Loss of RNA Chaperone Hfq Unveils a Toxic Pathway in Pseudomonas aeruginosa

Abstract

Hfq is an RNA chaperone that serves as a master regulator of bacterial physiology. Here we show that in the opportunistic pathogen Pseudomonas aeruginosa, the loss of Hfq can result in a dramatic reduction in growth in a manner that is dependent upon MexT, a transcription regulator that governs antibiotic resistance in this organism. Using a combination of chromatin immunoprecipitation with high-throughput sequencing and transposon insertion sequencing, we identify the MexT-activated genes responsible for mediating the growth defect of hfq mutant cells. These include a newly identified MexT-controlled gene that we call hilR. We demonstrate that hilR encodes a small protein that is acutely toxic to wild-type cells when produced ectopically. Furthermore, we show that hilR expression is negatively regulated by Hfq, offering a possible explanation for the growth defect of hfq mutant cells. Finally, we present evidence that the expression of MexT-activated genes is dependent upon GshA, an enzyme involved in the synthesis of glutathione. Our findings suggest that Hfq can influence the growth of P. aeruginosa by limiting the toxic effects of specific MexT-regulated genes. Moreover, our results identify glutathione to be a factor important for the in vivo activity of MexT. IMPORTANCE Here we show that the conserved RNA chaperone Hfq is important for the growth of the opportunistic pathogen Pseudomonas aeruginosa. We found that the growth defect of hfq mutant cells is dependent upon the expression of genes that are under the control of the transcription regulator MexT. These include a gene that we refer to as hilR, which we show is negatively regulated by Hfq and encodes a small protein that can be toxic when ectopically produced in wild-type cells. Thus, Hfq can influence the growth of P. aeruginosa by limiting the toxic effects of MexT-regulated genes, including one encoding a previously unrecognized small protein. We also show that MexT activity depends on an enzyme that synthesizes glutathione.

Hfq-Assisted RsmA Regulation Is Central to Pseudomonas aeruginosa Biofilm Polysaccharide PEL Expression

“…Because Hfq (like RsmA) is a global regulator, its absence can cause pleotropic effects. As previously reported by other laboratories ( Sonnleitner et al, 2003 ; Hill et al, 2019 ), lack of Hfq in PAO1 causes growth defects ( Supplementary Figure S5A ). These defects are complemented by WT Hfq ( Supplementary Figure S5B ).…”

Gene Expression Profiling of Pseudomonas aeruginosa Upon Exposure to Colistin and Tobramycin

“…Colistin caused a significant up-regulation of the PA14 genes PA14_22420, PA14_22740, PA14_28410, mexF, mexE, PA14_32480, PA14_32490, PA14_39060, PA14_39420, PA14_41990, PA14_56620, PA14_56640, and PA14_64530 (Table 1 and Supplementary Table 1), all of which belong to the MexT regulon (Tian et al, 2009a;Hill et al, 2019). MexT is a transcriptional regulator of the LysR family known to control the expression of pathogenicity, virulence and antibiotic resistance determinants in Pae (Köhler et al, 1997a(Köhler et al, ,b, 1999Tian et al, 2009a;Huang et al, 2019).…”

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“…Because Hfq (like RsmA) is a global regulator, its absence can cause pleotropic effects. As previously reported by other laboratories (Sonnleitner et al, 2003;Hill et al, 2019), lack of Hfq in PAO1 causes growth defects (Supplementary Figure S5A). These defects are complemented by WT Hfq (Supplementary Figure S5B).…”