Mario Huesca

Enterohemorrhagic Escherichia coli Induces Apoptosis Which Augments Bacterial Binding and Phosphatidylethanolamine Exposure on the Plasma Membrane Outer Leaflet

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) is a gastrointestinal pathogen that causes watery diarrhea and hemorrhagic colitis and can lead to serious and even fatal complications such as hemolytic uremic syndrome. We investigated the ability of EHEC to kill host cells using three human epithelial cell lines. Analysis of phosphatidylserine expression, internucleosomal cleavage of host cell DNA and morphological changes detected by electron microscopy changes revealed evidence of apoptotic cell death. The rates and extents of cell death were similar for both verotoxin-producing and nonproducing strains of EHEC as well as for a related gastrointestinal pathogen, enteropathogenic E. coli (EPEC). The induction of apoptosis by bacterial attachment was independent of verotoxin production and greater than that produced by a similar treatment with verotoxin alone. Expression of phosphatidylethanolamine, previously reported to bind EHEC and EPEC, was also increased on apoptotic cells but with little correlation to phosphatidylserine expression. Phosphatidylethanolamine levels but not phosphatidylserine levels on dying cells correlated with EHEC binding. Cells treated with phosphatidylethanolamine-containing liposomes also showed increased EHEC binding. These results suggest that bacterial induction of apoptosis offers an advantage for bacterial attachment by augmenting outer leaflet levels of the phosphatidylethanolamine receptor.

Variance in Fibronectin Binding and fnb Locus Polymorphisms in Staphylococcus aureus: Identification of Antigenic Variation in a Fibronectin Binding Protein Adhesin of the Epidemic CMRSA-1 Strain of Methicillin-Resistant S. aureus

ABSTRACT The fnbA and fnbB genes ofStaphylococcus aureus 8325-4 encode fibronectin (Fn) binding proteins FnBPA and FnBPB, which promote adherence to host tissues. Each adhesin contains three copies of a repeated D motif that binds Fn and is a target for vaccine development. In this study, we assess variability within the Fn-binding domain of the FnBP adhesins and evaluate factors that promote variance in Fn binding among clinical isolates. Based on variation in the number of fnb genes or the number of D motifs, we identified five polymorphism groups.S. aureus 8325-4 and 91% of methicillin-resistantS. aureus (MRSA) isolates belong to polymorphism group I, with two fnb genes and three copies of the D motif. Polymorphism group II contained one fnb gene with only two D motifs and was associated with the epidemic CMRSA-4 strain, which exhibited high protease activity and low Fn binding. Polymorphism group III was unique to the epidemic CMRSA-1 strain, defined by the presence of a fourth D motif that exhibited antigenic variation within a conserved sequence that is essential for Fn binding. However, the sequence of the D motifs was otherwise highly conserved among the other polymorphism groups. Variation in Fn binding among MRSA isolates was inversely related to protease activity but not to the number offnb genes or the number of D motifs. Therefore, thefnb locus is polymorphic in a small number of strains, but this does not contribute to variation in Fn binding. The antigenic variation that was observed only in the epidemic CMRSA-1 strain may have evolved in response to a host immune response encountered during successive cycles of colonization, transmission, and infection in the nosocomial environment.

Inhibition of Helicobacter pylori and Helicobacter mustelae Binding to Lipid Receptors by Bovine Colostrum

Helicobacter pylori, the etiologic agent of chronic-active gastritis and duodenal ulcers in humans, and Helicobacter mustelae, a gastric pathogen in ferrets, bind to phosphatidylethanolamine (PE), a constituent of host gastric mucosal cells, and to gangliotetraosylceramide (Gg4) and gangliotriaosylceramide (Gg3). The effect of a bovine colostrum concentrate (BCC) on the interaction of H. pylori and H. mustelae to their lipid receptors was examined. BCC blocked attachment of both species to Gg4, Gg3, and PE. Partial inhibition of binding was observed with native bovine and human colostra. BCC lacked detectable antibodies (by immunoblotting) to H. pylori surface proteins (adhesins). However, colostral lipid extracts contained PE and lyso-PE that bound H. pylori in vitro. These results indicate that colostrum can block the binding of Helicobacter species to select lipids and that binding inhibition is conferred, in part, by colostral PE or PE derivatives. Colostral lipids may modulate the interaction of H. pylori and other adhesin-expressing pathogens with their target tissues.

Adhesion and Virulence Properties of Epidemic Canadian Methicillin-Resistant Staphylococcus aureus Strain 1: Identification of Novel Adhesion Functions Associated with Plasmin-Sensitive Surface Protein

Epidemic Canadian methicillin-resistant Staphylococcus aureus strain 1 (CMRSA-1) comprises related subtypes that differ in phenotype and prevalence, with subtypes 1A, 1B, and 1D representing 1%, 71%, and 18%, respectively, of total CMRSA-1 isolates. The predominant CMRSA-1B subtype possesses a variant of the staphylococcal cassette chromosome mec, harboring pls, which encodes plasmin-sensitive surface protein (Pls). CMRSA-1B cells that express Pls exhibited poor adhesion to keratinocyte extracellular matrix. However, CMRSA-1B and purified Pls adhered to cellular lipids and glycolipids, and Pls promoted bacterial cell-cell interactions. Although exoprotein expression was restricted to a precursor form of lipase in CMRSA-1B, it was not attenuated in virulence relative to CMRSA-1A, which exhibits normal exoprotein expression. In contrast, CMRSA-1D exhibited a pleiotropic defect in exoprotein expression and attenuated virulence relative to CMRSA-1A. These data indicate that the high transmissibility of CMRSA-1B was not achieved at the expense of attenuated virulence and that Pls confers a novel adhesion mechanism.

Synthetic Peptide Immunogens Elicit Polyclonal and Monoclonal Antibodies Specific for Linear Epitopes in the D Motifs of Staphylococcus aureus Fibronectin-Binding Protein, Which Are Composed of Amino Acids That Are Essential for Fibronectin Binding

ABSTRACT A fibronectin (Fn)-binding adhesin of Staphylococcus aureus contains three tandem 37- or 38-amino-acid motifs (D1, D2, and D3), which function to bind Fn. Plasma from patients with S. aureus infections contain antibodies that preferentially recognize ligand induced binding sites in the D motifs and do not inhibit Fn binding (F. Casolini, L. Visai, D. Joh, P. G. Conaldi, A. Toniolo, M. Höök, and P. Speziale, Infect. Immun. 66:5433–5442, 1998). To eliminate the influence of Fn binding on antibody development, we used synthetic peptide immunogens D121–34 and D320–33, which each contain a conserved pattern of amino acids that is essential for Fn binding but which cannot bind Fn without N- or C-terminal extensions. The D320–33 immunogen promoted the production of polyclonal antibodies that were 10-fold more effective as inhibitors of Fn-binding to the D3 motif than antibodies obtained by immunizing with an extended peptide D316–36, which exhibits functional Fn binding. The D320–33 immunogen also facilitated the production of a monoclonal antibody, 9C3, which was highly specific for the epitope SVDFEED, and abolished Fn binding by the D3 motif. When mixed with polyclonal anti-D121–34 immunoglobulin G, 70% inhibition of Fn binding to the three tandem D motifs was achieved compared to no more than 30% inhibition with either antibody preparation alone. Therefore, by immunizing with short synthetic peptides that are unable to bind Fn, we have effectively stimulated the production of antibodies specific for epitopes comprised of amino acids that are essential for Fn binding. Although these epitopes occur within a conserved pattern of amino acids that is required for Fn binding, the antibodies recognized specific linear epitope sequences and not a conserved structure common to all repeated motifs.

Comparison of Helicobacter mustelae and Helicobacter pylori adhesion to eukaryotic cells in vitro

BACKGROUND & AIMS Bacterial adhesion to mucosal surfaces is an important pathogenic mechanism for Helicobacter-induced gastritis. The aims of this study were to compare binding of selected Helicobacter mustelae and Helicobacter pylori strains to lipids extracted from HEp-2, Chinese hamster ovary, human embryonic lung cells, and ferret gastrointestinal tissues as well as to intact tissue culture cells and to analyze the fatty acids of the receptor. METHODS Thin-layer chromatography overlay binding and a receptor-based immunoassay detected adhesion of bacteria to commercial lipids and to individual species within the lipid extracts. H. mustelae binding to tissue culture cells was performed by whole cell bacterial adhesion assay. RESULTS H. mustelae and H. pylori both bound to phosphatidylethanolamine and lysophosphatidylethanolamine. Adhesion of H. mustelae to intact eukaryotic cells correlated with the amount of phosphatidylethanolamine. Binding of helicobacters was greater to lipids derived from ferret antrum compared with colon (P < 0.05). Biochemical analysis suggested that heterogeneity in fatty acid composition of phosphatidylethanolamine could influence the degree of Helicobacter binding. CONCLUSIONS Adhesion of Helicobacter strains correlates with the quantity of phosphatidylethanolamine present in the epithelial cell and with the differences in the fatty acid profile of the lipid.

A novel small molecule with potent anticancer activity inhibits cell growth by modulating intracellular labile zinc homeostasis

ML-133 is a novel small molecule with potent antiproliferative activity, as shown in cancer cell lines and in a human colon tumor xenograft model. ML-133 reduces the concentration of intracellular labile zinc in HT-29 colon cancer cells, leading to induction of the Krüppel-like factor 4 transcription factor. Krüppel-like factor 4 displaces the positive regulator SP1 from the cyclin D1 promoter, thereby negatively regulating the expression of cyclin D1 and promoting the G1-S phase arrest of cell proliferation. The antiproliferative and antitumor activity of ML-133 described in the present study suggests modulation of intracellular zinc homeostasis as a potential strategy for the treatment of several cancer types, and ML-133 represents a promising new class of antitumor agents that deserves further development. [Mol Cancer Ther 2009;8(9):2586–96]

Potent antimicrobial activity of 3-(4,5-diaryl-1H-imidazol-2-yl)-1H-indole derivatives against methicillin-resistant Staphylococcus aureus

A new series of antimicrobial derivatives [3-(4,5-diaryl-1H-imidazol-2-yl)-1H-indole)] have been synthesized with potent activity against strains of Staphylococcus aureus, including methicillin-resistant strains (MRSA). Compound 17 [3-(4,5-bis(4-fluorophenyl)-1H-imidazol-2-yl)-5-bromo-1H-indole], the most active derivative was shown to inhibit the growth of all Gram-positive strains tested, including vancomycin resistant Enterococcus faecalis and Enterococcus faecium with no activity against Gram-negative bacteria.

Abstract 3710: Preclinical dose scheduling studies of LOR-253, a novel anticancer drug, in combination with chemotherapeutics in lung and colon cancers

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL LOR-253 is an anticancer small molecule that is currently in a Phase I clinical study in patients with advanced or metastatic solid tumors. LOR-253 has a novel anticancer mechanism based on chelation of intracellular labile zinc, leading to inhibition of angiogenesis as well as G1/S cell cycle arrest due to induction of the tumor suppressor Kruppel-like factor 4 (KLF4). In preclinical studies, LOR-253 has shown potent antitumor activity against several cancers, including non-small cell lung cancer (NSCLC) and colon cancer, without significant toxicity. To support the clinical development of LOR-253 in combination with chemotherapeutics, we investigated the effect of dose scheduling of LOR-253 plus chemotherapy agents on anticancer activity in NSCLC (H226) and colon cancer (SW620) cell lines. Treatment of H226 cells in vitro with docetaxel, paclitaxel, or cisplatin for two days followed by increasing doses of LOR-253 resulted in significantly higher anti-proliferative activity than either agent alone. Assessment of drug interactions by determination of the combination index (D) (Berenbaum MC, Adv Cancer Res., 1981; 35:269-335) showed that the anticancer activities of the combination of LOR-253 with these chemotherapy agents were synergistic (D < 1). In studies with LOR-253 and docetaxel, synergistic anticancer activity against H226 was maintained with either concurrent or sequential treatments of each agent. Anticancer synergy was also seen in SW620 cells treated with LOR-253 in combination with oxaliplatin, CPT-11, or Fluorouracil. The effects of dose scheduling on activity of LOR-253 and docetaxel in vivo was also examined. Nude mice with H226 tumor xenografts were treated with repeat cycles of docetaxel once per week, followed two days later by treatment with LOR-253 for three consecutive days. Docetaxel was administered at an experimentally-determined ED50 dose level (defined as the effective dose at which 50% of mice showed tumor growth inhibition), while LOR-253 was given at its maximum efficacious dose in this model (10 mg/kg) or sub-optimal dose (5 mg/kg). Sequential administration of docetaxel followed by LOR-253 showed significant antitumor activity when docetaxel was given at either ED50 or sub-ED50 doses plus 10 mg/kg of LOR-253, compared to either agent alone given at the same dose, or with concurrent administration of LOR-253 and docetaxel at these dose levels. Preliminary results also show significant antitumor activity of LOR-253 plus docetaxel against H226 tumors in mice treated with repeat cycles of the reverse order of these treatments (LOR-253 for three days, followed by docetaxel 1X/week). In summary, dose scheduling studies with LOR-253 plus chemotherapy drugs demonstrate strong anticancer activities in NSCLC and colon cancer, providing support for the design of LOR-253 combination strategies for treatment of these cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3710. doi:1538-7445.AM2012-3710

Abstract 667: Anticancer activity and tumor selectivity of LOR-253, a novel drug candidate, in lung carcinoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL LOR-253 is a novel drug candidate entering a first in man dose escalation Phase I clinical trial in patients with advanced solid tumors. LOR-253 was well tolerated in single and repeat-dose GLP-toxicology studies conducted in rats and dogs at significantly higher doses than equivalent efficacious doses determined in xenograft models in mice, which indicates a potential wide therapeutic window. Previously we reported that LOR-253 reduced the levels of intracellular labile zinc leading to cell cycle arrest, through the down regulation of the metal-responsive transcription factor 1 (MTF-1) and the induction of the Kruppel like factor 4 (KLF4). KLF4 is a G1/S cell cycle checkpoint gene with tumor suppressor function in a variety of cancers. A recent study demonstrated dramatically decreased levels of KLF4 expression in most primary lung tumors (Hu et al. Clin Cancer Res 2009; 15 (18): 5688-95). Anti-tumor activity of KLF4 was demonstrated when KLF4 expression was enforced in cancer cell lines with low basal KLF4 expression levels. In addition, tumor growth in animals was highly compromised when KLF4 gene was stably transfected. The aim of this study was to investigate differential effects of LOR-253 on normal lung fibroblasts vs lung cancer cell lines with different basal levels of KLF4 expression. We found an inverse correlation between LOR-253 sensitivity and basal KLF4 expression. LOR-253 was highly active in three different NSCLC cell lines (H1299, H322 and H226) as shown by much lower IC50 values. KLF4 expression levels in these cell lines were significantly lower than that in normal cells. In contrast, tumor cell lines with KLF4 expression levels similar or higher than normal cells were less sensitive to LOR-253 treatment. Similar results were obtained in in vivo xenograft models, which showed higher in vivo efficacy of LOR-253 in treated mice harboring low basal KLF4-expressing tumors. A time-course gene and protein expression of KLF4 and cyclin D1 in normal lung fibroblast (HFL1) and in NSCLC H226 cells treated with LOR-253 demonstrated a differential effect between normal vs cancer cells. A maximum expression of KLF4 and suppression of cyclin D1 in H226 cells were observed between 16 and 24 hours of treatment while there was no significant effect on HFL1 cells. Based on these results the cell line H226 was selected for further LOR-253 target validation and efficacy biomarker expression studies. A dose-response effect of LOR-253 treatment on tumor growth and KLF4 and cyclin D1 expression was observed in the H226 xenograft model using RT-PCR, Western blot and immunohistochemical analysis. Studies are underway to determine the expression levels of other biomarkers such as MTF-1, HIF-1 a and b-catenin in response to LOR-253 treatment, as well as the effect of in vivo gene knock down and over-expression of MTF-1, KLF4 and other biomarkers on the antitumor activity of LOR-253. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 667. doi:10.1158/1538-7445.AM2011-667