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1.
Infect Immun ; 92(8): e0027024, 2024 Aug 13.
Artículo en Inglés | MEDLINE | ID: mdl-39023271

RESUMEN

Bordetella pertussis, the bacterium responsible for whooping cough, remains a significant public health challenge despite the existing licensed pertussis vaccines. Current acellular pertussis vaccines, though having favorable reactogenicity and efficacy profiles, involve complex and costly production processes. In addition, acellular vaccines have functional challenges such as short-lasting duration of immunity and limited antigen coverage. Filamentous hemagglutinin (FHA) is an adhesin of B. pertussis that is included in all multivalent pertussis vaccine formulations. Antibodies to FHA have been shown to prevent bacterial attachment to respiratory epithelial cells, and T cell responses to FHA facilitate cell-mediated immunity. In this study, FHA's mature C-terminal domain (MCD) was evaluated as a novel vaccine antigen. MCD was conjugated to virus-like particles via SpyTag-SpyCatcher technology. Prime-boost vaccine studies were performed in mice to characterize immunogenicity and protection against the intranasal B. pertussis challenge. MCD-SpyVLP was more immunogenic than SpyTag-MCD antigen alone, and in Tohama I strain challenge studies, improved protection against challenge was observed in the lungs at day 3 and in the trachea and nasal wash at day 7 post-challenge. Furthermore, a B. pertussis strain encoding genetically inactivated pertussis toxin was used to evaluate MCD-SpyVLP vaccine immunity. Mice vaccinated with MCD-SpyVLP had significantly lower respiratory bacterial burden at both days 3 and 7 post-challenge compared to mock-vaccinated animals. Overall, these data support the use of SpyTag-SpyCatcher VLPs as a platform for use in vaccine development against B. pertussis and other pathogens.


Asunto(s)
Adhesinas Bacterianas , Anticuerpos Antibacterianos , Bordetella pertussis , Vacuna contra la Tos Ferina , Vacunas de Partículas Similares a Virus , Tos Ferina , Animales , Bordetella pertussis/inmunología , Ratones , Tos Ferina/prevención & control , Tos Ferina/inmunología , Vacuna contra la Tos Ferina/inmunología , Vacuna contra la Tos Ferina/administración & dosificación , Anticuerpos Antibacterianos/inmunología , Adhesinas Bacterianas/inmunología , Adhesinas Bacterianas/genética , Vacunas de Partículas Similares a Virus/inmunología , Vacunas de Partículas Similares a Virus/administración & dosificación , Femenino , Ratones Endogámicos BALB C , Factores de Virulencia de Bordetella/inmunología , Infecciones del Sistema Respiratorio/prevención & control , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/microbiología
2.
PLoS Pathog ; 18(5): e1010511, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35605029

RESUMEN

Hematogenous dissemination is a critical step in the evolution of local infection to systemic disease. The Lyme disease (LD) spirochete, which efficiently disseminates to multiple tissues, has provided a model for this process, in particular for the key early event of pathogen adhesion to the host vasculature. This occurs under shear force mediated by interactions between bacterial adhesins and mammalian cell-surface proteins or extracellular matrix (ECM). Using real-time intravital imaging of the Lyme spirochete in living mice, we previously identified BBK32 as the first LD spirochetal adhesin demonstrated to mediate early vascular adhesion in a living mouse; however, deletion of bbk32 resulted in loss of only about half of the early interactions, suggesting the existence of at least one other adhesin (adhesin-X) that promotes early vascular interactions. VlsE, a surface lipoprotein, was identified long ago by its capacity to undergo rapid antigenic variation, is upregulated in the mammalian host and required for persistent infection in immunocompetent mice. In immunodeficient mice, VlsE shares functional overlap with OspC, a multi-functional protein that displays dermatan sulfate-binding activity and is required for joint invasion and colonization. In this research, using biochemical and genetic approaches as well as intravital imaging, we have identified VlsE as adhesin-X; it is a dermatan sulfate (DS) adhesin that efficiently promotes transient adhesion to the microvasculature under shear force via its DS binding pocket. Intravenous inoculation of mice with a low-passage infectious B. burgdorferi strain lacking both bbk32 and vlsE almost completely eliminated transient microvascular interactions. Comparative analysis of binding parameters of VlsE, BBK32 and OspC provides a possible explanation why these three DS adhesins display different functionality in terms of their ability to promote early microvascular interactions.


Asunto(s)
Adhesinas Bacterianas , Variación Antigénica , Antígenos Bacterianos , Proteínas Bacterianas , Borrelia burgdorferi , Lipoproteínas , Enfermedad de Lyme , Microvasos , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/inmunología , Animales , Variación Antigénica/genética , Variación Antigénica/inmunología , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Adhesión Bacteriana/genética , Adhesión Bacteriana/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Borrelia burgdorferi/genética , Borrelia burgdorferi/inmunología , Dermatán Sulfato/inmunología , Lipoproteínas/genética , Lipoproteínas/inmunología , Enfermedad de Lyme/genética , Enfermedad de Lyme/inmunología , Enfermedad de Lyme/microbiología , Mamíferos , Ratones , Microvasos/inmunología , Microvasos/microbiología , Resistencia al Corte
3.
BMC Vet Res ; 20(1): 421, 2024 Sep 20.
Artículo en Inglés | MEDLINE | ID: mdl-39304865

RESUMEN

BACKGROUND: Mycoplasmal pneumonia of sheep and goats (MPSG) is an important infectious disease that threatens sheep and goat production worldwide, and Mycoplasma ovipneumoniae (Movi) is one of the major aetiological agents causing MPSG. The aim of this study was to investigate the immunological activity of the Hsp70‒P113 fusion protein derived from Movi and to develop a serological assay for the detection of Movi. METHODS: This study involved codon optimization of the dominant antigenic regions of Movi heat shock protein 70 (Hsp70) and adhesin P113. Afterwards, the optimized sequences were inserted into the prokaryotic expression vector pET-30a( +) through tandem linking with the aid of a linker. Once a positive recombinant plasmid (pET-30a-rHsp70-P113) was successfully generated, the expression conditions were further refined. The resulting double gene fusion target protein (rHsp70‒P113) was subsequently purified using ProteinIso® Ni-NTA resin, and the reactivity of the protein was confirmed via SDS‒PAGE and Western blot analysis. An indirect enzyme-linked immunosorbent assay (i-ELISA) technique was developed to detect Movi utilizing the fusion protein as the coating antigen. The specificity, sensitivity, and reproducibility of all methods were assessed after each reaction parameter was optimized. RESULTS: The resulting rHsp70-P113 protein had a molecular weight of approximately 51 kDa and was predominantly expressed in the supernatant. Western blot analysis demonstrated its favourable reactivity. The optimal parameters for the i-ELISA technique were as follows: the rHsp70-P113 protein was encapsulated at a concentration of 5 µg/mL; the serum was diluted at a ratio of 1:50; the HRP-labelled donkey anti-goat IgG was diluted at a ratio of 1:6,000. The results of the cross-reactivity assays revealed that the i-ELISA was not cross-reactive with other goat-positive sera against Mycoplasma mycodies subsp. capri (Mmc), Mycoplasma capricolum subsp. capripneumoniae (Mccp), Mycoplasma arginini (Marg), orf virus (ORFV) or enzootic nasal tumour virus of goats (ENTV-2). The sensitivity of this method is high, with a maximum dilution of up to 1:640. The results of the intra- and inter-batch replication tests revealed that the coefficients of variation were both less than 10%, indicating excellent reproducibility. The analysis of 108 clinical serum samples via i-ELISA and indirect haemagglutination techniques yielded significant findings. Among these samples, 43 displayed positive results, whereas 65 presented negative results, resulting in a positivity rate of 39.8% for the i-ELISA method. In contrast, the indirect haemagglutination technique identified 20 positive samples and 88 negative samples, resulting in a positivity rate of 18.5%. Moreover, a comparison between the two methods revealed a conformity rate of 78.7%. CONCLUSION: The results obtained in this study lay the groundwork for advancements in the use of an Movi antibody detection kit, epidemiological inquiry, and subunit vaccines.


Asunto(s)
Ensayo de Inmunoadsorción Enzimática , Enfermedades de las Cabras , Cabras , Proteínas HSP70 de Choque Térmico , Mycoplasma ovipneumoniae , Neumonía por Mycoplasma , Proteínas Recombinantes de Fusión , Enfermedades de las Ovejas , Animales , Mycoplasma ovipneumoniae/inmunología , Mycoplasma ovipneumoniae/genética , Proteínas HSP70 de Choque Térmico/inmunología , Proteínas HSP70 de Choque Térmico/genética , Enfermedades de las Cabras/diagnóstico , Enfermedades de las Cabras/inmunología , Enfermedades de las Cabras/microbiología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Ensayo de Inmunoadsorción Enzimática/métodos , Enfermedades de las Ovejas/inmunología , Enfermedades de las Ovejas/diagnóstico , Enfermedades de las Ovejas/microbiología , Ovinos , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/genética , Neumonía por Mycoplasma/veterinaria , Neumonía por Mycoplasma/diagnóstico , Neumonía por Mycoplasma/inmunología , Adhesinas Bacterianas/inmunología , Adhesinas Bacterianas/genética , Anticuerpos Antibacterianos/sangre , Sensibilidad y Especificidad , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/genética
4.
Proc Natl Acad Sci U S A ; 118(32)2021 08 10.
Artículo en Inglés | MEDLINE | ID: mdl-34344825

RESUMEN

Nontypeable Haemophilus influenzae (NTHi) is a common cause of localized respiratory tract disease and results in significant morbidity. The pathogenesis of NTHi disease begins with nasopharyngeal colonization, and therefore, the prevention of colonization represents a strategy to prevent disease. The NTHi HMW1 and HMW2 proteins are a family of conserved adhesins that are present in 75 to 80% of strains and have been demonstrated to play a critical role in colonization of the upper respiratory tract in rhesus macaques. In this study, we examined the vaccine potential of HMW1 and HMW2 using a mouse model of nasopharyngeal colonization. Immunization with HMW1 and HMW2 by either the subcutaneous or the intranasal route resulted in a strain-specific antibody response associated with agglutination of bacteria and restriction of bacterial adherence. Despite the specificity of the antibody response, immunization resulted in protection against colonization by both the parent NTHi strain and heterologous strains expressing distinct HMW1 and HMW2 proteins. Pretreatment with antibody against IL-17A eliminated protection against heterologous strains, indicating that heterologous protection is IL-17A dependent. This work demonstrates the vaccine potential of the HMW1 and HMW2 proteins and highlights the importance of IL-17A in protection against diverse NTHi strains.


Asunto(s)
Adhesinas Bacterianas/inmunología , Infecciones por Haemophilus/microbiología , Haemophilus influenzae/patogenicidad , Adhesinas Bacterianas/genética , Pruebas de Aglutinación , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Adhesión Bacteriana , Femenino , Infecciones por Haemophilus/inmunología , Infecciones por Haemophilus/prevención & control , Haemophilus influenzae/genética , Haemophilus influenzae/inmunología , Inmunización , Interleucina-17/sangre , Ratones Endogámicos BALB C , Nasofaringe/microbiología
5.
An Acad Bras Cienc ; 96(2): e20231208, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38747841

RESUMEN

The enterotoxigenic Escherichia coli (ETEC) strain is one of the most frequent causative agents of childhood diarrhea and travelers' diarrhea in low-and middle-income countries. Among the virulence factors secreted by ETEC, the exoprotein EtpA has been described as an important. In the present study, a new detection tool for enterotoxigenic E. coli bacteria using the EtpA protein was developed. Initially, antigenic sequences of the EtpA protein were selected via in silico prediction. A chimeric recombinant protein, corresponding to the selected regions, was expressed in an E. coli host, purified and used for the immunization of mice. The specific recognition of anti-EtpA IgG antibodies generated was evaluated using flow cytometry. The tests demonstrated that the antibodiesdeveloped were able to recognize the native EtpA protein. By coupling these antibodies to magnetic beads for the capture and detection of ETEC isolates, cytometric analyses showed an increase in sensitivity, specificity and the effectiveness of the method of separation and detection of these pathogens. This is the first report of the use of this methodology for ETEC separation. Future trials may indicate their potential use for isolating these and other pathogens in clinical samples, thus accelerating the diagnosis and treatment of diseases.


Asunto(s)
Anticuerpos Antibacterianos , Escherichia coli Enterotoxigénica , Proteínas de Escherichia coli , Citometría de Flujo , Animales , Femenino , Ratones , Anticuerpos Antibacterianos/inmunología , Escherichia coli Enterotoxigénica/inmunología , Proteínas de Escherichia coli/inmunología , Citometría de Flujo/métodos , Inmunoglobulina G/inmunología , Ratones Endogámicos BALB C , Sensibilidad y Especificidad , Adhesinas Bacterianas/inmunología
6.
Infect Immun ; 89(8): e0012121, 2021 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-34097470

RESUMEN

Salmonella enterica Typhimurium is a rod-shaped Gram-negative bacterium that mostly enters the human body through contaminated food. It causes a gastrointestinal disorder called salmonellosis in humans and typhoid-like systemic disease in mice. OmpV, an outer membrane protein of S. Typhimurium, helps in adhesion and invasion of bacteria to intestinal epithelial cells and thus plays a vital role in the pathogenesis of S. Typhimurium. In this study, we have shown that intraperitoneal immunization with OmpV is able to induce high IgG production and protection against systemic disease. Further, oral immunization with OmpV-incorporated proteoliposome (OmpV-proteoliposome [PL]) induces production of high IgA antibody levels and protection against gastrointestinal infection. Furthermore, we have shown that OmpV induces Th1 bias in systemic immunization with purified OmpV, but both Th1 and Th2 polarization in oral immunization with OmpV-proteoliposome (PL). Additionally, we have shown that OmpV activates innate immune cells, such as monocytes, macrophages, and intestinal epithelial cells, in a Toll-like receptor 2 (TLR2)-dependent manner. Interestingly, OmpV is recognized by the TLR1/2 heterodimer in monocytes, but by both TLR1/2 and TLR2/6 heterodimers in macrophages and intestinal epithelial cells. Further, downstream signaling involves MyD88, interleukin-1 receptor-associated kinase (IRAK)-1, mitogen-activated protein kinase (MAPK) (both p38 and Jun N-terminal protein kinase (JNK)), and transcription factors NF-κB and AP-1. Due to its ability to efficiently activate both the innate and adaptive immune systems and protective efficacy, OmpV can be a potential vaccine candidate against S. Typhimurium infection. Further, the fact that OmpV can be recognized by both TLR1/2 and TLR2/6 heterodimers increases its potential to act as good adjuvant in other vaccine formulations.


Asunto(s)
Adhesinas Bacterianas/inmunología , Antígenos Bacterianos/inmunología , Gastroenteritis/inmunología , Gastroenteritis/microbiología , Inmunidad , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/microbiología , Salmonella typhimurium/inmunología , Animales , Biomarcadores , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Interacciones Huésped-Patógeno/inmunología , Ratones , Transducción de Señal
7.
Infect Immun ; 89(5)2021 04 16.
Artículo en Inglés | MEDLINE | ID: mdl-33558320

RESUMEN

Enterotoxigenic Escherichia coli (ETEC) contributes significantly to the substantial burden of infectious diarrhea among children living in low- and middle-income countries. In the absence of a vaccine for ETEC, children succumb to acute dehydration as well as nondiarrheal sequelae related to these infections, including malnutrition. The considerable diversity of ETEC genomes has complicated canonical vaccine development approaches defined by a subset of ETEC pathovar-specific antigens known as colonization factors (CFs). To identify additional conserved immunogens unique to this pathovar, we employed an "open-aperture" approach to capture all potential conserved ETEC surface antigens, in which we mined the genomic sequences of 89 ETEC isolates, bioinformatically selected potential surface-exposed pathovar-specific antigens conserved in more than 40% of the genomes (n = 118), and assembled the representative proteins onto microarrays, complemented with known or putative colonization factor subunit molecules (n = 52) and toxin subunits. These arrays were then used to interrogate samples from individuals with acute symptomatic ETEC infections. Surprisingly, in this approach, we found that immune responses were largely constrained to a small number of antigens, including individual colonization factor antigens and EtpA, an extracellular adhesin. In a Bangladeshi cohort of naturally infected children <2 years of age, both EtpA and a second antigen, EatA, elicited significant serologic responses that were associated with protection from symptomatic illness. In addition, children infected with ETEC isolates bearing either etpA or eatA genes were significantly more likely to develop symptomatic disease. These studies support a role for antigens not presently targeted by vaccines (noncanonical) in virulence and the development of adaptive immune responses during ETEC infections. These findings may inform vaccine design efforts to complement existing approaches.


Asunto(s)
Inmunidad Adaptativa , Antígenos Bacterianos/inmunología , Escherichia coli Enterotoxigénica/inmunología , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/inmunología , Interacciones Huésped-Patógeno/inmunología , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/inmunología , Susceptibilidad a Enfermedades , Humanos , Virulencia , Factores de Virulencia/genética , Factores de Virulencia/inmunología
8.
BMC Immunol ; 22(1): 68, 2021 10 12.
Artículo en Inglés | MEDLINE | ID: mdl-34641798

RESUMEN

BACKGROUND: The necessity of the tetanus-reduced dose diphtheria-acellular pertussis (Tdap) vaccine in adolescence and adults has been emphasized since the resurgence of small-scale pertussis in Korea and worldwide due to the waning effect of the vaccine and variant pathogenic stains in the late 1990s. GreenCross Pharma (GC Pharma), a Korean company, developed the Tdap vaccine GC3111 in 2010. Recently, they enhanced the vaccine, GC3111, produced previously in 2010 to reinforce the antibody response against filamentous hemagglutinin (FHA). In this study, immunogenicity and efficacy of the enhanced Tdap vaccine compared and evaluated with two Tdap vaccines, GC3111 vaccine produced in 2010 previously and commercially available Tdap vaccine in a murine model. METHODS: Two tests groups and positive control group of Balb/c mice were primed with two doses of the diphtheria-tetanus-acellular pertussis (DTaP) vaccine followed by a single booster Tdap vaccine at 9 week using the commercially available Tdap vaccine or 2 Tdap vaccines from GC Pharma (GC3111, enhanced GC3111). Humoral response was assessed 1 week before and 2 and 4 weeks after Tdap booster vaccination. The enhanced GC3111 generated similar humoral response compare to the commercial vaccine for filamentous hemagglutinin (FHA). The interferon gamma (IFN-γ) (Th1), interleukin 5 (IL-5) (Th2) and interleukin 17 (IL-17) (Th17) cytokines were assessed 4 weeks after booster vaccination by stimulation with three simulators: heat inactivated Bordetella pertussis (hBp), vaccine antigens, and hBp mixed with antigens (hBp + antigen). A bacterial challenge test was performed 4 weeks after booster vaccination. RESULTS: Regarding cell-mediated immunity, cytokine secretion differed among the three simulators. However, no difference was found between two test groups and positive control group. All the vaccinated groups indicated a Th1 or Th1/Th2 response. On Day 5 post-bacterial challenge, B. pertussis colonies were absent in the lungs in two test groups and positive control group. CONCLUSIONS: Our results confirmed the immunogenicity of GC Pharma's Tdap vaccine; enhanced GC3111 was equivalent to the presently used commercial vaccine in terms of humoral response as well as cell-mediated cytokine expression.


Asunto(s)
Bordetella pertussis/fisiología , Vacuna contra Difteria, Tétanos y Tos Ferina/inmunología , Células TH1/inmunología , Tos Ferina/inmunología , Adhesinas Bacterianas/inmunología , Adolescente , Adulto , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Inmunidad Humoral , Inmunización Secundaria , Inmunogenicidad Vacunal , Interferón gamma/metabolismo , Corea (Geográfico) , Ratones , Ratones Endogámicos BALB C , Factores de Virulencia de Bordetella/inmunología
9.
PLoS Pathog ; 15(4): e1007713, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-31009507

RESUMEN

Staphylococcus aureus expresses a number of cell wall-anchored proteins that mediate adhesion and invasion of host cells and tissues and promote immune evasion, consequently contributing to the virulence of this organism. The cell wall-anchored protein clumping factor B (ClfB) has previously been shown to facilitate S. aureus nasal colonization through high affinity interactions with the cornified envelope in the anterior nares. However, the role of ClfB during skin and soft tissue infection (SSTI) has never been investigated. This study reveals a novel role for ClfB during SSTIs. ClfB is crucial in determining the abscess structure and bacterial burden early in infection and this is dependent upon a specific interaction with the ligand loricrin which is expressed within the abscess tissue. Targeting ClfB using a model vaccine that induced both protective humoral and cellular responses, leads to protection during S. aureus skin infection. This study therefore identifies ClfB as an important antigen for future SSTI vaccines.


Asunto(s)
Adhesinas Bacterianas/metabolismo , Infecciones Cutáneas Estafilocócicas/microbiología , Staphylococcus aureus/inmunología , Vacunas/inmunología , Factores de Virulencia/metabolismo , Virulencia , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/inmunología , Animales , Adhesión Bacteriana , Femenino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Infecciones Cutáneas Estafilocócicas/inmunología , Infecciones Cutáneas Estafilocócicas/metabolismo , Vacunas/administración & dosificación , Factores de Virulencia/genética , Factores de Virulencia/inmunología
10.
PLoS Pathog ; 15(5): e1007773, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-31107907

RESUMEN

Neutrophil-derived networks of DNA-composed extracellular fibers covered with antimicrobial molecules, referred to as neutrophil extracellular traps (NETs), are recognized as a physiological microbicidal mechanism of innate immunity. The formation of NETs is also classified as a model of a cell death called NETosis. Despite intensive research on the NETs formation in response to pathogens, the role of specific bacteria-derived virulence factors in this process, although postulated, is still poorly understood. The aim of our study was to determine the role of gingipains, cysteine proteases responsible for the virulence of P. gingivalis, on the NETosis process induced by this major periodontopathogen. We showed that NETosis triggered by P. gingivalis is gingipain dependent since in the stark contrast to the wild-type strain (W83) the gingipain-null mutant strain only slightly induced the NETs formation. Furthermore, the direct effect of proteases on NETosis was documented using purified gingipains. Notably, the induction of NETosis was dependent on the catalytic activity of gingipains, since proteolytically inactive forms of enzymes showed reduced ability to trigger the NETs formation. Mechanistically, gingipain-induced NETosis was dependent on proteolytic activation of protease-activated receptor-2 (PAR-2). Intriguingly, both P. gingivalis and purified Arg-specific gingipains (Rgp) induced NETs that not only lacked bactericidal activity but instead stimulated the growth of bacteria species otherwise susceptible to killing in NETs. This protection was executed by proteolysis of bactericidal components of NETs. Taken together, gingipains play a dual role in NETosis: they are the potent direct inducers of NETs formation but in the same time, their activity prevents P. gingivalis entrapment and subsequent killing. This may explain a paradox that despite the massive accumulation of neutrophils and NETs formation in periodontal pockets periodontal pathogens and associated pathobionts thrive in this environment.


Asunto(s)
Adhesinas Bacterianas/inmunología , Infecciones por Bacteroidaceae/inmunología , Cisteína Endopeptidasas/inmunología , Trampas Extracelulares/inmunología , Neutrófilos/inmunología , Peritonitis/inmunología , Porphyromonas gingivalis/inmunología , Porphyromonas gingivalis/patogenicidad , Receptor PAR-2/metabolismo , Adhesinas Bacterianas/metabolismo , Animales , Infecciones por Bacteroidaceae/metabolismo , Infecciones por Bacteroidaceae/microbiología , Infecciones por Bacteroidaceae/patología , Células Cultivadas , Cisteína Endopeptidasas/metabolismo , Trampas Extracelulares/microbiología , Femenino , Cisteína-Endopeptidasas Gingipaínas , Humanos , Ratones , Ratones Endogámicos C57BL , Neutrófilos/microbiología , Neutrófilos/patología , Peritonitis/metabolismo , Peritonitis/microbiología , Receptor PAR-2/inmunología , Transducción de Señal
11.
Microb Pathog ; 160: 105171, 2021 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-34481860

RESUMEN

Staphylococcus aureus infection is emerging as a global threat because of the highly debilitating nature of the associated disease's unprecedented magnitude of its spread and growing global resistance to antimicrobial medicines. Recently WHO has categorized these bacteria under the high global priority pathogen list and is one of the six nosocomial pathogens termed as ESKAPE pathogens which have emerged as a serious threat to public health worldwide. The development of a specific vaccine can stimulate an optimal antibody response, thus providing immunity against it. Therefore, in the present study efforts have been made to identify potential vaccine candidates from the Clumping factor surface proteins (ClfA and ClfB) of S. aureus. Employing the immunoinformatics approach, fourteen antigenic peptides including T-cell, B-cell epitopes were identified which were non-toxic, non-allergenic, high antigenicity, strong binding efficiency with commonly occurring MHC alleles. Consequently, a multi-epitope vaccine chimera was designed by connecting these epitopes with suitable linkers an adjuvant to enhance immunogenicity. Further, homology modeling and molecular docking were performed to construct the three-dimensional structure of the vaccine and study the interaction between the modeled structure and immune receptor (TLR-2) present on lymphocyte cells. Consequently, molecular dynamics simulation for 100 ns period confirmed the stability of the interaction and reliability of the structure for further analysis. Finally, codon optimization and in silico cloning were employed to ensure the successful expression of the vaccine candidate. As the targeted protein is highly antigenic and conserved, hence the designed novel vaccine construct holds potential against emerging multi-drug-resistant organisms.


Asunto(s)
Adhesinas Bacterianas/inmunología , Coagulasa/inmunología , Epítopos de Linfocito B , Epítopos de Linfocito T , Infecciones Estafilocócicas , Biología Computacional , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Péptidos , Reproducibilidad de los Resultados , Infecciones Estafilocócicas/prevención & control , Staphylococcus aureus , Vacunas de Subunidad
12.
Protein Expr Purif ; 178: 105782, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33122039

RESUMEN

Streptococcus pneumoniae is a gram-positive bacterial pathogen causing invasive pneumonia, meningitis, otitis media, and bacteremia. Owing to the current pitfalls of polysaccharide and polysaccharide-conjugate vaccines, protein vaccines are considered promising candidates against pneumonia. Pneumococcal surface protein A (PspA) and pneumococcal surface adhesin A (PsaA) are virulence proteins showing good immunogenicity and protective effects against S. pneumoniae strains in mice. In this study, we expressed the fusion protein PsaA-PspA, which consists of PsaA and the N-terminal region of PspA family 1 and 2, in Escherichia coli. We describe a novel and effective method to purify PsaA-PspA using hydroxyapatite and two-step chromatography. After determining the optimal induction conditions and a series of purification steps, we obtained PsaA-PspA fusion protein with over 95% purity at a final yield of 22.44% from the starting cell lysate. The molecular weight of PsaA-PspA was approximately 83.6 kDa and its secondary structure was evaluated by circular dichroism. Immunization with the purified protein induced high levels of IgG antibodies in mice. Collectively, these results demonstrate that our purification method can effectively produce high-purity PsaA-PspA fusion protein with biological activity and chemical integrity, which can be widely applied to the purification of other PspA subclass proteins.


Asunto(s)
Adhesinas Bacterianas , Anticuerpos Antibacterianos/inmunología , Proteínas Bacterianas , Inmunoglobulina G/inmunología , Proteínas Recombinantes de Fusión , Streptococcus pneumoniae/inmunología , Adhesinas Bacterianas/química , Adhesinas Bacterianas/inmunología , Adhesinas Bacterianas/aislamiento & purificación , Adhesinas Bacterianas/farmacología , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/farmacología , Escherichia coli , Femenino , Expresión Génica , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacología
13.
Helicobacter ; 26(1): e12758, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33259676

RESUMEN

BACKGROUND: Helicobacter pylori is a gram-negative bacterium involved in many gastric pathologies such as ulcers and cancers. Although the treatment for this infection has existed for several years, the development of a vaccine is nevertheless necessary to reduce the severe forms of the disease. For more than three decades, many advances have been made particularly in the understanding of virulence factors as well as the pathogenesis of gastric diseases caused by H. pylori. Among these key virulence factors, specific antigens have been identified: Urease, Vacuolating cytotoxin A (VacA), Cytotoxin-associated gene A (CagA), Blood group antigen-binding adhesin (BabA), H. pylori adhesin A (HpaA), and others. OBJECTIVES: This review will focus on H. pylori adhesins, in particular, on HpaA and on the current knowledge of H. pylori vaccines. METHODS: All of the information included in this review was retrieved from published studies on H. pylori adhesins in H. pylori infections. RESULTS: These proteins, used in their native or recombinant forms, induce protection against H. pylori in experimental animal models. CONCLUSION: H. pylori adhesins are known to be promising candidate vaccines against H. pylori. Future research should be carried out on adhesins, in particular, on HpaA.


Asunto(s)
Adhesinas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Infecciones por Helicobacter , Helicobacter pylori , Animales , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Infecciones por Helicobacter/prevención & control , Helicobacter pylori/inmunología , Ureasa/inmunología , Factores de Virulencia/inmunología
14.
Fish Shellfish Immunol ; 108: 80-85, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33285164

RESUMEN

The outer membrane protein U (OmpU) is a conserved outer membrane protein in a variety of pathogenic Vibrio species and has been considered as a vital protective antigen for vaccine development. Vibrio mimicus (V. mimicus) is the pathogen causing ascites disease in aquatic animals. In this study, the prokaryotically expressed and purified His-tagged OmpU of V. mimicus (His-OmpU) was used as a subunit vaccine. The formalin inactivated V. mimicus, purified His tag (His-tag), and PBS were used as controls. The vaccinated yellow catfish were challenged with V. mimicus at 28 days post-vaccination, and the results showed that the His-OmpU and inactivated V. mimicus groups exhibited much higher survival rates than the His-tag and PBS groups. To fully understand the underlying mechanism, we detected the expression levels of several immune-related genes in the spleen of fish at 28 days post-vaccination and 24 h post-challenge. The results showed that most of the detected immune-related genes were significantly upregulated in His-OmpU and inactivated V. mimicus groups. In addition, we performed the serum bactericidal activity assay, and the results showed that the serum from His-OmpU and inactivated V. mimicus groups exhibited much stronger bactericidal activity against V. mimicus than those of His-tag and PBS groups. Finally, the serum agglutination antibody was detected, and the antibody could be detected in His-OmpU and inactivated V. mimicus groups with the antibody titers increasing along with the time post-vaccination, but not in His-tag or PBS group. Our data reveal that the recombinant OmpU elicits potent protective immune response and is an effective vaccine candidate against V. mimicus in yellow catfish.


Asunto(s)
Adhesinas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Enfermedades de los Peces/inmunología , Inmunogenicidad Vacunal , Vibriosis/veterinaria , Vibrio mimicus/inmunología , Animales , Bagres , Vacunas de Subunidad/inmunología , Vibriosis/inmunología
15.
J Immunol ; 202(8): 2431-2450, 2019 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-30867241

RESUMEN

OmpU, one of the porins of Gram-negative bacteria Vibrio cholerae, induces TLR1/2-MyD88-NF-κB-dependent proinflammatory cytokine production by monocytes and macrophages of human and mouse origin. In this study, we report that in both the cell types, OmpU-induced proinflammatory responses involve activation of MAPKs (p38 and JNK). Interestingly, we observed that in OmpU-treated macrophages, p38 activation is TLR2 dependent, but JNK activation happens through a separate pathway involving reactive oxygen species (ROS) generation by NADPH oxidase complex and mitochondrial ROS. Further, we observed that OmpU-mediated mitochondrial ROS generation probably depends on OmpU translocation to mitochondria and NADPH oxidase-mediated ROS production is due to activation of scavenger receptor CD36. For the first time, to our knowledge, we are reporting that a Gram-negative bacterial protein can activate CD36 as a pattern recognition receptor. Additionally, we found that in OmpU-treated monocytes, both JNK and p38 activation is linked to the TLR2 activation only. Therefore, the ability of macrophages to employ multiple receptors such as TLR2 and CD36 to recognize a single ligand, as in this case OmpU, probably explains the very basic nature of macrophages being more proinflammatory than monocytes.


Asunto(s)
Adhesinas Bacterianas/inmunología , Antígenos CD36/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , Macrófagos/inmunología , Especies Reactivas de Oxígeno/inmunología , Vibrio cholerae/inmunología , Animales , Humanos , Macrófagos/patología , Ratones , Monocitos/inmunología , Monocitos/patología , Células RAW 264.7 , Células THP-1
16.
J Bacteriol ; 203(2)2020 12 18.
Artículo en Inglés | MEDLINE | ID: mdl-33077633

RESUMEN

Mycoplasma bovis causes serious infections in ruminants, leading to huge economic losses. Lipoproteins are key components of the mycoplasma membrane and are believed to function in nutrient acquisition, adherence, enzymatic interactions with the host, and induction of the host's immune response to infection. Many genes of M. bovis have not been assigned functions, in part because of their low sequence similarity with other bacteria, making it difficult to extrapolate gene functions. This study examined functions of a surface-localized leucine-rich repeat (LRR) lipoprotein encoded by mbfN of M. bovis PG45. Homologs of MbfN were detected as 48-kDa peptides by Western blotting in all the strains of M. bovis included in this study, with the predicted 70-kDa full-length polypeptide detected in some strains. Sequence analysis of the gene revealed the absence in some strains of a region encoding the carboxyl-terminal 147 amino acids found in strain PG45, which could account for the variation detected by immunoblotting. In silico analysis of MbfN suggested that it may have an adhesion-related function. In vitro binding assays confirmed MbfN to be a fibronectin and heparin-binding protein. Disruption of mbfN in M. bovis PG45 significantly reduced (P = 0.033) the adherence of M. bovis PG45 to MDBK cells in vitro, demonstrating the role of MbfN as an adhesin.IMPORTANCE Experimental validation of the putative functions of genes in M. bovis will advance our understanding of the basic biology of this economically important pathogen and is crucial in developing prevention strategies. This study demonstrated the extracellular matrix binding ability of a novel immunogenic lipoprotein of M. bovis, and the role of this protein in adhesion by M. bovis suggests that it could play a role in virulence.


Asunto(s)
Adhesinas Bacterianas/metabolismo , Matriz Extracelular/metabolismo , Lipoproteínas/metabolismo , Infecciones por Mycoplasma/veterinaria , Mycoplasma bovis/metabolismo , Adhesinas Bacterianas/química , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/inmunología , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Secuencia de Bases , Western Blotting/veterinaria , Bovinos , Biología Computacional , Electroforesis en Gel de Poliacrilamida/veterinaria , Matriz Extracelular/química , Fibronectinas/metabolismo , Lipoproteínas/química , Lipoproteínas/genética , Modelos Estructurales , Infecciones por Mycoplasma/microbiología , Mycoplasma bovis/genética , Proteolisis , Ratas , Ratas Sprague-Dawley , Rumiantes , Alineación de Secuencia/veterinaria
17.
Infect Immun ; 88(10)2020 09 18.
Artículo en Inglés | MEDLINE | ID: mdl-32661123

RESUMEN

Anaplasma phagocytophilum causes granulocytic anaplasmosis, a debilitating infection that can be fatal in the immunocompromised. It also afflicts animals, including dogs, horses, and sheep. No granulocytic anaplasmosis vaccine exists. Because A. phagocytophilum is an obligate intracellular bacterium, inhibiting microbe-host cell interactions that facilitate invasion can disrupt infection. The binding domains of A. phagocytophilum adhesins A. phagocytophilum invasion protein A (AipA), A. phagocytophilum surface protein (Asp14), and outer membrane protein A (OmpA) are essential for optimal bacterial entry into host cells, but their relevance to infection in vivo is undefined. In this study, C57BL/6 mice were immunized with a cocktail of keyhole limpet hemocyanin-conjugated peptides corresponding to the AipA, Asp14, and OmpA binding domains in alum followed by challenge with A. phagocytophilum The bacterial peripheral blood burden was pronouncedly reduced in immunized mice compared to controls. Examination of pre- and postchallenge sera from these mice revealed that immunization elicited antibodies against AipA and Asp14 peptides but not OmpA peptide. Nonetheless, pooled sera from pre- and postchallenge groups, but not from control groups, inhibited A. phagocytophilum infection of HL-60 cells. Adhesin domain immunization also elicited interferon gamma (IFN-γ)-producing CD8-positive (CD8+) T cells. A follow-up study confirmed that immunization against only the AipA or Asp14 binding domain was sufficient to reduce the bacterial peripheral blood load in mice following challenge and elicit antibodies that inhibit A. phagocytophilum cellular infection in vitro These data demonstrate that AipA and Asp14 are critical for A. phagocytophilum to productively infect mice, and immunization against their binding domains elicits a protective immune response.


Asunto(s)
Adhesinas Bacterianas/inmunología , Anaplasma phagocytophilum/inmunología , Vacunas Bacterianas/inmunología , Ehrlichiosis/prevención & control , Adhesinas Bacterianas/química , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Anticuerpos Bloqueadores/sangre , Anticuerpos Bloqueadores/inmunología , Carga Bacteriana , Vacunas Bacterianas/administración & dosificación , Linfocitos T CD8-positivos/inmunología , Modelos Animales de Enfermedad , Células HL-60 , Humanos , Inmunización , Interferón gamma/inmunología , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Dominios Proteicos/inmunología , Vacunas Conjugadas/administración & dosificación , Vacunas Conjugadas/inmunología , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunología
18.
Infect Immun ; 88(9)2020 08 19.
Artículo en Inglés | MEDLINE | ID: mdl-32540869

RESUMEN

Human rhinovirus (hRV) is frequently detected in the upper respiratory tract, and symptomatic infection is associated with an increased nasopharyngeal bacterial load, with subsequent development of secondary bacterial diseases. Nontypeable Haemophilus influenzae (NTHI) is a commensal bacterial species of the human nasopharynx; however, in the context of prior or concurrent upper respiratory tract viral infection, this bacterium commonly causes multiple diseases throughout the upper and lower respiratory tracts. The present study was conducted to determine the mechanism(s) by which hRV infection promotes the development of NTHI-induced diseases. We showed that hRV infection of polarized primary human airway epithelial cells resulted in increased adherence of NTHI, due in part to augmented expression of CEACAM1 and ICAM1, host cell receptors to which NTHI binds via engagement of multiple adhesins. Antibody blockade of these host cell receptors significantly reduced NTHI adherence. With a specific focus on the NTHI type IV pilus (T4P), which we have previously shown binds to ICAM1, an essential adhesin and virulence determinant, we next showed that T4P-directed antibody blockade significantly reduced NTHI adherence to hRV-infected airway cells and, further, that expression of this adhesin was required for the enhanced adherence observed. Collectively, these data provide a mechanism by which "the common cold" promotes diseases due to NTHI, and they add further support for the use of PilA (the majority subunit of T4P) as a vaccine antigen, since antibodies directed against PilA are expected to limit the notably increased bacterial load associated with hRV coinfection and thereby to prevent secondary NTHI-induced diseases of the respiratory tract.


Asunto(s)
Adhesinas Bacterianas/inmunología , Adhesión Bacteriana/inmunología , Células Epiteliales/inmunología , Proteínas Fimbrias/inmunología , Haemophilus influenzae/inmunología , Interacciones Huésped-Patógeno/inmunología , Rhinovirus/inmunología , Adhesinas Bacterianas/genética , Anticuerpos Neutralizantes/farmacología , Antígenos CD/genética , Antígenos CD/inmunología , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Células Epiteliales/microbiología , Células Epiteliales/virología , Proteínas Fimbrias/genética , Regulación de la Expresión Génica/inmunología , Haemophilus influenzae/crecimiento & desarrollo , Interacciones Huésped-Patógeno/genética , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/inmunología , Cultivo Primario de Células , Unión Proteica , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/genética , ARN Mensajero/inmunología , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/microbiología , Mucosa Respiratoria/virología , Rhinovirus/crecimiento & desarrollo , Transducción de Señal
19.
Infect Immun ; 88(9)2020 08 19.
Artículo en Inglés | MEDLINE | ID: mdl-32513852

RESUMEN

Human surfactant protein-A2 (hSP-A2) is a component of pulmonary surfactant that plays an important role in the lung's immune system by interacting with viruses, bacteria, and fungi to facilitate pathogen clearance and by downregulating inflammatory responses after an allergic challenge. Genetic variation in SP-A2 at position Gln223Lys is present in up to ∼30% of the population and has been associated with several lung diseases, such as asthma, pulmonary fibrosis, and lung cancer (M. M. Pettigrew, J. F. Gent, Y. Zhu, E. W. Triche, et al., BMC Med Genet 8:15, 2007, https://bmcmedgenet.biomedcentral.com/articles/10.1186/1471-2350-8-15; Y. Wang, P. J. Kuan, C. Zing, J. T. Cronkhite, et al., Am J Hum Genet 84:52-59, 2009, https://www.cell.com/ajhg/fulltext/S0002-9297(08)00595-8). Previous work performed by our group showed differences in levels of SP-A binding to non-live mycoplasma membrane fractions that were dependent on the presence of a lysine (K) or a glutamine (Q) at amino acid position 223 in the carbohydrate region of SP-A2. On the basis of these differences, we have derived 20-amino-acid peptides flanking this region of interest in order to test the ability of each to regulate various immune responses to live Mycoplasma pneumoniae in SP-A knockout mice and RAW 264.7 cells. In both models, the 20-mer containing 223Q significantly decreased both tumor necrosis factor alpha (TNF-α) mRNA levels and protein levels in comparison to the 20-mer containing 223K during M. pneumoniae infection. While neither of the 20-mer peptides (223Q and 223K) had an effect on p38 phosphorylation during M. pneumoniae infection, the 223Q-20mer peptide significantly reduced NF-κB p65 phosphorylation in both models. Taken together, our data suggest that small peptides derived from the lectin domain of SP-A2 that contain the major allelic variant (223Q) maintain activity in reducing TNF-α induction during M. pneumoniae infection.


Asunto(s)
Antiinflamatorios/farmacología , Interacciones Microbiota-Huesped/inmunología , Mycoplasma pneumoniae/inmunología , Péptidos/farmacología , Neumonía por Mycoplasma/tratamiento farmacológico , Proteína A Asociada a Surfactante Pulmonar/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/inmunología , Animales , Antiinflamatorios/síntesis química , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Interacciones Microbiota-Huesped/genética , Humanos , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mycoplasma pneumoniae/efectos de los fármacos , Mycoplasma pneumoniae/patogenicidad , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/microbiología , Péptidos/síntesis química , Neumonía por Mycoplasma/genética , Neumonía por Mycoplasma/inmunología , Neumonía por Mycoplasma/microbiología , Dominios Proteicos , Proteína A Asociada a Surfactante Pulmonar/química , Proteína A Asociada a Surfactante Pulmonar/deficiencia , Proteína A Asociada a Surfactante Pulmonar/genética , Células RAW 264.7 , ARN Mensajero/genética , ARN Mensajero/inmunología , Transducción de Señal , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/inmunología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología
20.
Cell Microbiol ; 21(2): e12949, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30171791

RESUMEN

Pathogenic Leptospira bacteria are the causative agents of leptospirosis, a zoonotic disease affecting animals and humans worldwide. These pathogenic species have the ability to rapidly cross host tissue barriers by a yet unknown mechanism. A comparative analysis of pathogens and saprophytes revealed a higher abundance of genes encoding proteins with leucine-rich repeat (LRR) domains in the genomes of pathogens. In other bacterial pathogens, proteins with LRR domains have been shown to be involved in mediating host cell attachment and invasion. One protein from the pathogenic species Leptospira interrogans, LIC10831, has been previously analysed via X-ray crystallography, with findings suggesting it may be an important bacterial adhesin. Herein we show that LIC10831 elicits an antibody response in infected animals, is actively secreted by the bacterium, and binds human E- and VE-cadherins. These results provide biochemical and cellular evidences of LRR protein-mediated host-pathogen interactions and identify a new multireceptor binding protein from this infectious Leptospira species.


Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Leptospira interrogans/metabolismo , Proteínas/metabolismo , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/inmunología , Adhesinas Bacterianas/metabolismo , Animales , Células CHO , Línea Celular , Cricetulus , Cobayas , Humanos , Leptospira interrogans/inmunología , Leptospirosis/microbiología , Proteínas Repetidas Ricas en Leucina
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