Synthetic Cyclic Peptomers as Type III Secretion System Inhibitors

Abstract

Antibiotic-resistant bacteria are an emerging threat to global public health. New classes of antibiotics and tools for antimicrobial discovery are urgently needed. Type III secretion systems (T3SS), which are required by dozens of Gram-negative bacteria for virulence but largely absent from nonpathogenic bacteria, are promising virulence blocker targets. The ability of mammalian cells to recognize the presence of a functional T3SS and trigger NF-κB activation provides a rapid and sensitive method for identifying chemical inhibitors of T3SS activity. In this study, we generated a HEK293 stable cell line expressing green fluorescent protein (GFP) driven by a promoter containing NF-κB enhancer elements to serve as a readout of T3SS function. We identified a family of synthetic cyclic peptide-peptoid hybrid molecules (peptomers) that exhibited dose-dependent inhibition of T3SS effector secretion in Yersinia pseudotuberculosis and Pseudomonas aeruginosa without affecting bacterial growth or motility. Among these inhibitors, EpD-3'N, EpD-1,2N, EpD-1,3'N, EpD-1,2,3'N, and EpD-1,2,4'N exhibited strong inhibitory effects on translocation of the Yersinia YopM effector protein into mammalian cells (>40% translocation inhibition at 7.5 μM) and showed no toxicity to mammalian cells at 240 μM. In addition, EpD-3'N and EpD-1,2,4'N reduced the rounding of HeLa cells caused by the activity of Yersinia effector proteins that target the actin cytoskeleton. In summary, we have discovered a family of novel cyclic peptomers that inhibit the injectisome T3SS but not the flagellar T3SS.

Keywords: Pseudomonas aeruginosa; T3SS; Yersinia; cyclic peptides; peptoids; peptomers; type III secretion system; virulence blocker.

FIG 1

A newly developed NF-κB-GFP reporter stable cell line can be used to identify chemical inhibitors of the Yersinia T3SS. A HEK293-based stable cell line developed for this study was infected with Y. pseudotuberculosis Δyop6 (T3SS⁺) or Δyop6 ΔyopB (T3SS⁻), and GFP fluorescence was quantified as a readout of T3SS-induced NF-κB activation in the presence or absence of a known T3SS inhibitor, piericidin A1. The results are representative of three independent replicates. Data were analyzed by one-way ANOVA with Dunnett's multiple comparison test. ****, P < 0.0001; ***, P < 0.001.

FIG 2

First-generation cyclic peptomers inhibit NF-κB activation in infected host cells and secretion of Yersinia T3SS effector proteins. (A) Structures of the first-generation cyclic peptomers, each of which carries one side chain modification compared to the parental compound, epiphepropeptin D (EpD). (B) HEK293-GFP cells were infected with Y. pseudotuberculosis Δyop6 in the presence or absence of the first-generation cyclic peptomers, and GFP fluorescence was quantified. The fold reductions of the GFP signals in the presence of cyclic peptomers compared to the signal with DMSO are shown as averages for three independent experiments. The dotted lines represent 2 standard deviations above the DMSO control level. (C and D) WT Y. pseudotuberculosis was grown under T3SS-inducing conditions with 60 μM cyclic peptomer, 71 μM piericidin A1, or an equivalent volume of DMSO, and secretion of T3SS cargo into the culture supernatant was assessed by precipitating the secreted proteins, visualizing them with Coomassie blue (C), and quantifying the YopE effector band intensity (D). The data were analyzed by one-way ANOVA with Dunnett's multiple comparison test. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; ns, not significant. For panel C, the lanes containing the ladder and samples shown were separated by unrelated samples in the intervening lanes of the same gel and are therefore shown divided by white borders.

FIG 3

Second-generation cyclic peptomers inhibit secretion of Yersinia T3SS effector proteins. (A) Structures of the second-generation cyclic peptomers, each of which carries two, three, or four side chain modifications compared to EpD. (B) WT Y. pseudotuberculosis was grown under T3SS-inducing conditions with 60 μM cyclic peptomer, 71 μM piericidin A1, or an equivalent volume of DMSO, and secretion of T3SS cargo into the culture supernatant was assessed by precipitating secreted proteins and visualizing with Coomassie blue. Data were analyzed by one-way ANOVA with Dunnett's multiple comparison test. ****, P < 0.0001.

FIG 4

Cyclic peptomers inhibit type III secretion in Yersinia pseudotuberculosis in a dose-dependent manner. WT Y. pseudotuberculosis was grown under T3SS-inducing conditions with increasing concentrations of peptomers. Secretion of T3SS cargo into the culture supernatant was assessed by precipitating secreted proteins and visualizing them with Coomassie blue. YopE band intensities were quantified and normalized to that of the DMSO control. The results are from two independent experiments. Nonlinear curve fitting is shown to depict the trend of inhibition. (A) EpD-1N, EpD-3′N, EpD-4′N, EpD-1,2N, EpD-1,3′N, EpD-1,2,3′N, EpD-1,2,4′N, and EpD-1,2,3′,4′N. (B) EpD and EpD-6N.

FIG 5

Cyclic peptomers do not affect Yersinia growth or motility. (A) Yersinia pYV⁻ was grown in low-calcium medium at 37°C in the presence of 60 μM cyclic peptomer, an equivalent volume of DMSO, or kanamycin. The bacterial density over time was measured by determining the absorbance at 600 nm. (B) Yersinia pYV⁻ was grown in low-calcium medium at 37°C in the presence of 120 μM cyclic peptomer, an equivalent volume of DMSO, or kanamycin for 24 h. MTT was added, and the absorbance at 570 nm was used as a readout of bacterial metabolic activity. (C) Cyclic peptomers were added to motility agar plates at 60 μM, and the diameter of WT Yersinia colonies was measured as a readout of flagellar motility. Averages for three independent experiments are shown. Data were analyzed by one-way ANOVA with Dunnett's multiple comparison test. ****, P < 0.0001; ***, P < 0.001.

FIG 6

Cyclic peptomers inhibit type III secretion in Pseudomonas aeruginosa. WT Pseudomonas PA103 was grown under T3SS-inducing conditions with 60 μM cyclic peptomers (A), cyclic peptomers at concentrations ranging from 1.9 μM to 120 μM (B), or an equivalent volume of DMSO. Secretion of T3SS cargo into the culture supernatant was assessed by precipitating secreted proteins and visualizing them with Coomassie blue. ExoU band intensities were quantified and normalized to that of the DMSO control. Piericidin A1, which does not inhibit T3SS secretion in P. aeruginosa, was used as a negative control. (A) Data were analyzed by one-way ANOVA with Dunnett's multiple comparison test. ****, P < 0.0001; ***, P < 0.001. (B) The results are averages for at least two independent experiments. Nonlinear curve fitting is shown to depict the trend of inhibition.

FIG 7

The majority of cyclic peptomers are not toxic to mammalian cells. HeLa cells were treated with increasing concentrations of cyclic peptomers or an equivalent volume of DMSO for 24 h. MTT was added to the culture, and the absorbance at 570 nm was used as a readout of cellular metabolism. TPEN, a known cytotoxic compound, was used as a control. Data shown are the averages for two independent biological replicates.

FIG 8

Cyclic peptomers block translocation of a T3SS effector protein into host cells and protect host cells from T3SS effector activity. (A) Yersinia carrying a Yop effector–β-lactamase fusion (Δyop6/YopM-Bla) was used to infect CHO-K1 cells at an MOI of 5 in the presence of 7.5 μM peptomer or an equivalent volume of DMSO. CCF2-AM was added, and the percentage of cells with cleaved CCF2-AM (blue) among the total number of cells (green) was quantified. Data shown are averages for 3 independent experiments. (B) HeLa cells were infected with WT Yersinia at an MOI of 40 in the presence of increasing concentrations of peptomers for 3.5 h. Yersinia lacking a T3SS (ΔyscNU) was used as a negative control. Total cell area was quantified as a measure of cell rounding, which is a readout of host actin cytoskeleton modulation by the Yersinia T3SS YopEHO effector proteins. Median total area values for all imaged cells were plotted. Data shown are averages for two independent replicates with three technical replicates each. (C) Micrographs of HeLa cells infected with the ΔyscNU mutant or WT Yersinia and treated with EpD-3′N, EpD-1,2,4′N, or DMSO.

FIG 9

Structure-activity relationships and structural descriptions of peptoid analogs of epiphepropeptin D. N and N′ peptoids differ by a single -CH₂ group in the peptoid chain. The YopE secretion assay was used to evaluate potency, with the first- and second-generation sets analyzed at 60 μM and 15 μM, respectively. Secretion inhibition was colored to highlight potency differences between compounds.