Dataset Information

Inactivation of Glutamine Synthetase-Coding Gene glnA Increases Susceptibility to Quinolones Through Increasing Outer Membrane Protein F in Salmonella enterica Serovar Typhi.

ABSTRACT: Ciprofloxacin is the choice treatment for infections caused by Salmonella Typhi, however, reduced susceptibility to ciprofloxacin has been reported for this pathogen. Considering the decreased approbation of new antimicrobials and the crisis of resistance, one strategy to combat this problem is to find new targets that enhances the antimicrobial activity for approved antimicrobials. In search of mutants with increased susceptibility to ciprofloxacin; 3,216 EZ-Tn5 transposon mutants of S. Typhi were screened. S. Typhi zxx::EZ-Tn5 mutants susceptible to ciprofloxacin were confirmed by agar diffusion and MIC assays. The genes carrying EZ-Tn5 transposon insertions were sequenced. Null mutants of interrupted genes, as well as inducible genetic constructs, were produced using site-directed mutagenesis, to corroborate phenotypes. SDS-PAGE and Real-time PCR were used to evaluate the expression of proteins and genes, respectively. Five mutants with increased ciprofloxacin susceptibility were found in the screening. The first confirmed mutant was the glutamine synthetase-coding gene glnA. Analysis of outer membrane proteins revealed increased OmpF, a channel for the influx of ciprofloxacin and nalidixic acid, in the glnA mutant. Expression of ompF increased four times in the glnA null mutant compared to WT strain. To understand the relationship between the expression of glnA and ompF, a strain with the glnA gene under control of the tetracycline-inducible Ptet promoter was created, to modulate glnA expression. Induction of glnA decreased expression of ompF, at the same time that reduced susceptibility to ciprofloxacin. Expression of sRNA MicF, a negative regulator of OmpF was reduced to one-fourth in the glnA mutant, compared to WT strain. In addition, expression of glnL and glnG genes (encoding the two-component system NtrC/B that may positively regulate OmpF) were increased in the glnA mutant. Further studies indicate that deletion of glnG decreases susceptibility to CIP, while deletion of micF gene increases susceptibility CIP. Our findings indicate that glnA inactivation promotes ompF expression, that translates into increased OmpF protein, facilitating the entry of ciprofloxacin, thus increasing susceptibility to ciprofloxacin through 2 possible mechanisms.

SUBMITTER: Millanao AR

PROVIDER: S-EPMC7103639 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Inactivation of Glutamine Synthetase-Coding Gene glnA Increases Susceptibility to Quinolones Through Increasing Outer Membrane Protein F in Salmonella enterica Serovar Typhi.

Millanao Ana R AR Mora Aracely Y AY Saavedra Claudia P CP Villagra Nicolás A NA Mora Guido C GC Hidalgo Alejandro A AA

Frontiers in microbiology 20200320

Ciprofloxacin is the choice treatment for infections caused by Salmonella Typhi, however, reduced susceptibility to ciprofloxacin has been reported for this pathogen. Considering the decreased approbation of new antimicrobials and the crisis of resistance, one strategy to combat this problem is to find new targets that enhances the antimicrobial activity for approved antimicrobials. In search of mutants with increased susceptibility to ciprofloxacin; 3,216 EZ-Tn5 transposon mutants ...[more]

PMID: 32265871

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Project description:BackgroundTyphoid fever is an acute systemic infection of humans caused by Salmonella enterica subspecies enterica serovar Typhi (S. Typhi). In chronic carriers, the bacteria survive the harsh environment of the gallbladder by producing biofilm. The phenotype of S. Typhi biofilm cells is significantly different from the free-swimming planktonic cells, and studies have shown that they are associated with antibiotic resistance, immune system evasion, and bacterial persistence. However, the mechanism of this transition and the events leading to biofilm formation are unknown. High throughput sequencing was performed to identify the genes involved in biofilm formation and to postulate the mechanism of action.ResultsPlanktonic S. Typhi cells were cultured using standard nutrient broth whereas biofilm cells were cultured in a stressful environment using high shearing-force and bile to mimic the gallbladder. Sequencing libraries were prepared from S. Typhi planktonic cells and mature biofilm cells using the Illumina HiSeq 2500 platform, and the transcriptome data obtained were processed using Cufflinks bioinformatics suite of programs to investigate differential gene expression between the two phenotypes. A total of 35 up-regulated and 29 down-regulated genes were identified. The identities of the differentially expressed genes were confirmed using NCBI BLAST and their functions were analyzed. The results showed that the genes associated with metabolic processes and biofilm regulations were down-regulated while those associated with the membrane matrix and antibiotic resistance were highly up-regulated.ConclusionsIt is proposed that the biofilm phenotype of S. Typhi allows the bacteria to increase production of the membrane matrix in order to serve as a physical shield and to adhere to surfaces, and enter an energy conservation state in response to the stressful environment. Conversely, the planktonic phenotype allows the bacteria to produce flagella and increase metabolic activity to enable the bacteria to migrate and form new colonies of infection. This data provide a basis for further studies to uncover the mechanism of biofilm formation in S. Typhi and to discover novel genes or pathways associated with the development of the typhoid carrier state.

Dataset Information

Inactivation of Glutamine Synthetase-Coding Gene glnA Increases Susceptibility to Quinolones Through Increasing Outer Membrane Protein F in Salmonella enterica Serovar Typhi.

Publications

Inactivation of Glutamine Synthetase-Coding Gene <i>glnA</i> Increases Susceptibility to Quinolones Through Increasing Outer Membrane Protein F in <i>Salmonella enterica</i> Serovar Typhi.

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