Salicylidene Acylhydrazides and Hydroxyquinolines Act as Inhibitors of Type Three Secretion Systems in Pseudomonas aeruginosa by Distinct Mechanisms

Abstract

Type 3 secretion systems (T3SSs) are major virulence factors in Gram-negative bacteria. Pseudomonas aeruginosa expresses two T3SSs, namely, an injectisome (iT3SS) translocating effector proteins in the host cell cytosol and a flagellum (fT3SS) ensuring bacterial motility. Inhibiting these systems is an appealing therapeutic strategy for acute infections. This study examines the protective effects of the salicylidene acylhydrazide INP0341 and of the hydroxyquinoline INP1750 (previously described as T3SS inhibitors in other species) toward cytotoxic effects of P. aeruginosain vitro Both compounds reduced cell necrosis and inflammasome activation induced by reference strains or clinical isolates expressing T3SS toxins or only the translocation apparatus. INP0341 inhibited iT3SS transcriptional activation, including in strains with constitutive iT3SS expression, and reduced the total expression of toxins, suggesting it targets iT3SS gene transcription. INP1750 inhibited toxin secretion and flagellar motility and impaired the activity of the YscN ATPase from Yersinia pseudotuberculosis (homologous to the ATPase present in the basal body of P. aeruginosa iT3SS and fT3SS), suggesting that it rather targets a T3SS core constituent with high homology among iT3SS and fT3SS. This mode of action is similar to that previously described for INP1855, another hydroxyquinoline, against P. aeruginosa Thus, although acting by different mechanisms, INP0341 and INP1750 appear as useful inhibitors of the virulence of P. aeruginosa Hydroxyquinolines may have a broader spectrum of activity by the fact they act upon two virulence factors (iT3SS and fT3SS).

Keywords: NLRC4 inflammasome; Pseudomonas aeruginosa; flagella; inhibitors; type three secretion system; virulence.

FIG 1

Chemical structure of the inhibitors used in this study.

FIG 2

Influence of INP0341 and INP1750 on iT3SS-mediated effects in phagocytic cells and epithelial cells. (A) Inhibition of CHA cytotoxicity by INP0341 and INP1750. Cell viability was assessed by measuring the release of LDH in the culture medium after 5 h (THP-1) or 7 h (A549) of incubation with CHA (10 bacteria/cell) in the presence of increasing concentrations of INP0341 or INP1750. Values are expressed in percentage of inhibition of the cytotoxicity of CHA (as measured in the absence of inhibitors) and are the means ± the standard errors of the mean (SEM) of three experiments performed in quadruplicates. Statistical analyses were performed (two-way analysis of variance [ANOVA], Bonferroni posttest; comparison between cells incubated with INP0341 and cells incubated with INP1750 [###, P < 0.001; ##, P < 0.01]). (B) Cytotoxicity induced by different P. aeruginosa strains in THP-1 monocytes (left) or A549 cells (right) after, respectively, 5 and 7 h of incubation in the presence of 0.8% DMSO (vehicle) or 80 μM INP0341 or INP1750. Values are expressed as the percentage of LDH release and are means ± the SEM of three experiments performed in quadruplicates. Statistical analyses (two-way ANOVA with Bonferroni posttest comparing each of the inhibitors with cells incubated with 0.8% DMSO [***, P < 0.001; **, P < 0.01] and comparing the effect of INP0341 and of INP1750 on each strain [#, P < 0.05]). (C) IL-1β secretion in the culture medium of THP-1 monocytes preincubated during 4 h in the presence of LPS (100 ng/ml), centrifuged, resuspended in fresh medium, and incubated for 5 h with different bacterial strains (10 bacteria/cell) or nigericin (0.02 μM) in the presence of 0.8% DMSO (vehicle) or 80 μM INP0341 or INP1750. Values are means ± the SEM of two experiments performed in duplicates. Statistical analyses were performed (two-way ANOVA with Bonferroni posttest comparing each of the inhibitors with cells incubated with DMSO [***, P < 0.001; **, P < 0.01]; multiple t test comparing the effect of INP0341 and of INP1750 on each strain [#, P < 0.05]).

FIG 3

Influence of INP0341 and INP1750 on iT3SS transcription. (Top panels) iT3SS transcriptional activation of the exsCEBA operon, followed by measuring the bioluminescence signal of the reporter strain CHA pC::lux over time, under control conditions (0.8% DMSO) or in the presence of 80 μM INP0341 or INP1750. Bacteria (10/cell) were cultured in the presence of THP-1 monocytes in RPMI 1640 medium (left) or of A549 epithelial cells in DMEM (right). Bioluminescence is expressed in arbitrary units (RLU), which correspond to 10⁻⁴-fold the actual readings. The dotted lines indicate the time selected in the bottom panels. (Bottom panels) Inhibition of the transcriptional activation of exsCEBA operon by increasing concentrations of INP0341 or INP1750, as measured after 5 h of incubation of CHA pC::lux (10 bacteria/cell) in the presence of THP-1 monocytes (left) or after 7 h of incubation in the presence of A549 cells (right). Values are expressed in percentage of inhibition of the bioluminescence signal recorded in the absence of inhibitors. All values are means ± the SEM of two or three experiments. Statistical analyses were performed (two-way ANOVA, Bonferroni posttest; comparison between cells incubated with INP0341 and cells incubated with INP1750 [###, P < 0.001; ##, P < 0.01]).

FIG 4

Influence of culture conditions on iT3SS transcriptional activation and its inhibition by INP0341 and INP1750. The iT3SS transcriptional activation of the exsCEBA operon was monitored by measuring the bioluminescence signal of the reporter strain CHA pC::lux after 9 h of incubation in different media and in the presence of 0.8% DMSO (vehicle) or of 80 μM INP0341 or INP1750. (A) RPMI 1640 medium containing 10% FCS and supplemented with increasing concentrations of Ca²⁺ so as to reach the concentration present in DMEM (circled values correspond to the concentrations present in each medium). (B) RPMI 1640 or DMEM supplemented with increasing amount of FCS (circled values correspond to the concentrations used in the routine). Statistical analyses were performed (two-way ANOVA with Bonferroni posttest comparing each of the inhibitors with cells incubated with DMSO [***, P < 0.001; **, P < 0.01; *, P < 0.05]; multiple t test comparing the effect of INP0341 and of INP1750 on each strain [###, P < 0.001; **, P < 0.01; #, P < 0.05]).

FIG 5

Influence of INP0341 and INP1750 on medium-dependent iT3SS mRNA expression levels. The influence of INP0341 and INP1750 on the expression levels of mRNA encoding iT3SS toxins (exoS and exoT), the iT3SS translocation apparatus (popB, popD, and pcrV), and the quorum sensing (QS) transcriptional activator (lasR and rhlR) was determined by real-time PCR. CHA, PA103, and PA103ΔexsE were grown from an OD₆₂₀ of 0.1 to an OD₆₂₀ of 0.8 with constant shaking in the presence of 0.8% DMSO (vehicle) or 80 μM INP0341 or INP1750, in eukaryotic cell culture medium (RPMI 1640 plus 10% FCS [top] or DMEM plus 10% FCS [bottom]). The results are expressed in mRNA relative expression compared to CHA (A) or PA103 (B and C) grown in the presence of DMSO. Dotted lines indicate the basal expression levels in PA103 in panel C. Values are means ± the SEM of two or three experiments performed in duplicates. Statistical analyses were performed (two-way ANOVA with Bonferroni posttest comparing each of the inhibitor with cells incubated with DMSO [***, P < 0.001; **, P < 0.01; *, P < 0.05] and comparing the effect of INP0341 and of INP1750 on each mRNA [###, P < 0.001; ##, P < 0.01; #, P < 0.05]).

FIG 6

Influence of INP0341 and INP1750 on ExoS secretion. CHA was grown from an OD₆₂₀ of 0.1 to an OD₆₂₀ of 0.8 with constant shaking in the presence of 0.8% DMSO (vehicle) or 80 μM INP0341 or INP1750 in low-calcium medium (LB broth, 5 mM EGTA, 20 mM MgCl₂) or in eukaryotic cell culture medium (RPMI 1640 plus 10% FCS or DMEM plus 10% FCS). ExoS was detected by Western blotting after separation by SDS-PAGE of 13 μl (supernatants) or 5 μg (lysates) of proteins. The band intensity was quantified by using ImageJ software.

FIG 7

Influence of INP0341 and INP1750 on bacterial motility and YscN-ATPase activity. (A) Effect of INP0341 and INP1750 on flagellar motility. (Top) Swarming (left) and swimming (right) motility. Bacteria were grown from an OD₆₂₀ of 0.1 to an OD₆₂₀ of 0.8 with constant shaking in the presence of 0.8% DMSO (vehicle) or 80 μM INP0341 or INP1750; then, 3-μl portions of these cultures were placed in the center of agar LB plates and grown overnight at 37°C. The area covered by bacteria was evaluated using QuantityOne software (Bio-Rad). Swarming was evaluated using 0.5% agar for CHA (illustrated in the pictures) and CHAΔexsA strains that were precultivated with INPs in low-calcium medium (LB broth, 5 mM EGTA, 20 mM MgCl₂) and swimming using 0.3% agar for CHA precultivated with INPs in low-calcium medium or in RPMI 1640 or DMEM added by 10% serum (right). (Bottom) Concentration effect relationship for swimming, with bacteria grown with increasing concentrations of INP1750 in RPMI 1640 or DMEM added by 10% serum, as described above. Values are means ± the SEM of two independent experiments performed in duplicate. Statistical analyses were performed (#, P < 0.05; ##, P < 0.01; ###, P < 0.001). A Student t test was used to compare values measured in the presence of INP0341 or in the presence of INP1750. (B) Effect of INP0341 and INP1750 on purified YscN T3SS ATPase. His-YscN (25 μg/ml) was incubated with INP0341 or INP1750 for 1 h; ATP (4 mM) was then added to the samples for 30 min, and the ATPase activity was measured as the amount of free phosphate liberated. Experiments were performed in duplicates. (Top) Percentage of inhibition of the enzymatic activity in the presence of 80 μM concentrations of each INP. (Bottom) Residual activity as a function of INP concentrations. Statistical analyes were performed (#, P < 0.05; ##, P < 0.01; ###, P < 0.001). A Student t test was used to compare values measured in the presence of INP0341 or in the presence of INP1750.

FIG 8

Influence of INP0341 and INP1750 on the cytotoxicity of P. aeruginosa clinical isolates. The percentage of LDH release from THP-1 monocytes (left) or A549 cells (right) exposed for 5 h (left) or 7 h (right) to clinical isolates (10 bacteria/cell) in the presence of 0.8% DMSO (control; open symbol) or 80 μM INP0341 or INP1750 was determined. Strains were grouped according to the expression of ExoU toxin (T3SS+ ExoU+ versus T3SS+ ExoU−) or of the ExoS toxin (ExoS++, level of expression higher than that detected in CHA; ExoS+, level of expression lower than that detected in CHA). Values are means ± the SEM of three independent experiments performed in quadruplicate (LDH). Statistical analyses were performed (***, P < 0.001; **, P < 0.01; *, P < 0.05 [two-way ANOVA, Bonferroni posttest]) comparing control conditions with INP0341 or INP1750.