scholarly journals Downregulation of Rv0189c, encoding a dihydroxyacid dehydratase, affects growth of Mycobacterium tuberculosis in vitro and in mice

Microbiology ◽  
2011 ◽  
Vol 157 (1) ◽  
pp. 38-46 ◽  
Author(s):  
Vinayak Singh ◽  
Deepak Chandra ◽  
Brahm S. Srivastava ◽  
Ranjana Srivastava
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Amino Acid ◽  
Ex Vivo ◽  
Functional Characterization ◽  
Acid Stress ◽  
Auxotrophic Mutant ◽  
Isoleucine Leucine ◽  
E Coli ◽  
Antisense Mrna

Dihydroxyacid dehydratase (DHAD), a key enzyme involved in branched-chain amino acid (BCAA) biosynthesis, catalyses the synthesis of 2-ketoacids from dihydroxyacids. In Mycobacterium tuberculosis, DHAD is encoded by gene Rv0189c, and it shares 40 % amino acid sequence identity and conserved motifs with DHAD of Escherichia coli encoded by ilvD. In this study, Rv0189c was overexpressed in E. coli and the resultant protein was characterized as a homodimer (∼155 kDa). Functional characterization of Rv0189c was established by biochemical testing and by genetic complementation of an intron-disrupted ilvD-auxotrophic mutant of E. coli to prototrophy. Growth of M. tuberculosis, E. coli BL21(DE3) and recombinant E. coli BL21(DE3) ΔilvD carrying Rv0189c was inhibited by transient nitric oxide (NO) exposure in minimal medium but growth was restored if the medium was supplemented with BCAA (isoleucine, leucine and valine). This suggested that inactivation of Rv0189c by NO probably inhibited bacterial growth. The role of Rv0189c in M. tuberculosis was elucidated by antisense and sense RNA constructs. Growth of M. tuberculosis transformed with a plasmid encoding antisense mRNA was markedly poor in the lungs of infected mice and in Middlebrook 7H9 broth compared to that of sense and vector-alone transformants, but growth was normal when the medium was supplemented with BCAA. Upregulation of Rv0189c was observed during the early exponential phase of growth, under acid stress and ex vivo, suggesting that Rv0189c has a role in the survival of M. tuberculosis during normal and stress conditions. It may be concluded that the DHAD encoded by Rv0189c is essential for the survival of M. tuberculosis and could be a potential drug/vaccine target, as it is absent in mammals.

scholarly journals Structural and Functional Characterization of Rv2966c Protein Reveals an RsmD-like Methyltransferase from Mycobacterium tuberculosis and the Role of Its N-terminal Domain in Target Recognition

2011 ◽  
Vol 286 (22) ◽  
pp. 19652-19661 ◽  
Author(s):  
Atul Kumar ◽  
Kashyap Saigal ◽  
Ketan Malhotra ◽  
Krishna Murari Sinha ◽  
Bhupesh Taneja
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Target Recognition ◽  
Functional Characterization ◽  
Cellular Growth ◽  
Functional Domain ◽  
E Coli ◽  
Terminal Domain ◽  
16 S Rrna

Nine of ten methylated nucleotides of Escherichia coli 16 S rRNA are conserved in Mycobacterium tuberculosis. All the 10 different methyltransferases are known in E. coli, whereas only TlyA and GidB have been identified in mycobacteria. Here we have identified Rv2966c of M. tuberculosis as an ortholog of RsmD protein of E. coli. We have shown that rv2966c can complement rsmD-deleted E. coli cells. Recombinant Rv2966c can use 30 S ribosomes purified from rsmD-deleted E. coli as substrate and methylate G966 of 16 S rRNA in vitro. Structure determination of the protein shows the protein to be a two-domain structure with a short hairpin domain at the N terminus and a C-terminal domain with the S-adenosylmethionine-MT-fold. We show that the N-terminal hairpin is a minimalist functional domain that helps Rv2966c in target recognition. Deletion of the N-terminal domain prevents binding to nucleic acid substrates, and the truncated protein fails to carry out the m2G966 methylation on 16 S rRNA. The N-terminal domain also binds DNA efficiently, a property that may be utilized under specific conditions of cellular growth.

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

scholarly journals Antagonism of Adherent Invasive E. coli LF82 With Human α-defensin 5 in the Follicle-associated Epithelium of Patients With Ileal Crohn’s Disease

2020 ◽  
Author(s):  
Lina Y Alkaissi ◽  
Martin E Winberg ◽  
Stéphanie DS Heil ◽  
Staffan Haapaniemi ◽  
Pär Myrelid ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Ex Vivo ◽  
Ussing Chambers ◽  
E Coli ◽  
Follicle Associated Epithelium ◽  
Vitro Model ◽  
Set Up

Abstract Background The first visible signs of Crohn’s disease (CD) are microscopic erosions over the follicle-associated epithelium (FAE). The aim of the study was to investigate the effects of human α-defensin 5 (HD5) on adherent-invasive Escherichia coli LF82 translocation and HD5 secretion after LF82 exposure in an in vitro model of human FAE and in human FAE ex vivo. Methods An in vitro FAE-model was set up by the coculture of Raji B cells and Caco-2-cl1 cells. Ileal FAE from patients with CD and controls were mounted in Ussing chambers. The effect of HD5 on LF82 translocation was studied by LF82 exposure to the cells or tissues with or without incubation with HD5. The HD5 secretion was measured in human FAE exposed to LF82 or Salmonella typhimurium. The HD5 levels were evaluated by immunofluorescence, immunoblotting, and ELISA. Results There was an increased LF82 translocation across the FAE-model compared with Caco-2-cl1 (P < 0.05). Incubation of cell/tissues with HD5 before LF82 exposure reduced bacterial passage in both models. Human FAE showed increased LF82 translocation in CD compared with controls and attenuated passage after incubation with sublethal HD5 in both CD and controls (P < 0.05). LF82 exposure resulted in a lower HD5 secretion in CD FAE compared with controls (P < 0.05), whereas Salmonella exposure caused equal secretion on CD and controls. There were significantly lower HD5 levels in CD tissues compared with controls. Conclusions Sublethal HD5 reduces the ability of LF82 to translocate through FAE. The HD5 is secreted less in CD in response to LF82, despite a normal response to Salmonella. This further implicates the integrated role of antimicrobial factors and barrier function in CD pathogenesis.

Loss of an Intimin-Like Protein Encoded on a Uropathogenic E. coli Pathogenicity Island Reduces Inflammation and Affects Interactions with the Urothelium

2021 ◽  
Author(s):  
Allyson E. Shea ◽  
Jolie A. Stocki ◽  
Stephanie D. Himpsl ◽  
Sara N. Smith ◽  
Harry L. T. Mobley
Keyword(s):  
Ex Vivo ◽  
Cell Adherence ◽  
Upec Strain ◽  
E Coli ◽  
Bladder Cell ◽  
Bladder Cells ◽  
Strain Cft073 ◽  
The Individual ◽  
Tract Infections

Uropathogenic Escherichia coli (UPEC) causes the majority of uncomplicated urinary tract infections (UTI), which affect nearly half of women worldwide. Many UPEC strains encode an annotated intimin-like adhesin ( ila ) locus in their genome related to a well-characterized virulence factor in diarrheagenic E. coli pathotypes. Its role in UPEC uropathogenesis, however, remains unknown. In prototype UPEC strain CFT073, there is an ila locus that encodes three predicted intimin-like genes sinH , sinI , and ratA . We used in silico approaches to determine the phylogeny and genomic distribution of this locus among uropathogens. We found that the currently annotated intimin-encoding proteins in CFT073 are more closely related to invasin proteins found in Salmonella . Deletion of the individual sinH , sinI , and ratA genes did not result in measurable effects on growth, biofilm formation, or motility in vitro . On average, sinH was more highly expressed in clinical strains during active human UTI than in human urine ex vivo . Unexpectedly, we found that strains lacking this ila locus had increased adherence to bladder cells in vitro , coupled with a decrease in bladder cell invasion and death. The sinH mutant displayed a significant fitness defect in the murine model of ascending UTI including reduced inflammation in the bladder. These data confirmed an inhibitory role in bladder cell adherence to facilitate invasion and inflammation; therefore, the ila locus should be termed invasin-like, rather than intimin-like. Collectively, our data suggest that loss of this locus mediates measurable interactions with bladder cells in vitro and contributes to fitness during UTI.

scholarly journals N-Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Stanislav Huszár ◽  
Vinayak Singh ◽  
Alica Polčicová ◽  
Peter Baráth ◽  
María Belén Barrio ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Target ◽  
Functional Characterization ◽  
Drug Candidate ◽  
Content Type ◽  
Chemical Libraries ◽  
Whole Cells ◽  
Encoding Gene

ABSTRACT The mycobacterial phosphoglycosyltransferase WecA, which initiates arabinogalactan biosynthesis in Mycobacterium tuberculosis, has been proposed as a target of the caprazamycin derivative CPZEN-45, a preclinical drug candidate for the treatment of tuberculosis. In this report, we describe the functional characterization of mycobacterial WecA and confirm the essentiality of its encoding gene in M. tuberculosis by demonstrating that the transcriptional silencing of wecA is bactericidal in vitro and in macrophages. Silencing wecA also conferred hypersensitivity of M. tuberculosis to the drug tunicamycin, confirming its target selectivity for WecA in whole cells. Simple radiometric assays performed with mycobacterial membranes and commercially available substrates allowed chemical validation of other putative WecA inhibitors and resolved their selectivity toward WecA versus another attractive cell wall target, translocase I, which catalyzes the first membrane step in the biosynthesis of peptidoglycan. These assays and the mutant strain described herein will be useful for identifying potential antitubercular leads by screening chemical libraries for novel WecA inhibitors.

scholarly journals Correlates between Models of Virulence for Mycobacterium tuberculosis among Isolates of the Central Asian Lineage: a Case for Lysozyme Resistance Testing?

2015 ◽  
Vol 83 (6) ◽  
pp. 2213-2223 ◽  
Author(s):  
Claire Pardieu ◽  
Nicola Casali ◽  
Simon O. Clark ◽  
Richard Hooper ◽  
Ann Williams ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Ex Vivo ◽  
Resistance Testing ◽  
Snp Analysis ◽  
Single Nucleotide ◽  
Content Type ◽  
Differential Outcomes ◽  
Central Asian ◽  
Strain H37rv

Virulence factors (VFs) contribute to the emergence of new humanMycobacterium tuberculosisstrains, are lineage dependent, and are relevant to the development ofM. tuberculosisdrugs/vaccines. VFs were sought withinM. tuberculosislineage 3, which has the Central Asian (CAS) spoligotype. Three isolates were selected from clusters previously identified as dominant in London, United Kingdom. Strain-associated virulence was studied in guinea pig, monocyte-derived macrophage, and lysozyme resistance assays. Whole-genome sequencing, single nucleotide polymorphism (SNP) analysis, and a literature review contributed to the identification of SNPs of interest. The animal model revealed borderline differences in strain-associated pathogenicity.Ex vivo, isolate C72 exhibited statistically significant differences in intracellular growth relative to C6 and C14. SNP candidates inducing lower fitness levels included 123 unique nonsynonymous SNPs, including three located in genes (lysX,caeA, andponA2) previously identified as VFs in the laboratory-adapted reference strain H37Rv and shown to confer lysozyme resistance. C72 growth was most affected by lysozymein vitro. A BLAST search revealed that all three SNPs of interest (C35F, P76Q, and P780R) also occurred in Tiruvallur, India, and in Uganda. Unlike C72, however, no single isolate identified through BLAST carried all three SNPs simultaneously. CAS isolates representative of three medium-sized human clusters demonstrated differential outcomes in models commonly used to estimate strain-associated virulence, supporting the idea that virulence varies within, not just across,M. tuberculosislineages. Three VF SNPs of interest were identified in two additional locations worldwide, which suggested independent selection and supported a role for these SNPs in virulence. The relevance of lysozyme resistance to strain virulence remains to be established.

scholarly journals Interaction of Enteropathogenic and Shiga Toxin-Producing Escherichia coli and Porcine Intestinal Mucosa: Role of Intimin and Tir in Adherence

2005 ◽  
Vol 73 (9) ◽  
pp. 6005-6016 ◽  
Author(s):  
Francis Girard ◽  
Isabelle Batisson ◽  
Gad M. Frankel ◽  
Josée Harel ◽  
John M. Fairbrother
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Actin Polymerization ◽  
Animal Species ◽  
Ex Vivo ◽  
Epec Strain ◽  
E Coli ◽  
In Vitro Organ Culture

ABSTRACT The ileal in vitro organ culture (IVOC) model using tissues originating from colostrum-deprived newborn piglets has proven to be an effective way to study the attaching and effacing (A/E) phenotype of porcine enteropathogenic Escherichia coli (EPEC) ex vivo. The aim of this study was to investigate the role of intimin subtype and Tir in the adherence of EPEC and Shiga-toxin-producing E. coli (STEC), isolated from different animal species, to porcine intestinal IVOC. Moreover, the role of intimin in Tir-independent adherence of the human EPEC strain E2348/69 was investigated using intimin and Tir-deficient derivatives. Our results demonstrated that A/E E. coli strains (AEEC) from various animal species and humans induce the A/E phenotype in porcine ileal IVOC and that intimin subtype influences intestinal adherence and tropism of AEEC strains. We also showed that a tir mutant of EPEC strain E2348/69 demonstrates close adherence to the epithelial cells of porcine ileal IVOC segments, with microvillous effacement but with no evidence of actin polymerization or pedestal formation, and that intimin seems to be involved in this phenotype. Overall, this study provides further evidence for the existence of one or more host-cell-encoded intimin receptor(s) in the pig gut.

scholarly journals Suspicion of quinolone active metabolite following discrepancy between predicted and experimental urine bactericidal activities.

1997 ◽  
Vol 41 (5) ◽  
pp. 927-930 ◽  
Author(s):  
L Aguilar ◽  
M J Giménez ◽  
J Costa ◽  
R Dal-Ré ◽  
J Prieto
Keyword(s):  
Antibacterial Activity ◽  
Active Metabolite ◽  
Ex Vivo ◽  
In Vitro Susceptibility ◽  
E Coli ◽  
Experimental Activity ◽  
Microbiological Parameters ◽  
Bactericidal Activities ◽  
Urine Levels

The prediction of urine antibacterial activity from pharmacological and microbiological parameters was assessed by using experimental urine levels and urine bactericidal titers determined up to 72 h after a 400-mg single dose of two quinolones in a phase I study. The area under the bactericidal curve (AUBC) was accurately predicted for norfloxacin but significantly (P < 0.001) underestimated for rufloxacin (actual value was four times higher than the predicted value against Escherichia coli and two times higher against Staphylococcus aureus). In vitro susceptibility differences between the two strains predicted the ex vivo AUBC differences for norfloxacin but not for rufloxacin, where ex vivo differences were greater than expected. Urine bactericidal titers for up to 72 h were accurately predicted for norfloxacin against E. coli and S. aureus and for rufloxacin against S. aureus, but experimental activity for up to 48 h was four times higher (P < 0.001) than the predicted activity for rufloxacin against E. coli. In the case of norfloxacin, the duration of adequate urine antibacterial activity against S. aureus was overestimated. Inaccurate estimations of ex vivo antibacterial activity of a suspected active metabolite (as with rufloxacin) when an adequate cutoff is not established may have dosing implications.

scholarly journals Isolation and Characterization of vicH, Encoding a New Pleiotropic Regulator in Vibrio cholerae

2000 ◽  
Vol 182 (7) ◽  
pp. 2026-2032 ◽  
Author(s):  
Christian Tendeng ◽  
Cyril Badaut ◽  
Evelyne Krin ◽  
Pierre Gounon ◽  
Saravuth Ngo ◽  
...  
Keyword(s):  
Amino Acid ◽  
Vibrio Cholerae ◽  
Genomic Library ◽  
Single Gene ◽  
Wild Type ◽  
Semisolid Medium ◽  
E Coli ◽  
Isolation And Characterization ◽  
Serovar Typhimurium

ABSTRACT During the last decade, the hns gene and its product, the H-NS protein, have been extensively studied in Escherichia coli. H-NS-like proteins seem to be widespread in gram-negative bacteria. However, unlike in E. coli and inSalmonella enterica serovar Typhimurium, little is known about their role in the physiology of those organisms. In this report, we describe the isolation of vicH, an hns-like gene in Vibrio cholerae, the etiological agent of cholera. This gene was isolated from a V. cholerae genomic library by complementation of different phenotypes associated with anhns mutation in E. coli. It encodes a 135-amino-acid protein showing approximately 50% identity with both H-NS and StpA in E. coli. Despite a low amino acid conservation in the N-terminal part, VicH is able to cross-react with anti-H-NS antibodies and to form oligomers in vitro. ThevicH gene is expressed as a single gene from two promoters in tandem and is induced by cold shock. A V. choleraewild-type strain expressing a vicHΔ92 gene lacking its 3′ end shows pleiotropic alterations with regard to mucoidy and salicin metabolism. Moreover, this strain is unable to swarm on semisolid medium. Similarly, overexpression of the vicH wild-type gene results in an alteration of swarming behavior. This suggests that VicH could be involved in the virulence process in V. cholerae, in particular by affecting flagellum biosynthesis.

scholarly journals Efflux Attenuates the Antibacterial Activity of Q203 in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Jichan Jang ◽  
Ryangyeo Kim ◽  
Minjeong Woo ◽  
Jinsun Jeong ◽  
Da Eun Park ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Efflux Pump ◽  
Ex Vivo ◽  
Drug Candidate ◽  
Content Type ◽  
Tb Treatment ◽  
Pump Activity ◽  
Efflux Pump Inhibitors ◽  
Combinatorial Strategy

ABSTRACT New and improved treatments for tuberculosis (TB) are urgently needed. Recently, it has been demonstrated that verapamil, an efflux inhibitor, can reduce bacterial drug tolerance caused by efflux pump activity when administered in combination with available antituberculosis agents. The aim of this study was to evaluate the effectiveness of verapamil in combination with the antituberculosis drug candidate Q203, which has recently been developed and is currently under clinical trials as a potential antituberculosis agent. We evaluated changes in Q203 activity in the presence and absence of verapamil in vitro using the resazurin microplate assay and ex vivo using a microscopy-based phenotypic assay for the quantification of intracellular replicating mycobacteria. Verapamil increased the potency of Q203 against Mycobacterium tuberculosis both in vitro and ex vivo, indicating that efflux pumps are associated with the activity of Q203. Other efflux pump inhibitors also displayed an increase in Q203 potency, strengthening this hypothesis. Therefore, the combination of verapamil and Q203 may be a promising combinatorial strategy for anti-TB treatment to accelerate the elimination of M. tuberculosis.

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