Recombination-based tools for introducing targeted genomic mutations in Mycobacterium tuberculosis are not efficient due to higher rate of illegitimate recombination compared with homologous DNA exchange. Moreover, involvement of multiple steps and specialized reagents make these tools cost ineffective. Here we introduce a novel clustered regularly interspaced short palindromic repeat (CRISPR) interference (CRISPRi) approach that efficiently represses expression of target genes in mycobacteria. CRISPRi system involves co-expression of the catalytically dead form of RNA-guided DNA endonuclease from the type II CRISPR system known as dCas9 and the small guide RNA specific to a target sequence, resulting in the DNA recognition complex that interferes with the transcription of corresponding DNA sequence. We show that co-expression of the codon-optimized dCas9 of S. pyogenes with sequence-specific guide RNA results in complete repression of individual or multiple targets in mycobacteria. CRISPRi thus offers a simple, rapid and cost-effective tool for selective control of gene expression in mycobacteria.
Hepatitis E virus (HEV) is a pathogen that is transmitted by the fecal-oral route. Owing to the lack of an efficient laboratory model, the life cycle of the virus is poorly understood. During the course of infection, interactions between the viral and host proteins play essential roles, a clear understanding of which is essential to decode the life cycle of the virus. In this study, we identified the direct host interaction partners of all HEV proteins and generated a PPI network. Our functional analysis of the HEV-human PPI network reveals a role of HEV proteins in modulating multiple host biological processes such as stress and immune responses, the ubiquitin-proteasome system, energy and iron metabolism, and protein translation. Further investigations revealed an essential role of several host factors in HEV replication. Collectively, the results from our study provide a vast resource of PPI data from HEV and its human host and identify the molecular components of the viral translation/replication machinery.
Bacterial P-loop GTPases belong to a family of proteins that selectively hydrolyze a small molecule guanosine tri-phosphate (GTP) to guanosine di-phosphate (GDP) and inorganic phosphate, and regulate several essential cellular activities such as cell division, chromosomal segregation and ribosomal assembly. A comparative genome sequence analysis of different mycobacterial species indicates the presence of multiple P-loop GTPases that exhibit highly conserved motifs. However, an exact function of most of these GTPases in mycobacteria remains elusive. In the present study we characterized the function of a P-loop GTPase in mycobacteria by employing an EngA homologue from Mycobacterium smegmatis, encoded by an open reading frame, designated as MSMEG_3738. Amino acid sequence alignment and phylogenetic analysis suggest that MSMEG_3738 (termed as EngAMS) is highly conserved in mycobacteria. Homology modeling of EngAMS reveals a cloverleaf structure comprising of α/β fold typical to EngA family of GTPases. Recombinant EngAMS purified from E. coli exhibits a GTP hydrolysis activity which is inhibited by the presence of GDP. Interestingly, the EngAMS protein is co-eluted with 16S and 23S ribosomal RNA during purification and exhibits association with 30S, 50S and 70S ribosomal subunits. Further studies demonstrate that GTP is essential for interaction of EngAMS with 50S subunit of ribosome and specifically C-terminal domains of EngAMS are required to facilitate this interaction. Moreover, EngAMS devoid of N-terminal region interacts well with 50S even in the absence of GTP, indicating a regulatory role of the N-terminal domain in EngAMS-50S interaction.
The preprotein translocase, YidC is an envelope protein which controls respiratory metabolism in Mycobacterium tuberculosis. Previously, we have established that depletion of yidC is deleterious for both extra- and intracellular proliferation of M. tuberculosis; however, it remains unclear how YidC expression is regulated under different growth conditions and whether its altered expression impact mycobacterial physiology. Herein, we show that yidC is expressed as an operon with upstream genes. Interestingly, expression analysis under various stress conditions reveals a distinct paradox in the profile of the yidC mRNA transcripts and the YidC protein. While YidC protein level is moderately elevated upon bacterial exposure to cell surface stresses, the corresponding mRNA transcript levels are significantly repressed under these conditions. In contrast, overexpression of M. tuberculosis yidC under a strong anhydrotetracycline-inducible promoter results in significant induction of YidC protein. Additionally, we also observe that overexpression of M. tuberculosis yidC, and not of its counterpart from fast-growing M. smegmatis, results in altered in vitro growth of bacteria, compromised integrity of bacterial cell envelope and differential expression of a small set of genes including those which are regulated under detergent stress. Overall findings of our study suggest that YidC proteins of slow- and fast-growing mycobacteria are functionally distinct despite exhibiting a great deal of identity.
India is hyperendemic to dengue virus and over 50% of the adults are seropositive but there is limited information on the association between prior dengue exposure with neutralizing antibody profiles and how this could influence virus evolution and vaccine development. In this work, we found that the dengue seropositivity increased with age and pre-existing antibody levels negatively correlated with viremia during acute phase of illness. Adults showed a higher levels of viremia which associated with lower levels of neutralizing antibodies as compared to children. The titers of neutralizing antibodies negatively influenced the dominance of circulating dengue serotypes with highest levels of the neutralizing antibodies against DENV-2 followed by DENV-1, DENV-3 and DENV-4. We observed minimal cross-reactivity of neutralizing antibodies with related flaviviruses such as Japanese encephalitis virus and West Nile virus and the antibodies elicited against Indian isolates show a reduced ability to neutralize international dengue isolates.
Delta and Omicron variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are remarkably contagious, and have been recognized as variants of concern (VOC). The acquisition of spontaneous substitutions or insertion–deletion mutations (indels) in the spike protein-encoding gene substantially increases the binding affinity of the receptor binding domain (RBD)-hACE2 complex and upsurges the transmission of both variants. In this study, we analyzed thousands of genome sequences representing 30 different SARS-CoV-2 variants and identified the Delta and Omicron variants specific nucleic acid signatures in the spike gene. Based on the variant-specific nucleic acid sequences, we synthesized different oligos and optimized a multiplex PCR (mPCR) assay that can identify and differentiate the Delta and Omicron variants of SARS-CoV-2. We further extended our work on this mPCR and translated it into a dipstick assay by adding a tag linker sequence to the 5’ end of the forward primer and biotin to the 3’ end of the oligos. Streptavidin-coated latex beads and the dipstick imprinted with a probe for the tag linker sequence in the test strips were used for the detection assay. Our dipstick-based assay, developed as a rapid point-of-care test for identifying and differentiating SARS-CoV-2 variants has the potential to be used in low-resource settings and scaled up to the population level.
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