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Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), which is still the leading cause of mortality from a single infectious disease worldwide. The development of novel anti-TB drugs and vaccines is severely hampered by the complicated and time-consuming genetic manipulation techniques for M. tuberculosis. Here, we harnessed an endogenous type III-A CRISPR/Cas10 system of M. tuberculosis for efficient gene editing and RNA interference (RNAi). This simple and easy method only needs to transform a single mini-CRISPR array plasmid, thus avoiding the introduction of exogenous protein and minimizing proteotoxicity. We demonstrated that M. tuberculosis genes can be efficiently and specifically knocked in/out by this system as confirmed by DNA high-throughput sequencing. This system was further applied to single- and multiple-gene RNAi. Moreover, we successfully performed genome-wide RNAi screening to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be extensively used for exploring the functional genomics of M. tuberculosis and facilitate the development of novel anti-TB drugs and vaccines., (Copyright © 2022. Published by Elsevier B.V.)

COVID-19 is a global pandemic, with attendant high morbidity and mortality. There is no previous documentation of its coinfection with Mycobacterium tuberculosis; the single most common cause of death from an infectious disease. Management and survival from this "cruel duel" in a low resource country will be daunting. We report the case of a middle-aged man who survived and the lessons learned from a COVID-19 treatment centre in the north-central of Nigeria. The patient presented with symptoms and clinical features of COVID-19 and Mycobacterium tuberculosis was confirmed with laboratory investigation. The patient commenced anti-tuberculous medications, received nutritional support and other supportive treatment for COVID-19 infection. He was discharged home to continue follow up at the medical outpatient and the DOTS clinic. Early recognition and prompt treatment are critical for a favourable clinical outcome.

Protein-protein interaction (PPI) network maintains proper function of all organisms. Simple high-throughput technologies are desperately needed to delineate the landscape of PPI networks. While recent state-of-the-art yeast two-hybrid (Y2H) systems improved screening efficiency, either individual colony isolation, library preparation arrays, gene barcoding or massive sequencing are still required. Here, we developed a recombination-based 'library vs library' Y2H system (RLL-Y2H), by which multi-library screening can be accomplished in a single pool without any individual treatment. This system is based on the phiC31 integrase-mediated integration between bait and prey plasmids. The integrated fragments were digested by MmeI and subjected to deep sequencing to decode the interaction matrix. We applied this system to decipher the trans-kingdom interactome between Mycobacterium tuberculosis and host cells and further identified Rv2427c interfering with the phagosome-lysosome fusion. This concept can also be applied to other systems to screen protein-RNA and protein-DNA interactions and delineate signaling landscape in cells.

Resistance to anti-tuberculosis drugs is a formidable obstacle to effective tuberculosis (TB) treatment and prevention globally. New forms of multidrug, extensive drug and total drug resistance Mycobacterium tuberculosis (Mtb) causing a serious threat to human as well as animal's population. Mtb shows diverse adaptability under stress conditions especially antibiotic treatment, however underlying physiological mechanism remained elusive. In present study, we investigated Mtb's response and adaptation with reference to gene expression during sub-lethal kanamycin exposure. Mtb were cultured under sub-lethal drug and control conditions, where half were sub-cultured every 3-days to observe serial adaptation under same conditions and the remaining were subjected to RNA-seq. We identified 98 up-regulated and 198 down-regulated responsive genes compared to control through differential analysis, of which Ra1750 and Ra3160 were the most responsive genes. In adaptive analysis, we found Ra1750, Ra3160, Ra3161, Ra3893 and Ra2492 up-regulation at early stage and gradually showed low expression levels at the later stages of drug exposure. The adaptive expression of Ra1750, Ra3160 and Ra3161 were further confirmed by real time qPCR. These results suggested that these genes contributed in Mtb's physiological adaptation during sub-lethal kanamycin exposure. Our findings may aid to edify these potential targets for drug development against drug resistance tuberculosis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Tuberculosis (TB) is an ancient disease caused by Mycobacterium tuberculosis ( Mtb ). TB is one of the world's deadliest diseases, with one-third of infected individuals falling ill each year especially in many developing countries. Upon invading host cells, such as macrophages, Mtb can replicate in infected cells by arresting phagosome maturation and then potentially escaping into the cytosol. Host cells have a mechanism to control intracellular Mtb by inducing autophagy, which is an elaborate cellular process to target intracellular pathogens for degradation in infected cells. However, some factors of Mtb are involved in defense against killing by autophagy. Thus, this review highlights the recent advances in the interactions between autophagy and Mtb .

Anti-tuberculosis drug resistance is considered as the main snag in the global elimination of tuberculosis by the end of 2030. One possible explanation to understand its pathogenesis is its inter-connected genome that makes an intricate network to generate the resistance in Mycobacterium tuberculosis (Mtb). Our hypothesis is to analyze the protein-DNA and protein-protein interaction data of Mtb to probe the candidate genes and then evaluate their resistance induction in Mtb against first line anti-TB drugs. The data of genome wide regulator-DNA interaction (Zeng et al., 2012) and global protein-protein interaction (Wang et al., 2010) were analyzed bioinformatically through cytoscape software to identify the candidate genes participating in drug resistance network. These were individually amplified, cloned and transformed into Mtb competent cells to make overexpression strains of these respective genes or (first define in full what is Egfp) eGFP. These strains were then screened with isoniazid (INH) and rifampicin (RIF) to identify their role in regulating drug resistance. Alamar blue dye color change was considered as the marker of resistance imparted by the candidate genes. Only 30 candidate genes were obtained and functionally categorized into small molecule metabolism, intermediate metabolism, information pathway, conserved hypothetical proteins, and cell wall-cell processes through mycrobrowser. After exposing these 30 overexpression strains to various concentrations of INH and RIF, only 7 strains Rv3416, Rv1267, Rv0165c, Rv1129c (metabolism regulatory proteins), Rv2669 (conserved hypothetical protein), Rv2703 (information pathway) and Rv2780 (intermediary metabolism) showed resistance by growing in the presence of 200 µg/ml INH and alamar blue color was not changed. There was absence of growth change and alamar blue color against rifampicin. Our results suggested that these genes might be the regulators of drug resistance network in Mtb. Targeting drug resistance network is a consideration in the development of new anti-TB drugs. [ABSTRACT FROM AUTHOR]

Reconnoitering the new genetics tools for identifying novel vaccine candidates and drug targets is the need of time to combat tuberculosis (TB). This will bring forth the comprehension of functional genomics, virulent genes, latency in host and resistance against drugs in the causative agent of TB i.e. Mycobacterium tuberculosis (Mtb). Keeping in view, Mtb endogenous CRISPR-Cas type III a system was exploited to envisage genome editing, mechanism of RNA interference, and throughout Mtb genome CRISPR interference (CRISPRi) screening using in-vitro and in-vivo. A repeat sequence of type IIIA CRISPR array was incorporated in pMV261-crRNA. Guide RNA for the target gene was designed and homology- directed repair (HDR) sequence containing GFP was employed for gene insertion- deletion (indel). For gene knock-down (RNA interference) mechanism was identified by RIP-qPCR. Guide RNA library (5658 in numbers) for the whole genome was designed from Twist Bioscience Company and high-throughput screening was performed for in-vitro and in-vivo growing Mtb using bioinformatics tools. qPCR declared the presence of type III A CRISPR array in Mtb. Using pMV261-crRNA and gRNA for the target gene successfully knocked-in GFP adjacent to gyrA gene and successfully knocked-out lpqE, lpqN, esxA and lpqD separately. For knock-down, katG, esxT, and dcd genes were successfully interfered at transcriptional level using CRISPRi. This was validated by RIP in which csm6-HA tag pull down CRISPR from bacteria having gRNA for katG or dcd or esxT. High-throughput screening analysis showed 228 growth promoting and 385 inhibiting genes in in-vitro culture and 29 growth promoting and 4 inhibiting genes in in-vivo environment. This is your futuristic vision, not a conclusion, please conclude your study in 1 sentence. This system can be used for genetic manipulation, exploring functional genomics of Mtb and identifying new vaccine candidates and drugs targets in Mtb. [ABSTRACT FROM AUTHOR]