RESUMEN

The emergence of multidrug-resistant (MDR) pathogens underscores the need for new antimicrobial agents to overcome the resistance mechanisms of these organisms. Cationic antimicrobial peptides (CAPs) provide a potential source of new antimicrobial therapeutics. We previously characterized a lytic base unit (LBU) series of engineered CAPs (eCAPs) of 12 to 48 residues demonstrating maximum antibacterial selectivity at 24 residues. Further, Trp substitution in LBU sequences increased activity against both P. aeruginosa and S. aureus under challenging conditions (e.g., saline, divalent cations, and serum). Based on these findings, we hypothesized that the optimal length and, therefore, the cost for maximum eCAP activity under physiologically relevant conditions could be significantly reduced using only Arg and Trp arranged to form idealized amphipathic helices. Hence, we developed a novel peptide series, composed only of Arg and Trp, in a sequence predicted and verified by circular dichroism to fold into optimized amphipathic helices. The most effective antimicrobial activity was achieved at 12 residues in length (WR12) against a panel of both Gram-negative and Gram-positive clinical isolates, including extensively drug-resistant strains, in saline and broth culture and at various pH values. The results demonstrate that the rational design of CAPs can lead to a significant reduction in the length and the number of amino acids used in peptide design to achieve optimal potency and selectivity against specific pathogens.

Asunto(s)

Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Diseño de Fármacos , Farmacorresistencia Bacteriana Múltiple , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/síntesis química , Arginina/química , Dicroismo Circular , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Triptófano/química

RESUMEN

BACKGROUND: Host-derived (LL-37) and synthetic (WLBU-2) cationic antimicrobial peptides (CAPs) are known for their membrane-active bactericidal properties. LL-37 is an important mediator for immunomodulation, while the mechanism of action of WLBU-2 remains unclear. OBJECTIVE: To determine if WLBU-2 induces an early proinflammatory response that facilitates bacterial clearance in cystic fibrosis (CF). METHODS: C57BL6 mice were given intranasal or intraperitoneal 1×10 (6) cfu/mL Pseudomonas aeruginosa (PA) and observed for 2h, followed by instillation of LL-37 or WLBU-2 (2-4mg/kg) with subsequent tissue collection at 24h for determination of bacterial colony counts and quantitative RT-PCR measurement of cytokine transcripts. CF airway epithelial cells (IB3-1, ΔF508/W1282X) were cultured in appropriate media with supplements. WLBU-2 (25µM) was added to the media with RT-PCR measurement of TNF-α and IL-1ß transcripts after 20, 30, and 60min. Flow cytometry was used to determine if WLBU-2 assists in cellular uptake of Alexa 488-labeled LPS. RESULTS: In murine lung exposed to intranasal or intraperitoneal WLBU-2, there was a reduction in the number of surviving PA colonies compared to controls. Murine lung exposed to intraperitoneal WLBU-2 showed fewer PA colonies compared to LL-37. After 24h WLBU-2 exposure, PA-induced IL-1ß transcripts from lungs showed a twofold decrease (p<0.05), while TNF-α levels were unchanged. LL-37 did not significantly change transcript levels. In IB3-1 cells, WLBU-2 exposure resulted in increased TNF-α and IL-1ß transcripts that decreased by 60min. WLBU-2 treatment of IB3-1 cells displayed increased LPS uptake, suggesting a potential role for CAPs in inducing protective proinflammatory responses. Taken together, the cytokine response, LPS uptake, and established antimicrobial activity of WLBU-2 demonstrate its ability to modulate proinflammatory signaling as a protective mechanism to clear infection. CONCLUSIONS: The immunomodulatory properties of WLBU-2 reveal a potential mechanism of its broad-spectrum antibacterial activity and warrant further preclinical evaluation to study bacterial clearance and rescue of chronic inflammation.

RESUMEN

BACKGROUND: Gram negative bacteria require iron for growth and virulence. It has been shown that certain pathogenic bacteria such as Neisseria gonorrhoeae possess a periplasmic protein called ferric binding protein (FbpA), which is a node in the transport of iron from the cell exterior to the cytosol. SCOPE OF REVIEW: The relevant literature is reviewed which establishes the molecular mechanism of FbpA mediated iron transport across the periplasm to the inner membrane. MAJOR CONCLUSIONS: Here we establish that FbpA may be considered a bacterial transferrin on structural and functional grounds. Data are presented which suggest a continuum whereby FbpA may be considered as a naked iron carrier, as well as a Fe-chelate carrier, and finally a member of the larger family of periplasmic binding proteins. GENERAL SIGNIFICANCE: An investigation of the molecular mechanisms of action of FbpA as a member of the transferrin super family enhances our understanding of bacterial mechanisms for acquisition of the essential nutrient iron, as well as the modes of action of human transferrin, and may provide approaches to the control of pathogenic diseases. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.

Asunto(s)

Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Haemophilus influenzae/metabolismo , Hierro/metabolismo , Neisseria gonorrhoeae/metabolismo , Neisseria meningitidis/metabolismo , Proteínas de Unión Periplasmáticas/química , Proteínas de Unión Periplasmáticas/metabolismo , Complejo Receptor de Transferrina Bacteriana/química , Complejo Receptor de Transferrina Bacteriana/metabolismo , Transporte Iónico , Modelos Moleculares , Estructura Terciaria de Proteína , Sideróforos/metabolismo , Transferrinas/química , Transferrinas/metabolismo

RESUMEN

Ferric binding protein A (FbpA) plays a central role in the iron acquisition processes of pathogenic Neisseria gonorrheae, Neisseria meningitidis, and Haemophilus influenzae. FbpA functions as an iron shuttle within the periplasmic space of these Gram-negative human pathogens. Iron is picked up by FbpA at the periplasmic aspect of the outer membrane with concomitant acquisition of a synergistic anion. Here we report the kinetics and mechanisms involved with loading of iron(III) into iron-free FbpA using iron(III) citrate as an iron source in the presence of excess citrate or phosphate (physiologically available anions) at pH 6.5. In the presence of excess phosphate, iron(III) citrate loads into apo-FbpA in three kinetically distinguishable steps, while in the presence of excess citrate, only two steps are discernible. A stable intermediate containing iron(III) citrate-bound FbpA is observed in each case. The observation of an additional kinetic step and moderate increase in apparent rate constants suggests an active role for phosphate in the iron insertion process. To further elucidate a mechanism for iron loading, we report on the sequestration kinetics of iron(III) citrate in the presence of phosphate with binding site mutant apo-FbpAs, H9E, E57D, E57Q, Q58A, Y195F, and Y196H. Tyrosine mutations drastically alter the kinetics and hamper iron sequestration ability. H9E, E57D, and E57Q have near native iron sequestration behavior; however, iron binding rates are altered, enabling assignment of sequential side chain interactions. Additionally, this investigation elaborates on the function of FbpA as a carrier for iron chelates as well as "naked" or free iron as originally proposed.

Asunto(s)

Ácido Cítrico/metabolismo , Compuestos Férricos/metabolismo , Proteínas de Unión a Hierro/metabolismo , Hierro/metabolismo , Fosfatos/metabolismo , Aniones/química , Aniones/metabolismo , Ácido Cítrico/química , Compuestos Férricos/química , Hierro/química , Proteínas de Unión a Hierro/genética , Cinética , Mutación , Neisseria/metabolismo , Fosfatos/química , Conformación Proteica , Ingeniería de Proteínas

RESUMEN

The bacterial transferrin ferric binding protein A (FbpA) requires an exogenous anion to facilitate iron sequestration, and subsequently to shuttle the metal across the periplasm to the cytoplasmic membrane. In the diverse conditions of the periplasm, numerous anions are known to be present. Prior in vitro experiments have demonstrated the ability of multiple anions to fulfill the synergistic iron-binding requirement, and the identity of the bound anion has been shown to modulate important physicochemical properties of iron-bound FbpA (FeFbpA). Here we address the kinetics and mechanism of anion exchange for the FeFbpA-nitrilotriacetate (NTA) assembly with several biologically relevant anions (citrate, oxalate, phosphate, and pyrophosphate), with nonphysiologic NTA serving as a representative synergistic anion/chelator. The kinetic data are consistent with an anion-exchange process that occurs in multiple steps, dependent on the identity of both the entering anion and the leaving anion. The exchange mechanism may proceed either as a direct substitution or through an intermediate FeFbpA-X* assembly based on anion (X) identity. Our kinetic results further develop an understanding of exogenous anion lability in the periplasm, as well as address the final step of the iron-free FbpA (apo-FbpA)/Fe(3+) sequestration mechanism. Our results highlight the kinetic significance of the FbpA anion binding site, demonstrating a correlation between apo-FbpA/anion affinity and the FeFbpA rate of anion exchange, further supporting the requirement of an exogenous anion to complete tight sequestration of iron by FbpA, and developing a mechanism for anion exchange within FeFbpA that is dependent on the identity of both the entering anion and the leaving anion.

Asunto(s)

Proteínas Bacterianas/química , Ácido Cítrico/química , Ácido Nitrilotriacético/química , Oxalatos/química , Periplasma/química , Fosfatos/química , Aniones/química , Proteínas de la Membrana Bacteriana Externa , Sitios de Unión , Cromatografía por Intercambio Iónico , Proteínas de Unión a Hierro , Cinética , Neisseria gonorrhoeae/química , Proteínas de Unión Periplasmáticas

RESUMEN

Antimicrobial proteins comprise a significant component of the acute innate immune response to infection. They are induced by pattern recognition receptors as well as by cytokines of the innate and adaptive immune pathways and play important roles in infection control and immunomodulatory homeostasis. Lipocalin 2 (siderocalin, NGAL, 24p3), a siderophore-binding antimicrobial protein, is critical for control of systemic infection with Escherichia coli; however, its role in mucosal immunity in the respiratory tract is unknown. In this study, we found that lipocalin 2 is rapidly and robustly induced by Klebsiella pneumoniae infection and is TLR4 dependent. IL-1beta and IL-17 also individually induce lipocalin 2. Mucosal administration of IL-1beta alone could reconstitute the lipocalin 2 deficiency in TLR4 knockout animals and rescue them from infection. Lipocalin 2-deficient animals have impaired lung bacterial clearance in this model and mucosal reconstitution of lipocalin 2 protein in these animals resulted in rescue of this phenotype. We conclude that lipocalin 2 is a crucial component of mucosal immune defense against pulmonary infection with K. pneumoniae.

Asunto(s)

Proteínas de Fase Aguda/inmunología , Proteínas de Fase Aguda/metabolismo , Infecciones por Klebsiella/inmunología , Infecciones por Klebsiella/metabolismo , Lipocalinas/inmunología , Lipocalinas/metabolismo , Proteínas Oncogénicas/inmunología , Proteínas Oncogénicas/metabolismo , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Fase Aguda/genética , Animales , Bronquios/metabolismo , Línea Celular , Epitelio/metabolismo , Humanos , Interleucina-17/farmacología , Interleucina-1beta/farmacología , Infecciones por Klebsiella/genética , Infecciones por Klebsiella/patología , Lipocalina 2 , Lipocalinas/genética , Ratones , Ratones Noqueados , Proteínas Oncogénicas/genética , Neumonía Bacteriana/genética , Neumonía Bacteriana/patología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/inmunología

RESUMEN

Neisseria gonorrhoeae has the capacity to acquire iron from its human host by removing this essential nutrient from serum transferrin. The transferrin binding proteins, TbpA and TbpB constitute the outer membrane receptor complex responsible for binding transferrin, extracting the tightly bound iron from the host-derived molecule, and transporting iron into the periplasmic space of this Gram-negative bacterium. Once iron is transported across the outer membrane, ferric binding protein A (FbpA) moves the iron across the periplasmic space and initiates the process of transport into the bacterial cytosol. The results of the studies reported here define the multiple steps in the iron transport process in which TbpA and TbpB participate. Using the SUPREX technique for assessing the thermodynamic stability of protein-ligand complexes, we report herein the first direct measurement of periplasmic FbpA binding to the outer membrane protein TbpA. We also show that TbpA discriminates between apo- and holo-FbpA; i.e. the TbpA interaction with apo-FbpA is higher affinity than the TbpA interaction with holo-FbpA. Further, we demonstrate that both TbpA and TbpB individually can deferrate transferrin and ferrate FbpA without energy supplied from TonB resulting in sequestration by apo-FbpA.

Asunto(s)

Hierro/metabolismo , Neisseria gonorrhoeae/metabolismo , Proteína A de Unión a Transferrina/metabolismo , Proteína B de Unión a Transferrina/metabolismo , Transferrina/metabolismo , Humanos , Hierro/química , Neisseria gonorrhoeae/química , Transferrina/química , Proteína A de Unión a Transferrina/química , Proteína B de Unión a Transferrina/química

RESUMEN

The obligate human pathogens Haemophilus influenzae, Neisseria gonorrhoeae, and N. meningitidis utilize a highly conserved, three-protein ATP-binding cassette transporter (FbpABC) to shuttle free Fe(3+) from the periplasm and across the cytoplasmic membrane. The periplasmic binding protein, ferric binding protein (FbpA), is capable of transporting other trivalent cations, including Ga(3+), which, unlike Fe(3+), is not redox-active. Because of a similar size and charge as Fe(3+), Ga(3+) is widely used as a non-redox-active Fe(3+) substitute for studying metal complexation in proteins and bacterial populations. The investigations reported here elucidate the similarities and differences in FbpA sequestration of Ga(3+) and Fe(3+), focusing on metal selectivity and the resulting transport function. The thermodynamic binding constant for Ga(3+) complexed with FbpA at pH 6.5, in 50 mM 4-morpholineethanesulfonic acid, 200 mM KCl, 5 mM KH(2)PO(4) was determined by UV-difference spectroscopy as log K'eff=13.7+/-0.6. This represents a 10(5)-fold weaker binding relative to Fe(3+) at identical conditions. The unfolding/refolding behavior of Ga(3+) and Fe(3+) holo-FbpA were also studied using a matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy technique, stability of unpurified proteins from rates of H/D exchange (SUPREX). This analysis indicates significant differences between Fe(3+) and Ga(3+) sequestration with regard to protein folding behavior. A series of kinetic experiments established the lability of the Ga(3+)FbpA-PO(4) assembly, and the similarities/differences of stepwise loading of Fe(3+) into apo- or Ga(3+)-loaded FbpA. These biophysical characterization data are used to interpret FbpA-mediated Ga(3+) transport and toxicity in cell culture studies.

Asunto(s)

Compuestos Férricos/química , Colorantes Fluorescentes/química , Galio/química , Proteínas de Unión a Hierro/química , Proteínas de Unión Periplasmáticas/química , Compuestos Férricos/metabolismo , Colorantes Fluorescentes/metabolismo , Colorantes Fluorescentes/farmacología , Galio/metabolismo , Galio/farmacología , Haemophilus influenzae/efectos de los fármacos , Haemophilus influenzae/crecimiento & desarrollo , Proteínas de Unión a Hierro/aislamiento & purificación , Proteínas de Unión a Hierro/metabolismo , Cinética , Pruebas de Sensibilidad Microbiana , Proteínas de Unión Periplasmáticas/aislamiento & purificación , Proteínas de Unión Periplasmáticas/metabolismo , Sensibilidad y Especificidad , Espectrofotometría Ultravioleta/métodos , Termodinámica

RESUMEN

Ferric binding protein, FbpA, is a member of the transferrin superfamily whose function is to move an essential nutrient, iron, across the periplasm and into the cytosol through formation of a ternary complex containing Fe (3+) and a synergistic anion, X. Here we utilize SUPREX ( stability of unpurified proteins from rates of H/D exchange) to determine the identification and distribution of the synergistic anion in FeFbpA-X species in periplasmic preparations from Gram-negative bacteria. SUPREX is a mass spectrometry-based technique uniquely suited for thermodynamic analyses of protein-ligand complexes in complex biological mixtures such as periplasmic preparations. Model binary mixtures of FeFbpA-Cit and FeFbpA-PO 4 were initially characterized by SUPREX due to the likely presence of citrate and phosphate ions in the periplasm. Ex vivo SUPREX analyses were performed on FeFbpA-X species overexpressed in an Escherichia coli cell line and on endogenous FeFbpA-X species in Neisseria gonorrheae. Detected in the E. coli periplasmic extract were two distinct populations of FbpA, including one in which the protein was unliganded (i.e., apoFbpA) and one in which the protein was bound to iron and the synergistic anion, phosphate (i.e., FeFbpA-PO 4). FeFbpA-PO 4 was the only population of FbpA molecules detected in the N. gonorrheae periplasmic extract. This work provides the first determination of the identity of the in vivo anion bound to FeFbpA-X in the periplasm and substantiates the hypothesis that the synergistic anion plays a structural and functional role in FbpA-mediated transport of iron across the periplasm and into the cytosol.

Asunto(s)

Proteínas Portadoras/metabolismo , Escherichia coli/metabolismo , Neisseria gonorrhoeae/metabolismo , Aniones/química , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Escherichia coli/química , Escherichia coli/genética , Neisseria gonorrhoeae/química , Neisseria gonorrhoeae/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

OBJECTIVES: We describe the antimicrobial activity against Pseudomonas aeruginosa of the de novo-derived antimicrobial peptide WLBU2 in an animal model of infection. METHODS: For this study, an intravenous (iv) model of P. aeruginosa infection was established. The minimum lethal murine dose of P. aeruginosa strain PA01 was determined to be 3 x 10(7) cfu when bacteria were administered iv. Increasing concentrations of WLBU2 were instilled either prior to or following PA01 septic exposure. RESULTS: For the mice given peptide post-bacterial infection, in the 1 mg/kg group, nine of nine animals died because of Pseudomonas sepsis; in the 3 mg/kg group, only one of nine succumbed to infection and in the 4 mg/kg group, all mice were protected (P < 0.0001). Similar results were obtained when WLBU2 was given 1 h prior to Pseudomonas infection. CONCLUSIONS: Although the therapeutic window in this model is narrow, the results nonetheless provide encouraging evidence for WLBU2 as a potential prophylactic or treatment of bacterial infection.

Asunto(s)

Péptidos Catiónicos Antimicrobianos/uso terapéutico , Bacteriemia/tratamiento farmacológico , Infecciones por Pseudomonas/tratamiento farmacológico , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/farmacología , Bacteriemia/microbiología , Progresión de la Enfermedad , Ratones , Datos de Secuencia Molecular , Infecciones por Pseudomonas/microbiología , Sepsis/microbiología , Sepsis/prevención & control , Sobrevida

RESUMEN

The obligate human pathogen Haemophilus influenzae utilizes a siderophore-independent (free) Fe(3+) transport system to obtain this essential element from the host iron-binding protein transferrin. The hFbpABC transporter is a binding protein-dependent ABC transporter that functions to shuttle (free) Fe(3+) through the periplasm and across the inner membrane of H. influenzae. This investigation focuses on the structure and function of the hFbpB membrane permease component of the transporter, a protein that has eluded prior characterization. Based on multiple-sequence alignments between permease orthologs, a series of site-directed mutations targeted at residues within the two conserved permease motifs were generated. The hFbpABC transporter was expressed in a siderophore-deficient Escherichia coli background, and effects of mutations were analyzed using growth rescue and radiolabeled (55)Fe(3+) transport assays. Results demonstrate that mutation of the invariant glycine (G418A) within motif 2 led to attenuated transport activity, while mutation of the invariant glycine (G155A/V/E) within motif 1 had no discernible effect on activity. Individual mutations of well-conserved leucines (L154D and L417D) led to attenuated and null transport activities, respectively. As a complement to site-directed methods, a mutant screen based on resistance to the toxic iron analog gallium, an hFbpABC inhibitor, was devised. The screen led to the identification of several significant hFbpB mutations; V497I, I174F, and S475I led to null transport activities, while S146Y resulted in attenuated activity. Significant residues were mapped to a topological model of the hFbpB permease, and the implications of mutations are discussed in light of structural and functional data from related ABC transporters.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/genética , Proteínas Bacterianas/genética , Haemophilus influenzae/genética , Hierro/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/fisiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/fisiología , Transporte Biológico/efectos de los fármacos , Transporte Biológico/genética , Análisis Mutacional de ADN , Galio/farmacología , Glicina/genética , Haemophilus influenzae/efectos de los fármacos , Haemophilus influenzae/metabolismo , Hierro/química , Leucina/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Homología de Secuencia de Aminoácido

RESUMEN

The efficacy of a novel synthetic antimicrobial peptide (WLBU2) was evaluated against three oral microorganisms (grown planktonically): Streptococcus gordonii, Fusobacterium nucleatum, and Porphyromonas gingivalis. WLBU2 killed all three species, with F. nucleatum being the most susceptible. WLBU2 also reduced the bacterial burden of S. gordonii and F. nucleatum biofilms.

Asunto(s)

Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Fusobacterium nucleatum/efectos de los fármacos , Péptidos/farmacología , Porphyromonas gingivalis/efectos de los fármacos , Streptococcus/efectos de los fármacos , Biopelículas/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana

RESUMEN

The ferric binding protein (FbpA) transports iron across the periplasmic space of certain Gram-negative bacteria and is an important component involved in iron acquisition by pathogenic Neisseria spp. (Neisseria gonorrheae and Neisseria meningitidis). Previous work has demonstrated that the synergistic anion, required for tight Fe(3+) sequestration by FbpA, also plays a key role in inserting Fe(3+) into the FbpA binding site. Here, we investigate the iron release process from various forms of holo-FbpA, Fe(3+)FbpA-X, during the course of a chelator competition reaction using EDTA and Tiron. Fe(3+)FbpA-X represents the protein assembly complex with different synergistic anions, X = PO(4)(3)(-) and NTA. Stepwise mechanisms of Fe(3+) release are proposed on the basis of kinetic profiles of these chelator competition reactions. Fe(3+)FbpA-PO(4) and Fe(3+)FbpA-NTA react differently with EDTA and Tiron during the Fe(3+)-exchange process. EDTA replaces PO(4)(3)(-) and NTA from the first coordination shell of Fe(3+) and acts as a synergistic anion to give a spectroscopically distinguishable intermediate, Fe(3+)FbpA-EDTA, prior to pulling Fe(3+) out of the protein. Tiron, on the other hand, does not act as a synergistic anion but is a more efficient competing chelator as it removes Fe(3+) from FbpA at rate much faster than EDTA. These results reaffirm the contribution of the synergistic anion to the FbpA iron transport process as the anion, in addition to playing a facilitative role in iron binding, appears to have a "gatekeeper" role, thereby modulating the Fe(3+) release process.

Asunto(s)

Proteínas Bacterianas/química , Proteínas de Transporte de Catión/química , Citosol/química , Compuestos Férricos/química , Proteínas de Unión a Hierro/química , Hierro/química , Periplasma/química , Sal Disódica del Ácido 1,2-Dihidroxibenceno-3,5-Disulfónico/química , Proteínas Bacterianas/metabolismo , Transporte Biológico Activo , Proteínas de Transporte de Catión/metabolismo , Citosol/metabolismo , Sinergismo Farmacológico , Ácido Edético/química , Compuestos Férricos/metabolismo , Humanos , Hierro/metabolismo , Quelantes del Hierro/química , Proteínas de Unión a Hierro/metabolismo , Cinética , Modelos Químicos , Neisseria/química , Neisseria/metabolismo , Ácido Nitrilotriacético/química , Periplasma/metabolismo , Espectrofotometría , Termodinámica , Transferrina/química , Transferrina/metabolismo

RESUMEN

Cationic amphipathic peptides have been extensively investigated as a potential source of new antimicrobials that can complement current antibiotic regimens in the face of emerging drug-resistant bacteria. However, the suppression of antimicrobial activity under certain biologically relevant conditions (e.g., serum and physiological salt concentrations) has hampered efforts to develop safe and effective antimicrobial peptides for clinical use. We have analyzed the activity and selectivity of the human peptide LL37 and the de novo engineered antimicrobial peptide WLBU2 in several biologically relevant conditions. The host-derived synthetic peptide LL37 displayed high activity against Pseudomonas aeruginosa but demonstrated staphylococcus-specific sensitivity to NaCl concentrations varying from 50 to 300 mM. Moreover, LL37 potency was variably suppressed in the presence of 1 to 6 mM Mg(2+) and Ca(2+) ions. In contrast, WLBU2 maintained its activity in NaCl and physiologic serum concentrations of Mg(2+) and Ca(2+). WLBU2 is able to kill P. aeruginosa (10(6) CFU/ml) in human serum, with a minimum bactericidal concentration of <9 microM. Conversely, LL37 is inactive in the presence of human serum. Bacterial killing kinetic assays in serum revealed that WLBU2 achieved complete bacterial killing in 20 min. Consistent with these results was the ability of WLBU2 (15 to 20 microM) to eradicate bacteria from ex vivo samples of whole blood. The selectivity of WLBU2 was further demonstrated by its ability to specifically eliminate P. aeruginosa in coculture with human monocytes or skin fibroblasts without detectable adverse effects to the host cells. Finally, WLBU2 displayed potent efficacy against P. aeruginosa in an intraperitoneal infection model using female Swiss Webster mice. These results establish a potential application of WLBU2 in the treatment of bacterial sepsis.

Asunto(s)

Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Bacteriemia/tratamiento farmacológico , Bacteriemia/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Antibacterianos/síntesis química , Antibacterianos/toxicidad , Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/toxicidad , Catelicidinas , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Femenino , Fibroblastos/fisiología , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Monocitos/fisiología , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/crecimiento & desarrollo

RESUMEN

The antimicrobial activity of the collective molecules comprising human milk reflects an evolutionarily successful paradigm for preventing and limiting microbial infection. Understanding the molecules that participate in this process and how they work can yield insight into potentially new antimicrobial therapies. Upon proteolytic processing, antimicrobial peptides can be derived from milk proteins, such as lactoferrin, casein, and lysozyme. Similarly, using the HIV-1 gp41 protein template, we have demonstrated that the 28-residue C-terminus, when produced as an independent peptide, exhibits selective toxicity for bacteria over eukaryotic cells. Upon optimizing this sequence for cationic charge and hydrophobic character presented as a alpha-helical structure, we show improved capability of the parent LLP1 sequence to selectively kill bacteria in the host environment and that this activity is increased by the inclusion of Trp residues on the hydrophobic face. We report that it is possible to (i) design de novo antimicrobial peptides that demonstrate optimal antimicrobial activity with minimal inflammatory activity and (ii) design antimicrobial peptides to function in a defined environment. In the end, we describe a de novo designed antimicrobial peptide, WLBU2, which is selectively toxic to microbial pathogens in complex environments and does not stimulate a significant immunomodulatory response. In spite of these properties, WLBU2 activity against Pseudomonas aeruginosa in human milk is inferior to the host peptide LL37 with regard to antimicrobial potency. These studies demonstrate that antimicrobial peptides can be engineered for greater potency in one medium but may not be optimal for working in a different medium such as human milk.

Asunto(s)

Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Leche Humana/inmunología , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/química , Humanos , Inmunidad Innata , Leche Humana/química , Leche Humana/microbiología , Datos de Secuencia Molecular

RESUMEN

Comparison of human immunodeficiency virus lentiviral lytic peptide 1 with other host-derived peptides indicates that antimicrobial properties of membrane-active peptides are markedly influenced by their cationic, hydrophobic, and amphipathic properties. Many common themes, such as Arg composition of the cationic face of an amphipathic helix and the importance of maintaining the hydrophobic face, have been deduced from these observations. These studies suggest that a peptide with these structural properties can be derived de novo by using only a few strategically positioned amino acids. However, the effects of length and helicity on antimicrobial activity and selectivity have not been objectively evaluated in the context of this motif. To address these structure-function issues, multimers of a 12-residue lytic base unit (LBU) peptide composed only of Arg and Val residues aligned to form idealized amphipathic helices were designed. Bacterial killing assays and circular dichroism analyses reveal a strong correlation between antibacterial activity, peptide length, and propensity to form a helix in solvent mimicking the environment of a membrane. Increasing peptide length beyond two LBUs (24-residue peptides) resulted in no appreciable increase in antimicrobial activity. Derivatives (WLBU) of the LBU series were further engineered by substituting Trp residues in the hydrophobic domains. The 24-residue WLBU2 peptide was active at physiologic NaCl concentrations against Staphylococcus aureus and mucoid and nonmucoid strains of Pseudomonas aeruginosa. Further, WLBU2 displayed the highest antibacterial selectivity of all peptides evaluated in the present study by using a coculture model of P. aeruginosa and primary human skin fibroblasts. These findings provide fundamental information toward the de novo design of an antimicrobial peptide useful for the management of infectious diseases.

Asunto(s)

Péptidos Catiónicos Antimicrobianos , Diseño de Fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/toxicidad , Células Cultivadas , Dicroismo Circular , Técnicas de Cocultivo , Fibroblastos/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Pseudomonas aeruginosa/crecimiento & desarrollo , Piel/citología , Staphylococcus aureus/crecimiento & desarrollo , Relación Estructura-Actividad , Triptófano

RESUMEN

SUPREX (stability of unpurified proteins from rates of H/D exchange) is a H/D exchange- and matrix-assisted laser desorption/ionization (MALDI)-based technique for characterizing the equilibrium unfolding/refolding properties of proteins and protein-ligand complexes. Here, we describe the application of SUPREX to the thermodynamic analysis of synergistic anion binding to iron-loaded ferric-binding protein (Fe(3+)FbpA-X, X = synergistic anion). The in vivo function of FbpA is to transport unchelated Fe(3+) across the periplasmic space of certain Gram-negative bacteria, a process that requires simultaneous binding of a synergistic anion. Our results indicate that Fe(3+)FbpA-X is not a so-called "ideal" protein system for SUPREX analyses because it does not exhibit two-state folding properties and it does not exhibit EX2 H/D exchange behavior. However, despite these nonideal properties of the Fe(3+)FbpA-X protein-folding/unfolding reaction, we demonstrate that the SUPREX technique is still amenable to the quantitative thermodynamic analysis of synergistic anion binding to Fe(3+)FbpA. As part of this work, the SUPREX technique was used to evaluate the DeltaDeltaG(f) values of four synergistic anion-containing complexes of Fe(3+)FbpA (i.e., Fe(3+)FbpA-PO(4), Fe(3+)FbpA-citrate, Fe(3+)FbpA-AsO(4), and Fe(3+)FbpA-SO(4)). The DeltaDeltaG(f) value obtained for Fe(3+)FbpA-citrate relative to Fe(3+)FbpA-PO(4) (1.45 +/- 0.44 kcal/mol), is in good agreement with that reported previously (1.98 kcal/mol). The value obtained for Fe(3+)FbpA-AsO(4) (0.58 +/- 0.45 kcal/mol) was also consistent with that reported previously (0.68 kcal/mol), but the measurement error is very close to the magnitude of the value. This work (i) demonstrates the utility of the SUPREX method for studying anion binding by FbpA, (ii) provides the first evaluation of a DeltaDeltaG(f) value for Fe(3+)FbpA-SO(4), -1.43 +/- 0.17 kcal/mol, and (iii) helps substantiate our hypothesis that the synergistic anion plays a role in controlling the lability of iron bound to FbpA in the transport process.

Asunto(s)

Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Proteína A de Unión a Transferrina/química , Aniones/química , Neisseria gonorrhoeae/metabolismo , Pliegue de Proteína , Termodinámica , Proteína A de Unión a Transferrina/metabolismo

RESUMEN

Two synergistic anions, p-nitrophenyl phosphate ester (NPP) and SO(4)(2-), were found to form new stable assemblies with Fe(3+) and a bacterial transferrin, FbpA (FbpA=ferric binding protein). Fe(3+)FbpA-SO(4) undergoes rapid anion exchange in the presence of NPP to form Fe(3+)FbpA-NPP. Formation of Fe(3+)FbpA-NPP was found to accelerate the rate of hydrolysis of the bound phosphate ester (k(hyd)=1.6 x 10(-6) s(-1) at 25 degrees C and pH 6.5) by >10(3) fold over the uncatalyzed reaction. These findings suggest a dual function for FbpA in vivo: transport of Fe(3+) across the periplasmic space to the inner membrane in certain gram-negative bacteria and hydrolysis of periplasmic polyphosphates.

Asunto(s)

Organofosfatos/química , Transferrina/metabolismo , Aniones , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Catálisis , Hierro/química , Espectrofotometría , Sulfatos/química

RESUMEN

Pathogenic Haemophilus influenzae, Neisseria spp. (Neisseria gonorrhoeae and N. meningitidis), Serratia marcescens, and other gram-negative bacteria utilize a periplasm-to-cytosol FbpABC iron transporter. In this study, we investigated the H. influenzae FbpABC transporter in a siderophore-deficient Escherichia coli background to assess biochemical aspects of FbpABC transporter function. Using a radiolabeled Fe3+ transport assay, we established an apparent Km=0.9 microM and Vmax=1.8 pmol/10(7)cells/min for FbpABC-mediated transport. Complementation experiments showed that hFbpABC is dependent on the FbpA binding protein for transport. The ATPase inhibitor sodium orthovanadate demonstrated dose-dependent inhibition of FbpABC transport, while the protonmotive-force-inhibitor carbonyl cyanide m-chlorophenyl hydrazone had no effect. Metal competition experiments demonstrated that the transporter has high specificity for Fe3+ and selectivity for trivalent metals, including Ga3+ and Al3+, over divalent metals. Metal sensitivity experiments showed that several divalent metals, including copper, nickel, and zinc, exhibited general toxicity towards E. coli. Significantly, gallium-induced toxicity was specific only to E. coli expressing FbpABC. A single-amino-acid mutation in the gene encoding the periplasmic binding protein, FbpA(Y196I), resulted in a greatly diminished iron binding affinity Kd=5.2 x 10(-4) M(-1), approximately 14 orders of magnitude weaker than that of the wild-type protein. Surprisingly, the mutant transporter [FbpA(Y196I)BC] exhibited substantial transport activity, approximately 35% of wild-type transport, with Km=1.2 microM and Vmax=0.5 pmol/10(7)cells/min. We conclude that the FbpABC complexes possess basic characteristics representative of the family of bacterial binding protein-dependent ABC transporters. However, the specificity and high-affinity binding characteristics suggest that the FbpABC transporters function as specialized transporters satisfying the strict chemical requirements of ferric iron (Fe3+) binding and membrane transport.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/fisiología , Compuestos Férricos/metabolismo , Haemophilus influenzae/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico Activo , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Cationes/metabolismo , Cobre/toxicidad , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Galio/toxicidad , Prueba de Complementación Genética , Haemophilus influenzae/efectos de los fármacos , Haemophilus influenzae/genética , Hierro/análisis , Hierro/metabolismo , Mutación Missense , Níquel/toxicidad , Proteínas de Unión Periplasmáticas/genética , Proteínas de Unión Periplasmáticas/metabolismo , Especificidad por Sustrato , Desacopladores/farmacología , Vanadatos/farmacología , Zinc/toxicidad

RESUMEN

Iron transport across the periplasmic space to the cytoplasmic membrane of certain Gram-negative bacteria is mediated by a ferric binding protein (Fbp). This requires Fe(3+) loading of Fbp at the inner leaflet of the outer membrane. A synergistic anion is required for tight Fe(3+) sequestration by Fbp. Although phosphate fills this role in the protein isolated from bacterial cell lysates, nitrilotriacetate anion (NTA) can also satisfy this requirement in vitro. Here, we report the kinetics and mechanism of Fe(3+) loading of Fbp from Fe(NTA)(aq) in the presence of phosphate at pH 6.5. The reaction proceeds in four kinetically distinguishable steps to produce Fe(3+)Fbp(PO(4)) as a final product. The first three steps exhibit half-lives ranging from ca. 20 ms to 0.5 min, depending on the concentrations, and produce Fe(3+)Fbp(NTA) as an intermediate product of significant stability. The rate for the first step is accelerated with an increasing phosphate concentration, while that of the third step is retarded by phosphate. Conversion of Fe(3+)Fbp(NTA) to Fe(3+)Fbp(PO(4)) in the fourth step is a slow process (half-life approximately 2 h) and is facilitated by free phosphate. A mechanism for the Fe(3+)-loading process is proposed in which the synergistic anions, phosphate and NTA, play key roles. These data suggest that not only is a synergistic anion required for tight Fe(3+) sequestration by Fbp, but also the synergistic anion plays a critical role in the process of inserting Fe(3+) into the Fbp binding site.

Asunto(s)

Proteínas Bacterianas/química , Proteínas de Unión a Hierro/química , Hierro/química , Modelos Químicos , Proteínas de Unión Periplasmáticas/química , Fosfatos/química , Aniones/química , Haemophilus influenzae/química , Cinética , Ligandos , Neisseria meningitidis/química , Ácido Nitrilotriacético/química , Termodinámica