Inhibition of Type IV Secretion Activity and Growth of by Cisplatin and Other Platinum Complexes.

Inhibition of Type IV Secretion Activity and Growth of Helicobacter pylori by Cisplatin and Other Platinum Complexes Clara Lettl 1,2† , Franziska Schindele 1,2† , Giambattista Testolin 3,4 , Alexander Bär 5 , Tobias Rehm 5 , Mark Brönstrup 3,4 , Rainer Schobert 5 , Ursula Bilitewski 3,4 , Rainer Haas 1,2 and Wolfgang Fischer 1,2 * 1 Max von Pettenkofer Institute of Hygiene and Medical Microbiology, Faculty of Medicine, LMU Munich, Munich, Germany, 2 German Center for Infection Research (DZIF), Munich Site, Munich, Germany, 3 Department of Chemical Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany, 4 German Center for Infection Research (DZIF), HannoverBraunschweig Site, Braunschweig, Germany, 5 Organic Chemistry Laboratory, University Bayreuth, Bayreuth, Germany Type IV secretion systems are protein secretion machineries that are frequently used by pathogenic bacteria to inject their virulence factors into target cells of their respective hosts. In the case of the human gastric pathogen Helicobacter pylori, the cytotoxinassociated gene (Cag) type IV secretion system is considered a major cause for severe disease, such as gastric cancer, and thus constitutes an attractive target for specific treatment options against H. pylori infections. Here, we have used a Cag type IV secretion reporter assay for screening a repurposing compound library for inhibitors targeting this system. We found that the antitumor agent cisplatin, a platinum coordination complex that kills target cells by formation of DNA crosslinks, is a potent inhibitor of the Cag type IV secretion system. Strikingly, we found that this inhibitory activity of cisplatin depends on a ligand exchange reaction which incorporates a solvent molecule (dimethylsulfoxide) into the complex, a modification which is known to be deleterious for DNA crosslinking, and for its anticancer activity. We extended our analysis to several analogous platinum complexes containing N-heterocyclic carbene, as well as DMSO or other ligands, and found varying inhibitory activities toward the Cag system which were not congruent with their DNAbinding properties, suggesting that protein interactions may cause the inhibitory effect. Inhibition experiments under varying conditions revealed effects on adherence and bacterial viability as well, and showed that the type IV secretion-inhibitory capacity of platinum complexes can be inactivated by sulfur-containing reagents and in complex bacterial growth media. Taken together, our results demonstrate DNA bindingindependent inhibitory effects of cisplatin and other platinum complexes against different H. pylori processes including type IV secretion. Keywords: Helicobacter pylori, type IV secretion system, Cag, cisplatin, protein secretion Frontiers in Cellular and Infection Microbiology | www.frontiersin.org December 2020 | Volume 10 | Article 6029581 Edited by: D. Scott Merrell, Uniformed Services University, United States Reviewed by: Javier Torres, Mexican Social Security Institute (IMSS), Mexico Timothy Cover, Vanderbilt University, United States *Correspondence: Wolfgang Fischer fi[email protected] † These authors have contributed equally to this work Specialty section: This article was submitted to Molecular Bacterial Pathogenesis, a section of the journal Frontiers in Cellular and Infection Microbiology Received: 07 September 2020 Accepted: 17 November 2020 Published: 18 December 2020 Citation: Lettl C, Schindele F, Testolin G, Bär A, Rehm T, Brönstrup M, Schobert R, Bilitewski U, Haas R and Fischer W (2020) Inhibition of Type IV Secretion Activity and Growth of Helicobacter pylori by Cisplatin and Other Platinum Complexes. Front. Cell. Infect. Microbiol. 10:602958. doi: 10.3389/fcimb.2020.602958 ORIGINAL RESEARCH published: 18 December 2020 doi: 10.3389/fcimb.2020.602958 INTRODUCTION The human gastric pathogen Helicobacter pylori is responsible for chronic gastritis, peptic ulcer disease, as well as gastric adenocarcinoma and MALT lymphoma, and thus represents one of the leading causes for infection-associated morbidity and mortality worldwide. It has been estimated that more than 4 billion individuals are infected with H. pylori, albeit with major regional variations (Hooi et al., 2017), and that more than 800,000 new cases of gastric cancer per year can be attributed to H. pylori infection (de Martel et al., 2020). Although consensus treatment strategies are available (Fallone et al., 2016), resistance rates against the therapeutically used antibiotics are increasing to an alarming extent (Savoldi et al., 2018). Because of this, H. pylori has been included on the WHO priority list for research and development of new antibiotics (Tacconelli et al., 2018). One possible approach toward novel treatment options is to identify potential inhibitors of important virulence properties, which might be utilized to complement established treatment regimes. One of the major factors involved in pathogenicity of H. pylori is the type IV protein secretion system encoded on the cytotoxinassociated gene (cag) pathogenicity island (Backert et al., 2017). The Cag type IV secretion system builds up a remarkable multiprotein complex composed of roughly 20 different proteins, which spans the bacterial inner and outer membranes and is able to translocate the bacterial CagA protein into the cytoplasm of gastric cells (Fischer, 2011;Chung et al., 2019;Hu et al., 2019). Furthermore, the Cag type IV secretion system is involved in inducing a strong proinflammatory response in gastric epithelial cells via NF-kB signaling (Zhang et al., 2020). The presence of the cag pathogenicity island increases the risk of developing severe disease, particularly of gastric adenocarcinoma (Wroblewski et al., 2010). Although many studies have addressed the composition of the secretion apparatus and the functions of individual components, and despite the availability of high-resolution structures of the Cag secretion apparatus (Frick-Cheng et al., 2016;Chung et al., 2019;Hu et al., 2019), details of the type IV secretion process are still only poorly understood. For example, the role of pilus-like structures associated with the type IV secretion system, and even their composition, are not clear (Backert et al., 2015;Chang et al., 2018). On the other hand, it is well-established that the Cag system, similar to other type IV secretion systems, contains three different putative ATPases that are all essential for the CagA secretion process. Several studies have reported small-molecule inhibitors with the potential of interfering with the Cag type IV secretion system. One study reported the identification of compounds that were able to reduce formation of secretion system-associated pili, and also type IV secretion itself (Shaffer et al., 2016). Other studies have described small-molecule inhibitors that are able to inhibit one of the ATPases, Caga(Hilleringmann et al., 2006;Sayer et al., 2014; Arya et al., 2019). In this study, we have used a recently described CagA translocation reporter assay (Schindele et al., 2016) to screen a small-compound repurposing library for molecules that are able to reduce this type IV secretion activity. Apart from a number of molecules with known or suspected antibacterial activities, we identified two anti-cancer drugs, cisplatin and carboplatin, for which antibacterial effects have been described previously. However, we show in detailed follow-up studies, also including other platinum complexes, that their activity against H. pylori does not depend on the typical DNA-binding properties required for anticancer activity of the platinum complexes, and thus differs widely from their activity toward other bacteria. MATERIALS AND METHODS Bacterial Strains, Cell Lines, and Culture Conditions H. pylori strains P12 and P12 [TEM-1–CagA] (Schindele et al., 2016) were grown on GC agar plates (Oxoid) supplemented with vitamin mix (1%) and horse serum (Life Technologies; 8%) (serum plates), and cultured for 16 to 60 h in a microaerobic atmosphere (85% N 2 , 10% CO 2 ,5%O 2 ) at 37°C. AGS cells were cultivated in RPMI (Gibco) supplemented with 10% FCS (heatinactivated; Life Technologies). Murine L929 fibroblasts were cultivated in DMEM (Gibco) supplemented with 10% FCS, 2 mM L-glutamine (Gibco) and 1 mM sodium pyruvate (Gibco) at 37°C in a 5% CO 2 incubator. Reagents The LOPAC 1280 library was obtained from Sigma (ordering no. LO1280). Cisplatin (no. P4394) and cis-dichlorido-bis(DMSO) platinum(II) (no. 767654) were purchased from Sigma. Transplatin was purchased from Alfa Aesar (no. 10472). Stock solutions of all platinum complexes were prepared in DMSO at 200-fold their final concentrations in the assays, except where indicated otherwise. The final DMSO concentration in the assays was thus always 0.5%. Antibodies, SDS-PAGE, Immunoblotting, and ELISA A polyclonal antiserum against the CagA EPIYA region (AK299) has been described previously (Schindele et al., 2016). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blotting was performed as described (Fischer et al., 2001). For the development of immunoblots, polyvinylidene difluoride (PVDF) filters were blocked with 5% non-fat milk powder in TBS (50 mM Tris–HCl, pH 7.5, 150 mM NaCl), 0.1% (v/v) Tween 20 (TBS-T), and incubated with the respective antisera at appropriate dilutions in TBS-T with 1% non-fat milk powder. Alkaline phosphatase-conjugated protein A was used to visualize bound antibody. Standard infections of AGS cells with H. pylori strains and subsequent preparations for phosphotyrosine immunoblotting were performed as described previously (Odenbreit et al., 2000). Briefly, cells seeded in 6-well plates (Falcon) were infected with bacteria at a multiplicity of infection of 100 for 4 h at 37°C, washed three times and suspended in PBS containing 1 mM Na 3 VO 4 , 1 mM PMSF, 10mgml −1 leupeptin, and 10 mgml −1 pepstatin. Cells with adherent bacteria were collected by centrifugation and resuspended in Lettl et al. Platinum Complex Activity Against H. pylori Frontiers in Cellular and Infection Microbiology | www.frontiersin.org December 2020 | Volume 10 | Article 6029582 SDS-PAGE sample solution. Tyrosine-phosphorylated proteins were analyzed by immunoblotting with the phosphotyrosine antibody PY99 (Santa Cruz Biotechnologies). Production of IL-8 by AGS cells after infection with H. pylori strains for 4 h was determinedfromco-incubationsupernatantsbyasandwichELISA as described elsewhere (Fischer et al., 2001). As a control, IL-8 production was induced with 20 ng/ml recombinant human TNF-a(Peprotech Inc.). TEM-1–CagA Translocation Assay The CagA translocation reporter assay with H. pylori strains producing TEM-1–CagA fusions was performed as described elsewhere (Schindele et al., 2016). Briefly, AGS cells were coincubated with H. pylori P12 [TEM-1-CagA] for 2.5 h in 384well, or in 96-well microtiter plates (black, clear bottom, tissue culture treated, 4titude) in PBS/10% FCS. After infection, cells were loaded with the fluorescent substrate CCF4-AM in a loading solution (LiveBLAzer-FRET B/G loading kit; Invitrogen) supplemented with 1 mM probenecid (Sigma) according to the manufacturer´s instructions. For fluorescence quantification by plate reading, infected cells were incubated with this loading solution at room temperature in the dark for 2 h, and then directly measured with a Clariostar reader (BMG Labtech) using an excitation wavelength of 405 nm, and emission wavelengths of 460 nm, or 530 nm. CagA translocation was calculated as the ratio of background-corrected (wells containing no cells or bacteria, but CCF4-AM loading solution) emission values at 460 to 530 nm, which were normalized to P12 [TEM-1– CagA] as a positive control, and P12 [TEM-1–CagA], DcagT as a negative control. For inhibition experiments, bacteria were preincubated with the respective concentrations of compounds obtained from the corresponding stock solutions for 30 min at 37°C in PBS/10% FCS, followed by infection for 2.5 h in the presence of the compound. Additionally, CagA translocation reporter assays were performed in brain heart infusion (BHI, BD), Brucella broth (BB, BD, supplemented with 10% FCS) or PBS/10% FCS mixed with the indicated amount of BB. To test the impact of amino acids on the inhibitory effect of cisplatin, stock solutions of L-cysteine (Serva), L-methionine (Merck) and L-alanine (Roth) were prepared in ddH 2 Oandmixedwith cisplatin (6.3 mM in DMSO). Cisplatin-amino acid mixtures were stored at −20°C until further use. Growth Assays H. pylori grown on serum plates were suspended to an optical density (OD 550 nm ) of 0.075 in BB/10% FCS and sub-cultured in 96-well microtiter plates (clear, flat-bottom, Costar, Corning Inc.). Compounds were added to the respective concentrations from corresponding stock solutions, and wells were sealed with a gas-permeable membrane (Breathe-Easy®sealing membrane, Diversified Biotech). Plates were incubated at 37°C, 10% CO 2 , 200 rpm in a plate reader (Clariostar, BMG Labtech) with an atmospheric control unit (BMG Labtech). OD 550 was automatically measured every 5 min until the stationary phase was reached. Growth curves were analyzed and processed using the MARS Data Analysis software 3.10 R5 (BMG Labtech). The effect of cisplatin on H. pylori viability in the presence of AGS cells was further assessed by incubating the bacteria (OD 550 nm = 0.1) together with AGS cells in PBS/10% FCS or BB/10% FCS supplemented with 100 µM cisplatin in DMSO, or with DMSO only. After 2 h at 37°C, 10% CO 2 , 5 µl of bacterial suspension were spotted on serum plates, and growth was checked after 24 h. Mass Spectrometry and NMR Analysis LCMS measurements were performed using an HPLC (Agilent technologies 1200 series) equipped with a Gemini-NX 3u C18 110A 50 × 2.0 mm column coupled to an ion trap mass spectrometer (Bruker amaZon SL). High resolution mass spectra were recorded by direct infusion into a Q-TOF mass spectrometer (Bruker maXis HD) using electrospray ionization (ESI) in the positive mode. 1 H and 195 Pt NMR spectra were recorded using a Bruker Advance-III HD 700 MHz spectrometer. Chemical shifts are reported as values in ppm, for the 1 H-NMR relative to residual solvent signal as internal standard and for the 195 Pt-NMR relative to the reference compound Na 2 PtCl 6 . Cytotoxicity Measurements The effect of compounds on eukaryotic cell proliferation and viability was assessed using the WST-1 cell proliferation assay (Roche Applied Science). Briefly, murine L929 fibroblasts were seeded into 96-well plates (3.0 × 10 5 cells/well, clear, flat-bottom, Costar, Corning Inc.) using Phenol red-free culture medium. After 24 h incubation at 37°C, 5% CO 2 , compounds were added in two-fold dilutions and incubation was continued for three days. WST-1 reagent was added according to the manufacturer’s protocol and plates were kept at 37°C, 5% CO 2 for 1 h. The absorbance at 450 nm and 690 nm (reference wavelength) was recorded in a plate reader (Clariostar, BMG Labtech). For evaluation, the difference of A 450nm and A 690nm was calculated, and the control value (medium and WST-1 only) was subtracted. Percental viability was normalized to the untreated control. Adherence Assays Cisplatin influence on adherence of H. pylori to AGS cells was essentially measured as described (Königer et al., 2016). Briefly, cells were infected with a GFP-producing variant of strain P12 (P12 [pHel12::gfp]; (Königer et al., 2016)), using an MOI of 60, and cisplatin was added at the time of infection, or 30 min later, from DMSO stock solutions of the corresponding concentrations. AGS cell infection was allowed to proceed until 1 h after infection at 37°C and 5% CO 2 .Afterthree washing steps with PBS, cells with adherent bacteria were collected by EDTA treatment, and analyzed in a flow cytometer (FACS CantoII, BD Biosciences). For analysis, the median fluorescence intensity of non-infected cells was subtracted from that of infected samples. Statistical Analysis Quantitative data sets shown are generally average values resulting from at least three independent experiments, with standard deviations. IC 50 values were calculated from at least three independent experiments by nonlinear regression using the Graphpad Prism5 software. Lettl et al. Platinum Complex Activity Against H. pylori Frontiers in Cellular and Infection Microbiology | www.frontiersin.org December 2020 | Volume 10 | Article 6029583 RESULTS Identification of Cisplatin and Carboplatin as Inhibitors of the Cag Type IV Secretion System In an attempt to identify small molecules that inhibit the Cag type IV secretion system, we screened the Library of Pharmacologically Active Compounds (LOPAC 1280 ;Sigma) with a TEM-1–CagA translocation reporter assay (Schindele et al., 2016) adapted to a microtiter plate format. This assay utilizes engineered H. pylori strains producing the TEM-1 blactamase fused to the N-terminus of CagA. Type IV secretion of the TEM-1–CagA fusion protein into target cells is monitored by loading the cells with the fluorescent b-lactamase substrate CCF4-AM, and CagA translocation is determined by calculating blue-to-green fluorescence ratios (see Experimental Procedures for details). Out of the 1280 compounds tested at a concentration of 50 µM, 151 resulted in a reduction of CagA translocation activity to less than 50%, as compared to infection with an untreated reporter strain. Since these compounds included several substances with suspected or previously demonstrated antibacterial activities against H. pylori, such as doxycycline and minocycline (Glupczynski et al., 1988), niclosamide (Tharmalingam et al., 2018), or clotrimazole, we applied a counterscreen for compounds inhibiting H. pylori growth. When all compounds that inhibited growth to more than 50% were removed, only 10 compounds remained (Table 1). Subsequently, we used the TEM-1–CagA reporter assay in a 96-well format to reproduce the type IV-inhibitory effects of these compounds at a slightly lower concentration (35 µM). In this assay, three compounds exhibited cytotoxic activities toward AGS cells, as indicated by CCF4-AM loading defects, and the initial inhibitory effects could not be reproduced for four further compounds at this concentration (Table 1). Thus, only the unspecific alkylating agent iodoacetamide, and the antitumor agents cisplatin and carboplatin remained as potential type IV secretion inhibitors. The activities of the latter compounds were confirmed in an orthogonal secondary assay, which measures CagA tyrosine phosphorylation following infection of AGS cells with H. pylori strain P12. While iodoacetamide did not inhibit CagA tyrosine phosphorylation (data not shown), cisplatin and carboplatin were able to strongly reduce or even block the appearance of tyrosine-phosphorylated CagA at a concentration of 50 µM, suggesting that they interfere with type IV secretion of CagA (Figure 1A). In contrast, both cisplatin and carboplatin apparently did not interfere with viability of H. pylori at these concentrations, as demonstrated by growth curves obtained in Brucella broth containing 10% FCS (BB/10% FCS; Figure 1B); however, the activity of these compounds depends on the media used (see below). Furthermore, levels of the chemokine interleukin-8 (IL-8), which is induced in AGS cells via the Cag type IV secretion system, were reduced in the presence of cisplatin in a dose-dependent manner (Figure 1C). In contrast, TNF-awas still able to induce an IL-8 response in AGS cells in the presence of 100 µM cisplatin (Figure 1C, right panel), indicating that the cisplatin effect on IL-8 induction was due to inhibition of the bacteria. Taken together, these observations suggested a specific interference of cisplatin and carboplatin with type IV secretion by H. pylori. Dependence of Cisplatin Activities on the Solvent Both cisplatin (cis-diamminedichloridoplatinum(II)) and carboplatin (cis-diammine-(1,1-cyclobutane-dicarboxylato)- platinum(II)) are planar platinum coordination complexes that are able to form intrastrand crosslinks between purine bases on DNA (Fichtinger-Schepman et al., 1985). For cisplatin, this crosslinking activity is thought to involve prior exchange of at least one chlorido ligand for a water molecule (aquation), which results in a cationic complex with higher affinity for DNA (Davies et al., 2000;Kelland, 2007). Other ligand exchange reactions may also take place, for example the replacement of a chlorido ligand for dimethylsulfoxide (DMSO), which coordinates platinum via its nucleophilic sulfur atom (Figure 2A)(Annibale et al., 1983). Since the stock solutions of the LOPAC 1280 library compounds were prepared in DMSO and then diluted in aqueous buffers, such complexes with DMSO ligands may have been present in our screening experiments. To examine this possibility, we compared the activity of cisplatin dissolved in DMSO with that of cisplatin diluted from stock solutions prepared in dimethylformamide (DMF), which does not seem to react with cisplatin (Hall et al., 2014). With cisplatin dissolved in DMSO, we obtained a dose-dependent inhibition of CagA translocation with a half-maximal inhibitory concentration (IC 50 ) of 5.98 µM, as measured by the TEM-1– CagA assay, whereas DMSO had no effect (Figure 2B). Using cisplatin from DMF stock solutions, however, we achieved inhibition only at high concentrations (>32 µM), clearly indicating a solvent-dependent activity of cisplatin (Figure 2C). We also obtained an inhibition of CagA translocation with cisdichlorido-bis(DMSO)platinum(II) dissolved in DMSO, albeit at a reduced efficiency in comparison to cisplatin (IC 50 = 29.5 µM; TABLE 1 | Properties of compounds identified during initial screening of the LOPAC 1280 library a . Compound Effect on type IV secretion (TEM-1–CagA assay) or H. pylori growth (% of untreated control) TEM-1–CagA, 384 well, 50 µM Growth, 100 µM TEM-1–CagA, 96 well, 35 µM Carboplatin 13.7 84.9 32.8 Cisplatin 3.4 88.8 7.6 Cantharidic acid 40.1 84.7 No CCF4 loading Cantharidin 32.7 74.6 No CCF4 loading Dihydroergocristine methanesulfonate 39.7 72.5 80.2 Iodoacetamide 47.3 74.7 49.2 Methysergide maleate 5.7 83.3 89.5 NF449 octasodium salt 42.9 102.7 103.0 Propantheline bromide 38.7 65.6 84.0 Wortmannin 35.1 94.0 Low CCF4 loading a Compounds were initially selected for inhibition of TEM-1–CagA activity by more than 50%, and H. pylori growth inhibition by less than 50% in comparison to untreated (DMSO) control. Lettl et al. Platinum Complex Activity Against H. pylori Frontiers in Cellular and Infection Microbiology | www.frontiersin.org December 2020 | Volume 10 | Article 6029584 Figure 2D), but not when the same complex was dissolved in DMF instead (IC 50 ≥100 µM; data not shown). Interestingly, when we measured H. pylori growth in a microplate format, we did not observe significant growth defects upon addition of cisplatin dissolved in DMSO (Figure 2E), or cis-dichlorido-bis (DMSO)platinum(II) dissolved in DMSO or in DMF (data not shown). However, cisplatin dissolved in DMF resulted in a clear dose-dependent growth inhibition (Figure 2E). This suggests that cisplatin dissolved in DMF exerts its expected toxic activity against H. pylori, but does not impact strongly on type IV secretion, whereas a DMSO ligand in the platinum complex modulates its activity toward Cag type IV secretion inhibition, while reducing the antibacterial effect. To examine whether cisplatin was subject to ligand exchange reactions in our DMSO stock solutions, we analyzed the complexes by mass spectrometry, and detected a cisplatin derivative with one chlorido ligand replaced by DMSO as the major molecular species in DMSO solution (m/z = 342.999 Da; Figure 3A). Upon dilution in water or acetonitrile containing 0.1% formic acid, this complex partly reacted further to give a monoammine-monochloro-bis-DMSO complex (m/z = 403.988 Da; Figure 3B). No ligand exchange reactions were observed with cisplatin dissolved in DMF (data not shown). Consistent with these results, 1 H-NMR and 195 Pt-NMR measurements of cisplatin dissolved in DMF-d 7 showed stable peaks at 4.17 ppm and −2090 ppm, respectively (Figure 3C). In contrast, cisplatin dissolved in DMSO-d 6 was subject to decomposition over time, with formation of a main species within 24 h, as observed by both 1 H-NMR and 195 Pt-NMR (Figure 3C). Particularly, the signals at time zero at 3.94 ppm and −2090 ppm for the 1 H and 195 Pt nuclei, respectively, decreased over time, with a concomitant increase of peaks at 4.73 ppm and −3138 ppm, respectively. Analysis of the NMR sample by mass spectrometry confirmed the exchange of a chlorido ligand for a DMSO-d 6 molecule (Figure 3D). Keeping the DMSO-d 6 sample at room temperature showed further conversion of the newly formed complex, which could be avoided by keeping the samples at 4°C between the analyses. At this or lower temperatures, the cisplatin-DMSO complex proved to be stable for at least several days. Taken together, NMR and mass spectrometry data indicate the formation of DMSO adduct derivatives of cisplatin in DMSOcontaining solutions. AB C FIGURE 1 | Effects of cisplatin and carboplatin on type IV secretion and on H. pylori growth. (A) Bacteria were pre-incubated for 30 min at 37°C, 10% CO 2 in PBS/ 10% FCS in the presence or absence of the indicated compounds at 50 µM, and subsequently used for co-incubation with AGS cells for 4 h in the same medium. Translocation of CagA was determined by its subsequent tyrosine phosphorylation using phosphotyrosine (PTyr) or CagA immunoblots. (B) H. pylori P12 was grown in BB/10% FCS in a microtiter plate in the presence of 0.5% DMSO (control), or of the indicated compounds at 50 µM. (C) Supernatants of AGS cells co-incubated with H. pylori under the same conditions as in (A), but with different amounts of cisplatin (CisPt), were analyzed for IL-8 concentrations by sandwich ELISA (left panel). The indicated values were normalized to the values obtained with strain P12 treated with 0.5% DMSO alone. As a control, AGS cells were treated with 20 ng/ ml TNF-ain the presence or absence of 100 µM cisplatin, and IL-8 values were normalized to those obtained for TNF-awithout cisplatin (right panel). All data represent mean values of four independent experiments with standard deviations. Lettl et al. Platinum Complex Activity Against H. pylori Frontiers in Cellular and Infection Microbiology | www.frontiersin.org December 2020 | Volume 10 | Article 6029585 Interestingly, incorporation of DMSO as a ligand in the platinum complex has been described as a functional inactivation with respect to DNA interaction, since DMSO represents a poor leaving group (Annibale et al., 1983). Therefore, dissolving cisplatin in DMSO is strongly discouraged in order not to lose its cytotoxic activity (Hall et al., 2014). Using a WST-1 cytotoxicity assay in L929 murine fibroblasts, we confirmed that cisplatin dissolved in DMF has a cytotoxic effect, whereas cisplatin dissolved in DMSO, or cisdichlorido-bis(DMSO)platinum(II), dissolved in either DMF or DMSO, showed no cytotoxic effects at the same concentrations (Figure 2F). This suggests that the inhibitory effect of cisplatin on type IV secretion is not a consequence of its DNAbinding capability. Effects of Other Platinum Complexes on Type IV Secretion Inhibition and H. pylori Growth It has been shown before that cisplatin has cytotoxic effects on E. coli and other bacteria, particularly if these have defects in their DNA damage repair pathways (Bhattacharya and Beck, 2002), suggesting that the DNA-binding properties of cisplatin are responsible for this finding. To further evaluate whether the observed type IV secretion-inhibitory effects may depend on DNA interaction, we extended our experiments to other platinum complexes with various ligands, structures, and oxidation states of platinum. First, we made use of dichloridoplatinum(II) complexes containing DMSO and Nheterocyclic carbene (NHC) ligands, which exhibit variable DNA-binding properties depending on steric shielding of their leaving groups (Muenzner et al., 2015;Rehm et al., 2018). Different dichloridoplatinum(II) complexes with imidazol-2ylidene or benzimidazol-2-ylidene ligands in combination with DMSO were found to inhibit CagA translocation, as analyzed with the TEM-1–CagA assay (Figure 4A). Some of these compounds had additional effects on H. pylori growth, but only at higher concentrations (Figure 4B). To examine the role of DMSO in these complexes, we next tested corresponding NHC complexes in which the DMSO ligand is replaced by a triphenylphosphane ligand, a variation which results in slower DNA binding due to increased shielding of the chloride leaving group, and altered responses of cancer cells (Muenzner et al., 2015). Interestingly, those NHC-triphenylphosphane complexes which did not interfere with H. pylori growth, also did not substantially inhibit CagA translocation (Figure 4C), indicating that the DMSO ligand cannot be functionally replaced by a A B DE F C FIGURE 2 | Solvent influence on type IV secretion inhibition and cytotoxicity by platinum complexes. (A) Scheme illustrating the potential chlorido to DMSO ligand exchange reaction upon solution of cisplatin in DMSO. (B) H. pylori P12 producing a TEM-1-CagA fusion was pre-incubated for 30 min with different concentrations of cisplatin diluted from corresponding stock solutions in DMSO (so that the final DMSO concentration was 0.5% in each case), subsequently co-incubated with AGS cells for 150 min, and then analyzed via the TEM-1-CagA reporter assay for translocation of CagA. Mean values of inhibition experiments were subjected to nonlinear regression analysis, resulting in the indicated IC 50 value for CagA translocation inhibition. The indicated bars represent mean values including standard deviations of six independent experiments normalized to P12 [TEM-1-CagA] left untreated, which was set to 100%. (C, D) The same experiments as in (B) were performed with cisplatin diluted from stock solutions in DMF (C), or with a cis-dichlorido-bis(DMSO)platinum(II) complex (cis-dcbd-platinum(II)) diluted from stock solutions in DMSO (D). The indicated bars represent mean values including standard deviations of four (C), or seven (D) independent experiments, respectively, normalized to P12 [TEM-1-CagA] treated with 0.5% DMSO only, which was set to 100%. Note that addition of 0.5% DMF alone reduces CagA translocation by about 20%. (E) Growth curves under standard growth conditions of H. pylori P12 in the absence (Control with 0.5% DMSO) or presence of the indicated compound concentrations taken from the respective stock solutions. Representative curves are shown. (F) Cytotoxicity of the indicated compounds, diluted to the respective concentrations from stock solutions in either DMSO or DMF, toward L929 cells, as determined by a WST-1 assay. The indicated values represent mean values including standard deviations of three independent experiments normalized to untreated control. Lettl et al. Platinum Complex Activity Against H. pylori Frontiers in Cellular and Infection Microbiology | www.frontiersin.org December 2020 | Volume 10 | Article 6029586 A B D C FIGURE 3 | Analysis of platinum complexes formed in different solvents. (A) Mass spectrum (MS) and high-resolution mass spectrum (HRMS) showing the correct isotopic pattern of the main component in the sample of cisplatin dissolved in DMSO. (B) MS and HRMS showing the isotopic pattern of cisplatin dissolved in DMSO and diluted with water/acetonitrile 70/30 containing 0.1% HCOOH. (C) Stacked 1 H-NMR and 195 Pt-NMR spectra, recorded at the indicated time points, of cisplatin dissolved either in DMF-d 7 or in DMSO-d 6 . In contrast to the DMSO-d 6 solution, cisplatin in DMF-d 7 is stable over several days. In the time between the analyses, the samples were kept at 4°C. (D) MS and HRMS spectra of the cisplatin DMSO-d6 solution analyzed by NMR. Lettl et al. Platinum Complex Activity Against H. pylori Frontiers in Cellular and Infection Microbiology | www.frontiersin.org December 2020 | Volume 10 | Article 6029587 A B D C FIGURE 4 | Inhibitory properties of NHC-platinum complexes toward type IV secretion and growth. (A) The indicated stable platinum benzimidazol-2-ylidene or imidazol-2-ylidene complexes with DMSO ligands were added to H. pylori P12 [TEM-1-CagA] pre-incubation suspensions at the indicated concentrations, and type IV translocation of CagA was determined with the TEM-1-CagA reporter assay. (B) The same complexes were added to H. pylori P12 suspensions in BB/10% FCS, and optical densities (550 nm) were recorded for 13-14 h in a microtiter plate. Growth curves are shown as optical density differences. (C) Analogous complexes with triphenylphosphane ligands instead of DMSO ligands, or complexes containing two different NHC ligands, were added at the indicated concentrations, and examined with the TEM-1-CagA reporter assay as in (A) (left panel). Growth curves in the presence or absence of the same complexes at the indicated concentrations (right panel). Some compounds partially precipitated in growth medium when added at 100 µM, and are therefore shown at 10 µM only. (D) An octahedral tetrachloridoplatinum(IV) complex with two NHC ligands, or transplatin were analyzed with the TEM-1-CagA assay at the indicated concentrations (left panel) Growth curves in the presence of the same complexes (right panels). Due to partial precipitation at 100 µM, TR425 is only shown at a concentration of 10 µM. All indicated bars represent mean values including standard deviations of at least 3 independent experiments normalized to P12 [TEM-1-CagA] treated with 0.5% DMSO, which was set to 100%. Representative growth curves are shown. Lettl et al. Platinum Complex Activity Against H. pylori Frontiers in Cellular and Infection Microbiology | www.frontiersin.org December 2020 | Volume 10 | Article 6029588 triphenylphosphane ligand. However, when we tested complexes in which the DMSO ligand was replaced by a second NHC ligand in cis configuration (Rehm et al., 2016), we found some with clear antibacterial effects toward H. pylori (data not shown), but also others which had similar properties as cisplatin or the DMSOcontaining complexes (Figure 4C). Finally, we analyzed the effect of an analogous platinum(IV) complex with two NHC ligands, which exhibits reduced DNA-binding properties and only moderate cytotoxicity (Rehm et al., 2019). Here, we also observed a substantial inhibitory potential with respect to CagA type IV secretion for compound TR425, while H. pylori growth was not strongly affected (Figure 4D). This result confirms that the strength of interaction with DNA does not correlate with type IV secretion inhibition. In the same line, we found that transplatin (trans-dichloridodiammineplatinum(II)), which does not show DNA binding-dependent cytotoxicity, but is subject to ligand exchange when dissolved in DMSO (Hall et al., 2014), still inhibits CagA translocation, albeit with a reduced efficiency in comparison to cisplatin (IC 50 = 10.9 µM; Figure 4D). Antibacterial Effects of Cisplatin on H. pylori and Influence on Adherence The results shown so far indicated that some platinum complexes with DMSO or other ligands exert inhibitory effects toward type IV secretion, but do not inhibit bacterial growth, whereas others also gave rise to growth defects. Since measuring growth curves requires the use of complex media, whereas the type IV secretion reporter assay is routinely carried out in phosphate buffer supplemented with fetal calf serum, in which the bacteria do not grow readily, we next tested whether these different media might influence the observed platinum complex effects, in addition to the solvent used for generating stock solutions. When we examined H. pylori that had been treated with cisplatin/DMSO in PBS/10% FCS during co-incubation withAGScellsfor2h,fortheirviabilitybyplatingon standard media, we noticed only weak growth (Figure 5A). This antibacterial effect of cisplatin/DMSO was not detected after treatment and co-incubation in BB/10% FCS (Figure 5A). Therefore, we carried out the TEM-CagA translocation assay in A B D C FIGURE 5 | Activities of platinum complexes in different media and during infection of AGS cells. (A) H. pylori P12 was co-incubated with AGS cells for 2 h with 100 µM cisplatin or 0.5% DMSO in the indicated media, and subsequently spotted on serum agar plates. Growth was monitored after 24 h. (B) H. pylori P12 [TEM-1-CagA] was pre-incubated and co-incubated with AGS cells in the presence of 100 µM cisplatin in PBS/10% FCS, BB/10% FCS, or mixtures of both, as indicated, and CagA translocation was determined by the TEM-1-CagA reporter assay. The same tests were also performed with BB or BHI medium alone. (C) NHC-platinum complexes TR425 and TR382 were added at the indicated concentrations to bacterial suspensions in either PBS/10% FCS, or BB/10% FCS, and CagA translocation was determined after AGS cell infection as in (B).(D) H. pylori P12 [TEM-1-CagA] was incubated in PBS/10% FCS together with 32 µM cisplatin/DMSO, or additionally with the indicated concentrations of cysteine, methionine, or alanine, and CagA translocation was determined as in (B). All bars represent mean values including standard deviations of at least 3 independent experiments normalized to P12 [TEM-1-CagA] treated with 0.5% DMSO, which was set to 100%. Lettl et al. Platinum Complex Activity Against H. pylori Frontiers in Cellular and Infection Microbiology | www.frontiersin.org December 2020 | Volume 10 | Article 6029589