Tackling recalcitrant Pseudomonas aeruginosa infections in critical illness via anti-virulence monotherapy

Abstract

Intestinal barrier derangement allows intestinal bacteria and their products to translocate to the systemic circulation. Pseudomonas aeruginosa (PA) superimposed infection in critically ill patients increases gut permeability and leads to gut-driven sepsis. PA infections are challenging due to multi-drug resistance (MDR), biofilms, and/or antibiotic tolerance. Inhibition of the quorum-sensing transcriptional regulator MvfR(PqsR) is a desirable anti-PA anti-virulence strategy as MvfR controls multiple acute and chronic virulence functions. Here we show that MvfR promotes intestinal permeability and report potent anti-MvfR compounds, the N-Aryl Malonamides (NAMs), resulting from extensive structure-activity-relationship studies and thorough assessment of the inhibition of MvfR-controlled virulence functions. This class of anti-virulence non-native ligand-based agents has a half-maximal inhibitory concentration in the nanomolar range and strong target engagement. Using a NAM lead in monotherapy protects murine intestinal barrier function, abolishes MvfR-regulated small molecules, ameliorates bacterial dissemination, and lowers inflammatory cytokines. This study demonstrates the importance of MvfR in PA-driven intestinal permeability. It underscores the utility of anti-MvfR agents in maintaining gut mucosal integrity, which should be part of any successful strategy to prevent/treat PA infections and associated gut-derived sepsis in critical illness settings. NAMs provide for the development of crucial preventive/therapeutic monotherapy options against untreatable MDR PA infections.

Fig. 1. Current view of the P. aeruginosa MvfR QS system impact on acute and chronic functions.

MvfR (PqsR), in the presence of its ligands/inducers PQS or HHQ, binds and activates the transcription of the pqs operon, whose encoded proteins catalyze the biosynthesis of ∼60  low molecular weight molecules, including HHQ, HQNO, and 2-AA. HHQ in turn is converted to PQS via PqsH. HQNO promotes pro-acute and pro-persistent phenotypes, while HHQ and PQS promote acute phenotypes. 2-AA’s immunomodulatory action and epigenetic regulation and its ability to promote the accumulation of lasR mutants and formation of AT/P cells that survive antibiotic killing contribute to persistent infections. Moreover, MvfR impacts the production of several virulence factors, including pyocyanin, and binds and directly regulates the expression of 35 loci across the P. aeruginosa genome, including major regulators and virulence factors, such as the QS regulators LasR and RhlR, and genes involved in protein secretion, translation, and response to oxidative stress. Agents that bind and inhibit MvfR function are a successful strategy to control the multiple virulence functions under MvfR control.

Fig. 2. Dose-dependent inhibition of pyocyanin production and PqsA-GFP expression.

IC₅₀ determination was performed in the presence and absence of each of the 10 NAM compounds at 13 different concentrations ranging from 0.01–50 µM. Graphs are showing IC₅₀ values for pyocyanin (blue) and PqsA-GFP (green) for D41, D42, D43, D57, D69, D77, D80, D88, D95, D100. IC₅₀ curves were plotted as a percentage of pyocyanin production and pqsA-GFP expression of the indicated compounds. The percentage of compound inhibition was calculated by comparing PA14 cells grown in the presence of the vehicle control. The IC₅₀ values for each compound were calculated using the GraphPad Prism 9.2.0 software. Data represent the mean value of biological replicates, and the number (n) of replicates is indicated on the graph. The error bars denote ±SEM. n = number of biological replicates; NAM = N-Aryl Malonamide.

Fig. 3. NAMs are potent agents against the formation of antibiotic tolerant, persisters (AT/P) cells and biofilm.

a AT/P cells formation in PA14 and isogenic ∆mvfR in the presence or absence of the indicated compounds. Cells were grown in the presence of 10 μg mL⁻¹ meropenem with or without 10 µM of the compounds for 24 h. Values were normalized to the cells grown for 4 h in the absence of antibiotics and compound. The PA14 cells grown with antibiotics and vehicle (DMSO) were considered control, and the percentage values were calculated compared to control. b Initiation of biofilm formation of PA14 and ∆mvfR cells with or without compound. Biofilm was grown in the 96 well microtiter plate at 37 °C for 24 h containing M63 minimal media in the presence of 10 µM of the compounds or vehicle. After 24 h, the wells were washed to remove planktonic cells, and the biofilm was stained with 0.1% crystal violet. The stained biofilm was washed and solubilized in ethanol: acetone (80:20). OD was measured at 570 nm. The biofilm grown with the vehicle was considered as a control. The percentage value was calculated in comparison to the PA14 control. a, b Data represent at least n = 3 biological replicates, each dot on the graph represents one replicate, and the number of biological replicates for each compound is depicted in the graph. The error bars denote ±SEM. One-way ANOVA followed by Tukey post-test was applied. ∗, ∗∗ and ∗∗∗ indicate significant differences from the control at P < 0.05, P < 0.01, and P < 0.001, respectively.

Fig. 4. In vitro assessment of NAMs efficacy indicates no off-target effect.

a Effect of the selected compounds on the MvfR-independent HAQs, 2-AA, and DHQ production. The production of the MvfR-regulated small molecules 2-AA, HHQ, PQS, DHQ, and HQNO was measured in the cultures at OD_600nm = 3.0 in the presence of 50 µM of the indicated compounds using a MvfR mutant strain that constitutively expresses the pqsABCDE genes. The cells were grown with or without (vehicle only) compound, and the small molecules production was measured using liquid chromatography-mass spectrophotometry (LC-MS). The percentage production was calculated compared to cells grown with the vehicle control. b AT/P cell formation in the PA14 isogenic mutant ∆mvfR in the presence of the indicated compounds. Cells were grown in the presence of 10 μg mL⁻¹ meropenem, with or without 10 µM of the compounds for 24 h. Values were normalized to cells grown for 4 h in the absence of antibiotics and compounds. The ∆mvfR cells grown with antibiotics and vehicles were considered control, and the percentage values were calculated compared to control. c Biofilm formation of mutant ∆mvfR cells with or without compound. Biofilm was grown in the 96-well microtiter plate at 37 °C for 24 h containing M63 minimal media in the presence of 10 µM of the compounds or vehicle. The biofilm grown with the vehicle was considered as a control. The percentage value was calculated in comparison to the PA14 control. a–c Data represent at least n = 3, each dot on the graph represents one biological replicate. The number of biological replicates for each compound and strain is depicted in the graph. The error bars denote ±SEM. HAQs = 4-hydroxy-2-alkylquinolines; 2-AA = 2-aminoacetophenone; DHQ = 2,4-dihydroxyquinoline; PQS = 3,4-dihydroxy-2-heptylquinoline; DHQ = 2,4-dihydroxyquinoline; HQNO = 4-hydroxy-2-heptylquinoline N-oxide.

Fig. 5. IC₅₀ measurements, in vitro engagement, and molecular docking studies support the prioritization of compound D88.

a Dose-dependent inhibition of HHQ, PQS, HQNO, 2-AA, and DHQ production was measured in PA14 cultures at OD_600nm = 3.0. The cells grown with the compound’s vehicle were considered the control. b Dose-dependent production of AA in the presence of D88 in PA14 cultures at OD_600nm = 3.0. AA is the primary precursor of all the MvfR-regulated small molecules assessed in A. GraphPad Prism 9.2.0 software plotted the IC₅₀ curves against percent inhibition of the HAQs, 2-AA, DHQ, and AA production at each concentration. a, b Data represent the mean value of n = 3 biological replicates. Error bars denote ±SEM. c D88 inhibition of MvfR binding to the pqsA promoter. Overnight grown culture of PA14 expressing MvfR-VSV-G was diluted to OD 600_nm 0.01 and grown at 37 °C with and without D88 (50 µM) and/or PQS (38 µM) until OD 600 _nm 1.0. Thereafter MvfR-DNA complex were cross-linked and isolated via chromatin immunoprecipitation (ChIP). Coprecipitated DNA was purified and quantified using quantitative real-time polymerase chain reaction qPCR. MvfR binding to the pqsA promoter was calculated using the input method. Data reprsent n = 3 biological replicates, and the error bars denote ±SEM. Statistical analysis was carried out using GraphPad Prism 9.2.0 software. Unpaired t test was applied between the compared group. ∗ indicate significant differences from the control at P < 0.05 (Control vs. PQS = 0.038; PQS vs. PQS + D88 = 0.012). d The representative structure of the MvfR ligand-binding domain (LBD) complex with D88 (magenta sticks) was obtained from the docking analysis using AutoDock Vina. Tyr258 related to pi-interaction and Val170, Leu189, Ile236, and Ile263 related to hydrophobic interaction are shown as blue and green sticks, respectively. e Two-dimensional diagram of MvfR-D88 docking analyzed using PoseView program. A Green dashed line connecting two green dots indicates pi interaction. The solid green line indicates hydrophobic interactions made by hydrophobic residues (Val170, Leu189, Ile236, and Ile263) surrounding D88. AA = anthranilic acid; HHQ = 4-hydroxy-2-heptylquinoline; PQS = 3,4-dihydroxy-2-heptylquinoline; HQNO = ; 2-AA = 2-aminoacetophenone; DHQ = 2,4-dihydroxyquinoline; MvfR-VSV-G = MvfR fused to a vesicular stomatitis virus glycoprotein; Val = ; LeTyr = Tyrosine; Val = Valine; Leu = Leucine; Ile= Isoleucine.

Fig. 6. MvfR promotes intestinal permeability. Its pharmacologic inhibition mitigates the host intestinal barrier damage, ameliorates bacterial dissemination, and abolishes the production of the small molecules.

a Schematic representation of the burn-site infection model and treatment plan. The color of syringes indicates administration: Green bacterial inoculum; blue D88; yellow FITC-dextran; and red blood collection. b Fluorescein Isothiocyanate-Dextran (FITC-dextran) 3-5 kDa levels in the serum 22 h post-infection. FITC-dextran 3-5 kDa was gavaged 18 hr post-burn and infection. Blood was collected 4 h following gavage (22 h post-burn and infection), and the FITC-dextran fluorescence intensity was measured using fluorescent spectrophotometry (excitation, 480 nm, and emission, 520 nm). c Effect of D88 on the bacterial dissemination to the ileum and colon and bacterial load at the site of infection. Small and large intestinal tissues and muscle underlying the burn eschar and infection site from mice of each group were collected at 22 h post-burn and infection. Sample homogenates were serially diluted and plated on Pseudomonas isolation agar plates. Bacterial colony-forming units (CFUs) were counted and normalized by the tissue weight. d D88 inhibits the production of PQS, HHQ, HQNO, DHQ, and 2-AA in the infected mice. Production was measured in samples of the underlying muscle at the site of infection. Tissue was collected at 12 h and 22 h post-infection, and these molecules were quantified using liquid chromatography-mass spectrophotometry (LC-MS). b–d Data represent at least n = 5 biological replicates, each dot represent data from one mouse. The exact number of mice used in each condition is indicated in each bar. The error bars denote ±SEM. b–d One-way ANOVA followed by Tukey post-test was applied. The no-treatment group data (Burn + PA14) were compared to the vehicle and the D88 treated groups. ∗, ∗∗, and ∗∗∗ indicates significant differences compared to the control at P < 0.05, P < 0.01, and P < 0.001, respectively. ns represents no significant difference.

Fig. 7. D88 mitigates the morphologic alterations of the intestinal lining and attenuates intestinal inflammation.

a Representative confocal image (n = 2) of distal ileum with Claudin-1 immunofluorescence staining. The experiment was repeated independently two times with similar results. Samples for confocal imaging were harvested 22 h post-burn and infection. Green fluorescence represents Claudin-1, and blue fluorescence represents the DAPI stain, the white line represents the scale bar (100 µm). b Levels of tumor necrosis factor (TNF-α) in the distal ileum 22 h post-burn and infection. The total protein was isolated from the distal ileum, and the concentration of TNF-α in the sample was quantified using ELISA. c The levels of interleukin (IL-6) in the distal ileum were also quantified using ELISA. b, c Data represent at least n = 5, each dot in the bars represents data from one mouse. The error bars denote ± SEM. One-way ANOVA followed by Tukey post-test was applied. The no-treatment group data (Burn + PA14) were compared to the vehicle-treated and the D88-treated groups. ∗, ∗∗, and ∗∗∗ indicates significant differences compared to the control at P < 0.05, P < 0.01, and P < 0.001, respectively. ns represents no significant difference.