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Targeting of Uropathogenic Escherichia coli papG gene using CRISPR-dot nanocomplex reduced virulence of UPEC.

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.

SUBMITTER: Gupta S

PROVIDER: S-EPMC8423837 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundUropathogenic Escherichia coli (UPEC) are amongst the most frequent causes of urinary tract infections. We report a systematic review and meta-analysis of virulence factors and antimicrobial resistance of UPEC isolated from urinary tract infections.MethodsA systematic review and meta-analysis were performed using PRISMA guidelines (Research Registry ref. 5874). Data were extracted from PubMed/MEDLINE and ScienceDirect databases for studies published from January 1, 2000 to December 31, 2019. Studies reporting antimicrobial resistance and virulence factors of UPEC isolated in confirmed urinary tract infections (≥105CFU/ml) were eligible. Prevalence of antimicrobial resistance and virulence factors of UPEC were estimated using random-effects meta-analysis model. Estimates with 95% confidence intervals, I-square (I2) statistic, and Cochran's Q test were computed using the score statistic and the exact binomial method by incorporating the Freeman-Tukey double arcsine transformation of proportions.ResultsOur search returned 2504 hits, of which 13 studies were included in the meta-analysis, totalling 1888 UPEC isolates. Highest antimicrobial resistance rates were observed among the antibiotic class of tetracycline in 69.1% (498/721), followed by sulphonamides in 59.3% (1119/1888), quinolones in 49.4% (1956/3956), and beta-lactams in 36.9% (4410/11964). Among beta-lactams, high resistance was observed in aminopenicillins in 74.3% (1157/1557) and first generation cephalosporins in 38.8% (370/953). Meanwhile, virulence factors with highest prevalence were immune suppressors (54.1%) followed by adhesins (45.9%). Taken individually, the most observed virulence genes were shiA (92.1%), CSH (80.0%), fimH/MSHA (75.3%), traT (75.1%), sisA (72.2%), iucD (65.7%), iutA (61.8%), kpsMTII (60.6%), and PAI (55.2%).ConclusionsThe increased antibiotic resistance of UPEC isolates was demonstrated and suggested a need for reassessment of empirical therapies in urinary tract infections treatment caused by this pathogen. In addition, this pathotype exhibited diverse surface and secreted virulence factors.

Project description:UNLABELLED:Chaperone-usher pathway (CUP) pili are extracellular organelles produced by Gram-negative bacteria that mediate bacterial pathogenesis. Small-molecule inhibitors of CUP pili, termed pilicides, were rationally designed and shown to inhibit type 1 or P piliation. Here, we show that pilicide ec240 decreased the levels of type 1, P, and S piliation. Transcriptomic and proteomic analyses using the cystitis isolate UTI89 revealed that ec240 dysregulated CUP pili and decreased motility. Paradoxically, the transcript levels of P and S pilus genes were increased during growth in ec240, even though the level of P and S piliation decreased. In contrast, the most downregulated transcripts after growth in ec240 were from the type 1 pilus genes. Type 1 pilus expression is controlled by inversion of the fimS promoter element, which can oscillate between phase on and phase off orientations. ec240 induced the fimS phase off orientation, and this effect was necessary for the majority of ec240's inhibition of type 1 piliation. ec240 increased levels of the transcriptional regulators SfaB and PapB, which were shown to induce the fimS promoter phase off orientation. Furthermore, the effect of ec240 on motility was abolished in the absence of the SfaB, PapB, SfaX, and PapX regulators. In contrast to the effects of ec240, deletion of the type 1 pilus operon led to increased S and P piliation and motility. Thus, ec240 dysregulated several uropathogenic Escherichia coli (UPEC) virulence factors through different mechanisms and independent of its effects on type 1 pilus biogenesis and may have potential as an antivirulence compound. IMPORTANCE:CUP pili and flagella play active roles in the pathogenesis of a variety of Gram-negative bacterial infections, including urinary tract infections mediated by UPEC. These are extremely common infections that are often recurrent and increasingly caused by antibiotic-resistant organisms. Preventing piliation and motility through altered regulation and assembly of these important virulence factors could aid in the development of novel therapeutics. This study increases our understanding of the regulation of these virulence factors, providing new avenues by which to target their expression.

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Targeting of Uropathogenic Escherichia coli papG gene using CRISPR-dot nanocomplex reduced virulence of UPEC.

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