Biology subjects

Swaminath, S.

Publications and source records attributed to Swaminath, S..

4 recordsLinked to original sources

bioRxiv2024

Novel CRITR-seq approach reveals influenza transcription is modulated by NELF and is a key event precipitating an interferon response

Transcription of interferons upon viral infection is critical for cell-intrinsic innate immunity. This process is influenced by many host and viral factors. To identify host factors that modulate interferon induction within cells infected by influenza A virus, we developed CRISPR with Transcriptional Readout (CRITR-seq). CRITR-seq is a method linking CRISPR guide sequence to activity at a promoter of interest. Employing this method, we find that depletion of the Negative Elongation Factor (NELF) complex increases both flu transcription and interferon expression. We find that the process of flu transcription, both in the presence and absence of viral replication, is a key contributor to interferon induction. Taken together, our findings highlight innate immune ligand concentration as a limiting factor in triggering an interferon response, identify NELF as an important interface with the flu life cycle, and validate CRITR-seq as a tool for genome-wide screens for phenotypes of gene expression. Significance StatementNearly every cell in the human body has the ability to detect and respond to viral infection by producing interferons. The timing and magnitude of the interferon response impacts the course of disease, and both hosts and viruses have evolved mechanisms to regulate interferon induction within infected cells. It has previously been challenging to comprehensively search for regulators of interferon expression using selection-based screens. Here we developed a CRISPR screening strategy to measure the effects of gene edits on transcription at a promoter of interest. Applying this method to study interferon transcription during influenza infection, we identified an interface between human and influenza transcription machinery that modulates the viral life cycle and influences the interferon response.

molecular biology↗

bioRxiv2024

Efficient genome replication in influenza A virus requires NS2 and sequence beyond the canonical promoter

Influenza A virus encodes promoters in both the sense and antisense orientations. These support the generation of new genomes, antigenomes, and mRNA transcripts. Using minimal replication assays--transfections with viral polymerase, nucleoprotein, and a genomic template--the influenza promoter sequences were identified as 13nt at the 5 end of the viral genomic RNA (U13) and 12nt at the 3 end (U12). Other than the fourth 3 nucleotide, the U12 and U13 sequences are identical between all eight RNA molecules that comprise the segmented influenza genome. Despite possessing identical promoters, individual segments can exhibit different transcriptional dynamics during infection. However flu promoter sequences were defined in experiments without influenza NS2, a protein which modulates transcription and replication differentially between genomic segments. This suggests that the identity of the "complete" promoter may depend on NS2. Here we assess how internal sequences of two genomic segments, HA and PB1, may contribute to NS2-dependent replication as well as map such interactions down to individual nucleotides in PB1. We find that the expression of NS2 significantly alters sequence requirements for efficient replication beyond the identical U12 and U13 sequence, providing a mechanism for the divergent replication and transcription dynamics across the influenza A virus genome.

microbiology↗

bioRxiv2020

The rifampicin-inactivating mono-ADP-ribosyl transferase of Mycobacterium smegmatis significantly influences reactive oxygen species levels in the actively growing cells

A classic example of antibiotic inactivating function in bacteria is the Mycobacterium smegmatis (Msm) encoded rifampicin-inactivating mono-ADP-ribosyl transferase (arr). Since its probable biological role has been proposed to be in DNA damage response, which is inflicted by reactive oxygen species (ROS), in the present study, we examined whether Msm Arr influences ROS levels. For this purpose, the levels of the ROS, hydroxyl radical and superoxide, were determined in the mid-log phase (MLP) cells of Msm arr knockout (arr-KO) strain, in comparison to those in the equivalently grown Msm arr+ wild-type (WT) strain. The MLP arr-KO cells generated significantly elevated levels of superoxide and hydroxyl radical, unlike the equivalently grown WT MLP cells. Complementation of arr-KO with arr, but not with empty vector, restored the ROS levels comparable to those in the WT strain. Elevated ROS levels in the arr-KO strain enabled selection of rifampicin-resistant mutants at 10-7 cfu/ml from the rifampicin-unexposed MLP cells of arr-KO, which is one-log10 higher than that for WT cells (10-8). Upon prolonged exposure to rifampicin, the susceptibility, persister formation, generation of elevated levels of hydroxyl radical by the persisters, rifampicin-resister generation frequency of the persisters and regrowth of the rifampicin-resistant mutants from the respective persisters were all comparable between the arr-KO and WT strains. These observations revealed that Arr influences ROS levels in the actively growing M. smegmatis cells but not in the rifampicin-exposed cells. We proposed the probable pathway through which Arr might be influencing ROS levels in the actively growing M. smegmatis cells. IMPORTANCEDiverse genera of bacteria consisting of pathogens, opportunistic pathogens and non-pathogens, possess Arr-type activities that confer equally efficient rifampicin resistance, thereby posing serious health hazard. Acquisition of this function by other bacteria through horizontal gene transfer enhances the hazard posed by the bacteria possessing it. M. smegmatis is an opportunistic human pathogen that causes infections of skin and soft tissues. Moreover, M. smegmatis is a genetically tractable model organism for M. tuberculosis with the potential to function even as tuberculosis vaccine. In view of these significant aspects of Arr and M. smegmatis, the study to find out the natural physiological role of Arr in M. smegmatis, gains importance for designing strategies to prevent antibiotic inactivation and to target the cellular function to contain the bacterium. Above all, the three-dimensional structure of M. smegmatis Arr reveals significant structural homology with eukaryotic ADP-ribosyltransferases and bacterial toxins, thereby giving the study broad significance.

microbiology↗

bioRxiv2019

Reduced Permeability to Rifampicin by Capsular Thickening as a Mechanism of Antibiotic Persistence in Mycobacterium tuberculosis

Persisters constitute a subpopulation of bacteria that can tolerate lethal concentrations of antibiotics. Multiple mechanisms have been suggested for bacterial persistence against antibiotics. With mycobacteria being no exception to this behaviour, we had reported the de novo emergence of genetically antibiotic-resistant Mycobacterium tuberculosis from persister cells upon prolonged exposure to microbicidal concentrations of the anti-tuberculosis drugs, rifampicin and moxifloxacin. Here, we present evidence for reduced permeability to rifampicin as a mechanism for persistence of Mycobacterium tuberculosis in vitro. We observed that rifampicin persistent M. tuberculosis cells developed a thick outer layer (TOL) capsule. The TOL restricted the entry of fluorochrome-conjugated rifampicin, 5-carboxyfluorescein-rifampicin (5-FAM-rifampicin), which retained only 2.5% of its original bactericidal activity, but high levels of permeability, on actively growing mid-log phase cells. Gentle mechanical removal of TOL significantly enhanced 5-FAM-rifampicin entry into the persister cells. The level of 5-FAM-rifampicin in the persister cells was not affected by the pre-incubation of the cells with verapamil, a drug efflux pump inhibitor, ruling out the involvement of efflux pumps in the reduced intracellular concentration of 5-FAM-rifampicin. GC-MS analysis of TOL showed the presence of [~]7-fold, [~]5-fold and [~]2- fold higher levels of -D-glucopyranoside, 1,2,5-linked-mannitol, and 3,4-linked mannose, respectively, among [~]2-fold higher levels of derivatives of several other types of sugars such as arabinose and galactose. Taken together, the present study reveals that rifampicin-persistent M. tuberculosis cells develop TOL that enables the bacilli to restrict entry of rifampicin and thereby remain tolerant to the antibiotic in vitro.

microbiology↗