RESUMEN
Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.
Asunto(s)
Antibacterianos/farmacología , Bacteriemia , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Espacio Intracelular/microbiología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/efectos de los fármacos , Vancomicina/farmacología , Animales , Antibacterianos/uso terapéutico , Bacteriemia/tratamiento farmacológico , Bacteriemia/microbiología , Portador Sano/tratamiento farmacológico , Portador Sano/microbiología , Diseño de Fármacos , Femenino , Inmunoconjugados/química , Espacio Intracelular/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Ratones , Pruebas de Sensibilidad Microbiana , Fagosomas/efectos de los fármacos , Fagosomas/metabolismo , Fagosomas/microbiología , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/patología , Staphylococcus aureus/patogenicidad , Vancomicina/uso terapéuticoRESUMEN
Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.
Asunto(s)
Proteínas Bacterianas/inmunología , Glicosiltransferasas/inmunología , Interacciones Huésped-Patógeno/inmunología , Staphylococcus aureus Resistente a Meticilina/fisiología , Infecciones Estafilocócicas/inmunología , Staphylococcus epidermidis/fisiología , Factores de Virulencia/inmunología , Animales , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Adhesión Bacteriana/genética , Adhesión Bacteriana/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catepsina G/genética , Catepsina G/inmunología , Catepsina G/metabolismo , Línea Celular Tumoral , Pared Celular/enzimología , Pared Celular/genética , Pared Celular/inmunología , Epítopos/genética , Epítopos/inmunología , Epítopos/metabolismo , Femenino , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Interacciones Huésped-Patógeno/genética , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Masculino , Ratones , Secuencias Repetitivas de Aminoácido , Infecciones Estafilocócicas/enzimología , Infecciones Estafilocócicas/genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Due to the increasing need of new treatment options against bacterial lung infections, novel antimicrobial peptides (AMPs) are under development. Local bioavailability and less systemic exposure lead to the inhalation route of administration. Combining AMPs with nanocarriers (NCs) into nanosystems (NSs) might be a technique for improved results. An air-liquid interface (ALI) in vitro inhalation model was set up including a human alveolar lung cell line (A549) and an optimized exposure system (P.R.I.T.® ExpoCube®) to predict acute local lung toxicity. The approach including aerosol controls (cupper-II-sulfate and lactose) delivered lowest observable adverse effect levels (LOAELs). Different combinations of AMPs (AA139, M33) and NCs (polymeric nanoparticles (PNPs), micelles and liposomes) were tested under ALI and submerged in vitro conditions. Depending on the nature of AMP and NCs, packing of AMPs into NSs reduced the AMP-related toxicity. Large differences were found between the LOAELs determined by submerged or ALI testing with the ALI approach indicating higher sensitivity of the ALI model. Since aerosol droplet exposure is in vivo relevant, it is assumed that ALI based results represents the more significant source than submerged testing for in vivo prediction of local acute lung toxicity. In accordance with the current state-of-the-art view, this study shows that ALI in vitro inhalation models are promising tools to further develop in vitro methods in the field of inhalation toxicology.
Asunto(s)
Antibacterianos/toxicidad , Nanopartículas/toxicidad , Péptidos/toxicidad , Células A549 , Aerosoles , Antibacterianos/administración & dosificación , Infecciones Bacterianas/tratamiento farmacológico , Supervivencia Celular/efectos de los fármacos , Humanos , Liposomas , Pulmón/efectos de los fármacos , Enfermedades Pulmonares/tratamiento farmacológico , Metacrilatos/administración & dosificación , Metacrilatos/toxicidad , Micelas , Nanopartículas/administración & dosificación , Nylons/toxicidad , Péptidos/administración & dosificaciónRESUMEN
Peptide antibiotics are an abundant and synthetically tractable source of molecular diversity, but they are often cationic and can be cytotoxic, nephrotoxic and/or ototoxic, which has limited their clinical development. Here we report structure-guided optimization of an amphipathic peptide, arenicin-3, originally isolated from the marine lugworm Arenicola marina. The peptide induces bacterial membrane permeability and ATP release, with serial passaging resulting in a mutation in mlaC, a phospholipid transport gene. Structure-based design led to AA139, an antibiotic with broad-spectrum in vitro activity against multidrug-resistant and extensively drug-resistant bacteria, including ESBL, carbapenem- and colistin-resistant clinical isolates. The antibiotic induces a 3-4 log reduction in bacterial burden in mouse models of peritonitis, pneumonia and urinary tract infection. Cytotoxicity and haemolysis of the progenitor peptide is ameliorated with AA139, and the 'no observable adverse effect level' (NOAEL) dose in mice is ~10-fold greater than the dose generally required for efficacy in the infection models.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Bacterias Gramnegativas/efectos de los fármacos , Animales , Carbapenémicos/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Colistina/farmacología , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Femenino , Proteínas del Helminto/química , Proteínas del Helminto/farmacología , Humanos , Masculino , Ratones , Pruebas de Sensibilidad Microbiana , Peritonitis/tratamiento farmacológico , Peritonitis/microbiología , Neumonía/tratamiento farmacológico , Neumonía/microbiología , Infecciones Urinarias/tratamiento farmacológico , Infecciones Urinarias/microbiologíaRESUMEN
Micron scale latex beads are well established as highly biocompatible reagents. Imbibing two fluorescent dyes into the interior of the beads enables the creation of a family of combinatorially colored labels. Previous use of such beads, in flow cytometry for example, has focused on beads of approximately 5 microm diameter. We show here that 280 nm combinatorially labeled particles can be used to create ELISA-style assays in 200 microm scale virtual wells, using digital microscopy as the readout. The utility of this technique is illustrated by profiling the secreted cytokine footprints of peripheral blood mononuclear cells in a multiparametric version of the popular Elispot assay. Doing so reveals noncanonical classes of T lymphocytes. We further show that the secreting cell type can be concurrently identified by surface staining with a cell type specific antibody conjugated to the same multiplexed beads.
Asunto(s)
Anticuerpos/química , Citometría de Flujo/métodos , Inmunoensayo/métodos , Microesferas , Linfocitos T/citología , Linfocitos T/inmunología , Anticuerpos/inmunología , Citocinas/análisis , Citocinas/inmunología , HumanosRESUMEN
The secreted immunoglobulin footprint of single hybridoma cells, containing ~10 fg of antibody purified in situ, has been probed for 9 properties concurrently by use of detection labels comprising 280 nm combinatorially colored fluorescent latex beads functionalized with proteins. Specificity of each individual hybridoma cell's product has thereby been assessed in a primary screen. Varying the density of antigen on beads to modulate the avidity of the interaction between bead and secreted antibody footprint allowed rank ordering by affinity in the same primary screen. As more criteria were added to the selection process, the frequency of positive cells went down; in some cases, the favorable cell was present at <1/50,000. Recovery of the cell of interest was accomplished by plating the cells in a viscous medium on top of a membrane. After collecting the antibody footprint on a capture surface beneath the membrane, the immobilized cells were transferred to an incubator while the footprints were analyzed to locate the hybridoma cells of interest. The desired cells were then cloned by picking them from the corresponding locations on the membrane.
Asunto(s)
Afinidad de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Hibridomas/citología , Hibridomas/inmunología , Inmunoensayo/métodos , Inmunoglobulinas/inmunología , Animales , Hibridomas/metabolismo , Inmunoglobulinas/metabolismo , Ratones , Ratones Endogámicos BALB C , MicroesferasRESUMEN
Discovery of therapeutic antibodies is a field of intense development, where immunization of rodents remains a major source of antibody candidates. However, high orthologue protein sequence homology between human and rodent species disfavors generation of antibodies against functionally conserved binding epitopes. Chickens are phylogenetically distant from mammals. Since chickens generate antibodies from a restricted set of germline genes, the possibility of adapting the Symplex antibody discovery platform to chicken immunoglobulin genes and combining it with high-throughput humanization of antibody frameworks by "mass complementarity-determining region grafting" was explored. Hence, wild type chickens were immunized with an immune checkpoint inhibitor programmed cell death 1 (PD1) antigen, and a repertoire of 144 antibodies was generated. The PD1 antibody repertoire was successfully humanized, and we found that most humanized antibodies retained affinity largely similar to that of the parental chicken antibodies. The lead antibody Sym021 blocked PD-L1 and PD-L2 ligand binding, resulting in elevated T-cell cytokine production in vitro. Detailed epitope mapping showed that the epitope recognized by Sym021 was unique compared to the clinically approved PD1 antibodies pembrolizumab and nivolumab. Moreover, Sym021 bound human PD1 with a stronger affinity (30 pM) compared to nivolumab and pembrolizumab, while also cross-reacting with cynomolgus and mouse PD1. This enabled direct testing of Sym021 in the syngeneic mouse in vivo cancer models and evaluation of preclinical toxicology in cynomolgus monkeys. Preclinical in vivo evaluation in various murine and human tumor models demonstrated a pronounced anti-tumor effect of Sym021, supporting its current evaluation in a Phase 1 clinical trial. Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; CD, cluster of differentiation; CDC, complement-dependent cytotoxicity; CDR, complementarity determining region; DC, dendritic cell; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence activated cell sorting; FR, framework region; GM-CSF, granulocyte-macrophage colony-stimulating factor; HRP, horseradish peroxidase; IgG, immunoglobulin G; IL, interleukin; IFN, interferon; mAb, monoclonal antibody; MLR, mixed lymphocyte reaction; NK, natural killer; PBMC, peripheral blood mono-nuclear cell; PD1, programmed cell death 1; PDL1, programmed cell death ligand 1; RT-PCR, reverse transcription polymerase chain reaction; SEB, Staphylococcus Enterotoxin B; SPR, surface Plasmon Resonance; VL, variable part of light chain; VH, variable part of heavy chain.
Asunto(s)
Anticuerpos Monoclonales Humanizados/genética , Anticuerpos Monoclonales/genética , Proteínas Aviares/genética , Pollos/fisiología , Ingeniería de Proteínas/métodos , Linfocitos T/inmunología , Animales , Antígeno B7-H1/metabolismo , Células Cultivadas , Citocinas/metabolismo , Mapeo Epitopo , Humanos , Epítopos Inmunodominantes/genética , Activación de Linfocitos , Macaca fascicularis , Ratones , Ratones Endogámicos C57BL , Proteína 2 Ligando de Muerte Celular Programada 1/metabolismo , Receptor de Muerte Celular Programada 1/inmunología , Unión ProteicaRESUMEN
Colistin is an antimicrobial peptide (AMP) used as a drug of last resort, although plasmid-mediated colistin resistance (MCR) has been reported. AA139 and SET-M33 are novel AMPs currently in development for the treatment of multidrug-resistant (MDR) Gram-negative bacterial infections. As many AMPs have a similar mode of action to colistin, potentially leading to cross-resistance, the antimicrobial activity of AA139 and SET-M33 was investigated against a collection of 50 clinically and genotypically diverse Klebsiella pneumoniae isolates with differing antibiotic resistance profiles, including colistin-resistant strains. The collection was genotypically characterised and susceptibility to clinically relevant antibiotics was determined. Susceptibility to AA139 and SET-M33 did not differ among the collection despite differences in underlying mechanisms of resistance or susceptibility to colistin. For three colistin-susceptible and three colistin-resistant strains with distinct MDR profiles as well as an additional MCR-producing strain, the bactericidal activity of AA139, SET-M33 and colistin during 24 h of exposure was examined. Following 24 h of exposure to AA139, SET-M33 or colistin, the seven strains were tested for changes in susceptibility to the respective AMPs. AA139 and SET-M33 showed a concentration-dependent bactericidal effect irrespective of bacterial susceptibility to colistin. Exposure to low colistin concentrations resulted in the development of colistin resistance in colistin-susceptible strains, whereas susceptibility to AA139 and SET-M33 following exposure to the respective AMPs was maintained. The two novel AMPs remained effective against colistin-resistant strains and may be promising novel drugs for the treatment of clinically and genotypically diverse MDR K. pneumoniae infections, including infections associated with colistin-resistant bacteria.
Asunto(s)
Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Klebsiella pneumoniae/efectos de los fármacos , Farmacorresistencia Bacteriana , Variación Genética , Genotipo , Klebsiella pneumoniae/clasificación , Klebsiella pneumoniae/genética , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacosRESUMEN
The worldwide occurrence of resistance to standard antibiotics and lack of new antibacterial drugs demand new strategies to treat complicated infections. Hence, the aim of this study was to examine the antibacterial activities of an antimicrobial peptide, arenicin-3 derivative AA230, and ethylenediaminetetraacetic acid (EDTA) as well as the two compounds in combination against Gram-negative bacteria. AA230 showed strong antibacterial activity against all of the studied standard strains and clinical isolates, with minimum inhibitory concentrations ranging between 1 µg/mL and 8 µg/mL. AA230 exhibited a bactericidal mode of action. EDTA inhibited the growth of Acinetobacter baumannii at 500â»1000 µg/mL. Strains of Acinetobacter baumannii were found to be more susceptible to EDTA than Pseudomonas aeruginosa or Escherichia coli. The antibacterial effects of both AA230 and EDTA were independent of the antibiotic resistance patterns. Indifference to synergistic activity was observed for AA230 and EDTA combinations using checkerboard titration. In time-kill studies, a substantial synergistic interaction between AA230 and EDTA was detected against all of the tested strains. The addition of EDTA enabled a 2â»4-fold decrease in the AA230 dose. In conclusion, AA230 could have potential applications in the treatment of infections caused by Gram-negative organisms, and its effect can be potentiated by EDTA.