USE OF PHENOTHIAZINE DERIVATIVE IN THE TREATMENT OF INFECTION CAUSED BY BACTERIA CARRYING TYPE IV PILI

Abstract

The present invention provides a phenothiazine derivative of formula (I) for use in preventing and/or treating infection caused by bacteria carrying Type IV pili, such as N. meningitidis, and more specifically for use in preventing and/or treating meningitis. The present invention further relates to a composition for the use in preventing and/or treating infection caused by bacteria carrying Type IV pili, such as purpura fulminans and meningitis, comprising a phenothiazine derivative of formula (I) and an antibiotic selected from the group consisting of beta-lactams and aminoglycosides, and/or dexamethasone.

##STR00001##

Claims

1. A method for treating and/or preventing meningitis caused by bacteria carrying Type IV pili in patients diagnosed with or at risk of developing such infection, comprising the step of administering a therapeutically effective amount of a phenothiazine derivative or a pharmaceutical salt thereof, wherein said phenothiazine derivative is a compound of formula (I): ##STR00014## X.sub.1 is a C.sub.1-C.sub.6 alkyl substituted by R.sub.1; wherein R.sub.1 is YR.sub.3R.sub.4, wherein Y is C or N, If Y is N then R.sub.3 and R.sub.4 are independently of each other H, (C.sub.1-C.sub.6)alkyl, or R3 and R4 form together with Y, a ring selected from an heteroaryl or an heterocyclyl group, said ring being optionally substituted by one or more groups selected from: (C.sub.1-C.sub.6)alkyl optionally substituted by OH or O(CO)(C1-C10)alkyl; or an amide group, and If Y is C then R.sub.3 and R.sub.4 form together with Y, a ring selected from an heteroaryl or an heterocyclyl group, said ring being optionally substituted by (C.sub.1-C.sub.6)alkyl groups optionally substituted by OH; R.sub.2 is H, an halogen (preferably Cl or F), CF.sub.3, a (C.sub.1-C.sub.6)alkoxy, S(O)(C.sub.1-C.sub.6)alkyl, SO.sub.2 (C.sub.1-C.sub.6) alkyl, SO.sub.3H, CN, a (C.sub.1-C.sub.6)alkyl, a (C.sub.1-C.sub.6)thioalkoxy, NO.sub.2 X.sub.2 is S or SO.sub.2.

2. The method of claim 1, wherein said phenothiazine derivative is selected from the group consisting of promazine, thioridazine, mesoridazine, trifluoperazine, prochlorperazine, fluphenazine, and perphenazine.

3. The method of claim 1, wherein said phenothiazine derivative is thioridazine, mesoridazine, or trifluoperazine.

4. The method of claim 1, wherein said bacteria carrying Type IV pili are selected from the group consisting of Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus sp., notably Haemophilus influenzae, and Listeria monocytogenes, and is preferentially Neisseria meningitidis.

5. The method of claim 1, wherein vascular damages are prevented.

6. The method of claim 1, wherein circulatory collapse is prevented.

7. The method of claim 1, wherein said phenothiazine derivative is administered intravenously or intra-muscularly.

8. The method of claim 1, comprising the further administration of an antibiotic.

9. The method of claim 1, wherein said antibiotic is selected from the group consisting of beta-lactams and aminoglycosides, and/or dexamethasone.

10. The method of claim 9, wherein said antibiotic is a beta-lactam, preferably a cephalosporin of third generation.

11. The method of claim 8, wherein said phenothiazine derivative and said antibiotic are administered simultaneously, sequentially, or separately.

12. A method for treating and/or preventing meningitis caused by bacteria carrying Type IV pili in patients diagnosed or at risk of developing such infection, comprising the step of administering a pharmaceutical composition comprising a phenothiazine derivative and an antibiotic selected in the group consisting of beta-lactams and aminoglycosides, and/or dexamethasone.

13. The method according to claim 12, wherein meningitis is caused by bacteria carrying Type IV pili selected from the group consisting of Streptococcus pneumonia, Neisseria meningitidis, Haemophilus sp., notably Haemophilus influenza, and Listeria monocytogenes, and is preferably Neisseria meningitidis.

14. A kit comprising a phenothiazine derivative and an antibiotic selected in the group consisting of beta-lactams and aminoglycosides, and/or dexamethasone.

Description

LEGENDS OF THE FIGURES

[0145] FIG. 1: Antimicrobial activity of Trifluoperazine on N. meningitidis in vitro.

N. meningitidis Nm2C4.3 strain grown in liquid culture at 10.sup.7 CFU/ml were treated with increasing concentrations of Trifluoperazine (10 to 50 M) or Gentamicin (150 g/ml) for 15 minutes (A) Bactericidal activity was determined by the count of colony forming units on GCB agar plates 24 h after treatment. (B) Images of the non-treated bacteria or bacteria treated with 30 M TFP grown for 24 h on GCB agar plates.

[0146] FIG. 2: Trifluoperazine inhibits meningococcal aggregation.

N. meningitidis 2C4.3 was grown in liquid culture at 10.sup.7 CFU/ml for 2 h to form bacterial aggregates and were treated with increasing concentrations of Trifluoperazine (10 to 40 M), Gentamicin (150 g/ml) or cefotaxim (20 g/ml) for 20 minutes and bacterial aggregates were immediately visualized using a phase-contrast microscope. (A) Representatives images of the bacterial aggregates observed in phase-contrast microscope in non-treated or treated conditions. (B) Quantification of the bacterial aggregation was performed using Image J software.

[0147] FIG. 3: Trifluoperazine rapidly inhibits the aggregation of meningococcal wild type strain whereas it does not affect the aggregation of a PilT derivative mutant.

N. meningitidis 2C4.3 wild type strain and the isogenic derivative mutant PilT (PiIT) were grown in liquid culture at 10.sup.7 CFU/ml for 2 h to form bacterial aggregates and then treated with 50 M Trifluoperazine. Time lapse phase-contrast video microscopy was performed to visualize the effect on bacterial aggregates over time. Images were taken at the indicated time points of the video.

[0148] FIG. 4: The effect of Trifluoperazine on meningococcal aggregation is reversible/transient.

N. meningitidis 2C4.3 was grown in liquid culture at 10.sup.7 CFU/ml for 2 h to form bacterial aggregates and were treated with 30 M Trifluoperazine for 30 minutes to induce bacterial dispersion or PBS as a control. The medium was replaced to remove the Trifluoperazine and the reformation of bacterial aggregates were visualized overtime using a phase-contrast microscope.

[0149] FIG. 5: Trifluoperazine induces the loss of bacterial type IV pili

N. meningitidis 2C4.3 wild type strain and the isogenic derivative mutant PilT (PiIT) were grown in liquid culture at 10.sup.7 CFU/ml for 2 h to form bacterial aggregates and then treated with 50 M Trifluoperazine for 15 minutes before analysis by transmission electron microscopy. Arrows point at bundles of type IV pili expressed at the bacterial surface.

[0150] FIG. 6: Chemical Structure of phenothiazine derivatives used in this study.

Aliphatic compounds (Promazine, Chlorpromazine, Triflupromazine, Levomepromazine), piperidines (Mesoridazine, Thioridazine) and piperazines (Trifluoperazine, Fluphenazine, Prochlorperazine and Perphenazine)

[0151] FIG. 7: Dose-dependent effect of phenothiazine derivatives on the dispersion of meningococcal aggregates. Bacteria were grown in suspension for 2 h then Phenothiazine derivatives were added at the indicated concentrations. The effect on bacterial aggregation was observed 30 min after treatment by phase contrasts microscopy.

[0152] FIG. 8: Trifluoperazine prevents meningococcal adhesion to endothelial cells.

Nm2C4.3 grown in suspension were pretreated in the absence or in the presence of 30 M Trifluoperazine for 30 minutes before adhesion to human dermal microvascular endothelial cells (HDMECs) for 30 minutes. Infection was allowed to proceed for further 30, 60 or 90 minutes before fixation and immunostaining using anti-Nm2C4.3 antibody and Alexa Fluor 633 Phalloidin. (A) Representative fluorescence microscopy showing bacterial colony (white dots) formed at the surface of the endothelial cells. (B) Quantification of the bacterial colonization was performed using Image J software.

[0153] FIG. 9: Trifluoperazine induces the dispersion of compact meningococcal microcolonies formed at the surface of infected human endothelial cells.

Human bone marrow microvascular endothelial cells (HBMEC) were infected with Nm2C4.3 for 1 h 30, treated with Trifluoperazine (10-40 M) for 30 minutes, before fixation and immunofluorescence analysis using anti-Nm2C4.3 antibody and Alexa Fluor 633 Phalloidin. (A) Representative fluorescence microscopy showing bacterial colony (white dots) formed at the surface of the endothelial cells. (B) Quantification of the bacterial colonization was performed using Image J software.

[0154] FIG. 10: Trifluoperazine exerts a cytoprotective effect on endothelial cells infected by N. meningitidis: Effect on cytoskeleton remodelling and endothelial cell junction integrity.

(A) Experimental procedure: HDMEC were infected for 2 h with Nm2C4.3, treated for 1 h with gentamicin (150 g/ml) and then 15 min with Trifluoperazine (50 M) (or solvent alone as a control) and left in medium for 1 h before fixation. (B) Upper panels: Representative fluorescence microscopy showing the effect on the endothelial cell cytoskeleton remodelling analysed by immunostaining of Ezrin and Actin. Lower panels: Effect on the endothelial cell junction integrity was analysed by immunostaining of Actin and VE-cadherin. (C) Quantification of the bacterial colonization (upper panel) Ezrin recruitment and cortical actin polymerization at sites of bacterial adhesion (lower panel) was performed using Image J software.

[0155] FIG. 11: Trifluoperazine exerts a cytoprotective effect on endothelial cells infected by N. meningitidis: Trifluoperazine inhibits PECAM-1 recruitment at the bacterial adhesion sites.

(A) Monolayers of HBMECs were non-infected or infected for 2 h with meningococci and the effect on the localisation of the endothelial cell junction proteins PECAM-1 and -catenin was analysed by immunofluorescence analysis. Arrows point at PECAM-1 molecules recruited at bacterial adhesion sites (B) HBMECs were infected for 2 h with meningococci in the presence or in the absence of 5 M Trifluoperazine and the effect on junctional PECAM-1 and on the endothelial cell cytoskeleton remodelling (Ezrin, Actin) was analysed by immunofluorescence analysis. (C) Quantification of Actin and PECAM-1 recruitment at sites of bacterial adhesion was performed using Image J software.

[0156] FIG. 12: Trifluoperazine exerts a cytoprotective effect on endothelial cells infected by N. meningitidis: Effect on basement membrane remodelling.

(A) Experimental procedure: HBMECs grown on a fluorescent matrix (Gelatin-Fitc) were colonized by N. meningitidis for 6 h. They were then treated with 150 g/ml gentamicin for 1 h, then 15 min with Trifluoperazine (50 M) (or solvent alone as control) and incubated for further 15 h in the presence of 150 g/ml gentamicin. (B) Fluorescence microscopy was used to visualize cells stained with labelled phalloidin and the fluorescent matrix. The presence of dark zones corresponds to zones of matrix degradation. (C) Quantification of the percentage of matrix degradation, using Image J software.

[0157] FIG. 13: Trifluoperazine induces the dispersion of compact EPEC microcolonies formed at the surface of human endothelial cells.

Human bone marrow microvascular endothelial cells (HBMEC) were infected with a GFP-expressing mutant Enteropathogenic Escherichia coli, which lacks the ATPase escN (EPEC escN-GFP) for 2 h, then treated with Trifluoperazine (10-50 M) for 30 minutes, before fixation and immunofluorescence analysis using Alexa Fluor 633 Phalloidin. (A) Representative fluorescence microscopy showing bacterial colony (white dots) formed at the surface of the endothelial cells. (B) Quantification of the bacterial colonization was performed using Image J software.

[0158] FIG. 14: Trifluoperazine induces the dispersion of compact meningococcal microcolonies formed at the surface of infected human brain endothelial cells.

A. HBMEC were infected with Nm2C4.3 for 1 h 30, treated with Trifluoperazine (10-40 M) for 30 minutes, before fixation and immunofluorescence analysis using anti-Nm2C4.3 antibody and Alexa Fluor 633 Phalloidin. B. Quantification of the bacterial colonization was performed using Image J software.

[0159] FIG. 15: Trifluoperazine induces the dispersion of compact meningococcal microcolonies formed during in situ infection of human brain vessels

A. Immunofluorescence analysis of CD31 (green), N. meningitidis colonies (red) and nucleus (blue) in the cortical region of serial brain sections from the same donor infected with Nm2C4.3 wild-type strain for 1 h, then treated with Trifluoperazine (40 M for 30 min) or PBS alone, as a control. Scale bars, 20 m. Images are representative of 2 different human brain sections. B. Means.e.m. of the vascular colonization index. Analysis of 50 sections per brain section, n=2 sections per condition. ***P<0.001, one-way ANOVA.

EXAMPLES

Example 1: Trifluoperazine Exerts a Moderate Bactericidal Effect on Meningococci

Materials and Methods

[0160] Nm2C4.3, a piliated capsulated Opa.sup.Opc.sup. variant of the serogroup C meningococcal clinical isolate 8013, was cultured in Dulbecco's Modified Eagle Medium (DMEM) 4.5 g/L-Glutamax media 0.1% BSA during two hours at 37 C. 5% CO2. A bacterial suspension at optical density (OD) 0.1 was then distributed in 24-well plate (1 ml/well). After 1 hour of incubation, Trifluoperazine (Sigma #T8516, stock solution in PBS) was added to obtain final concentrations of 10, 20, 30, 40, and 50 M. Gentamicin (150 g/ml in DMEM) and PBS alone were used as controls. After 15 min treatment, serial dilutions were performed in physiological water and 100 l of each dilution were plated on Petri dishes containing GCB solid media (BD, Difco GC media) containing supplements, incubated overnight and the colony forming units were counted.

Results

[0161] Previous studies showed that Trifluoperazine was a broad-spectrum bactericide for Gram-positive and Gram-negative bacteria, especially active on staphylococci and vibrios (Mazumber et al., 2001). When tested on N. meningitidis, Trifluoperazine also showed some significant antimicrobial activity at concentrations ranging from 10 to 50 M: the viable count of the culture that contained 10.sup.7 CFU/ml was reduced to 10.sup.6 CFU/ml at 10 to 30 M and dropped to 10.sup.3 CFU/ml at 50 M (FIG. 1). However, this effect was moderate in comparison to antibiotic treatment such as Gentamicin, which killed all bacteria (FIG. 1).

Conclusions

[0162] Trifluoperazine exerts a moderate bactericidal effect on meningococci.

Example 2: Trifluoperazine Rapidly Induced the Dispersal of Meningococcal Aggregates

Materials and Methods

[0163] N. meninigitidis 2C4.3 strain was grown in suspension in wells of a 24 well plate, containing 1 ml of DMEM medium supplemented with 10% heat-inactivated fetal calf serum. After 2 h of growth, Trifluoperazine (or control vehicle) was added at various concentrations ranging from 10 to 40 M for 20 min and the bacterial aggregates were visualized over time using a phase-contrast microscope.

Results

[0164] When grown in suspension in liquid culture, meningococci form bacterial aggregates due to interbacterial interactions promoted by their type IV pili (Helaine et al., 2005; Pelicic, 2008). When added to bacterial aggregates, which were pre-formed for two hours, Trifluoperazine induced their dispersion (FIG. 2). This effect was dose-dependent (between 10-40 M) (FIG. 2), and time laps video microscopy showed that this effect occurs within minutes (10-15 min) after addition of Trifluoperazine (FIG. 3). Upon removal of Trifluoperazine this effect was reversible (FIG. 4). In contrast to Trifluoperazine, addition of conventional antibiotics used in the treatment of meningococcaemia, such as Gentamicin (150 g/ml) or Cefotaxim (20 g/ml), did not significantly induce the dispersion of bacterial aggregates formed in suspension.

Conclusions

[0165] In contrast to conventional antibiotics used for the treatment of meningococcemia (Cefotaxim or Gentamicin), Trifluoperazine induced the dispersal of bacterial aggregates formed in suspension.

Example 3: Trifluoperazine Induces the Loss of Bacterial Type IV Pili

Materials and Methods

[0166] N. meninigitidis 2C4.3 strain and the isogenic derivative mutant PilT, where the pilT gene was interrupted by an erythromycin-resistance cassette (Pujol et al., 1999), were grown in suspension in DMEM medium supplemented with 10% heat-inactivated fetal calf serum. PilT belongs to a highly conserved protein family homologous to AAA-type motor proteins and is proposed to cause the retraction of type IV pili by disassembling the pilin subunits at the base of the fiber (Morand et al., 2004). After 2 h of growth, Trifluoperazine (or control vehicle) was added at 50 M for 20 min. The effect on bacterial aggregates were visualized over time using a phase-contrast microscope or the bacterial suspensions were fixed in 4% Paraformaldehyde for 10 min, centrifuged at 1000 rpm for 5 minutes and the bacterial pellets washed in PBS. After negative staining with 1% phosphotungstic acid, bacteria were analysed by transmission electron microscopy, using a JEOL 1011 microscope.

Results

[0167] Results showed that Trifluoperazine exerts a rapid effect on meningococcal aggregation, a function carried by the type IV pili. These polymeric pilus fibers are highly dynamic molecular structures that switch between elongation and retraction. The hexameric ATPase PilT is required for type IV pilus retraction. Interestingly, Trifluoperazine treatment had no effect on the aggregation of a derivative PilT mutant strain, unable to retract its type IV pili (FIG. 3). We therefore examined the effect of Trifluoperazine on the piliation status of meningococci by transmission electron microscopy. As shown in FIG. 5, while control 2C4.3 meningococci (treated with solvent alone) express type IV pili on their surface, (i.e. long filamentous appendages assembled in bundles, pointed by arrows), these structures were no longer observed on bacteria treated with 50 M Trifluoperazine for 30 minutes. In contrast, Trifluoperazine treatment of the PilT mutant did not affect its surface expression of type IV pili (FIG. 5).

Conclusions

[0168] Trifluoperazine induces a drastic loss of the surface expression of meningococcal type IV pili. Trifluoperazine affects the pilus dynamics by exerting a direct or indirect effect on the PilT ATPase, responsible for Type IV pilus retraction.

Example 4: The Phenothiazine Derivatives Compounds, Piperidines and Piperazines, all Induce the Dispersal of Meningococcal Aggregates

Materials and Methods

[0169] N. meninigitidis 2C4.3 strain was grown in suspension in wells of a 24 well plate, containing 1 ml of DMEM medium supplemented with 10% heat-inactivated fetal calf serum. After 2 h of growth, various concentrations (0.5 to 80 M) of phenothiazine-derivative compounds or control vehicle (PBS or DMSO) were added to the wells for 30 min and the bacterial aggregates were visualized using a phase-contrast microscope. Were tested phenothiazine-derivative compounds of: [0170] the Piperazine group: Trifluoperazine (Sigma #T8516), Fluphenazine (Sigma #F4765), Prochlorperazine (Sigma #P9178) Perphenazine (Sigma #P6402); [0171] the Piperidine group: Thioridazine (Sigma #T9025) and Mesoridazin (Sigma #M4068); [0172] the Aliphatic group: Chlorpromazine (Sigma #C8138), Promazine (Sigma #P6656), Triflupromazine (Sigma #1686003) and levomepromazine (Sigma #L0500000).

Results

[0173] Trifluoperazine belongs to a large family of phenothiazine derivatives classified into three groups that differ with respect to the substituent on nitrogen: the aliphatic compounds (bearing acyclic groups), the piperidines (bearing piperidine-derived groups), and the piperazine (bearing piperazine-derived substituents) (FIG. 6). We addressed here the effect of the other derivatives compounds on the dispersion of meningococcal aggregates.

[0174] All piperazine and piperidine compounds tested induced the dispersion of meningococcal aggregates formed in suspension, in a dose-dependent manner and with varying efficiencies:

[0175] While piperazines induced the dispersal at concentrations ranging from 10 to 50 M, Piperidines were efficient at 1 to 4 M (FIG. 7; Table 1).

[0176] By contrast, no effects were observed with the aliphatic compounds (Chlorpromazine, Levomepromazine Triflupromazine) in this range of concentrations excepted for Promazine, which inhibited bacterial aggregation with better efficacy than Trifluoperazine (FIG. 7; Table 1).

TABLE-US-00001 TABLE 1 Effect of the different phenothiazine-derivative compounds on meningococcal dispersal. Concentration promoting efficient meningococcal Structure Name dispersal (~80%) [00004] Thioridazine 4 M [00005] Mesoridazine 6 M [00006] Promazine 10 M [00007] Trifluoperazine 30 M [00008] Prochlorperazine 30 M [00009] Fluphenazine 40 M [00010] Perphenazine 40 M [00011] Chlorpromazine No effect [00012] Triflupromazine No effect [00013] Levomepromazine No effect

Conclusions

[0177] The effect of Trifluoperazine on the dispersion of meningococcal aggregates is common to the tested members of the piperazine and piperidine groups of phenothiazine derivatives.

Example 5: Trifluoperazine Induces the Dispersion of Compact Meningococcal Microcolonies Formed at the Surface of Infected Human Endothelial Cells

Materials and Methods

[0178] HBMEC, a human endothelial cell line isolated from bone marrow capillaries (Schweitzer et al., 1997) were grown in Dulbecco's Modified Eagle Medium (DMEM) 4.5 g/L-Glutamax (ThermoFischer) 10% FBS. Cells were grown on Thermanox coverslips coated with gelatin 2% (BD Difco #214340) for 2 days to reach confluency. Cells were then infected with a suspension of Nm2C4.3 (10.sup.7 CFU/ml) in DMEM/FBS during 30 minutes to allow bacterial adhesion. Cells were then washed three times with medium to remove non-adherent bacteria and infection was pursued for 1 h 30: adherent bacteria grew at the endothelial cell surface and formed micro-colonies. Trifluoperazine was then added to obtain final concentrations of 10, 20, 30 and 40 M. After incubation for 30 minutes, cells were washed and fixed in 4% Paraformaldehyde for 10 min, washed three times with PBS, and permeabilized with 0.1% Triton X-100 in PBS for 10 min. Cells were blocked for 30 min with 3% BSA in PBS and were incubated for 2 h with the primary antibodies (Rabbit polyclonal anti-Nm2C4.3 strain, kindly provided by Dr X. Nassif, INEM, Paris). After three washes with PBS, cells were incubated for 1 h with Alexa Fluor 491-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories), together with Alexa Fluor 633 Phalloidin (Thermofischer) and DAPI (0.5 mg ml-1, Sigma Aldrich) to stain Actin and nuclei, respectively. Labelled preparations were mounted in Glycergel media (DAKO) and image acquisitions were performed with a DM16000 microscope (Leica, X40). Quantification was done with ImageJ software (NIH). Results are presented as a vascular colonisation index corresponding to the area occupied by the fluorescently labelled bacteria per fields in relation to the area occupied by the fluorescently labelled human endothelial cells (Actin staining). Statistical analysis were performed with Student t test.

Results

[0179] After their initial adhesion on human endothelial cells, meningococci rapidly proliferate at the endothelial cell surface and form compact microcolonies, a process referred to as vascular colonization (Melican and Dumenil, 2012). This intimate interaction of meningococci with endothelial cells leads to deregulated inflammatory and coagulation processes, endothelial dysfunction and, ultimately, the breach of endothelial barriers and bacterial dissemination into perivascular tissues (Coureuil et al., 2014; Join-Lambert et al., 2013). As expected, when pre-treated with 30 M Trifluoperazine bacteria did no longer adhere to and/or form bacterial colony at the endothelial cells surface (FIG. 8). More interestingly, when Trifluoperazine was applied to compact meningococcal microcolonies already established at the endothelial cell surface, treatment for 30 min induced their dispersion. This effect was dose-dependent (between 10-40 M) and observed on both a human bone marrow endothelial cell line (FIG. 9) and on primary human endothelial cells isolated from dermal microvessels (FIG. 10). In contrast to Trifluoperazine, addition of conventional antibiotics used in the treatment of meningococcaemia, such as Gentamicin (150 g/ml) or Cefotaxim (20 g/ml), poorly induced the dispersion of these microcolonies.

Conclusions

[0180] In contrast to conventional antibiotics used for the treatment of meningococcemia (Cefotaxim or Gentamicin), Trifluoperazine induces the dispersal of bacterial microcolonies that form at the endothelial cell surface.

Example 6: Trifluoperazine Exerts a Cytoprotective Effect on Endothelial Cells Infected by N. meningitidis: Effect on Cytoskeleton Remodelling and Endothelial Cell Junction Integrity

Materials and Methods

[0181] HDMECs were grown in their specific culture medium (Promocell #C-12210) and confluent monolayers were infected with 2C4.3. Briefly, bacteria were precultured in prewarmed cell culture medium for 1 h 30 min at 37 C. 5% CO.sub.2. The OD.sub.600 was adjusted to 0.1 and HDMECs were then overlaid with bacteria for 30 min (MOI of 100). Unbound bacteria were removed by three washes in cell culture media and infection was allowed to proceed for 1 h at 37 C. 5% CO.sub.2. Gentamicin was added at 150 g/ml for 1 h, then after three washes in cell culture media, Trifluoperazine was applied where mentioned at 50 M for 20 min. After three washes in cell culture media, cells were allowed to recover for 1 h in cell culture media. At the indicated time, cells were fixed with 4% Paraformaldehyde for 10 min. Immunolabelling was then performed as above described in example 4, using DAPI, Alexa Fluor 633 Phalloidin, Polyclonal antisera raised against Ezrin, obtained from Pr P. Mangeat (CRBM, Montpellier, France) and anti-human VE-Cadherin (eBioscience #BMS158).

[0182] When indicated, HBMECs were grown on Thermanox coverslips coated with gelatin 2% for 2 days to reach confluency. Cells were then infected as above described in the presence or in the absence of 5 M Trifluoperazine for 2 h. Cells were washed and fixed in 4% paraformaldehyde and immunolabelling was performed with a polyclonal antisera raised anti-Nm2C4.3 strain, anti-human PECAM-1 mouse monoclonal, clone HEC7 (ABCAM ab119339) or rabbit polyclonal 177 raised against PECAM-1, obtained from WA Muller (Northwestern University, Chicago, Ill., US), anti-0 catenin (05-482, UBI), polyclonal antisera raised against Ezrin, Alexa Fluor 633 Phalloidin. Secondary antibodies were from Jackson ImmunoResearch Laboratories. Image acquisitions were performed on a Leica DM16000 with Yokogawa CSU-X1M1 system and CoolSnap HQ2 (Photometrics). Quantifications were done using ImageJ software (NIH).

Results

[0183] After adhesion, N. meningitidis promotes host cell signalling events, involving Ezrin, Src and Cortactin as main organizers of actin polymerization and receptor clustering (Eugene et al., 2002; Hoffmann et al., 2001; Lambotin et al., 2005; Merz et al., 1999; Soyer et al., 2014). These events promote formation of membrane protrusions that surround bacteria and increase the membrane surface to which the bacteria adhere. This step is critical to resist the shear stress conditions that prevail in vivo (Mikaty et al., 2009). Furthermore, bacteria promote signalling events leading to the delocalization of cell-cell junction molecules such as VE-cadherin, ZO-1 or Claudin-5, at the sites of bacterial adhesion where these proteins are sequestered (Coureuil et al., 2010; Coureuil et al., 2009). VE-cadherin is an endothelial specific cell-cell adhesion molecule that plays a pivotal role in the formation, maturation and remodelling of the vascular wall. These events result in the destabilization of the endothelial cell-cell junctions, increased permeability and bacterial diffusion within surrounding tissues (Coureuil et al., 2014; Dupin et al., 2012). These bacteria-to-cell signaling events are dependent on the interaction between Type IV and endothelial cell receptors (Bernard et al., 2014; Coureuil et al., 2010). We therefore addressed whether Trifluoperazine, by inducing the loss of meningocococcal Type IV pili and the subsequent dispersion of the bacteria that colonize the endothelial cell surface, would prevent the subsequent vascular damages promoted by bacteria-induced signalling events.

[0184] Indeed, in control infected conditions, meningococcal microcolonies formed at the endothelial cell surface induced a strong recruitment of Ezrin, and an important cortical actin polymerization at the bacterial adhesion sites, accompanied by the loss of the continuous staining of VE-cadherin at the endothelial cell junctions and the formation of gaps between cells (pointed by arrows, FIG. 10). Upon treatment with antibiotics (Gentamicin 150 g/ml for 1 h), although it reduced the number of colonies present at the cell surface, residual colonies (still induced Ezrin recruitment, cortical actin polymerization at bacterial adhesion sites, accompanied by the discontinuous distribution of VE-cadherin and the occurrence of gaps between the endothelial cells (FIG. 10), indicating that antibiotic-treated bacteria still promoted signalling events leading to vascular alterations. When used in combination with gentamicin, Trifluoperazine, induced the dispersion of most of the bacterial colonies at the endothelial cell surface. Few Ezrin recruitment was observed under residual bacteria, F-actin was polymerized at the periphery of the cells, accompanied with a continuous staining of VE-cadherin at the intercellular junctions and the absence of gap between cells, therefore indicating that the clearance of bacteria promoted by Trifluoperazine preserved endothelial cell junction integrity (FIG. 10).

[0185] Interestingly, we also observed that upon adhesion to endothelial cells, meningococci promote the massive delocalization of PECAM-1/CD31 from the intercellular junctions of the endothelial cells to the bacterial adhesion sites (FIG. 11A). Treatment with low concentrations of Trifluoperazine (5 M) did not affect bacterial colonization, nor prevented Ezrin recruitment or Actin polymerization at bacterial sites; however, it prevented PECAM-1 recruitment at bacterial adhesion sites (FIGS. 11B, 11C). These results indicate that, besides its effect on bacterial clearance obtained at concentrations ranged from 10 to 50 M that preserved endothelial cell junction integrity, Trifluoperazine may further improve vascular protection at lower concentrations by acting directly on infected endothelial cells (i.e. by inhibiting the massive PECAM-1 delocalization from the endothelial cell junctions).

Conclusions

[0186] In contrast to conventional antibiotics used for the treatment of meningococcemia, Trifluoperazine can stop the endothelial cells from receiving intracellular signals that results in their large scale systemic dysregulation. These compounds exert a vasculoprotective effect on infected endothelial cells by acting both on bacteria and on infected cells.

Example 7: Trifluoperazine Exerts a Cytoprotective Effect on Endothelial Cells Infected by N. meningitidis: Effect on Basement Membrane Remodelling

Materials and Methods

[0187] Plastic coverslips (13 mm diameter) (Nalgen #174950) were washed in ethanol 70%, dried then coated with poly-L-lysine 1 mg/ml for 20 min at room temperature. After one wash in sterile PBS, 0.5% Glutaraldehyde was added for 15 min at room temperature. After three washes in PBS, Gelatin-FITC (0.2 mg/ml Invitrogen #G13187) was added for 10 minutes at room temperature in the dark. Coverslip were then washed with sterile PBS and treated with 5 mg/ml sodium borohydride for 3 min. After three washes in sterile PBS, 10.sup.5 HDMECs (PromoCell #C-12210) were seeded per well in their PromoCell culture medium and incubated overnight at 37 C. 5% CO.sub.2. The day after, cells were infected with N. meningitidis Nm2C4.3, as above described. Infection was allowed to proceed for 5 h 30 min before Gentamicin treatment at 150 mg/ml for 1 h. After three washes in cell culture medium, Trifluoperazine was added for 20 min, washed with the culture medium and were incubated overnight in cell culture media containing 15 mg/ml Gentamicin. Cells were then fixed in 4% Paraformaldehyde for 10 min, stained with Phalloidin 633 for 1 hour and mounted in glycergel (DAKO). Image acquisitions were performed on a Leica DMI6000 with Yokogawa CSU-X1M1 system and CoolSnap HQ2 (Photometrics). Quantifications were performed using ImageJ software (NIH).

Results

[0188] To visualize a potential protective effect of Trifluoperazine on the basement membrane integrity, HDMECs were grown on a fluorescent gelatin matrix (Gelatin-FITC), the appearance of dark areas corresponding to the zones of matrix degradation (FIG. 12). In the absence of infection, a homogenous fluorescence was observed, showing the integrity of this basement membrane, only few zones of degradation were observed (<5% of the basement membrane). Because it takes 16 to 24 h to observe matrix degradation, the time that metalloproteinases involved are synthesized and degrade the matrix, antibiotics treatment was applied after few hours of infection to avoid bacterial overload and subsequent cell death, due to culture medium consumption. Despite antibiotic treatment with 150 g/ml Gentamicin, infection of endothelial cells was accompanied by a degradation of about 20% of the basement membrane 24 h post-infection. Treatment with Trifluoperazine reduced this drastic degradation of the matrix in a dose-dependent manner (by 30 to 60%) (FIG. 12).

Conclusions

[0189] All these results demonstrate that treatment with Trifluoperazine induces a cyprotective effect on endothelial cells, by preserving the integrity of their basement membrane.

Example 8: Trifluoperazine Induces the Dispersion of Compact Microcolonies Formed by Enteropathogenic Escherichia coli at the Surface of Infected Human Endothelial Cells

Materials and Methods

[0190] HBMEC were grown on Thermanox coverslips coated with gelatin 2% for 2 days to reach confluency. Cells were then infected for 1 h with a GFP-expressing mutant Enteropathogenic Escherichia coli (EPEC), which lacks the ATPase escN. This strain is unable to translocate effector proteins and is more prone to form pilus-dependent microcolony at the host cell surface (Jensen et al., 2015). Cells were then washed three times with medium to remove non-adherent bacteria and infection was pursued for 1 extra hour, to allow bacterial growth at the endothelial cell surface to form microcolonies. Trifluoperazine was then added to obtain final concentrations of 10, 20, 30 and 50 M. After incubation for 30 minutes, cells were washed and fixed in 4% Paraformaldehyde for 10 min, washed three times with PBS. Cells were incubated for 1 h with Alexa Fluor 633 Phalloidin (Thermofischer) together with DAPI (0.5 mg ml-1, Sigma Aldrich) to stain Actin and nuclei, respectively. Labelled preparations were mounted in Glycergel media (DAKO) and image acquisitions were performed with a DM16000 microscope (Leica, X20). Quantification was done with ImageJ software (NIH). Results are presented as a vascular colonization index corresponding to the area occupied by the fluorescently labelled bacteria per fields in relation to the area occupied.

Results

[0191] To address whether trifluoperazine would affect vascular colonization by other piliated pathogens, we used Enteropathogenic Escherichia coli (EPEC), as, similarly to N. meningitidis, EPEC requires Type IV bundle-forming pili to autoaggregate and to form microcolonies on human cells (Bieber et al., 1998; Moreira et al., 2006). Type IV pili are also essential for EPEC virulence, as EPEC mutants hindered for microcolony formation have been shown to be highly attenuated for virulence in human volunteers (Bieber et al., 1998).

[0192] Human endothelial cells were infected for 1 h with a GFP-expressing mutant Enteropathogenic Escherichia coli (EPEC), which lacks the ATPase escN, as this strain, unable to translocate effector proteins and to induce pedestal formation, is more prone to form pilus-dependent microcolony at the host cell surface (Jensen et al., 2015).

[0193] After their initial adhesion on human endothelial cells, escN EPEC-GFP rapidly proliferated at the endothelial cell surface and formed compact microcolonies. Treatment for 30 min with Trifluoperazine (10 to 50 M) induced the dispersion of these microbial colonies in a dose-dependent manner (FIG. 13).

Conclusions

[0194] The effect of Trifluoperazine on the dispersal of bacterial microcolonies is not limited to N. meningitidis but also apply to other bacterial pathogens that require type IV pili to colonize human cells.

Example 9: Trifluoperazine Induces the Dispersion of Compact Microcolonies Established at the Surface of Human Brain Endothelial Cells

Materials and Methods

[0195] HCMEC/D3, a well-established human brain endothelial cell line (Weksler et al, 2013) was grown in EBM-2 basal medium (Lonza, Walkersville, Md., USA) supplemented with 5% Fetal Bovine Serum Gold, 10 mM HEPES (PM Laboratories GmbH, Pasching, Austria), 1% Penicillin-Streptomycin, 1% chemically defined lipid concentrate (Invitrogen Ltd, Paisley, UK), 1.4 M hydrocortisone, 5 g.Math.ml.sup.1 ascorbic acid and 1 ng.Math.ml.sup.1 bFGF (Sigma-Aldrich, St. Louis, Mo.). Cells were grown on Thermanox coverslips coated with rat collagen I for 4 days at 37 C. in a humidified incubator in 5% CO.sub.2. Cells were then infected with a suspension of Nm2C4.3 (10.sup.7 CFU/ml) in EBM2/FBS during 30 minutes to allow bacterial adhesion. Cells were then washed three times with medium to remove non-adherent bacteria and infection was pursued for 1 h 30: adherent bacteria grew at the endothelial cell surface and formed micro-colonies. Trifluoperazine was then added to obtain final concentrations of 10, 20, 30 and 40 M. After incubation for 30 minutes, cells were washed and fixed in 4% Paraformaldehyde for 10 min, washed three times with PBS, and permeabilized with 0.1% Triton X-100 in PBS for 10 min. Cells were blocked for 30 min with 3% BSA in PBS and were incubated for 2 h with the primary antibodies (Rabbit polyclonal anti-Nm2C4.3 strain, kindly provided by Dr X. Nassif, INEM, Paris). After three washes with PBS, cells were incubated for 1 h with Alexa Fluor 491-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories), together with Alexa Fluor 633 Phalloidin (Thermofischer) and DAPI (0.5 mg ml-1, Sigma Aldrich) to stain Actin and nuclei, respectively. Labelled preparations were mounted in Glycergel media (DAKO) and image acquisitions were performed with a DM16000 microscope (Leica, X40). Quantification was done with ImageJ software (NIH). Results are presented as a vascular colonisation index corresponding to the area occupied by the fluorescently labelled bacteria per fields in relation to the area occupied by the fluorescently labelled human endothelial cells (Actin staining). Statistical analysis were performed with Student t test.

Results

[0196] Trifluoperazine was applied to compact meningococcal microcolonies already established at the surface of human brain endothelial cells, treatment for 30 min induced their dispersion. This effect was dose-dependent (between 10-40 M) (FIG. 14).

Conclusion

[0197] Trifluoperazine induces the dispersal of bacterial microcolonies that form at the surface of human brain endothelial cells.

Example 10: Effect of Trifluoperazine on the Colonization of Human Brain Vessels, Using an In Situ Meningococcal Infection Model of Fresh Human Frontal Brain Tissues Obtained from Deceased Normal Subjects

Materials and Methods

[0198] In situ infection of fresh human brain sections was performed as described previously (Bernard et al, 2014). Fresh human brain sections were obtained from frontal lobe specimens of macroscopically and histologically normal brain (confirmed by a neuropathologist) of individuals referred to the Department of Forensic Medicine for unexplained out-of-hospital sudden death (consent forms ML1094, PFS 10-008, ClinicalTrials.gov NCT00320099 from The Institutional Review Boards of the Poincar Hospital, Versailles-Saint Quentin University and the French Agence de la Biomdecine). After freezing of the brain tissue with isopentane cooled in liquid nitrogen, the sections, 7 m thick, containing leptomeninges, cortical ribbon and the underlying white matter were immobilised on Superfrost plus microscope slides and stored at 80 C. Defrosted sections were rehydrated in PBS for 5 min and incubated for 1 h with medium containing 0.1% BSA prior to infection with suspensions of bacteria (210.sup.7 bacteria in 150 l of medium containing 0.1% BSA) for 1 h at 37 C. Sections were then treated for 30 min Trrifluoperazine 40 M or PBS alone as a control and were then gently washed horizontally 5 times and fixed in PAF 4% for 10 min at RT. Adherent meningococci were detected by immunofluorescence analysis: brain sections were incubated with the following primary antibodies for 2 h in PBS/BSA 0.1%: monoclonal anti-human CD31/PECAM-1 mouse monoclonal antibody (clone HEC7, ABCAM, ab119339) and a rabbit polyclonal serum anti-Nm 2C4.3 strain (1:3000). Alexa-conjugated phalloidin and DAPI (0.5 mg/ml) were added to Alexa-conjugated secondary antibodies for 1 h. After additional washing, coverslips were mounted in glycergel (Dako). Entire samples were scanned using a Lamina (Perkin Helmer) and were further analysed using confocal microscopy (spinning disk Leica, 63x). Quantification analysis of the images (n=50 per section) was performed using ImageJ software (NIH). Results are presented as a vascular colonisation index corresponding to the area occupied by the fluorescently labelled bacteria in relation to the human vessel area delineated by the anti-PECAM-1 staining, from 2 independent sections per condition.

Results

[0199] The effect of trifluoperazine on the colonization of human brain vessels were further analyzed using an in situ meningococcal infection model of fresh human frontal brain tissues obtained from deceased normal subjects, as described previously (Bernard et al, 2014). In this setting, histological and anatomical characteristics of the brain vessels are conserved. Meningococci incubated with tissue sections established specific tight association with brain vessels, reminiscent of neuropathological findings in patients with meningococcal meningitis and adhesion relied on the expression of type IV pili (Bernard et al, 2014).

[0200] Upon infection, meningococci developed microcolonies immediately adjacent to CD31-positive endothelial cells (FIG. 15. A.). Consistent with in vitro cellular models, treatment of infected brain sections with Trifloperazine 40 M for 30 min induced the dispersal of these meningococcal microcolonies (FIG. 15. A et B.), reducing by 80% the vascular colonization of the human brain vessels.

[0201] Therefore, trifluoperazine reduced in situ infection of human brain vessels, indicating that it might reduce the signs of meningitis.

Conclusion

[0202] In this study, trifluoperazine and related phenothiazines were identified to blocked all the functions carried by the type IV pili (bacterial competence, twitching motility, aggregation and adhesion to inert surface or host endothelial cells) in different bacterial pathogens.

[0203] Trifluoperazine has shown to induce within minutes the retraction of the meningococcal Type IV pili. In contrast to conventional antibiotics used for the treatment of meningococcemia (Cefotaxim or Gentamicin), trifluoperazine promotes the dispersal of compact microcolonies already formed at the surface of peripheral and brain endothelial cells in vitro and reduce subsequent endothelial alteration. Trifluoperazine induces the dispersal of compact microcolonies formed in situ in a meningococcal infection model of human frontal brain tissues. Finally, when used in vivo, in mice engrafted with human skin, trifluoperazine prevents the massive colonization of the human dermal vasculature, reduces the signs of intravascular coagulation, reduces incidence of vascular alteration. Importantly, while cefotaxime treatment increased by 2 fold this inflammatory response, most likely by promoting the release of various Pathogen-associated molecular patterns that activate innate immune response, trifluoperazine alone or in combination with cefotaxime, drastically reduced the hallmark of vascular inflammation. By inducing bacterial clearance, trifluoperazine can prevent an overwhelming inflammatory response, therefore conferring a potential advantage over antibiotics treatment.

[0204] These findings reveal an unexpected outcome of phenothiazine therapy for the targeting of a major bacterial virulence factor that could be developed to prevent and treat bacterial diseases, in particular, as antibiotic adjuvant therapy for treatment of meningococcal diseases.

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