C910 chemical compound inhibits the traffiking of several bacterial AB toxins with cross-protection against influenza virus

Abstract

The development of anti-infectives against a large range of AB-like toxin-producing bacteria includes the identification of compounds disrupting toxin transport through both the endolysosomal and retrograde pathways. Here, we performed a high-throughput screening of compounds blocking Rac1 proteasomal degradation triggered by the Cytotoxic Necrotizing Factor-1 (CNF1) toxin, which was followed by orthogonal screens against two toxins that hijack the endolysosomal (diphtheria toxin) or retrograde (Shiga-like toxin 1) pathways to intoxicate cells. This led to the identification of the molecule C910 that induces the enlargement of EEA1-positive early endosomes associated with sorting defects of CNF1 and Shiga toxins to their trafficking pathways. C910 protects cells against eight bacterial AB toxins and the CNF1-mediated pathogenic Escherichia coli invasion. Interestingly, C910 reduces influenza A H1N1 and SARS-CoV-2 viral infection in vitro. Moreover, parenteral administration of C910 to mice resulted in its accumulation in lung tissues and a reduction in lethal influenza infection.

Keywords: Biological sciences; Microbiology.

Graphical abstract

Figure 1

High-throughput screen of broad-spectrum antitoxin inhibitors (A) General scheme and results of the screening pipeline developed to isolate small chemical compounds inhibiting the CNF1-mediated cell depletion of Rac1 followed by orthogonal screens with AB toxins hijacking the endolysosomal (DT) or retrograde (Stx1) pathways. Chemical structure of N-(3,3-diphenylpropyl)-1-propyl-4-piperidinamine, referred to as C910. (B) ChemBridge library compounds were screened in HUVECs. Upper black dots represent positive controls (100% Rac1 signal in control HUVECs), whereas lower black dots correspond to Rac1 levels set to 0% in HUVECs exposed to 10-nM CNF1 for 6 h (negative controls). The red dots correspond to HUVECs incubated with chemicals in the presence of 10-nM CNF1 for 6 h. Cutoff (blue dashed line) was set to 30% of the Rac1 cellular signal. (C) and (D) graphs showing the dose-dependent protective effects conferred by C910 against protein biosynthesis inhibition at increasing concentrations of DT. (C and D) Graphs showing the dose-dependent protective effects conferred by C910 against protein biosynthesis inhibition at increasing concentrations of DT (C) and Stx1 (D). HeLa cells were pretreated for 1 h in DMEM with C910 at the indicated concentrations (filled colored circles) or DMSO vehicle (black circles) prior to the addition of DT (C) or Stx1 (D) for 6 h and 18 h, respectively. Each point represents the mean of duplicates ± SD from one representative experiment (n = 3). (E) Rac1 levels in CNF1-treated cells in the presence of increasing concentrations of C910. HUVECs were intoxicated with 10-nM CNF1 for 6 h in the absence or presence of C910 at the indicated concentrations. Each point represents the mean of duplicates ± SD from one representative experiment (n = 3). See also Figure S1.

Figure 2

Absence of an impact from C910 on CNF1 and STxB entry into cells (A) HUVECs were pretreated with DMSO vehicle or 40-μM C910 for 2 h and incubated with CNF1-Cy3 (1/500) on ice for 30 min in the presence of DMSO vehicle or 40-μM C910 before flow cytometry analysis. The signal from the control DMSO-treated cells was used as a background. The MFI (mean fluorescence intensity) of C910-treated cells was normalized to that of DMSO-treated cells. The histogram represents the mean ± SEM of three independent experiments. ns, not significant, paired two-tailed t test. (B and C) Representative images (B) and quantification (C) showing that C910 does not affect the internalization of CNF1 or STxB. (B) Fifteen minutes of internalization of CNF1-Cy3 (red) in HUVECs and Alexa Fluor 488-STxB (green) in HeLa cells in the presence of DMSO vehicle or 40-μM C910. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. (C) Quantification of the signal intensity of CNF1-Cy3 or Alexa Fluor 488-STxB associated with the cells. The mean values ±SD of the signal intensity measured for n > 25 cells per condition from one representative experiment are shown, n = 3. ns, not significant, unpaired t test. (D) Representative images showing the signal intensity of cleaved DQ-BSA in HUVECs 4 h or 16 h after endocytosis under treatment with DMSO vehicle or 20-μM C910. Bafilomycin A1 (Baf A1) treatment was used as a positive control to inhibit acid pH-dependent endosomal proteases. Scale bar, 20 μm. (E) Signal intensity quantification of cleaved DQ-BSA. The mean values ±SD of the signal intensity measured for n > 25 cells per condition from one representative experiment are shown, n = 3. ∗∗∗p < 0.001, ∗∗p < 0.01, ns, not significant, one-way ANOVA. (F) Representative images and percentage of LysoTracker-positive cells after treatment with DMSO vehicle, C910 (10, 20, 40, and 60 μM) or BafA1 (100 nM) for 4 h. Data show the mean ± SEM from three independent experiments, 12 repeats in total with n > 800 cells per condition. ∗∗∗p < 0.001, ns, not significant, one-way ANOVA. Scale bar, 20 μm. See also Figure S2.

Figure 3

C910 alters the morphology and function of EEA1-positive early endosomes (A and B) Immunofluorescence analysis showing the impact of C910 on early endosome morphology. A: HUVECs were treated with control DMSO vehicle or 40-μM C910 for 45 min and labeled for the indicated intracellular compartment markers. Nuclei were labeled with DAPI (blue). Scale bar, 10 μm. (B) Enlargement of the EEA1/Rab5-positive compartments in an HUVEC treated as in (A). (C) Representative electron micrographs showing a BSA-gold-positive compartment after 15 min of endocytosis in a DMSO vehicle- or C910-treated HUVEC. White arrows indicate BSA-gold. Scale bar, 100 nm. (D) Plot quantifying the sizes of the BSA-gold positive compartments from HUVECs as in (C). Each point represents an individual compartment, n = 87 (DMSO) and 77 (C910) from one representative experiment with the mean ± SD shown in red. ∗∗∗∗p < 0.0001, unpaired two-tailed t test. (E–H) Representative images and quantification of the distribution of CNF1-Cy3 in HUVECs (E–F) or Alexa Fluor 488-STxB (STxB) in HeLa cells (G and H) after two different times of endocytosis. Nuclei were labeled with DAPI (blue). Scale bar, 10 μm. (E) HUVECs were pretreated with DMSO vehicle or 40-μM C910 for 30 min followed by incubation with CNF1-Cy3 (red) for 30 min or 90 min in the presence of compound. Cells were labeled for EEA1 (green) or Rab7 (green). (F) Graph showing the Pearson’s coefficient values of the cellular colocalization of the CNF1 and Rab7 signals for each cell (open circles), n = 60 (DMSO) and 51 (C910) with the mean ± SD in red. ∗∗∗∗p < 0.0001, unpaired two-tailed t test. (G) HeLa cells were pretreated with DMSO vehicle or C910 40-μM before endocytosis of STxB (green) for 10 or 55 min in the presence of compound. Cells were then labeled for EEA1 (red) or Giantin (magenta). (H) Graphs showing the Mander’s overlap coefficient values with the mean ± SD in red between the STxB and Giantin signals (left), as well as the STxB and EEA1 signals (right) for each cell (open circles), n = 44 (DMSO) and 53 (C910). Data from three independent experiments are expressed as arbitrary units (a.u.). ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, unpaired two-tailed t test. See also Figures S3 and S4.

Figure 4

Extended spectrum of protection conferred by C910 against AB toxins (A) Dose-dependent protective effect conferred by C910 against protein synthesis inhibition by PE from P. aeruginosa. L929 cells were pretreated for 1 h with control DMSO vehicle (black circles) or C910 at the indicated concentrations (filled colored circles) before the addition of PE for 18 h. Each point represents the mean of duplicates ±SD from one representative experiment (n = 3). See also Figure S1D. (B) Immunoblots showing the kinetics of MEK2 cleavage at two concentrations of LT (PA: 0.3 μg/mL, LF: 0.1 μg/mL (LT) or diluted 1/10 (LT_1/10) in the absence or presence of 40-μM C910. Anti-GAPDH immunoblots were used as loading control. The blots are representative of four independent experiments. (C) Non-linear regression curves of the normalized MEK2 levels as a function of time (h) (R² > 0.9) under the conditions described in (B). The curves show the percentages of the MEK2 signal as a function of time in HUVECs intoxicated with LT (red circles) or LT_1/10 (orange circles) in the presence of DMSO vehicle (filed circles) or 40-μM C910 (open circles). Each point represents the mean ± SD from four independent experiments. (D) Vero cells intoxicated with eight cytotoxic units (cu) of TcdA or TcdB for 8 h in the presence of DMSO vehicle or C910 at the indicated concentrations. Scale bar, 10 μm. (E) Anti-Rac1 immunoblots showing the non-glucosylated form of Rac1 in Vero cells treated as in (D). The histogram corresponds to the Rac1 signals normalized to GAPDH and set to 100% for the control conditions. Data are expressed as the mean ± SD, n = 3 independent experiments. Two-way ANOVA with Tukey’s multiple comparison test was used to compare toxin-treated cells with non-intoxicated controls in the presence of C910 at different concentrations, ∗∗∗∗p < 0.0001; ns, not significant.

Figure 5

C910 protects cells from UPEC invasion mediated by CNF1 (A–F) Measurements of cell invasion by uropathogenic E. coli UTI89ΔhlyAΔcnf1 (UPEC) (A, B, and C) and Salmonella enterica serovar Typhimurium SL1344 (Salmonella) (D, E, and F). HUVECs were preincubated for 30 min with DMSO vehicle or 15-μM C910 before UPEC infection for 2.5 h in the absence or presence of 1-nM CNF1. HUVECs were preincubated for 3 h with DMSO vehicle or 15-μM C910 before infection with Salmonella in the absence or presence of C910 for 1 h. Graphs show the number of viable bacteria associated with the cells (A and D) or viable intracellular bacteria resistant to gentamicin treatment (B and E). Values correspond to colony-forming units (CFU)/mL, presented as the mean ± SEM from three independent experiments. One-way ANOVA was performed overall (p < 0.0001) with Tukey’s multiple comparison test to compare the treated conditions in (A) and (B), ∗∗∗∗p < 0.0001; ns, not significant. Data in (D) and (E) were analyzed by an unpaired two-tailed t test, ns, not significant. (C) Immunoblots of anti-Rac1 and anti-RhoA showing that C910 blocked CNF1-mediated Rac1 and RhoA depletion during cell infection by UPEC, as described in (B). (F) Immunoblot of anti-Rac1 showing that C910 and/or Salmonella infection, as described in (E), did not affect Rac1 cellular levels. Anti-GAPDH was used as a loading control.

Figure 6

C910 inhibits IAV H1N1 and SARS-CoV-2 cellular infection (A) C910 inhibits a single H1N1 IAV cycle. A549 cells were pretreated with DMSO vehicle or C910 (30 μM) for 1 h at 37°C before infection with the H1N1_WSN mCitrin reporter virus at an MOI of 5. At 6 h post-infection, the cells were analyzed by flow cytometry. mCitrin fluorescence (left) is shown for non-infected (NI) or infected cells (Inf). The histogram (right) represents the percentage of infected cells, which was calculated as the mean ± SEM of six replicates from one representative experiment (n = 2). ∗∗∗∗p < 0.0001, unpaired two-tailed t test. (B) C910 inhibits H1N1 IAV virion production. A549 cells were infected with H1N1_pdm09 at an MOI of 0.001 in the presence of DMSO vehicle or 30-μM C910. Viral production was titrated at 24 h post-infection from the cell supernatant with a plaque-forming assay. The histogram represents the mean ± SD of three replicates in one representative experiment (n = 2). ∗∗p = 0.005, unpaired two-tailed t test. (C) Schematic protocol of SARS-CoV-2 infection. U2OS-ACE2 GFP1-10 and GFP11 cells were preincubated with C910 for 2 h, and then SARS-CoV-2 at MOI = 0.1 was added for 20 h. The relative infection efficiency was calculated as the ratio between the GFP area and the total number of cells stained with DAPI. (D) Data are shown as the mean ± SD from duplicate wells, one representative experiment (n = 3).

Figure 7

C910 protects mice against IAV H1N1 infection (A and D) Timeline of the procedure. (B and C) Survival curves (B) and weight loss (C) following intranasal infection with PR/8, as described in (A) for mice treated with control DMSO vehicle (n = 8) or C910 (n = 8). (E and F) Survival curves (E) and weight loss (F) following intranasal infection with PR/8 as described in (D) for mice treated with control DMSO vehicle (n = 10) or C910 (n = 11). The Mantel–Cox test was performed to compare the survival curves between the groups injected with DMSO or C910. Weight loss data are shown as the mean ± SEM, ∗p < 0.05, multiple t test. See also Figure S6.