Browsing by Author "Chakravarty, Shubham"

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    DNA alternate polymerase PolB mediates inhibition of type III secretion in Pseudomonas aeruginosa
    (Elsevier, 2021) Chakravarty, Shubham; Ramos-Hegazy, Layla; Gasparovic, Abigail; Anderson, Gregory G.; Biology, School of Science
    Opportunistic pathogen Pseudomonas aeruginosa uses a variety of virulence factors to cause acute and chronic infections. We previously found that alternate DNA polymerase gene polB inhibits P. aeruginosa pyocyanin production. We investigated whether polB also affects T3SS expression. polB overexpression significantly reduced T3SS transcription and repressed translation of the master T3SS regulator ExsA, while not affecting exsA mRNA transcript abundance. Further, polB does not act through previously described genetic pathways that post-transcriptionally regulate ExsA. Our results show a novel T3SS regulatory component which may lead to development of future drugs to target this mechanism.
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    Molecular mechanisms of cytotoxicity regulation in pseudomonas aeruginosa by the magnedium transporter MGTE
    (2017-07) Chakravarty, Shubham; Anderson, Gregory G.
    The Gram-negative bacterium Pseudomonas aeruginosa causes numerous acute and chronic opportunistic infections in humans. One of its most formidable weapons is a type III secretion system (T3SS), a multi-protein molecular syringe that injects powerful toxins directly into host cells. The toxins lead to cell dysfunction and, ultimately, cell death. Identification of regulatory pathways that control T3SS gene expression may lead to the discovery of novel therapeutics to treat P. aeruginosa infections. In a previous study, it was found that expression of the magnesium transporter gene mgtE inhibits T3SS gene transcription. MgtE-dependent inhibition appeared to interfere with the synthesis or function of the master T3SS transcriptional activator ExsA, although the exact mechanism was unclear. In this work, we demonstrate that mgtE expression acts through the GacAS two-component system to activate transcription of the small regulatory RNAs RsmY and RsmZ. This event ultimately leads to inhibition of exsA translation. Moreover, our data reveal that MgtE acts solely through this pathway to regulate T3SS gene transcription. Our study reveals an important mechanism that may allow P. aeruginosa to fine-tune T3SS activity in response to certain environmental stimuli. In addition, a previous study has shown that the P. aeruginosa gene algR abrogates mgtE mediated regulation of cytotoxicity. AlgR has pleiotropic effects in P. aeruginosa, including regulation of synthesis of the exopolysaccharide alginate. In the second part of my thesis, I show that algR and mgtE genetically crosstalk to inhibit ExsA driven T3SS gene transcription. This genetic interaction between algR and mgtE seems to be specifically directed towards regulation of T3SS gene expression rather than having an indiscriminate effect on multiple virulence attributes in P. aeruginosa. Additionally, we have further demonstrated that AlgR inhibits mgtE transcription. These studies suggest the presence of a T3SS inhibitor that is inhibited by both AlgR and MgtE. Future work will involve transcriptomic and proteomic analysis to identify such an inhibitor. Taken together, this study provides important insight into the molecular mechanisms of mgtE expression and function in P. aeruginosa. We have established that mgtE has pleiotropic effects on cytotoxicity in P. aeruginosa. Thus, given the role that cytotoxicity regulation plays in shaping P. aeruginosa pathogenesis and associated clinical outcomes, mgtE might be an interesting drug target, though extensive future studies are required to validate this proposition. Nevertheless, this research, provides clues for identification of novel therapeutic targets in P. aeruginosa. Hence this work, in the long run, serve to ameliorate the morbidity and mortality in patients infected with P. aeruginosa.
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    Phosphoglycerate mutase affects Stenotrophomonas maltophilia attachment to biotic and abiotic surfaces
    (Elsevier, 2020) Ramos-Hegazy, Layla; Chakravarty, Shubham; Anderson, Gregory G.; Biology, School of Science
    Stenotrophomonas maltophilia biofilm formation is of increasing medical concern, particularly for lung infections. However, the molecular mechanisms facilitating the biofilm lifestyle in S. maltophilia are poorly understood. We generated and screened a transposon mutant library for mutations that lead to altered biofilm formation compared to wild type. One of these mutations, in the gene for glycolytic enzyme phosphoglycerate mutase (gpmA), resulted in impaired attachment on abiotic and biotic surfaces. As adherence to a surface is the initial step in biofilm developmental processes, our results reveal a unique factor that could affect S. maltophilia biofilm initiation and, possibly, subsequent development.
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    Pseudomonas aeruginosa Magnesium Transporter MgtE Inhibits Type III Secretion System Gene Expression by Stimulating rsmYZ Transcription
    (American Society for Microbiology, 2017-10-31) Chakravarty, Shubham; Melton, Cameron N.; Bailin, Adam; Yahr, Timothy L.; Anderson, Gregory G.; Biology, School of Science
    Pseudomonas aeruginosa causes numerous acute and chronic opportunistic infections in humans. One of its most formidable weapons is a type III secretion system (T3SS), which injects powerful toxins directly into host cells. The toxins lead to cell dysfunction and, ultimately, cell death. Identification of regulatory pathways that control T3SS gene expression may lead to the discovery of novel therapeutics to treat P. aeruginosa infections. In a previous study, we found that expression of the magnesium transporter gene mgtE inhibits T3SS gene transcription. MgtE-dependent inhibition appeared to interfere with the synthesis or function of the master T3SS transcriptional activator ExsA, although the exact mechanism was unclear. We now demonstrate that mgtE expression acts through the GacAS two-component system to activate rsmY and rsmZ transcription. This event ultimately leads to inhibition of exsA translation. This inhibitory effect is specific to exsA as translation of other genes in the exsCEBA operon is not inhibited by mgtE Moreover, our data reveal that MgtE acts solely through this pathway to regulate T3SS gene transcription. Our study reveals an important mechanism that may allow P. aeruginosa to fine-tune T3SS activity in response to certain environmental stimuli.IMPORTANCE The type III secretion system (T3SS) is a critical virulence factor utilized by numerous Gram-negative bacteria, including Pseudomonas aeruginosa, to intoxicate and kill host cells. Elucidating T3SS regulatory mechanisms may uncover targets for novel anti-P. aeruginosa therapeutics and provide deeper understanding of bacterial pathogenesis. We previously found that the magnesium transporter MgtE inhibits T3SS gene transcription in P. aeruginosa In this study, we describe the mechanism of MgtE-dependent inhibition of the T3SS. Our report also illustrates how MgtE might respond to environmental cues, such as magnesium levels, to fine-tune T3SS gene expression.
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    The Regulatory Protein AlgR Influences Pseudomonas aeruginosa Pathogenesis on Airway Cells
    (Office of the Vice Chancellor for Research, 2013-04-05) Chakravarty, Shubham; Anderson, Gregory G.
    One of the primary causes of high mortality in patients with Cystic Fibrosis (CF) is lung infection caused by the Gram-negative bacterium Pseudomonas aeruginosa. In the lungs of such patients, this bacterium forms lifelong infections, characterized by biofilm formation. Our goal is to identify factors that influence P. aeruginosa biofilm formation in the CF airways. Using a novel model of biofilm formation on cultured human CF airway cells, we found that mutation of the gene algR resulted in significant reduction in the ability to form biofilms as well as adhere to CF airway cells. When algR gene activity was restored in these deletion mutant strains, by an algR complementation plasmid, adherence improved to a level similar to that of the wild type P. aeruginosa strain, and biofilm production was also restored significantly. Additionally, e observed the effects of AlgR on transcription of the Type III Secretion System (T3SS), and found that AlgR might be influence regulation of T3SS activity through the magnesium transporter protein MgtE. Altogether, our results point to a role for AlgR in biofilm formation on CF airway cells through modulation of T3SS as well as adherence. Through this and additional studies, such as investigation of cross-talk between AlgR and other genes such as MgtE, which is a putative virulence modulator in P. aeruginosa, we aim to get a more lucid understanding of the molecular mechanisms responsible for persistence of lifelong P. aeruginosa biofilms in lungs of CF patients.