RESUMEN
Several vaccines have been introduced to combat the coronavirus infectious disease-2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current SARS-CoV-2 vaccines include mRNA-containing lipid nanoparticles or adenoviral vectors that encode the SARS-CoV-2 Spike (S) protein of SARS-CoV-2, inactivated virus, or protein subunits. Despite growing success in worldwide vaccination efforts, additional capabilities may be needed in the future to address issues such as stability and storage requirements, need for vaccine boosters, desirability of different routes of administration, and emergence of SARS-CoV-2 variants such as the Delta variant. Here, we present a novel, well-characterized SARS-CoV-2 vaccine candidate based on extracellular vesicles (EVs) of Salmonella typhimurium that are decorated with the mammalian cell culture-derived Spike receptor-binding domain (RBD). RBD-conjugated outer membrane vesicles (RBD-OMVs) were used to immunize the golden Syrian hamster ( Mesocricetus auratus ) model of COVID-19. Intranasal immunization resulted in high titers of blood anti-RBD IgG as well as detectable mucosal responses. Neutralizing antibody activity against wild-type and Delta variants was evident in all vaccinated subjects. Upon challenge with live virus, hamsters immunized with RBD-OMV, but not animals immunized with unconjugated OMVs or a vehicle control, avoided body mass loss, had lower virus titers in bronchoalveolar lavage fluid, and experienced less severe lung pathology. Our results emphasize the value and versatility of OMV-based vaccine approaches.
RESUMEN
Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite for pneumococcal transmission and disease. Current vaccines protect only against disease and colonization caused by a limited number of serotypes, consequently allowing serotype replacement and transmission. Therefore, the development of a broadly protective vaccine against colonization, transmission and disease is desired but requires a better understanding of pneumococcal adaptation to its natural niche. Hence, we measured the levels of free and protein-bound transition metals in human nasal fluid, to determine the effect of metal concentrations on the growth and proteome of S. pneumoniae. Pneumococci cultured in medium containing metal levels comparable to nasal fluid showed a highly distinct proteomic profile compared to standard culture conditions, including the increased abundance of nine conserved, putative surface-exposed proteins. AliA, an oligopeptide binding protein, was identified as the strongest protective antigen, demonstrated by the significantly reduced bacterial load in a murine colonization and a lethal mouse pneumonia model, highlighting its potential as vaccine antigen.
Asunto(s)
Antígenos Bacterianos/aislamiento & purificación , Proteínas de la Membrana/aislamiento & purificación , Metales/farmacología , Vacunas Neumococicas/inmunología , Streptococcus pneumoniae/efectos de los fármacos , Adulto , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Medios de Cultivo/química , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Proteínas de la Membrana/inmunología , Metales/análisis , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Líquido del Lavado Nasal/química , Nasofaringe/microbiología , Infecciones Neumocócicas/inmunología , Infecciones Neumocócicas/prevención & control , Vacunas Neumococicas/administración & dosificación , Adulto JovenRESUMEN
Bacterial outer membrane vesicles (OMVs) attract increasing interest as immunostimulatory nanoparticles for the development of vaccines and therapeutic agents. We previously engineered the autotransporter protein Hemoglobin protease (Hbp) into a surface display carrier that can be expressed to high density on the surface of Salmonella OMVs. Moreover, we implemented Tag-Catcher protein ligation technology, to obtain dense display of single heterologous antigens and nanobodies on the OMVs through coupling to the distal end of the Hbp passenger domain. Here, we aimed to further expand the versatility of the Hbp platform by enabling the coupling of heterologous proteins to internal sites of the Hbp passenger. Inserted SpyTags were shown to be accessible at the Salmonella OMV surface and to efficiently couple SpyCatcher-equipped fusion proteins. Next, we combined distally placed SnoopCatcher or SnoopTag sequences with internal SpyTags in a single Hbp molecule. This allowed the coupling of two heterologous proteins to a single Hbp carrier molecule without obvious steric hindrance effects. Since coupling occurs to Hbp that is already exposed on the OMVs, there are no limitations to the size and complexity of the partner proteins. In conclusion, we constructed a versatile modular platform for the development of bivalent recombinant OMV-based vaccines and therapeutics.
RESUMEN
The Escherichia coli virulence factor hemoglobin protease (Hbp) has been engineered into a surface display system that can be expressed to high density on live E. coli and Salmonella enterica serovar Typhimurium cells or derived outer membrane vesicles (OMVs). Multiple antigenic sequences can be genetically fused into the Hbp core structure for optimal exposure to the immune system. Although the Hbp display platform is relatively tolerant, increasing the number, size, and complexity of integrated sequences generally lowers the expression of the fused constructs and limits the density of display. This is due to the intricate mechanism of Hbp secretion across the outer membrane and the efficient quality control of translocation-incompetent chimeric Hbp molecules in the periplasm. To address this shortcoming, we explored the coupling of purified proteins to the Hbp carrier after its translocation across the outer membrane using the recently developed SpyTag/SpyCatcher protein ligation system. As expected, fusion of the small SpyTag to Hbp did not hamper display on OMVs. Subsequent addition of purified proteins fused to the SpyCatcher domain resulted in efficient covalent coupling to Hbp-SpyTag. Using in addition the orthogonal SnoopTag/SnoopCatcher system, multiple antigen modules could be coupled to Hbp in a sequential ligation strategy. Not only antigens proved suitable for Spy-mediated ligation but also nanobodies. Addition of this functionality to the platform might allow the targeting of live bacterial or OMV vaccines to certain tissues or immune cells to tailor immune responses.IMPORTANCE Outer membrane vesicles (OMVs) derived from Gram-negative bacteria attract increasing interest in the development of vaccines and therapeutic agents. We aim to construct a semisynthetic OMV platform for recombinant antigen presentation on OMVs derived from attenuated Salmonella enterica serovar Typhimurium cells displaying an adapted Escherichia coli autotransporter, Hbp, at the surface. Although this autotransporter accepts substantial modifications, its capacity with respect to the number, size, and structural complexity of the antigens genetically fused to the Hbp carrier is restricted. Here we describe the application of SpyCatcher/SpyTag protein ligation technology to enzymatically link antigens to Hbp present at high density in OMVs. Protein ligation was apparently unobstructed by the membrane environment and allowed a high surface density of coupled antigens, a property we have shown to be important for vaccine efficacy. The OMV coupling procedure appears versatile and robust, allowing fast production of experimental vaccines and therapeutic agents through a modular plug-and-display procedure.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biotecnología/métodos , Escherichia coli/metabolismo , Proteínas de la Membrana/metabolismo , Salmonella typhimurium/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: Heterologous protein production in Escherichia coli often suffers from bottlenecks such as proteolytic degradation, complex purification procedures and toxicity towards the expression host. Production of proteins in an insoluble form in inclusion bodies (IBs) can alleviate these problems. Unfortunately, the propensity of heterologous proteins to form IBs is variable and difficult to predict. Hence, fusing the target protein to an aggregation prone polypeptide or IB-tag is a useful strategy to produce difficult-to-express proteins in an insoluble form. RESULTS: When screening for signal sequences that mediate optimal targeting of heterologous proteins to the periplasmic space of E. coli, we observed that fusion to the 39 amino acid signal sequence of E. coli TorA (ssTorA) did not promote targeting but rather directed high-level expression of the human proteins hEGF, Pla2 and IL-3 in IBs. Further analysis revealed that ssTorA even mediated IB formation of the highly soluble endogenous E. coli proteins TrxA and MBP. The ssTorA also induced aggregation when fused to the C-terminus of target proteins and appeared functional as IB-tag in E. coli K-12 as well as B strains. An additive effect on IB-formation was observed upon fusion of multiple ssTorA sequences in tandem, provoking almost complete aggregation of TrxA and MBP. The ssTorA-moiety was successfully used to produce the intrinsically unstable hEGF and the toxic fusion partner SymE, demonstrating its applicability as an IB-tag for difficult-to-express and toxic proteins. CONCLUSIONS: We present proof-of-concept for the use of ssTorA as a small, versatile tag for robust E. coli-based expression of heterologous proteins in IBs.
Asunto(s)
Escherichia coli/genética , Cuerpos de Inclusión/metabolismo , Interleucina-3/biosíntesis , Señales de Clasificación de Proteína/genética , Proteínas Portadoras , Factor de Crecimiento Epidérmico/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Vectores Genéticos , Humanos , Cuerpos de Inclusión/química , Interleucina-3/genética , Proteínas de Unión Periplasmáticas/química , Proteínas de Unión Periplasmáticas/genética , Proteínas de Unión Periplasmáticas/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Solubilidad , Tiorredoxinas/química , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMEN
Cell division in Escherichia coli involves a set of essential proteins that assembles at midcell to form the so-called divisome. The divisome regulates the invagination of the inner membrane, cell wall synthesis, and inward growth of the outer membrane. One of the divisome proteins, FtsQ, plays a central but enigmatic role in cell division. This protein associates with FtsB and FtsL, which, like FtsQ, are bitopic inner membrane proteins with a large periplasmic domain (denoted FtsQp, FtsBp, and FtsLp) that is indispensable for the function of each protein. Considering the vital nature and accessible location of the FtsQBL complex, it is an attractive target for protein-protein interaction inhibitors intended to block bacterial cell division. In this study, we expressed FtsQp, FtsBp, and FtsLp individually and in combination. Upon co-expression, FtsQp was co-purified with FtsBp and FtsLp from E. coli extracts as a stable trimeric complex. FtsBp was also shown to interact with FtsQp in the absence of FtsLp albeit with lower affinity. Interactions were mapped at the C terminus of the respective domains by site-specific cross-linking. The binding affinity and 1:1:1 stoichiometry of the FtsQpBpLp complex and the FtsQpBp subcomplex were determined in complementary surface plasmon resonance, analytical ultracentrifugation, and native mass spectrometry experiments.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/citología , Escherichia coli/metabolismo , Complejos Multiproteicos/metabolismo , Secuencia de Aminoácidos , Técnicas Biosensibles , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , División Celular , Reactivos de Enlaces Cruzados/metabolismo , Proteínas Inmovilizadas/metabolismo , Luz , Espectrometría de Masas , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Péptidos/química , Péptidos/metabolismo , Periplasma/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Solubilidad , Relación Estructura-Actividad , UltracentrifugaciónRESUMEN
Escherichia coli cell division is effected by a large assembly of proteins called the divisome, of which a subcomplex consisting of three bitopic inner membrane proteins, FtsQ, FtsB, and FtsL, is an essential part. These three proteins, hypothesized to link cytoplasmic to periplasmic events during cell division, contain large periplasmic domains that are of major importance for function and complex formation. The essential nature of this subcomplex, its low abundance, and its multiple interactions with key divisome components in the relatively accessible periplasm make it an attractive target for the development of protein-protein interaction inhibitors. Although the crystal structure of the periplasmic domain of FtsQ has been solved, the structure of the FtsQBL complex is unknown, with only very crude indications of the interactions in this complex. In this study, we used in vivo site-specific photo cross-linking to probe the surface of the FtsQ periplasmic domain for its interaction interfaces with FtsB and FtsL. An interaction hot spot for FtsB was identified around residue Ser-250 in the C-terminal region of FtsQ and a membrane-proximal interaction region for both proteins around residue Lys-59. Sequence alignment revealed a consensus motif overlapping with the C-terminal interaction hot spot, underlining the importance of this region in FtsQ. The identification of contact sites in the FtsQBL complex will guide future development of interaction inhibitors that block cell division.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de la Membrana/química , Complejos Multiproteicos/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , División Celular/fisiología , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mapeo Peptídico/métodos , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Several Lactococcus lactis strains produce the lantibiotic nisin. The dedicated enzymes NisB and NisC and the transporter NisT modify and secrete the ribosomally synthesized nisin precursor peptide. NisB can function in the absence of the cyclase NisC, yielding the dehydrated prenisin that lacks the thioether rings. A kinetic analysis of nisin production by L. lactis NZ9700 demonstrated that the prenisin was released from the cell into the medium before the processing of the leader sequence occurred. Upon the deletion of nisC, the production of prenisin was reduced by 70%, while in the absence of nisB, the production of prenisin was nearly completely abolished. In cells lacking nisT, no secretion was observed, while the expression of nisABC in these cells resulted in considerable growth rate inhibition caused by the intracellular accumulation of active nisin. Overall, these data indicate that the efficiency of prenisin transport by NisT is markedly enhanced by NisB, suggesting a channeling mechanism of prenisin transfer between the nisin modification enzymes and the transporter.
Asunto(s)
Proteínas Bacterianas/metabolismo , Lactococcus lactis/enzimología , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Nisina/biosíntesis , Precursores de Proteínas/biosíntesis , Proteínas Bacterianas/genética , Bacteriocinas/biosíntesis , Transporte Biológico/genética , Genes Bacterianos , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genéticaRESUMEN
LmrCD is an ABC-type multidrug transporter in Lactococcus lactis. LmrR encodes a putative transcriptional regulator. In a DeltalmrR strain, lmrCD is up-regulated. LmrR binds the promoter region of lmrCD and interacts with drugs that cause lmrCD up-regulation. This suggests that LmrR is a drug-dependent transcriptional regulator of lmrCD expression.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/biosíntesis , Regulación Bacteriana de la Expresión Génica/fisiología , Lactococcus lactis/fisiología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/biosíntesis , Proteínas Represoras/fisiología , ADN Bacteriano/metabolismo , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica/genética , Lactococcus lactis/genética , Unión Proteica , Proteínas Represoras/genéticaRESUMEN
The fluorescent compound Hoechst 33342 is a substrate for many multidrug resistance (MDR) transporters and is widely used to characterize their transport activity. We have constructed mutants of the adenosine triphosphate (ATP) binding cassette (ABC)-type MDR transporter LmrA of Lactococcus lactis that are defective in ATP hydrolysis. These mutants and wild-type LmrA exhibited an atypical behavior in the Hoechst 33342 transport assay. In membrane vesicles, Hoechst 33342 transport was shown to be independent of the ATPase activity of LmrA, and it was not inhibited by orthovanadate but sensitive to uncouplers that collapse the proton gradient and to N,N'-dicyclohexylcarbodiimide, an inhibitor of the F0F1-ATPase. In contrast, transport of Hoechst 33342 by the homologous, heterodimeric MDR transporter LmrCD showed a normal ATP dependence and was insensitive to uncouplers of the proton gradient. With intact cells, expression of LmrA resulted in an increased rate of Hoechst 33342 influx while LmrCD caused a decrease in the rate of Hoechst 33342 influx. Cellular toxicity assays using a triple knockout strain, i.e., L. lactis delta lmrA delta lmrCD, demonstrate that expression of LmrCD protects cells against the growth inhibitory effects of Hoechst 33342, while in the presence of LmrA, cells are more susceptible to Hoechst 33342. Our data demonstrate that the LmrA-mediated Hoechst 33342 transport in membrane vesicles is influenced by the transmembrane pH gradient due to a pH-dependent partitioning of Hoechst 33342 into the membrane.
Asunto(s)
Proteínas Bacterianas/fisiología , Bencimidazoles/metabolismo , Lactococcus lactis/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/fisiología , Fuerza Protón-Motriz/fisiología , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/fisiología , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bencimidazoles/farmacología , Transporte Biológico/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Diciclohexilcarbodiimida/farmacología , Colorantes Fluorescentes/metabolismo , Colorantes Fluorescentes/farmacología , Concentración de Iones de Hidrógeno , Ionóforos/farmacología , Lactococcus lactis/efectos de los fármacos , Lactococcus lactis/genética , Liposomas , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/fisiología , Metilaminas/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Mutagénesis Sitio-Dirigida , Nigericina/farmacología , ATPasas de Translocación de Protón/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Valinomicina/farmacología , Vanadatos/farmacologíaRESUMEN
The human MDR1 gene is induced by cadmium exposure although no resistance to this metal is observed in human cells overexpressing hMDR1. To access the role of MDR proteins in cadmium resistance, human MDR1, Lactococcus lactis lmrA, and Oenococcus oeni omrA were expressed in an Escherichia coli tolC mutant strain which proved to be hypersensitive to cadmium. Both the human and bacterial MDR genes conferred cadmium resistance to E. coli up to 0.4 mM concentration. Protection was abolished by 100 microM verapamil. Quantification of intracellular cadmium concentration by atomic absorption spectrometry showed a reduced cadmium accumulation in cells expressing the MDR genes. Inside-out membrane vesicles of L. lactis overexpressing lmrA displayed an ATP-dependent (109)Cd(2+) uptake that was stimulated by glutathione. An evolutionary model is discussed in which MDR proteins have evolved independently from an ancestor protein displaying both organic xenobiotic- and divalent metal-extrusion abilities.