MSMEG_0311 is a conserved essential polar protein involved in mycobacterium cell wall metabolism.

Cell wall synthesis and cell division are two closely linked pathways in a bacterial cell which distinctly influence the growth and survival of a bacterium. This requires an appreciable coordination between the two processes, more so, in case of mycobacteria with an intricate multi-layered cell wall structure. In this study, we investigated a conserved gene cluster using CRISPR-Cas12 based gene silencing technology to show that knockdown of most of the genes in this cluster leads to growth defects. Investigating conserved genes is important as they likely perform vital cellular functions and the functional insights on such genes can be extended to other mycobacterial species. We characterised one of the genes in the locus, MSMEG_0311. The repression of this gene not only imparts severe growth defect but also changes colony morphology. We demonstrate that the protein preferentially localises to the polar region and investigate its influence on the polar growth of the bacillus. A combination of permeability and drug susceptibility assay strongly suggests a cell wall associated function of this gene which is also corroborated by transcriptomic analysis of the knockdown where a number of cell wall associated genes, particularly iniA and sigF regulon get altered. Considering the gene is highly conserved across mycobacterial species and appears to be essential for growth, it may serve as a potential drug target.

Harnessing CRISRP-Cas9 as an anti-mycobacterial system.

Rapid emergence of drug resistance has posed new challenges to the treatment of mycobacterial infections. As the pace of development of new drugs is slow, alternate treatment approaches are required. Recently, CRISPR-Cas systems have emerged as potential antimicrobials. These sequence-specific nucleases introduce double strand cuts in the target DNA, which if left unrepaired, prove fatal to the host. For most bacteria, homologous recombination repair (HRR) is the only pathway for repair and survival. Mycobacteria is one of the few bacteria which possesses the non-homologous end joining (NHEJ) system in addition to HRR for double strand break repair. To assess the antimicrobial potential of CRISPR-system, Cas9-induced breaks were introduced in the genome of Mycobacterium smegmatis and the survival was studied. While the single strand breaks were efficiently repaired, the organism was unable to repair the double strand breaks efficiently. In a mixed population of antibiotic-resistant and sensitive mycobacterial cells, selectively targeting a factor that confers hygromycin resistance, turned the entire population sensitive to the drug. Further, we demonstrate that the sequence-specific targeting could also be used for curing plasmids from mycobacterium cells. Considering the growing interest in nucleic acid-based therapy to curtail infections and combat antimicrobial resistance, our data shows that CRISPR-systems hold promise for future use as an antimicrobial against drug-resistant mycobacterial infections.

Host directed therapies: COVID-19 and beyond.

The global spread of SARS-CoV-2 has necessitated the development of novel, safe and effective therapeutic agents against this virus to stop the pandemic, however the development of novel antivirals may take years, hence, the best alternative available, is to repurpose the existing antiviral drugs with known safety profile in humans. After more than one year into this pandemic, global efforts have yielded the fruits and with the launch of many vaccines in the market, the world is inching towards the end of this pandemic, nonetheless, future pandemics of this magnitude or even greater cannot be denied. The preparedness against viruses of unknown origin should be maintained and the broad-spectrum antivirals with activity against range of viruses should be developed to curb future viral pandemics. The majority of antivirals developed till date are pathogen specific agents, which target critical viral pathways and lack broad spectrum activity required to target wide range of viruses. The surge in drug resistance among pathogens has rendered a compelling need to shift our focus towards host directed factors in the treatment of infectious diseases. This gains special relevance in the case of viral infections, where the pathogen encodes a handful of genes and predominantly depends on host factors for their propagation and persistence. Therefore, future antiviral drug development should focus more on targeting molecules of host pathways that are often hijacked by many viruses. Such cellular proteins of host pathways offer attractive targets for the development of broad-spectrum anticipatory antivirals. In the present article, we have reviewed the host directed therapies (HDTs) effective against viral infections with a special focus on COVID-19. This article also discusses the strategies involved in identifying novel host targets and subsequent development of broad spectrum HDTs.