, by altering membrane rigidity or cell shape such that the bacteria could not engage with macrophage membrane receptors.
The team carried out a detailed dot blot analysis on the TB bacterial cell membrane using protein transfer membranes, a technique that allows qualitative and quantitative analysis of the proteins present in a sample. The strength of using dot blot analysis for studies of this type is that it enables rapid, high-throughput screening and is more economical than Western blot. Choosing the right protein transfer membrane is critical to the success of the experiment and users should consider the nature of the protein being bound, the solvents being used, the capacity of the membrane to retain protein, and the level of non-specific protein and ligand binding present. These factors all affect the success of protein transfer.
The Toulouse team selected the Pall BioTraceTM NT transfer membranes which are made from pure nitrocellulose unsupported media. BioTrace NT are designed with a high protein binding capacity (209µg/cm2) and exhibit lower protein burn through than competitors during electrophoretic transfer. Membrane pore size is 0.2 μm, with a typical thickness ranging from 101.6 - 190.5 μm. BioTrace membranes are available as 7 x 8.5 cm sheets, 20 x20 cm sheets, or a 30 cm x 3 m roll.
Determining the role of rv0180c in bacterial infection
Using protein transfer analysis of rv0180c wild type and mutant strains, the researchers confirmed the hypothesis that bacterial cell envelope proteins that control bacterial shape are altered in the rv0180c mutant. This alteration of bacterial shape impairs engagement with the macrophage membrane receptors that the bacteria use to invade host cells. This finding represents a novel mechanism of action in bacterial infection and provides new insight into the function of bacterial genes that significantly affect tuberculosis virulence and spreading. Understanding these functions will help provide new and better answers, and ultimately contribute to ending the global TB epidemic, as well as providing insight into the mechanisms of infection of this and other bacterial diseases.
You can find out more about the BioTrace NT transfer membranes used in this study as well as the other Pall technologies that help scientists solve complex research challenges every day.
1 World Health Organization. Tuberculosis. [online] Available at:https://www.who.int/news-room/fact-sheets/detail/tuberculosis [Accessed 26 Jan 2022]
2 Payros D, et al. Rv0180c contributes to Mycobacterium tuberculosis cell shape and to infectivity in mice and macrophages. PLOS Pathogens. 2021.17(11) November e1010020. https://doi.org/10.1371/journal.ppat.1010020