AUTHOR=Millanao Ana R. , Mora Aracely Y. , Saavedra Claudia P. , Villagra Nicolás A. , Mora Guido C. , Hidalgo Alejandro A. 

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

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.00428

DOI=10.3389/fmicb.2020.00428

ISSN=1664-302X

ABSTRACT=<p>Ciprofloxacin is the choice treatment for infections caused by <italic>Salmonella</italic> 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-Tn<italic>5</italic> transposon mutants of <italic>S</italic>. Typhi were screened. <italic>S.</italic> Typhi <italic>zxx</italic>::EZ-Tn<italic>5</italic> mutants susceptible to ciprofloxacin were confirmed by agar diffusion and MIC assays. The genes carrying EZ-Tn<italic>5</italic> 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 <italic>glnA</italic>. Analysis of outer membrane proteins revealed increased OmpF, a channel for the influx of ciprofloxacin and nalidixic acid, in the <italic>glnA</italic> mutant. Expression of <italic>ompF</italic> increased four times in the <italic>glnA</italic> null mutant compared to WT strain. To understand the relationship between the expression of <italic>glnA</italic> and <italic>ompF</italic>, a strain with the <italic>glnA</italic> gene under control of the tetracycline-inducible P<sup>tet</sup> promoter was created, to modulate <italic>glnA</italic> expression. Induction of <italic>glnA</italic> decreased expression of <italic>ompF</italic>, 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 <italic>glnA</italic> mutant, compared to WT strain. In addition, expression of <italic>glnL</italic> and <italic>glnG</italic> genes (encoding the two-component system NtrC/B that may positively regulate OmpF) were increased in the <italic>glnA</italic> mutant. Further studies indicate that deletion of <italic>glnG</italic> decreases susceptibility to CIP, while deletion of <italic>micF</italic> gene increases susceptibility CIP. Our findings indicate that <italic>glnA</italic> inactivation promotes <italic>ompF</italic> expression, that translates into increased OmpF protein, facilitating the entry of ciprofloxacin, thus increasing susceptibility to ciprofloxacin through 2 possible mechanisms.</p>