The Mla system and its role in maintaining outer membrane barrier function in Stenotrophomonas maltophilia

Abstract

Stenotrophomonas maltophilia are ubiquitous Gram-negative bacteria found in both natural and clinical environments. It is a remarkably adaptable species capable of thriving in various environments, thanks to the plasticity of its genome and a diverse array of genes that encode a wide range of functions. Among these functions, one notable trait is its remarkable ability to resist various antimicrobial agents, primarily through mechanisms that regulate the diffusion across cell membranes. We have investigated the Mla ABC transport system of S. maltophilia, which in other Gram-negative bacteria is known to transport phospholipids across the periplasm and is involved in maintaining outer membrane homeostasis. First, we structurally and functionally characterized the periplasmic substrate-binding protein MlaC, which determines the specificity of this system. The predicted structure of the S. maltophilia MlaC protein revealed a hydrophobic cavity of sufficient size to accommodate the phospholipids commonly found in this species. Moreover, recombinant MlaC produced heterologously demonstrated the ability to bind phospholipids. Gene knockout experiments in S. maltophilia K279a revealed that the Mla system is involved in baseline resistance to antimicrobial and antibiofilm agents, especially those with divalent-cation chelating activity. Co-culture experiments with Pseudomonas aeruginosa also showed a significant contribution of this system to the cooperation between both species in the formation of polymicrobial biofilms. As suggested for other Gram-negative pathogenic microorganisms, this system emerges as an appealing target for potential combined antimicrobial therapies.

Keywords: Mla system; Stenotrophomonas maltophilia; biofilm; chelating agents; membrane permeability.

Figure 1

AlphaFold prediction for the 3D structure of MlaC (Smlt4673) of S. maltophilia from residue A28 to the last protein residue. (A) The five models generated by AlphaFold superimposed to the top-ranked model and colored according to the per-residue pLDDT score (Model Confidence). (B) CASTp-predicted ligand-binding cavity for the Smlt4673 AlphaFold models for which the cavity volume is highest (upper figure) and lowest (lower figure).

Figure 2

MlaC of S. maltophilia is a promiscuous PL-binding protein. (A) Determination of PL species co-purified with MlaC expressed in E. coli before (upper panel) and after (lower panel) delipidation of the protein by HPLC. MALDI-TOF/TOF mass spectra under denaturing conditions in negative ion mode. (B) Relationship of m/z values to the most probable PL species in E. coli. Abundant [M-H]− ions were observed for two glycerophospholipid classes: phosphatidylglycerols (PGs) and phosphatidylethanolamines (PEs). (C) T_m shift of MlaC protein with PLs, compared with protein control in 1% DMSO, as determined by nanoDSF. PE14/14 (PE C14:0/C14:0) shows a shift in the edge of significance (red lines at +/- 0.5°C from control). (D, E) K_d determination with PE14/14 and PE16/16 (PE C16:0/C16:0) by MST confirmed potential binding of PE14/14, with no evidence of binding for PE16/16, as also observed with nanoDSF. The normalized fluorescence (Fnorm%) is indicated in the y-axis.

Figure 3

The Mla system is required to maintain the homeostasis of the OM barrier. (A) Plating efficiency assay on MacConkey and LB agar plates. The ten-fold dilutions are indicated above each plate. K279a ΔmlaF-B and ΔmlaA mutants were more sensitive to bile salts. Complementation of each mutant restored the wild-type phenotype. Complementation plasmids pBBR1MCS1-mlaF-B and pBBR1-BAD-Cm-mlaA are indicated as pmlaF-B and pmlaA respectively. (B) NPN uptake, represented by the increase in fluorescence compared to cells not treated with NPN, without any supplementation. ΔmlaF-B showed increased cell permeability, which was partially restored by complementation. (C) NPN uptake assay in the presence of 0.5 mM EDTA. Asterisks represent 2-way ANOVA with post hoc Bonferroni test results for each time point versus the wild-type strain (P<0.05).

Figure 4

Cells with deleted Mla components showed an altered cell morphology in the presence of EDTA. SEM analysis of bacterial cells without EDTA treatment (A) or treated with 0.5 mM EDTA (B). Putative bacterial ghost cells are indicated by white arrowheads. Empty cell envelopes without cytoplasmic and nuclear contents as shown by TEM and marked with black arrowheads (C, D). The scale bars in panels (A, B) represent 1 µm, and the scale bars in panels (C, D) correspond to 0.2 µm and 0.5 µm, respectively. Complementation plasmid pBBR1MCS1-mlaF-B is indicated as pmlaF-B.

Figure 5

Biofilm formation is reduced in the K279a ΔmlaF-B mutant when grown in modified BM2 minimal media. Cells were grown in a polystyrene microtiter plate in BM2-glucose minimal medium supplemented with casamino acids (CAA) or 0.5 × BHI at 30°C and without and with 0.5 mM EDTA. The amount of each biofilm was quantified by crystal violet staining (OD₅₅₀ value) after 24 hours of incubation under static conditions. Complementation plasmid pBBR1MCS1-mlaF-B is indicated as pmlaF-B. Data are means ± SD (n = 6). Two-way ANOVA with post hoc Bonferroni’s multiple comparison test was used to determine the significance of the data between groups (**P < 0.01).

Figure 6

Mutation in the Mla system of S. maltophilia reduces the biomass of mixed biofilm with P. aeruginosa. (A) CLSM images of dual species biofilms of sfGFP-labelled S. maltophilia K279a strains (green) and P. aeruginosa PAO1::tdTomato (red) formed after incubation for 72 hours in 0.5 × BHI at 30°C. Complementation plasmid pBBR1MCS1-mlaF-B is indicated as pmlaF-B. (B) Quantification of fluorescence signals derived from CLSM 3D images. Data are means ± SD (n = 3). Two-way ANOVA with post hoc Bonferroni’s multiple comparison test was used to determine the significance of the data between groups (**P < 0.01; ***P < 0.001).