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• W2300329086 abstract "•Inhibitors of L. monocytogenes infectivity reduce virulence gene expression•Binding of inhibitor to the PrfA regulator reduces affinity for its DNA motif•First crystal structure of a Crp family regulator with an inhibitor•Provides rationale for screening with Crp family transcriptional regulators The transcriptional activator PrfA, a member of the Crp/Fnr family, controls the expression of some key virulence factors necessary for infection by the human bacterial pathogen Listeria monocytogenes. Phenotypic screening identified ring-fused 2-pyridone molecules that at low micromolar concentrations attenuate L. monocytogenes cellular uptake by reducing the expression of virulence genes. These inhibitors bind the transcriptional regulator PrfA and decrease its affinity for the consensus DNA-binding site. Structural characterization of this interaction revealed that one of the ring-fused 2-pyridones, compound 1, binds at two separate sites on the protein: one within a hydrophobic pocket or tunnel, located between the C- and N-terminal domains of PrfA, and the second in the vicinity of the DNA-binding helix-turn-helix motif. At both sites the compound interacts with residues important for PrfA activation and helix-turn-helix formation. Ring-fused 2-pyridones represent a new class of chemical probes for studying virulence in L. monocytogenes. The transcriptional activator PrfA, a member of the Crp/Fnr family, controls the expression of some key virulence factors necessary for infection by the human bacterial pathogen Listeria monocytogenes. Phenotypic screening identified ring-fused 2-pyridone molecules that at low micromolar concentrations attenuate L. monocytogenes cellular uptake by reducing the expression of virulence genes. These inhibitors bind the transcriptional regulator PrfA and decrease its affinity for the consensus DNA-binding site. Structural characterization of this interaction revealed that one of the ring-fused 2-pyridones, compound 1, binds at two separate sites on the protein: one within a hydrophobic pocket or tunnel, located between the C- and N-terminal domains of PrfA, and the second in the vicinity of the DNA-binding helix-turn-helix motif. At both sites the compound interacts with residues important for PrfA activation and helix-turn-helix formation. Ring-fused 2-pyridones represent a new class of chemical probes for studying virulence in L. monocytogenes. In light of increasing antibiotic resistance, novel therapies are required to potentiate or succeed our current selection of therapeutic options (Davies and Davies, 2010Davies J. Davies D. Origins and evolution of antibiotic resistance.Microbiol. Mol. Biol. Rev. 2010; 74: 417-433Crossref PubMed Scopus (3322) Google Scholar). An alternative to classical antibiotics are drugs inhibiting the virulence of pathogenic bacteria. The first step in establishing this as a viable and effective therapeutic strategy is to understand how the virulence of pathogenic bacteria can be controlled (Allen et al., 2014Allen R.C. Popat R. Diggle S.P. Brown S.P. Targeting virulence: can we make evolution-proof drugs?.Nat. Rev. Microbiol. 2014; 12: 300-308Crossref PubMed Scopus (353) Google Scholar, Clatworthy et al., 2007Clatworthy A.E. Pierson E. Hung D.T. Targeting virulence: a new paradigm for antimicrobial therapy.Nat. Chem. Biol. 2007; 3: 541-548Crossref PubMed Scopus (974) Google Scholar, Rasko and Sperandio, 2010Rasko D.A. Sperandio V. Anti-virulence strategies to combat bacteria-mediated disease.Nat. Rev. Drug Discov. 2010; 9: 117-128Crossref PubMed Scopus (933) Google Scholar). The Gram-positive bacterium Listeria monocytogenes is a saprophyte responsible for the severe disease listeriosis in humans upon ingestion (Freitag et al., 2009Freitag N.E. Port G.C. Miner M.D. Listeria monocytogenes - from saprophyte to intracellular pathogen.Nat. Rev. Microbiol. 2009; 7: 623-628Crossref PubMed Scopus (417) Google Scholar, Vázquez-Boland et al., 2001Vázquez-Boland J.A. Kuhn M. Berche P. Chakraborty T. Domınguez-Bernal G. Goebel W. González-Zorn B. Wehland J. Kreft J. Listeria pathogenesis and molecular virulence determinants.Clin. Microbiol. Rev. 2001; 14: 584-640Crossref PubMed Scopus (1692) Google Scholar). Its ability to grow at low temperatures, in high-salt and low-oxygen conditions, makes L. monocytogenes one of the most problematic foodborne pathogens. Although the incidence rate is low, L. monocytogenes is capable of crossing key protective barriers within the body (e.g., intestinal, placental, and blood-brain) and causing severe diseases (e.g., bacteremia, meningitis, and meningoencephalitis) (Drevets and Bronze, 2008Drevets D.A. Bronze M.S. Listeria monocytogenes: epidemiology, human disease, and mechanisms of brain invasion.FEMS Immunol. Med. Microbiol. 2008; 53: 151-165Crossref PubMed Scopus (226) Google Scholar). Pregnant, immunocompromised, and other at-risk patients are vulnerable to invasive listeriosis, and the high mortality rates within these subpopulations (∼20%–40%) are a stark demonstration of the clinical difficulty in dealing with these infections (Drevets and Bronze, 2008Drevets D.A. Bronze M.S. Listeria monocytogenes: epidemiology, human disease, and mechanisms of brain invasion.FEMS Immunol. Med. Microbiol. 2008; 53: 151-165Crossref PubMed Scopus (226) Google Scholar, Hamon et al., 2006Hamon M. Bierne H. Cossart P. Listeria monocytogenes: a multifaceted model.Nat. Rev. Microbiol. 2006; 4: 423-434Crossref PubMed Scopus (459) Google Scholar, Jackson et al., 2010Jackson K.A. Iwamoto M. Swerdlow D. Pregnancy-associated listeriosis.Epidemiol. Infect. 2010; 138: 1503-1509Crossref PubMed Scopus (111) Google Scholar, Vázquez-Boland et al., 2001Vázquez-Boland J.A. Kuhn M. Berche P. Chakraborty T. Domınguez-Bernal G. Goebel W. González-Zorn B. Wehland J. Kreft J. Listeria pathogenesis and molecular virulence determinants.Clin. Microbiol. Rev. 2001; 14: 584-640Crossref PubMed Scopus (1692) Google Scholar). The virulence factors governing host invasion and infection by L. monocytogenes have been well elucidated (Vázquez-Boland et al., 2001Vázquez-Boland J.A. Kuhn M. Berche P. Chakraborty T. Domınguez-Bernal G. Goebel W. González-Zorn B. Wehland J. Kreft J. Listeria pathogenesis and molecular virulence determinants.Clin. Microbiol. Rev. 2001; 14: 584-640Crossref PubMed Scopus (1692) Google Scholar). The bacterium adheres to and enters both phagocytic and non-phagocytic cells, by using specific adhesins (e.g., InlA and InlB), depending on the cell type. With the aid of listeriolysin O (LLO) and a phospholipase (PlcA), the bacterium lyses the phagosome and proceeds to replicate intracellularly (Schnupf and Portnoy, 2007Schnupf P. Portnoy D.A. Listeriolysin O: a phagosome-specific lysin.Microbes Infect. 2007; 9: 1176-1187Crossref PubMed Scopus (278) Google Scholar). Once in the cytoplasm, the bacterium uses the ActA protein to recruit the Arp2/3 complex facilitating the formation of an actin comet-tail, by which L. monocytogenes is able to move to an adjacent cell without exposing itself to the extracellular environment. ActA has also been shown to play a role during bacterial attachment and uptake into epithelial cells (Garcia-Del Portillo and Pucciarelli, 2012Garcia-Del Portillo F. Pucciarelli M.G. Remodeling of the Listeria monocytogenes cell wall inside eukaryotic cells.Commun. Integr. Biol. 2012; 5: 160-162Crossref PubMed Google Scholar, Suarez et al., 2001Suarez M. Gonzalez-Zorn B. Vega Y. Chico-Calero I. Vazquez-Boland J.A. A role for ActA in epithelial cell invasion by Listeria monocytogenes.Cell Microbiol. 2001; 3: 853-864Crossref PubMed Scopus (105) Google Scholar). Within the next cell, the bacterium degrades the double-membrane vacuole and perpetuates the infection cycle (Hamon et al., 2006Hamon M. Bierne H. Cossart P. Listeria monocytogenes: a multifaceted model.Nat. Rev. Microbiol. 2006; 4: 423-434Crossref PubMed Scopus (459) Google Scholar, Portnoy et al., 2002Portnoy D.A. Auerbuch V. Glomski I.J. The cell biology of Listeria monocytogenes infection: the intersection of bacterial pathogenesis and cell-mediated immunity.J. Cell Biol. 2002; 158: 409-414Crossref PubMed Scopus (335) Google Scholar). The expression of the majority of virulence genes required for these processes is regulated by PrfA, a transcriptional activator from the Crp/Fnr family of regulators (Scortti et al., 2007Scortti M. Monzo H.J. Lacharme-Lora L. Lewis D.A. Vazquez-Boland J.A. The PrfA virulence regulon.Microbes Infect. 2007; 9: 1196-1207Crossref PubMed Scopus (174) Google Scholar). Members of this family bind as homodimers to consensus DNA sequences found in the promoter region of regulated genes (Won et al., 2009Won H.S. Lee Y.S. Lee S.H. Lee B.J. Structural overview on the allosteric activation of cyclic AMP receptor protein.Biochim. Biophys. Acta. 2009; 1794: 1299-1308Crossref PubMed Scopus (47) Google Scholar). PrfA positively regulates the expression of the above and other Listerial virulence factors (Freitag et al., 2009Freitag N.E. Port G.C. Miner M.D. Listeria monocytogenes - from saprophyte to intracellular pathogen.Nat. Rev. Microbiol. 2009; 7: 623-628Crossref PubMed Scopus (417) Google Scholar, Scortti et al., 2007Scortti M. Monzo H.J. Lacharme-Lora L. Lewis D.A. Vazquez-Boland J.A. The PrfA virulence regulon.Microbes Infect. 2007; 9: 1196-1207Crossref PubMed Scopus (174) Google Scholar), and a ΔprfA strain is avirulent (Andersson et al., 2015Andersson C. Gripenland J. Johansson J. Using the chicken embryo to assess virulence of Listeria monocytogenes and to model other microbial infections.Nat. Protoc. 2015; 10: 1155-1164Crossref PubMed Scopus (18) Google Scholar, Chakraborty et al., 1992Chakraborty T. Leimeister-Wächter M. Domann E. Hartl M. Goebel W. Nichterlein T. Notermans S. Coordinate regulation of virulence genes in Listeria monocytogenes requires the product of the prfA gene.J. Bacteriol. 1992; 174: 568-574PubMed Google Scholar, Freitag et al., 1993Freitag N.E. Rong L. Portnoy D.A. Regulation of the prfA transcriptional activator of Listeria monocytogenes: multiple promoter elements contribute to intracellular growth and cell-to-cell spread.Infect. Immun. 1993; 61: 2537-2544PubMed Google Scholar, Gripenland et al., 2014Gripenland J. Andersson C. Johansson J. Exploring the chicken embryo as a possible model for studying Listeria monocytogenes pathogenicity.Front. Cell. Infect. Microbiol. 2014; 4: 170PubMed Google Scholar). Structurally, each monomer of PrfA comprises an N-terminal eight-stranded β-barrel domain connected by an α helix linker to a C-terminal α/β domain (Eiting et al., 2005Eiting M. Hageluken G. Schubert W.D. Heinz D.W. The mutation G145S in PrfA, a key virulence regulator of Listeria monocytogenes, increases DNA-binding affinity by stabilizing the HTH motif.Mol. Microbiol. 2005; 56: 433-446Crossref PubMed Scopus (76) Google Scholar, Vega et al., 2004Vega Y. Rauch M. Banfield M.J. Ermolaeva S. Scortti M. Goebel W. Vazquez-Boland J.A. New Listeria monocytogenes prfA* mutants, transcriptional properties of PrfA* proteins and structure-function of the virulence regulator PrfA.Mol. Microbiol. 2004; 52: 1553-1565Crossref PubMed Scopus (64) Google Scholar). The C-terminal region contains the winged helix-turn-helix (HTH) motif responsible for binding to consensus promoter sequences (Eiting et al., 2005Eiting M. Hageluken G. Schubert W.D. Heinz D.W. The mutation G145S in PrfA, a key virulence regulator of Listeria monocytogenes, increases DNA-binding affinity by stabilizing the HTH motif.Mol. Microbiol. 2005; 56: 433-446Crossref PubMed Scopus (76) Google Scholar, Vega et al., 2004Vega Y. Rauch M. Banfield M.J. Ermolaeva S. Scortti M. Goebel W. Vazquez-Boland J.A. New Listeria monocytogenes prfA* mutants, transcriptional properties of PrfA* proteins and structure-function of the virulence regulator PrfA.Mol. Microbiol. 2004; 52: 1553-1565Crossref PubMed Scopus (64) Google Scholar). While most Crp family members require a small molecule cofactor for DNA binding (e.g., cAMP for Crp in Escherichia coli) (Won et al., 2009Won H.S. Lee Y.S. Lee S.H. Lee B.J. Structural overview on the allosteric activation of cyclic AMP receptor protein.Biochim. Biophys. Acta. 2009; 1794: 1299-1308Crossref PubMed Scopus (47) Google Scholar), PrfA is capable of binding to its DNA consensus sequences with low affinity even in the absence of a cofactor (Eiting et al., 2005Eiting M. Hageluken G. Schubert W.D. Heinz D.W. The mutation G145S in PrfA, a key virulence regulator of Listeria monocytogenes, increases DNA-binding affinity by stabilizing the HTH motif.Mol. Microbiol. 2005; 56: 433-446Crossref PubMed Scopus (76) Google Scholar). Nonetheless, the activity of PrfA is known to increase under inducible conditions, and this has led to the hypothesis that the intercellular mechanism of activation may be regulated by a host-derived or host-regulated cofactor (Freitag et al., 2009Freitag N.E. Port G.C. Miner M.D. Listeria monocytogenes - from saprophyte to intracellular pathogen.Nat. Rev. Microbiol. 2009; 7: 623-628Crossref PubMed Scopus (417) Google Scholar, Scortti et al., 2007Scortti M. Monzo H.J. Lacharme-Lora L. Lewis D.A. Vazquez-Boland J.A. The PrfA virulence regulon.Microbes Infect. 2007; 9: 1196-1207Crossref PubMed Scopus (174) Google Scholar). Recently, it was suggested that PrfA activation follows a two-step process, where PrfA needs to be in a reduced form for DNA binding, followed by interaction of PrfA with reduced glutathione for transcriptional activity at targets genes (Reniere et al., 2015Reniere M.L. Whiteley A.T. Hamilton K.L. John S.M. Lauer P. Brennan R.G. Portnoy D.A. Glutathione activates virulence gene expression of an intracellular pathogen.Nature. 2015; 517: 170-173Crossref PubMed Scopus (168) Google Scholar). Structural evidence toward an allosteric mode of activation for PrfA in vivo has been provided by the crystal structure of the constitutively active PrfA mutant PrfAG145S (Eiting et al., 2005Eiting M. Hageluken G. Schubert W.D. Heinz D.W. The mutation G145S in PrfA, a key virulence regulator of Listeria monocytogenes, increases DNA-binding affinity by stabilizing the HTH motif.Mol. Microbiol. 2005; 56: 433-446Crossref PubMed Scopus (76) Google Scholar, Ripio et al., 1997Ripio M.T. Dominguez-Bernal G. Lara M. Suarez M. Vazquez-Boland J.A. A Gly145Ser substitution in the transcriptional activator PrfA causes constitutive overexpression of virulence factors in Listeria monocytogenes.J. Bacteriol. 1997; 179: 1533-1540Crossref PubMed Scopus (137) Google Scholar, Vega et al., 2004Vega Y. Rauch M. Banfield M.J. Ermolaeva S. Scortti M. Goebel W. Vazquez-Boland J.A. New Listeria monocytogenes prfA* mutants, transcriptional properties of PrfA* proteins and structure-function of the virulence regulator PrfA.Mol. Microbiol. 2004; 52: 1553-1565Crossref PubMed Scopus (64) Google Scholar). This amino acid substitution repositions the HTH motif in an ordered and exposed “active” conformation with increased DNA-binding affinity, in contrast to PrfAWT where the HTH motif remains flexible and partially disordered (Eiting et al., 2005Eiting M. Hageluken G. Schubert W.D. Heinz D.W. The mutation G145S in PrfA, a key virulence regulator of Listeria monocytogenes, increases DNA-binding affinity by stabilizing the HTH motif.Mol. Microbiol. 2005; 56: 433-446Crossref PubMed Scopus (76) Google Scholar). We previously developed ring-fused 2-pyridone scaffolds which contain a peptidomimetic backbone (Svensson et al., 2001Svensson A. Larsson A. Emtenäs H. Hedenström M. Fex T. Hultgren S.J. Pinkner J.S. Almqvist F. Kihlberg J. Design and evaluation of pilicides: potential novel antibacterial agents directed against uropathogenic Escherichia coli.ChemBioChem. 2001; 2: 915-918Crossref PubMed Scopus (119) Google Scholar). Originally designed to mimic interactions between subunits in the chaperone-usher pathway responsible for pilus assembly in uropathogenic bacteria, we subsequently developed inhibitors with discrete substitution patterns from this scaffold, which both interact with structures important for E. coli adhesion to eukaryotic cells (Cegelski et al., 2009Cegelski L. Pinkner J.S. Hammer N.D. Cusumano C.K. Hung C.S. Chorell E. Åberg V. Walker J.N. Seed P.C. Almqvist F. et al.Small-molecule inhibitors target Escherichia coli amyloid biogenesis and biofilm formation.Nat. Chem. Biol. 2009; 5: 913-919Crossref PubMed Scopus (322) Google Scholar, Emtenäs et al., 2002Emtenäs H. Åhlin K. Pinkner J.S. Hultgren S.J. Almqvist F. Design and parallel solid-phase synthesis of ring-fused 2-pyridinones that target pilus biogenesis in pathogenic bacteria.J. Comb. Chem. 2002; 4: 630-639Crossref PubMed Scopus (63) Google Scholar, Pinkner et al., 2006Pinkner J.S. Remaut H. Buelens F. Miller E. Aberg V. Pemberton N. Hedenstrom M. Larsson A. Seed P. Waksman G. et al.Rationally designed small compounds inhibit pilus biogenesis in uropathogenic bacteria.Proc. Natl. Acad. Sci. USA. 2006; 103: 17897-17902Crossref PubMed Scopus (244) Google Scholar) and had broader impact on virulence regulation (Greene et al., 2014Greene S.E. Pinkner J.S. Chorell E. Dodson K.W. Shaffer C.L. Conover M.S. Livny J. Hadjifrangiskou M. Almqvist F. Hultgren S.J. Pilicide ec240 disrupts virulence circuits in uropathogenic Escherichia coli.MBio. 2014; 5: e02038Crossref Scopus (56) Google Scholar). We were therefore interested to investigate whether 2-pyridones could affect L. monocytogenes virulence-associated phenotypes. In this study, we have conducted a phenotypic screen and identified several ring-fused 2-pyridones that attenuate L. monocytogenes uptake into epithelial cells and decrease virulence gene expression. We describe how these inhibitors interact directly with the transcriptional regulator PrfA and weaken its DNA-binding capacity. Furthermore, we provide the first structural detail of a Crp family protein with a bound inhibitor by presenting the crystal structure of PrfA in complex with one 2-pyridone, and propose possible modes of action. Using flow cytometry, we performed an infection screen based on HeLa cells infected with GFP-carrying L. monocytogenes. The putative virulence-inhibiting ability of ring-fused 2-pyridones from our in-house collection was assessed, and several close analogs that significantly reduced the relative infection at 100 μM were identified (Figures S1A and S1B). At a concentration of 10 μM, two 1-naphthyl derivatives effectively reduced L. monocytogenes uptake by HeLa cells by 80%–90% (C10 [1] and KSK 67 [2]), whereas a related 3-quinoline analog, KSK 29 (3), was less effective (Figure S1B). Viable count experiments verified these flow cytometry results, with compounds 1 or 2 reducing the level of L. monocytogenes uptake relative to untreated controls at 10 and 1 μM (Figure 1A ). Furthermore, when we performed a time-course experiment monitoring the infection dynamics more closely, we observed an inability of L. monocytogenes to replicate within Caco-2 cells after treatment with compound 2, which displayed the largest efficiency in the uptake experiment (Figure 1B, left panel). Although the generation time of L. monocytogenes was slightly longer in presence of compounds 1 or 2 at 100 μM compared with the DMSO control (45, 50, and 43 min, respectively), it is unlikely that this would be sufficient to explain the dramatic decrease in infectivity observed with these compounds at 100 μM and at lower concentrations (Figures S1C and 1). The generation time in the presence of compound 3 was unaffected compared with the DMSO control (43 min, Figure S1C). To understand how the compounds attenuated the infectivity of L. monocytogenes, the expression of virulence genes was analyzed in bacteria grown with or without 100 μM of 1, 2, or 3. We initially investigated the expression of the hly gene which encodes the hemolysin LLO, and found it was downregulated by 1 or 2 compared with the untreated control, but not affected by compound 3 (Figure S2). Together with most virulence genes in L. monocytogenes, hly is positively regulated by the transcriptional activator PrfA (Freitag et al., 2009Freitag N.E. Port G.C. Miner M.D. Listeria monocytogenes - from saprophyte to intracellular pathogen.Nat. Rev. Microbiol. 2009; 7: 623-628Crossref PubMed Scopus (417) Google Scholar, Scortti et al., 2007Scortti M. Monzo H.J. Lacharme-Lora L. Lewis D.A. Vazquez-Boland J.A. The PrfA virulence regulon.Microbes Infect. 2007; 9: 1196-1207Crossref PubMed Scopus (174) Google Scholar), therefore we examined whether the expression of other PrfA-regulated genes was affected by treatment with compounds 1 and 2. A reduction in the levels of the virulence genes hly, actA, and plcA transcripts could be observed after treatment with compounds 1 and 2 (but not 3), whereas the expression of inlA and inlB was unaffected (Figure S2), possibly because basal expression of the latter are also controlled by other regulatory factors, such as the stress sigma factor σB (Stritzker et al., 2005Stritzker J. Schoen C. Goebel W. Enhanced synthesis of internalin A in aro mutants of Listeria monocytogenes indicates posttranscriptional control of the inlAB mRNA.J. Bacteriol. 2005; 187: 2836-2845Crossref PubMed Scopus (32) Google Scholar). We next investigated whether the virulence protein levels of the two major virulence factors, LLO and ActA, were affected by compound treatment. Of the 2-pyridones tested, compounds 1 and 2 displayed similar properties. At 100 μM of 1 or 2, expression of LLO and ActA was abolished, without a concomitant effect on PrfA protein levels or on the expression of the non-PrfA-regulated virulence factor P60 (Figures 2A and 2B). The reduction in LLO and ActA expression following treatment with 1 and 2 was dose dependent, with decreased levels down to ≤3.3 μM (Figures 2A and 2B). In contrast, compound 3 did not reduce LLO or ActA expression even at the highest concentration tested of 100 μM, in agreement with the lack of efficacy of 3 in abrogating hly or actA gene expression (Figures 2C and S2). Across all concentrations of 1, 2, or 3, PrfA expression remained unchanged, indicating that the virulence attenuation of 1 and 2 was not mediated through reduced PrfA protein levels (Figures 2A–2C). Once a host cell is invaded by L. monocytogenes, ActA expression is massively induced in a PrfA-dependent manner (Freitag et al., 2009Freitag N.E. Port G.C. Miner M.D. Listeria monocytogenes - from saprophyte to intracellular pathogen.Nat. Rev. Microbiol. 2009; 7: 623-628Crossref PubMed Scopus (417) Google Scholar, Scortti et al., 2007Scortti M. Monzo H.J. Lacharme-Lora L. Lewis D.A. Vazquez-Boland J.A. The PrfA virulence regulon.Microbes Infect. 2007; 9: 1196-1207Crossref PubMed Scopus (174) Google Scholar). We therefore analyzed whether ActA expression was affected at different time points when 100 μM of compound 2 was added 30 min post infection. The induction of intracellular ActA levels observed in the DMSO control 4 hr post infection, was weakened in compound-treated cells, although not completely abolished (Figure 2D). This indicates that the effectiveness of the compounds decreased post infection. We next examined whether ring-fused 2-pyridones could also reduce virulence factor expression in other L. monocytogenes strain backgrounds or serotypes. Addition of compound 1 effectively reduced LLO expression in L. monocytogenes serotypes associated with sporadic cases (10403S [serotype 1/2a] and LO28 [serotype 1/2c]) as well as a serotype associated with epidemic outbreaks (F2365 [serotype 4b]), whereas the levels of PrfA were unaffected in these strains (Figure 3A ). In addition, compound 2 effectively inhibited uptake of the F2365 strain into Caco-2 cells (Figure 3B).Figure 3Compounds 1 and 2 Reduce Virulence Factor Expression and Uptake of Multiple Serotypes of L. monocytogenesShow full caption(A) Compound 1 reduces LLO expression in different L. monocytogenes strain backgrounds. Protein extracts were isolated from indicated L. monocytogenes strain in the absence (C, equivalent volume DMSO) or presence of 100 μM of 1 and the specified proteins (LLO PrfA, or P60 (control)) were detected by western blot using specific antibodies. Upper panels show secreted fractions (Sup) of indicated samples; lower panels show whole-cell fractions (WC) of indicated samples.(B) Compound 2 inhibits the uptake of an L. monocytogenes strain of serovar 4b. L. monocytogenes strain F2365 was allowed to infect Caco-2 cells for 2 hr with 2 (50 μM). All samples were correlated to the DMSO-treated control (C, gray bar) which was arbitrarily set at 100%. Error bars show SDs. Significance was tested using Student's t test (two-tailed, significant differences are shown by asterisks; ***p < 0.001) and Dunnett's test (significant differences to the control were shown by #).View Large Image Figure ViewerDownload (PPT) (A) Compound 1 reduces LLO expression in different L. monocytogenes strain backgrounds. Protein extracts were isolated from indicated L. monocytogenes strain in the absence (C, equivalent volume DMSO) or presence of 100 μM of 1 and the specified proteins (LLO PrfA, or P60 (control)) were detected by western blot using specific antibodies. Upper panels show secreted fractions (Sup) of indicated samples; lower panels show whole-cell fractions (WC) of indicated samples. (B) Compound 2 inhibits the uptake of an L. monocytogenes strain of serovar 4b. L. monocytogenes strain F2365 was allowed to infect Caco-2 cells for 2 hr with 2 (50 μM). All samples were correlated to the DMSO-treated control (C, gray bar) which was arbitrarily set at 100%. Error bars show SDs. Significance was tested using Student's t test (two-tailed, significant differences are shown by asterisks; ***p < 0.001) and Dunnett's test (significant differences to the control were shown by #). The reduced levels of the virulence factors LLO and ActA, but not of PrfA (Figures 2A and 2B), suggested that 1 and 2 directly affected the activity of PrfA. Introducing a G145S amino acid substitution in PrfA generates a constitutively active mutant that contains a stabilized HTH DNA-binding motif, as opposed to the structurally undefined HTH present in PrfAWT (Eiting et al., 2005Eiting M. Hageluken G. Schubert W.D. Heinz D.W. The mutation G145S in PrfA, a key virulence regulator of Listeria monocytogenes, increases DNA-binding affinity by stabilizing the HTH motif.Mol. Microbiol. 2005; 56: 433-446Crossref PubMed Scopus (76) Google Scholar, Ripio et al., 1997Ripio M.T. Dominguez-Bernal G. Lara M. Suarez M. Vazquez-Boland J.A. A Gly145Ser substitution in the transcriptional activator PrfA causes constitutive overexpression of virulence factors in Listeria monocytogenes.J. Bacteriol. 1997; 179: 1533-1540Crossref PubMed Scopus (137) Google Scholar). Examining the effect of the compounds on PrfAG145S in comparison with PrfAWT would indicate whether 1 and 2 directly inhibited the PrfA activation process, or if their effects were mediated after formation of the HTH DNA-binding motif. To test this, a prfAG145S allele was introduced at its native site on the chromosome, such that PrfAG145S would be produced, and this strain was treated with 1, 2, or 3 at 100 μM. In the PrfAG145S-expressing strain, virulence gene expression as well as virulence protein levels remained essentially unaltered in the presence of the compounds (Figures 4 and S2). Once again, the levels of PrfA and P60 proteins did not vary considerably after treatment with the different compounds. We next challenged whether this mutation could also overcome the inhibitory effects of these compounds in a time-course cellular infection assay, with the most effective compound from the cellular uptake experiments, compound 2. While L. monocytogenes containing PrfAWT was unable to replicate within Caco-2 cells in presence of 2 at 100 μM (Figure 1B, left panel), a strain expressing PrfAG145S overcame the inhibitory effects and established an infection comparable with the untreated control (Figure 1B, right panel). Collectively, these data strongly indicated that the 2-pyridones 1 and 2 reduced L. monocytogenes virulence by attenuating PrfA activity, and furthermore suggested that this process occurred prior to formation of the DNA-binding HTH motif, since the PrfAG145S protein carrying the stabilized HTH motif was not affected by the compounds. We next determined whether 1 and 2 directly bound to PrfA via isothermal titration calorimetry (ITC). This in vitro method, with purified PrfA protein, allows measurement of the thermodynamic parameters of binding from the heat development that occurs upon ligand-protein interactions (Ladbury et al., 2010Ladbury J.E. Klebe G. Freire E. Adding calorimetric data to decision making in lead discovery: a hot tip.Nat. Rev. Drug Discov. 2010; 9: 23-27Crossref PubMed Scopus (321) Google Scholar). The data presented in Figure 5A show that binding of 1 to PrfAWT occurs with negative enthalpy in a 1 to 1 stoichiometry per monomer of homodimeric PrfA. From analysis of the integrated heat peaks as a function of ligand-to-protein ratio using a 1 to 1 binding model, a dissociation constant (KD) value for 1 binding to PrfA of ≈1 μM was determined (Table 1). Compound 2, and unexpectedly 3, also exhibited micromolar affinities for PrfAWT and interacted in 1 to 1 stoichiometry (Table 1 and Figure S3). We examined the binding of compounds 1–3 to the constitutively active PrfA mutant PrfAG145S and found that all three ligands bound to PrfAG145S with comparable affinity for 2 and reduced affinity for 1 and 3 (Table 1). These data suggested that the compounds do not bind at the DNA-binding HTH motif, since this region differs in conformation between the mutant (folded) versus the wild-type (WT) (partially disordered) (Eiting et al., 2005Eiti" @default.
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• W2300329086 title "Attenuating Listeria monocytogenes Virulence by Targeting the Regulatory Protein PrfA" @default.
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• W2300329086 cites W1966612320 @default.
• W2300329086 cites W1973843350 @default.
• W2300329086 cites W1981202673 @default.
• W2300329086 cites W1982845801 @default.
• W2300329086 cites W1984146298 @default.
• W2300329086 cites W1985108480 @default.
• W2300329086 cites W1985983024 @default.
• W2300329086 cites W1987453614 @default.
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• W2300329086 cites W1996633470 @default.
• W2300329086 cites W2005476432 @default.
• W2300329086 cites W2007839979 @default.
• W2300329086 cites W2020646005 @default.
• W2300329086 cites W2026701302 @default.
• W2300329086 cites W2047909468 @default.
• W2300329086 cites W2059206113 @default.
• W2300329086 cites W2060816398 @default.
• W2300329086 cites W2062688434 @default.
• W2300329086 cites W2063776073 @default.
• W2300329086 cites W2078341793 @default.
• W2300329086 cites W2078739770 @default.
• W2300329086 cites W2086036297 @default.
• W2300329086 cites W2088966542 @default.
• W2300329086 cites W2091213797 @default.
• W2300329086 cites W2091237831 @default.
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• W2300329086 cites W2094183205 @default.
• W2300329086 cites W2098089372 @default.
• W2300329086 cites W2101830323 @default.
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• W2300329086 cites W2108638835 @default.
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• W2300329086 cites W2115906498 @default.
• W2300329086 cites W2116967113 @default.
• W2300329086 cites W2125926815 @default.
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