PA3225 Is a Transcriptional Repressor of Antibiotic Resistance Mechanisms in Pseudomonas aeruginosa

Abstract

The tssABC1 locus is part of the Hcp secretion island I (HSI-I) type VI secretion system (T6SS) in Pseudomonas aeruginosa. Previous work implicated the tssC1 gene in P. aeruginosa biofilm-specific antibiotic resistance, and tssC1 is preferentially expressed in biofilms compared to planktonic cells. Using a DNA-dependent protein pulldown approach, we discovered that PA3225, an uncharacterized LysR-type transcriptional regulator, specifically bound to the tssABC1 upstream regulatory region. The deletion of PA3225 led to a 2-fold decrease in tssA1 expression levels in planktonic cells compared to the wild type, and tssA1 expression was slightly reduced in ΔPA3225 biofilms compared to wild-type biofilms. Intriguingly, further investigations revealed that the ΔPA3225 mutant was less susceptible to multiple, structurally unrelated antibiotics with various mechanisms of action when grown planktonically. The ΔPA3225 mutant was additionally more resistant to ciprofloxacin when grown in a biofilm. The decreased antibiotic susceptibility of the ΔPA3225 strain was linked to the transcriptional upregulation of the MexAB-OprM efflux pump. By using transcriptome sequencing (RNA-seq), other PA3225-regulated genes were identified, and the products of these genes, such as the putative ABC transporter PA3228, may also contribute to antibiotic resistance.

Evolution of Antibiotic Resistance in Biofilm and Planktonic Pseudomonas aeruginosa Populations Exposed to Subinhibitory Levels of Ciprofloxacin

“…Mutations in genes involved in the regulation of MexAB-OprM and MexCD-OprD were also present in some lineages of the planktonic populations, though different types of mutations were identified under the two growth conditions (Table S5). Mutations in the known regulators nalD and nalC of the MexAB-OprM efflux pump, as well as in PA3228, a recently described transcriptional regulator of antibiotic resistance in P. aeruginosa (34), were present only in the CIP-evolved planktonic populations. Interestingly, many of these regulators of multidrug efflux pumps were identified as pathoadaptive genes for P. aeruginosa evolution in the CF lungs (35), suggesting that antibiotic stress is one of the main drivers of evolution in biofilms in vivo.…”

The LysR-type transcriptional regulator BsrA (PA2121) controls vital metabolic pathways in Pseudomonas aeruginosa

“…In the first sequenced P. aeruginosa genome of reference strain PAO1 (23), 113 genes are annotated as encoding LysR-type transcriptional regulators, but their functions remain largely unknown. P. aeruginosa LTTRs with known roles include PA0133 (BauR) (24), PA0739 (SdsB1) (25), PA1413 (26), PA1422 (GbuR) (27), PA1998 (DhcR) (28), PA2076 (OdsR) (29), PA2206 (30), PA2258 (PtxR) (31), PA2432 (BexR) (32), PA2838 (33), PA3225 (34), PA3587 (MetR) (35), PA3630 (GfnR) (36), PA4109 (AmpR) (37), PA4203 (38), PA5437 (PycR) (39), PA1003 (MvfR, also called PqsR) (40–42), PA5344 (OxyR) (43–45) and PA2492 (MexT) (46, 47). The membrane-associated multiple virulence factor regulator MvfR was shown to be necessary for P. aeruginosa virulence (40).…”

Two Regulators, PA3898 and PA2100, Modulate the Pseudomonas aeruginosa Multidrug Resistance MexAB-OprM and EmrAB Efflux Pumps and Biofilm Formation

“…However, the MexAB-OprM operon, on the other hand, shows a unique spatial expression pattern with relatively high expression levels on the surface of the biofilm and downregulated expression in the middle, and the expression level in the interior of the biofilm is then comparable to that of planktonic growth. This unique spatial expression pattern of mexAB-oprM is likely the result of its complex regulatory network, involving multiple regulators working together to fine-tune the spatial expression of this important efflux system, as previous studies have revealed (24)(25)(26)(29)(30)(31).…”

“…Thus far, the control of the second transcriptional start site has not been assigned to any regulator(s), although the TetR family regulator NalD, the MerR family regulator BrlR, and the response regulator CpxR also bind directly to the promoter of the operon (25)(26)(27)(28). Other regulators have also been shown to affect the expression of mexAB-oprM indirectly, including NalC (29), ArmR (30), and PA3225 (31). It is currently unclear if an additional regulator(s) directly controls the expression of mexAB-oprM, via the second transcriptional start site downstream of the known binding sites of MexR and NalD.…”