| 1. |
- Gupta, Surbhi, et al.
(författare)
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Targeting of Uropathogenic Escherichia coli papG gene using CRISPR-dot nanocomplex reduced virulence of UPEC
- 2021
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Urinary tract infections (UTI) are the most common infectious diseases in the world. It is becoming increasingly tough to treat because of emergence of antibiotic resistance. So, there is an exigency to develop novel anti-virulence therapeutics to combat multi-drug resistance pathogenic strains. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) discovery has revolutionized the gene editing technology for targeted approach. The greatest obstacle for CRISPR/Cas9 is cargo delivery systems and both viral and plasmid methods have disadvantages. Here, we report a highly efficient novel CRISPR based gene editing strategy, CRISPR-dots for targeting virulence factor Fimbrial Adhesion (papG gene), the bacterial adhesion molecule. Carbon quantum dots (CQD) were used as a delivery vehicle for Cas9 and gRNA into CFT073, a UPEC strain. CQDs were covalently conjugated to cas9 and papG-targeted guide RNA (gRNA) forming a nanocomplex CRISPR-dots (Cri-dots) as confirmed by DLS and transmission electron microscopy. Cri-dots-papG significantly targeted papG as demonstrated by decrease in the expression of papG.Further papG deficient UPEC had significantly reduced adherence ability and biofilm forming ability as demonstrated by fluorescence microscopy and scanning electron microscopy. Also, papG deficient UPEC had reduced virulence as shown by significantly increased survival of Caenorhabditis elegans (C. elegans) worms compared to UPEC. Our findings suggest that targeting of papG gene using Cri-dots nanocomplexes significantly reduced the pathogenicity of UPEC. Thus, Cri-dots nanocomplex offer a novel anti-bacterial strategy against multi-drug resistant UPEC.
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| 2. |
- Kapoor, Manali, et al.
(författare)
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Impact of Conditioning Prior to Dilute Acid Deconstruction of Biomass for the Production of Fermentable Sugars
- 2017
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Ingår i: ACS Sustainable Chemistry and Engineering. - : American Chemical Society (ACS). - 2168-0485. ; 5:5, s. 4285-4292
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Tidskriftsartikel (refereegranskat)abstract
- Cost of cellulases is a major impediment in commercialization of cellulosic ethanol. To reduce the enzyme doses for the production of fermentable sugars from rice straw (RS), a series of alkali conditioning experiments were conducted prior to dilute acid (DA) pretreatment. This approach resulted in removal of a majority of extractives, ash, acetic acid, and part lignin, and thus resulted in lowering pseudolignin formation thereby increasing enzymatic hydrolysis yields. Glucan hydrolysis of 69.8%, 74.0%, and 83.5% was obtained at 10 wt % water insoluble solid (WIS) using 8 FPU enzyme/g WIS of biomass conditioned using 0.2, 0.4, and 0.5 wt % alkali prior to pretreatment, which is 14–37% higher than the control (61.0%). The overall sugar recovery in these experiments were 69.2%, 70.2%, and 68.5% at 15 wt % WIS resulting in a sugar concentration greater than 120 g/L, which in turn can produce approximately 5–6% w/v ethanol concentration in fermentation broth. It was found that this approach resulted in a decrease of the enzyme consumption vis-a-vis the conventional process by 46.4% to recover the same amount of sugars. This lowering of enzyme consumption has resulted in net savings, after taking into account the cost of alkali used in the conditioning steps.
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| 3. |
- Rathi, Bhawna, et al.
(författare)
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Anti-biofilm activity of caffeine against uropathogenic E. coli is mediated by curli biogenesis
- 2022
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Biofilms are assemblages of sessile microorganisms that form an extracellular matrix around themselves and mediate attachment to surfaces. The major component of the extracellular matrix of Uropathogenic E. coli and other Enterobacteriaceae are curli fibers, making biofilms robust and resistant to antimicrobials. It is therefore imperative to screen antibiofilm compounds that can impair biofilm formation. In the present study, we investigated the curli-dependent antibiofilm activity of caffeine against UPEC strain CFT073 and commensal strain E. coli K-12MG1655.Caffeine significantly reduced the biofilm formation of both UPEC and E. coli K-12 by 86.58% and 91.80% respectively at 48 mM caffeine as determined by Crystal Violet assay. These results were further confirmed by fluorescence microscopy and Scanning Electron Microscope (SEM). Caffeine significantly reduced the cytotoxicity and survivability of UPEC. Molecular docking analysis revealed a strong interaction between caffeine and curli regulator protein (Csg D) of E. coli. The qRT-PCR data also showed significant downregulation in the expression of CsgBA and the CsgDEFG operon at both 24 mM and 48 mM caffeine. The findings revealed that caffeine could inhibit E. coli biofilm formation by regulating curli assembly and thus may be used as an alternative therapeutic strategy for the treatment of chronic E. coli biofilm-related infections.
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| 4. |
- Verma, Vivek, et al.
(författare)
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α-Hemolysin of uropathogenic E. coli regulates NLRP3 inflammasome activation and mitochondrial dysfunction in THP-1 macrophages
- 2020
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Hemolysin expressing UPEC strains have been associated with severe advanced kidney pathologies, such as cystitis and pyelonephritis, which are associated with an inflammatory response. Macrophages play an important role in regulating an inflammatory response during a urinary tract infection. We have studied the role of purified recombinant α-hemolysin in inducing inflammatory responses and cell death in macrophages. Acylation at lysine residues through HlyC is known to activate proHlyA into a fully functional pore-forming toxin, HlyA. It was observed that active α-hemolysin (HlyA) induced cleavage of caspase-1 leading to the maturation of IL-1β, while inactive α-hemolysin (proHlyA) failed to do so in THP-1 derived macrophages. HlyA also promotes deubiquitination, oligomerization, and activation of the NLRP3 inflammasome, which was found to be dependent on potassium efflux. We have also observed the co-localization of NLRP3 within mitochondria during HlyA stimulations. Moreover, blocking of potassium efflux improved the mitochondrial health in addition to a decreased inflammatory response. Our study demonstrates that HlyA stimulation caused perturbance in potassium homeostasis, which led to the mitochondrial dysfunction followed by an acute inflammatory response, resulting in cell death. However, the repletion of intracellular potassium stores could avoid HlyA induced macrophage cell death. The findings of this study will help to understand the mechanism of α-hemolysin induced inflammatory response and cell death.
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