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1.
Nature ; 584(7821): 479-483, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32788728

RESUMEN

Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter6-9, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme10-12. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.


Asunto(s)
Membrana Celular/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/patogenicidad , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Amidohidrolasas/química , Amidohidrolasas/metabolismo , Secuencias de Aminoácidos , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Sitios de Unión , Membrana Celular/metabolismo , Estabilidad de Enzimas , Escherichia coli/citología , Escherichia coli/efectos de los fármacos , Genes Esenciales , Hidrolasas/química , Hidrolasas/metabolismo , Lípido A/química , Lípido A/metabolismo , Lipopolisacáridos/biosíntesis , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Periplasma/química , Periplasma/metabolismo , Unión Proteica , Virulencia
2.
J Bacteriol ; 203(13): e0014921, 2021 06 08.
Artículo en Inglés | MEDLINE | ID: mdl-33875545

RESUMEN

Lipoprotein diacylglyceryl transferase (Lgt) catalyzes the first step in the biogenesis of Gram-negative bacterial lipoproteins which play crucial roles in bacterial growth and pathogenesis. We demonstrate that Lgt depletion in a clinical uropathogenic Escherichia coli strain leads to permeabilization of the outer membrane and increased sensitivity to serum killing and antibiotics. Importantly, we identify G2824 as the first-described Lgt inhibitor that potently inhibits Lgt biochemical activity in vitro and is bactericidal against wild-type Acinetobacter baumannii and E. coli strains. While deletion of a gene encoding a major outer membrane lipoprotein, lpp, leads to rescue of bacterial growth after genetic depletion or pharmacologic inhibition of the downstream type II signal peptidase, LspA, no such rescue of growth is detected after Lgt depletion or treatment with G2824. Inhibition of Lgt does not lead to significant accumulation of peptidoglycan-linked Lpp in the inner membrane. Our data validate Lgt as a novel antibacterial target and suggest that, unlike downstream steps in lipoprotein biosynthesis and transport, inhibition of Lgt may not be sensitive to one of the most common resistance mechanisms that invalidate inhibitors of bacterial lipoprotein biosynthesis and transport. IMPORTANCE As the emerging threat of multidrug-resistant (MDR) bacteria continues to increase, no new classes of antibiotics have been discovered in the last 50 years. While previous attempts to inhibit the lipoprotein biosynthetic (LspA) or transport (LolCDE) pathways have been made, most efforts have been hindered by the emergence of a common mechanism leading to resistance, namely, the deletion of the gene encoding a major Gram-negative outer membrane lipoprotein lpp. Our unexpected finding that inhibition of Lgt is not susceptible to lpp deletion-mediated resistance uncovers the complexity of bacterial lipoprotein biogenesis and the corresponding enzymes involved in this essential outer membrane biogenesis pathway and potentially points to new antibacterial targets in this pathway.


Asunto(s)
Escherichia coli/metabolismo , Lipoproteínas/metabolismo , Transferasas/metabolismo , Animales , Antibacterianos/farmacología , Ácido Aspártico Endopeptidasas , Proteínas Bacterianas , Escherichia coli/genética , Femenino , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Ratones , Peptidoglicano/metabolismo , Transferasas/química , Transferasas/genética , Escherichia coli Uropatógena/genética , Escherichia coli Uropatógena/metabolismo
3.
Proc Natl Acad Sci U S A ; 115(14): 3692-3697, 2018 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-29555747

RESUMEN

The folding and insertion of integral ß-barrel membrane proteins into the outer membrane of Gram-negative bacteria is required for viability and bacterial pathogenesis. Unfortunately, the lack of selective and potent modulators to dissect ß-barrel folding in vivo has hampered our understanding of this fundamental biological process. Here, we characterize a monoclonal antibody that selectively inhibits an essential component of the Escherichia coli ß-barrel assembly machine, BamA. In the absence of complement or other immune factors, the unmodified antibody MAB1 demonstrates bactericidal activity against an E. coli strain with truncated LPS. Direct binding of MAB1 to an extracellular BamA epitope inhibits its ß-barrel folding activity, induces periplasmic stress, disrupts outer membrane integrity, and kills bacteria. Notably, resistance to MAB1-mediated killing reveals a link between outer membrane fluidity and protein folding by BamA in vivo, underscoring the utility of this antibody for studying ß-barrel membrane protein folding within a living cell. Identification of this BamA antagonist highlights the potential for new mechanisms of antibiotics to inhibit Gram-negative bacterial growth by targeting extracellular epitopes.


Asunto(s)
Antibacterianos/farmacología , Anticuerpos Antibacterianos/farmacología , Anticuerpos Monoclonales/farmacología , Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Proteínas de Escherichia coli/antagonistas & inhibidores , Escherichia coli/efectos de los fármacos , Fluidez de la Membrana/efectos de los fármacos , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/inmunología , Membrana Celular/metabolismo , Escherichia coli/inmunología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/inmunología , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformación Proteica , Pliegue de Proteína
4.
J Bacteriol ; 201(1)2019 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-30322857

RESUMEN

Integral ß-barrel membrane proteins are folded and inserted into the Gram-negative bacterial outer membrane by the ß-barrel assembly machine (BAM). This essential complex, composed of a ß-barrel protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE, resides in the outer membrane, a unique asymmetrical lipid bilayer that is difficult to recapitulate in vitro Thus, the probing of BAM function in living cells is critical to fully understand the mechanism of ß-barrel folding. We recently identified an anti-BamA monoclonal antibody, MAB1, that is a specific and potent inhibitor of BamA function. Here, we show that the inhibitory effect of MAB1 is enhanced when BAM function is perturbed by either lowering the level of BamA or removing the nonessential BAM lipoproteins, BamB, BamC, or BamE. The disruption of BAM reduces BamA activity, increases outer membrane (OM) fluidity, and activates the σE stress response, suggesting the OM environment and BAM function are interconnected. Consistent with this idea, an increase in the membrane fluidity through changes in the growth environment or alterations to the lipopolysaccharide in the outer membrane is sufficient to provide resistance to MAB1 and enable the BAM to tolerate these perturbations. Amino acid substitutions in BamA at positions in the outer membrane spanning region or the periplasmic space remote from the extracellular MAB1 binding site also provide resistance to the inhibitory antibody. Our data highlight that the outer membrane environment is a critical determinant in the efficient and productive folding of ß-barrel membrane proteins by BamA.IMPORTANCE BamA is an essential component of the ß-barrel assembly machine (BAM) in the outer membranes of Gram-negative bacteria. We have used a recently described inhibitory anti-BamA antibody, MAB1, to identify the molecular requirements for BAM function. Resistance to this antibody can be achieved through changes to the outer membrane or by amino acid substitutions in BamA that allosterically affect the response to MAB1. Sensitivity to MAB1 is achieved by perturbing BAM function. By using MAB1 activity and functional assays as proxies for BAM function, we link outer membrane fluidity to BamA activity, demonstrating that an increase in membrane fluidity sensitizes the cells to BAM perturbations. Thus, the search for potential inhibitors of BamA function must consider the membrane environment in which ß-barrel folding occurs.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Lipoproteínas/metabolismo , Fluidez de la Membrana , Pliegue de Proteína , Multimerización de Proteína , Sustitución de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/genética , Análisis Mutacional de ADN , Escherichia coli/genética , Proteínas de Escherichia coli/genética
5.
J Biol Chem ; 293(16): 6022-6038, 2018 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-29496999

RESUMEN

Germline-encoded receptors recognizing common pathogen-associated molecular patterns are a central element of the innate immune system and play an important role in shaping the host response to infection. Many of the innate immune molecules central to these signaling pathways are evolutionarily conserved. LysMD3 is a novel molecule containing a putative peptidoglycan-binding domain that has orthologs in humans, mice, zebrafish, flies, and worms. We found that the lysin motif (LysM) of LysMD3 is likely related to a previously described peptidoglycan-binding LysM found in bacteria. Mouse LysMD3 is a type II integral membrane protein that co-localizes with GM130+ structures, consistent with localization to the Golgi apparatus. We describe here two lines of mLysMD3-deficient mice for in vivo characterization of mLysMD3 function. We found that mLysMD3-deficient mice were born at Mendelian ratios and had no obvious pathological abnormalities. They also exhibited no obvious immune response deficiencies in a number of models of infection and inflammation. mLysMD3-deficient mice exhibited no signs of intestinal dysbiosis by 16S analysis or alterations in intestinal gene expression by RNA sequencing. We conclude that mLysMD3 contains a LysM with cytoplasmic orientation, but we were unable to define a physiological role for the molecule in vivo.


Asunto(s)
Eliminación de Gen , Animales , Autoantígenos/análisis , Infecciones Bacterianas/genética , Infecciones Bacterianas/inmunología , Sistemas CRISPR-Cas , Femenino , Inmunidad Innata , Inflamación/genética , Inflamación/inmunología , Masculino , Proteínas de la Membrana/análisis , Ratones , Micosis/genética , Micosis/inmunología , Filogenia , Virosis/genética , Virosis/inmunología
6.
Immunol Rev ; 265(1): 112-29, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25879288

RESUMEN

Inflammasomes are multi-protein signaling platforms that upon activation trigger the maturation of the pro-inflammatory cytokines, interleukin-1ß (IL-1ß) and IL-18, and cell death. Inflammasome sensors detect microbial and host-derived molecules. Here, we review the mechanisms of inflammasome activation triggered by bacterial infection, primarily focusing on two model intracellular bacterial pathogens, Francisella novicida and Salmonella typhimurium. We discuss the complex relationship between bacterial recognition through direct and indirect detection by inflammasome sensors. We highlight regulation mechanisms that potentiate or limit inflammasome activation. We discuss the importance of caspase-1 and caspase-11 in host defense, and we examine the downstream consequences of inflammasome activation within the context of bacterial infections.


Asunto(s)
Francisella/inmunología , Infecciones por Bacterias Gramnegativas/inmunología , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Salmonella typhimurium/inmunología , Animales , Caspasa 1/metabolismo , Caspasas/metabolismo , Caspasas Iniciadoras/metabolismo , Humanos , Inmunidad , Inflamasomas/inmunología , Interleucina-18/metabolismo , Ratones , Receptores de Reconocimiento de Patrones/metabolismo
7.
Proc Natl Acad Sci U S A ; 112(36): 11353-8, 2015 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-26311845

RESUMEN

Biofilm formation is a complex, ordered process. In the opportunistic pathogen Pseudomonas aeruginosa, Psl and Pel exopolysaccharides and extracellular DNA (eDNA) serve as structural components of the biofilm matrix. Despite intensive study, Pel's chemical structure and spatial localization within mature biofilms remain unknown. Using specialized carbohydrate chemical analyses, we unexpectedly found that Pel is a positively charged exopolysaccharide composed of partially acetylated 1→4 glycosidic linkages of N-acetylgalactosamine and N-acetylglucosamine. Guided by the knowledge of Pel's sugar composition, we developed a tool for the direct visualization of Pel in biofilms by combining Pel-specific Wisteria floribunda lectin staining with confocal microscopy. The results indicate that Pel cross-links eDNA in the biofilm stalk via ionic interactions. Our data demonstrate that the cationic charge of Pel is distinct from that of other known P. aeruginosa exopolysaccharides and is instrumental in its ability to interact with other key biofilm matrix components.


Asunto(s)
Biopelículas , ADN Bacteriano/metabolismo , Polisacáridos Bacterianos/metabolismo , Pseudomonas aeruginosa/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cationes/química , ADN Bacteriano/química , ADN Bacteriano/genética , Matriz Extracelular/metabolismo , Espacio Extracelular/genética , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Immunoblotting , Microscopía Confocal , Mutación , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Polisacáridos Bacterianos/química , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Receptores N-Acetilglucosamina/química , Receptores N-Acetilglucosamina/metabolismo , Coloración y Etiquetado/métodos
8.
J Immunol ; 194(7): 3236-45, 2015 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-25710914

RESUMEN

Type I IFN production is an important host immune response against viral and bacterial infections. However, little is known about the ligands and corresponding host receptors that trigger type I IFN production during bacterial infections. We used a model intracellular pathogen, Francisella novicida, to begin characterizing the type I IFN response to bacterial pathogens. F. novicida replicates in the cytosol of host cells and elicits a robust type I IFN response that is largely TLR independent, but is dependent on the adapter molecule STING, suggesting that the type I IFN stimulus during F. novicida infection is cytosolic. In this study, we report that the cytosolic DNA sensors, cyclic GMP-AMP synthase (cGAS) and Ifi204, are both required for the STING-dependent type I IFN response to F. novicida infection in both primary and immortalized murine macrophages. We created cGAS, Ifi204, and Sting functional knockouts in RAW264.7 macrophages and demonstrated that cGAS and Ifi204 cooperate to sense dsDNA and activate the STING-dependent type I IFN pathway. In addition, we show that dsDNA from F. novicida is an important type I IFN stimulating ligand. One outcome of cGAS-STING signaling is the activation of the absent in melanoma 2 inflammasome in response to F. novicida infection. Whereas the absent in melanoma 2 inflammasome is beneficial to the host during F. novicida infection, type I IFN signaling by STING and IFN regulatory factor 3 is detrimental to the host during F. novicida infection. Collectively, our studies indicate that cGAS and Ifi204 cooperate to sense cytosolic dsDNA and F. novicida infection to produce a strong type I IFN response.


Asunto(s)
Francisella/inmunología , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/metabolismo , Interferón Tipo I/metabolismo , Proteínas Nucleares/metabolismo , Nucleotidiltransferasas/metabolismo , Fosfoproteínas/metabolismo , Animales , Células de la Médula Ósea/metabolismo , Línea Celular , Citosol/inmunología , Citosol/metabolismo , ADN/inmunología , Modelos Animales de Enfermedad , Expresión Génica , Regulación de la Expresión Génica , Infecciones por Bacterias Gramnegativas/genética , Infecciones por Bacterias Gramnegativas/mortalidad , Inflamasomas/metabolismo , Factor 3 Regulador del Interferón/deficiencia , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Proteínas Nucleares/genética , Nucleotidiltransferasas/genética , Fosfoproteínas/genética , Unión Proteica , Interferencia de ARN , Transducción de Señal
9.
J Crohns Colitis ; 2024 Jan 24.
Artículo en Inglés | MEDLINE | ID: mdl-38267224

RESUMEN

BACKGROUND AND AIMS: The goal was to identify microbial drivers of IBD, by investigating mucosal-associated bacteria and their detrimental products in IBD patients. METHODS: We directly cultured bacterial communities from mucosal biopsies from pediatric gastrointestinal patients and examined for pathogenicity-associated traits. Upon identifying C. perfringens as toxigenic bacteria present in mucosal biopsies, we isolated strains and further characterized toxicity and prevalence. RESULTS: Mucosal biopsy microbial composition differed from corresponding stool samples. C. perfringens was present in 8 of 9 patients' mucosal biopsies, correlating with hemolytic activity, while not in all corresponding stool samples. Large IBD datasets showed higher C. perfringens prevalence in stool samples of IBD adults (18.7-27.1%) versus healthy (5.1%). In vitro, C. perfringens supernatants were toxic to cell types beneath the intestinal epithelial barrier, including endothelial, neuroblasts, and neutrophils, while impact on epithelial cells was less pronounced, suggesting C. perfringens may be damaging particularly when barrier integrity is compromised. Further characterization using purified toxins and genetic insertion mutants confirmed PFO toxin was sufficient for toxicity. Toxin RNA signatures were found in the original patient biopsies by PCR, suggesting intestinal production. C. perfringens supernatants also induced activation of neuroblast and dorsal root ganglion neurons in vitro, suggesting C. perfringens in inflamed mucosal tissue may directly contribute to abdominal pain, a frequent IBD symptom. CONCLUSIONS: Gastrointestinal carriage of certain toxigenic C. perfringens may have an important pathogenic impact on IBD patients. These findings support routine monitoring of C. perfringens and PFO toxins and potential treatment in patients.

10.
ACS Omega ; 8(13): 12558-12564, 2023 Apr 04.
Artículo en Inglés | MEDLINE | ID: mdl-37033834

RESUMEN

The search for antibiotics that function through novel mechanisms of action is ongoing, and recent progress in our lab identified the tricarboxylic acid cycle as a viable option. Promysalin is a secondary metabolite capable of species-specific inhibition of Pseudomonas aeruginosa, a common opportunistic pathogen. Promysalin disrupts primary metabolism in this bacterium by competitively inhibiting succinate dehydrogenase at the ubiquinone binding site. However, the activity of promysalin in cellulo is marred potentially by its chemical instability and/or propensity for efflux. To assess the success of these novel analogues, a novel strain of P. aeruginosa harboring gene deletions of eight efflux pumps and porins was developed and implemented. Herein, we disclose the synthesis and biological investigation of six promysalin analogues to overcome these liabilities and demonstrate that efflux likely plays a significant role in tolerating the effect of the inhibitor.

11.
Cell Rep ; 34(8): 108782, 2021 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-33626358

RESUMEN

In cystic fibrosis (CF) airways, Pseudomonas aeruginosa forms cellular aggregates called biofilms that are thought to contribute to chronic infection. To form aggregates, P. aeruginosa can use different mechanisms, each with its own pathogenic implications. However, how they form in vivo is controversial and unclear. One mechanism involves a bacterially produced extracellular matrix that holds the aggregates together. Pel and Psl exopolysaccharides are structural and protective components of this matrix. We develop an immunohistochemical method to visualize Pel and Psl in CF sputum. We demonstrate that both exopolysaccharides are expressed in the CF airways and that the morphology of aggregates is consistent with an exopolysaccharide-dependent aggregation mechanism. We reason that the cationic exopolysaccharide Pel may interact with some of the abundant anionic host polymers in sputum. We show that Pel binds extracellular DNA (eDNA) and that this interaction likely impacts current therapies by increasing antimicrobial tolerance and protecting eDNA from digestion.


Asunto(s)
Fibrosis Quística/microbiología , Pulmón/microbiología , Polisacáridos Bacterianos/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/metabolismo , Infecciones del Sistema Respiratorio/microbiología , Antibacterianos/uso terapéutico , Biopelículas/crecimiento & desarrollo , Fibrosis Quística/tratamiento farmacológico , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Farmacorresistencia Bacteriana , Expectorantes/uso terapéutico , Humanos , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crecimiento & desarrollo , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Esputo/microbiología
12.
Nat Commun ; 12(1): 4174, 2021 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-34234105

RESUMEN

The folding of ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the ß-barrel assembly machinery (BAM). How lateral opening in the ß-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Hidrolasas/metabolismo , Liposomas/metabolismo , Pliegue de Proteína , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Proteínas de la Membrana Bacteriana Externa/ultraestructura , Microscopía por Crioelectrón , Dispersión Dinámica de Luz , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas de Escherichia coli/ultraestructura , Hidrolasas/genética , Hidrolasas/aislamiento & purificación , Hidrolasas/ultraestructura , Metabolismo de los Lípidos , Liposomas/ultraestructura , Simulación de Dinámica Molecular , Conformación Proteica en Lámina beta , Proteolípidos/metabolismo , Proteolípidos/ultraestructura , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura
13.
mBio ; 12(3): e0020221, 2021 06 29.
Artículo en Inglés | MEDLINE | ID: mdl-34061593

RESUMEN

Pseudomonas aeruginosa causes life-threatening infections that are associated with antibiotic failure. Previously, we identified the antibiotic G2637, an analog of arylomycin, targeting bacterial type I signal peptidase, which has moderate potency against P. aeruginosa. We hypothesized that an antibody-antibiotic conjugate (AAC) could increase its activity by colocalizing P. aeruginosa bacteria with high local concentrations of G2637 antibiotic in the intracellular environment of phagocytes. Using a novel technology of screening for hybridomas recognizing intact bacteria, we identified monoclonal antibody 26F8, which binds to lipopolysaccharide O antigen on the surface of P. aeruginosa bacteria. This antibody was engineered to contain 6 cysteines and was conjugated to the G2637 antibiotic via a lysosomal cathepsin-cleavable linker, yielding a drug-to-antibody ratio of approximately 6. The resulting AAC delivered a high intracellular concentration of free G2637 upon phagocytosis of AAC-bound P. aeruginosa by macrophages, and potently cleared viable P. aeruginosa bacteria intracellularly. The molar concentration of AAC-associated G2637 antibiotic that resulted in elimination of bacteria inside macrophages was approximately 2 orders of magnitude lower than the concentration of free G2637 required to eliminate extracellular bacteria. This study demonstrates that an anti-P. aeruginosa AAC can locally concentrate antibiotic and kill P. aeruginosa inside phagocytes, providing additional therapeutic options for antibiotics that are moderately active or have an unfavorable pharmacokinetics or toxicity profile. IMPORTANCE Antibiotic treatment of life-threatening P. aeruginosa infections is associated with low clinical success, despite the availability of antibiotics that are active in standard microbiological in vitro assays, affirming the need for new therapeutic approaches. Antibiotics often fail in the preclinical stage due to insufficient efficacy against P. aeruginosa. One potential strategy is to enhance the local concentration of antibiotics with limited inherent anti-P. aeruginosa activity. This study presents proof of concept for an antibody-antibiotic conjugate, which releases a high local antibiotic concentration inside macrophages upon phagocytosis, resulting in potent intracellular killing of phagocytosed P. aeruginosa bacteria. This approach may provide new therapeutic options for antibiotics that are dose limited.


Asunto(s)
Antibacterianos/farmacología , Anticuerpos Monoclonales/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/inmunología , Animales , Antibacterianos/química , Antibacterianos/inmunología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Humanos , Macrófagos/microbiología , Ratones , Viabilidad Microbiana/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Prueba de Estudio Conceptual , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/metabolismo , Células RAW 264.7 , Ratas
14.
Elife ; 82019 06 25.
Artículo en Inglés | MEDLINE | ID: mdl-31237236

RESUMEN

Outer membrane proteins (OMPs) in Gram-negative bacteria dictate permeability of metabolites, antibiotics, and toxins. Elucidating the structure-function relationships governing OMPs within native membrane environments remains challenging. We constructed a diverse library of >3000 monoclonal antibodies to assess the roles of extracellular loops (ECLs) in LptD, an essential OMP that inserts lipopolysaccharide into the outer membrane of Escherichia coli. Epitope binning and mapping experiments with LptD-loop-deletion mutants demonstrated that 7 of the 13 ECLs are targeted by antibodies. Only ECLs inaccessible to antibodies were required for the structure or function of LptD. Our results suggest that antibody-accessible loops evolved to protect key extracellular regions of LptD, but are themselves dispensable. Supporting this hypothesis, no α-LptD antibody interfered with essential functions of LptD. Our experimental workflow enables structure-function studies of OMPs in native cellular environments, provides unexpected insight into LptD, and presents a method to assess the therapeutic potential of antibody targeting.


Asunto(s)
Anticuerpos Monoclonales/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Animales , Antibacterianos/farmacología , Sitios de Unión , Mapeo Epitopo , Epítopos/metabolismo , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Ratones Endogámicos BALB C , Estructura Secundaria de Proteína , Ratas Sprague-Dawley , Relación Estructura-Actividad
15.
Sci Rep ; 8(1): 7136, 2018 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-29740124

RESUMEN

Outer membrane proteins (OMPs) in Gram-negative bacteria are essential for a number of cellular functions including nutrient transport and drug efflux. Escherichia coli BamA is an essential component of the OMP ß-barrel assembly machinery and a potential novel antibacterial target that has been proposed to undergo large (~15 Å) conformational changes. Here, we explored methods to isolate anti-BamA monoclonal antibodies (mAbs) that might alter the function of this OMP and ultimately lead to bacterial growth inhibition. We first optimized traditional immunization approaches but failed to identify mAbs that altered cell growth after screening >3000 hybridomas. We then developed a "targeted boost-and-sort" strategy that combines bacterial cell immunizations, purified BamA protein boosts, and single hybridoma cell sorting using amphipol-reconstituted BamA antigen. This unique workflow improves the discovery efficiency of FACS + mAbs by >600-fold and enabled the identification of rare anti-BamA mAbs with bacterial growth inhibitory activity in the presence of a truncated lipopolysaccharide layer. These mAbs represent novel tools for dissecting the BamA-mediated mechanism of ß-barrel folding and our workflow establishes a new template for the efficient discovery of novel mAbs against other highly dynamic membrane proteins.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/aislamiento & purificación , Proteínas de la Membrana Bacteriana Externa/inmunología , Escherichia coli/inmunología , Proteínas de Escherichia coli/inmunología , Inmunización , Conformación Proteica , Pliegue de Proteína , Transporte de Proteínas/genética , Transporte de Proteínas/inmunología , Vacunación
16.
Nat Protoc ; 10(11): 1820-41, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26492139

RESUMEN

Allelic exchange is an efficient method of bacterial genome engineering. This protocol describes the use of this technique to make gene knockouts and knock-ins, as well as single-nucleotide insertions, deletions and substitutions, in Pseudomonas aeruginosa. Unlike other approaches to allelic exchange, this protocol does not require heterologous recombinases to insert or excise selective markers from the target chromosome. Rather, positive and negative selections are enabled solely by suicide vector-encoded functions and host cell proteins. Here, mutant alleles, which are flanked by regions of homology to the recipient chromosome, are synthesized in vitro and then cloned into allelic exchange vectors using standard procedures. These suicide vectors are then introduced into recipient cells by conjugation. Homologous recombination then results in antibiotic-resistant single-crossover mutants in which the plasmid has integrated site-specifically into the chromosome. Subsequently, unmarked double-crossover mutants are isolated directly using sucrose-mediated counter-selection. This two-step process yields seamless mutations that are precise to a single base pair of DNA. The entire procedure requires ∼2 weeks.


Asunto(s)
Marcación de Gen/métodos , Genoma Bacteriano , Biología Molecular/métodos , Pseudomonas aeruginosa/genética , Ingeniería Genética , Vectores Genéticos , Recombinación Homóloga
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