A Linkage between SmeIJK Efflux Pump, Cell Envelope Integrity, and σ^E-Mediated Envelope Stress Response in <i>Stenotrophomonas maltophilia</i>

<div><p>Resistance nodulation division (RND) efflux pumps, such as the SmeIJK pump of <i>Stenotrophomonas maltophilia</i>, are known to contribute to the multidrug resistance in Gram-negative bacteria. However, some RND pumps are constitutively expressed even though no antimicrobial stresses occur, implying that there should be some physical implications for these RND pumps. In this study, the role of SmeIJK in antimicrobials resistance, envelope integrity, and σ^E-mediated envelope stress response (ESR) of <i>S. maltophilia</i> was assessed. SmeIJK was involved in the intrinsic resistance of <i>S. maltophilia</i> KJ to aminoglycosides and leucomycin. Compared with the wild-type KJ, the <i>smeIJK</i> deletion mutant exhibited growth retardation in the MH medium, an increased sensitivity to membrane-damaging agents (MDAs), as well as activation of an σ^E-mediated ESR. Moreover, the expression of <i>smeIJK</i> was further induced by sub-lethal concentrations of MDAs or surfactants in an σ^E-dependent manner. These data collectively suggested an alternative physiological role of <i>smeIJK</i> in cell envelope integrity maintenance and σ^E-mediated ESR beyond the efflux of antibiotics. Because of the necessity of the physiological role of SmeIJK in protecting <i>S. maltophilia</i> from the envelope stress, <i>smeIJK</i> is constitutively expressed, which, in turn, contributes the intrinsic resistance to aminoglycoside and leucomycin. This is the first demonstration of the linkage among RND-type efflux pump, cell envelope integrity, and σ^E-mediated ESR in <i>S. maltophilia</i>.</p></div

Determination of <i>P_rpoE</i> activities in different strains and culture conditions.

<p>Plasmid containing a transcriptional fusion of the upstream region of <i>rpoE</i> to the <i>xylE</i> gene (pRpoE_xylE) was transferred into the wild-type KJ and its derived mutants. The C23O activities of the logarithmic-phase cultures of these strains were determined. Each bar represents the mean of three independent experiments. Error bars, where visible, indicate the average deviation. *, <i>p</i>≤0.01 significance calculated by a Student's <i>t</i>-test. (A) The impacts of <i>resA</i> mutant and <i>rseA/rpoE</i> double mutant on the promoter activity of <i>rpoE</i> gene. (B) The impacts of <i>smeIJK</i> mutant and culture media on the promoter activity of <i>rpoE</i> gene.</p

The role of SmeIJK pump in the tolerance to hypo-osomolarity and casein hydrolysate.

<p>The data are the average of the measurements made in triplicate. (A) The relative survival of KJΔIJK, KJΔJ, and KJΔK to the wild-type KJ in the LB medium containing different concentrations of NaCl. The relative survival percentage of individual mutant to the wild-type KJ, at each cultured condition, was calculated using the OD_{450 nm} of the wild-type KJ as 100%. (B) The relative survival of KJΔIJK, KJΔJ, and KJΔK to the wild-type KJ in the MH medium containing different concentrations of NaCl. The relative survival percentage of individual mutant to the wild-type KJ, at each cultured condition, was calculated using the OD_{450 nm} of the wild-type KJ as 100%. (C) The sensitivities of KJ to SDS in LB or LB containing casein hydrolysate, beef infusion, or starch were determined by the OD_{450 nm} measurement. The percentage of survival was defined as the OD_{450 nm} ratio of the SDS-additive group to the SDS-free counterpart. (D) Growth curves of KJΔIJK grown in the media of the LB, the LB without NaCl, the LB with casein hydrolysate, and the MH, respectively.</p

Growth of KJ, KJΔJ, KJΔK, and KJΔIJK in Luria-Bertani (LB) and Mueller-Hinton (MH) media.

<p>(A) Growth curves of KJ, KJΔJ, KJΔK, and KJΔIJK in LB and MH broth. (B) The growth curve of 24-h MH-cultured KJ and KJΔIJK cells subcultured into the MH or LB media.</p

Assessment of the cell envelope integrity of <i>S. maltophilia</i> KJ and its derived mutants in different cultured conditions.

<p>(A) Sodium dodecyl sulfate (SDS) survival analysis. The survival of KJ, KJΔIJK, KJΔJ, and KJΔK in LB or MH broth without or with 0.02% SDS was determined by the <i>A_{450 nm}</i> measurement. The percentage of survival was defined as the <i>A_{450 nm}</i> ratio of the SDS-additive group to the SDS-free counterpart. (B) Polymyxin E susceptibility. The polymyxin E susceptibility of KJ, KJΔIJK, KJΔJ, and KJΔK in LB or MH broth was determined by the disc diffusion assay.</p

Antimicrobial susceptibilities of <i>S. maltophilia</i> KJ and its derived deletion mutants.

<p>Antimicrobial susceptibilities of <i>S. maltophilia</i> KJ and its derived deletion mutants.</p

Determination of <i>P_smeI</i> activities in different strains.

<p>Plasmid containing a transcriptional fusion of the upstream region of <i>smeI</i> to the <i>xylE</i> gene (pSmeI_xylE) was transferred into the wild-type KJ and its derived mutants. The C23O activities of the logarithmic-phase cultures of these strains were determined. Each bar represents the mean of three independent experiments. Error bars, where visible, indicate the average deviation. *, <i>p</i>≤0.01 significance calculated by a Student's <i>t</i>-test. (A) The impacts of <i>resA</i> mutant and <i>rseA/rpoE</i> double mutant on the promoter activity of <i>smeIJK</i> operon. (B) The impacts of MDAs treatment on the promoter activity of <i>smeIJK</i> operon in the wild-type and <i>rpoE</i> mutant.</p

The involvement of <i>smeIJK</i> and membrane damaging agents (MDAs) in the σ^E-mediated envelope stress response (ESR) of <i>S. maltophilia</i>.

<p>(A) Deletion of <i>smeIJK</i> compromises the cell envelope integrity and activates the <i>rpoE</i> to alleviate the envelope stress. (B) The treatment of MDAs on the wild-type cells activates <i>rpoE</i> system and upregualtes <i>smeIJK</i> expression. <i>SmeIJK</i> operon is a member of <i>rpoE</i> regulon.</p

The expression of <i>smeIJK</i> operon in different stresses and culture media.

<p>Plasmid containing a transcriptional fusion of the upstream region of <i>smeI</i> to the <i>xylE</i> gene (pSmeI_xylE) was transferred into the wild-type KJ. The C23O activities of the logarithmic-phase cultures of KJ(pSmeI_xylE) were determined. Each bar represents the mean of three independent experiments. Each bar represents the mean of three independent experiments. *, <i>p</i>≤0.01 significance calculated by a Student's <i>t</i>-test. (A) The expression of <i>smeIJK</i> operon in different stresses. The concentrations of the stressors added were: Triton X-100, 100 µg/ml; benzalkonium chloride (BC), 10 µg/ml; cetyltributylammonium bromide (CTAB), 10 µg/ml; gentamicin (Gm), 1 µg/ml; amikacin (Ami), 1 µg/ml; and leucomycin (Leu), 0.5 µg/ml. (B) The expression of <i>smeIJK</i> operon in different culture media, including the LB, the LB without NaCl, the LB with casein hydrolysate, and the MH.</p