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1.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205064

RESUMO

Cortactin is a well-known regulatory protein of the host actin cytoskeleton and represents an attractive target of microbial pathogens like Helicobacter pylori. H. pylori manipulates cortactin's phosphorylation status by type-IV secretion-dependent injection of its virulence protein CagA. Multiple host tyrosine kinases, like FAK, Src, and Abl, are activated during infection, but the pathway(s) involved is (are) not yet fully established. Among them, Src and Abl target CagA and stimulate tyrosine phosphorylation of the latter at its EPIYA-motifs. To investigate the role of cortactin in more detail, we generated a CRISPR/Cas9 knockout of cortactin in AGS gastric epithelial cells. Surprisingly, we found that FAK, Src, and Abl kinase activities were dramatically downregulated associated with widely diminished CagA phosphorylation in cortactin knockout cells compared to the parental control. Together, we report here a yet unrecognized cortactin-dependent signaling pathway involving FAK, Src, and Abl activation, and controlling efficient phosphorylation of injected CagA during infection. Thus, the cortactin status could serve as a potential new biomarker of gastric cancer development.


Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Quinase 1 de Adesão Focal/genética , Infecções por Helicobacter/genética , Helicobacter pylori/genética , Proteínas Oncogênicas v-abl/genética , Regulação Bacteriana da Expressão Gênica/genética , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Fosforilação/genética , Quinases da Família src/genética
2.
BMC Res Notes ; 14(1): 216, 2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34059110

RESUMO

OBJECTIVE: Helicobacter pylori is one of the most common causes of gastric infections in humans. It is estimated that approximately 50% of people around the world are infected with this bacterium. This study aimed to determine the antibiotic resistance pattern, as well as the frequency of cagA and vacA genes in H. pylori isolates obtained from patients in the clinical centers in Tabriz city, Iran. RESULTS: The culture method detected 100 (45.25%) H. pylori isolates from 221 biopsy samples during 3 years. The results showed that 63% and 81% of the isolates were positive for cagA and vacA genes, respectively. The highest resistance of isolates was seen against metronidazole (79%) and amoxicillin (36%), respectively. Also, the isolates showed the least resistance to tetracycline (8%).


Assuntos
Helicobacter pylori , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Resistência Microbiana a Medicamentos , Genótipo , Helicobacter pylori/genética , Humanos , Irã (Geográfico)/epidemiologia
3.
BMC Infect Dis ; 21(1): 463, 2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34020607

RESUMO

BACKGROUND: Streptococcus pyogenes causes a profound global burden of morbidity and mortality across its diverse clinical spectrum. To support a new controlled human infection ('challenge') model seeking to accelerate S. pyogenes vaccine development, we aimed to develop an accurate and reliable molecular method for quantifying bacterial load from pharyngeal swabs collected during experimental human pharyngitis. METHODS: Combined sequential RNA + DNA extraction from throat swabs was compared to traditional separate RNA-only and DNA-only extractions. An emm-type specific qPCR was developed to detect the emm75 challenge strain. Results from the qPCR were compared to culture, using throat swab samples collected in a human challenge study. RESULTS: The qPCR was 100% specific for the emm75 challenge strain when tested against a panel of S. pyogenes emm-types and other respiratory pathogens. Combined RNA + DNA extraction had similar yield to traditional separate extractions. The combined extraction method and emm75 qPCR had 98.8% sensitivity compared to culture for throat swabs collected from challenge study participants. CONCLUSIONS: We have developed a reliable molecular method for measuring S. pyogenes bacterial load from throat swabs collected in a controlled human infection model of S. pyogenes pharyngitis. TRIAL REGISTRATION: NCT03361163 on 4th December 2017.


Assuntos
Antígenos de Bactérias/genética , Carga Bacteriana , Proteínas da Membrana Bacteriana Externa/genética , Faringite/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genética , Adulto , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Seguimentos , Voluntários Saudáveis , Humanos , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , Sensibilidade e Especificidade , Streptococcus pyogenes/classificação , Streptococcus pyogenes/isolamento & purificação
4.
Sheng Wu Gong Cheng Xue Bao ; 37(4): 1081-1091, 2021 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-33973426

RESUMO

The enterobacterial common antigen (ECA) is a polysaccharide composed of polysaccharide repeats that are located in the outer membrane of almost all Enterobacteriaceae bacteria and has diverse biological functions. ECA is synthesized by the synergistic action of multiple genes that are present in clusters on the genome of Enterobacteriaceae bacteria, forming the ECA antigen gene cluster, an important virulence factor that plays a role in host invasion and survival of Enterobacteriaceae in vivo. ECA also plays an important role in the maintenance of the bacterial outer membrane permeability barrier, flagella gene expression, swarming motility, and bile salts resistance. In addition, ECALPS, anchored in the core region of bacterial lipopolysaccharide, is an important surface antigen for bacteria, stimulating high levels of antibody production in the host and could be a target for vaccine research. This review summarizes ECA purification, genes involved in ECA biosynthesis, its immunological characteristics, biological functions and clinical applications.


Assuntos
Antígenos de Bactérias , Enterobacteriaceae , Antígenos de Bactérias/genética , Enterobacteriaceae/genética , Lipopolissacarídeos , Polissacarídeos
5.
Toxins (Basel) ; 13(3)2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33804551

RESUMO

The tumor necrosis factor-α (TNF-α)-inducing protein (tipα) gene family, comprising Helicobacter pylori membrane protein 1 (hp-mp1) and tipα, has been identified as a tumor promoter, contributing to H. pylori carcinogenicity. Tipα is a unique H. pylori protein with no similarity to other pathogenicity factors, CagA, VacA, and urease. American H. pylori strains cause human gastric cancer, whereas African strains cause gastritis. The presence of Tipα in American and Euro-Asian strains suggests its involvement in human gastric cancer development. Tipα secreted from H. pylori stimulates gastric cancer development by inducing TNF-α, an endogenous tumor promoter, through its interaction with nucleolin, a Tipα receptor. This review covers the following topics: tumor-promoting activity of the Tipα family members HP-MP1 and Tipα, the mechanism underlying this activity of Tipα via binding to the cell-surface receptor, nucleolin, the crystal structure of rdel-Tipα and N-terminal truncated rTipα, inhibition of Tipα-associated gastric carcinogenesis by tumor suppressor B-cell translocation gene 2 (BTG2/TIS21), and new strategies to prevent and treat gastric cancer. Thus, Tipα contributes to the carcinogenicity of H. pylori by a mechanism that differs from those of CagA and VacA.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Infecções por Helicobacter/microbiologia , Helicobacter pylori/metabolismo , Neoplasias Gástricas/microbiologia , Animais , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Transição Epitelial-Mesenquimal , Infecções por Helicobacter/complicações , Infecções por Helicobacter/diagnóstico , Infecções por Helicobacter/terapia , Helicobacter pylori/genética , Helicobacter pylori/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Proteínas Imediatamente Precoces/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Risco , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Neoplasias Gástricas/prevenção & controle , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Supressoras de Tumor/metabolismo
6.
mBio ; 12(2)2021 04 20.
Artigo em Inglês | MEDLINE | ID: covidwho-1195824

RESUMO

New vaccines are urgently needed against Mycobacterium tuberculosis (Mtb), which kills more than 1.4 million people each year. CD4 T cell differentiation is a key determinant of protective immunity against Mtb, but it is not fully understood how host-pathogen interactions shape individual antigen-specific T cell populations and their protective capacity. Here, we investigated the immunodominant Mtb antigen, MPT70, which is upregulated in response to gamma interferon (IFN-γ) or nutrient/oxygen deprivation of in vitro-infected macrophages. Using a murine aerosol infection model, we compared the in vivo expression kinetics of MPT70 to a constitutively expressed antigen, ESAT-6, and analyzed their corresponding CD4 T cell phenotype and vaccine protection. For wild-type Mtb, we found that in vivo expression of MPT70 was delayed compared to ESAT-6. This delayed expression was associated with induction of less differentiated MPT70-specific CD4 T cells but, compared to ESAT-6, also reduced protection after vaccination. In contrast, infection with an MPT70-overexpressing Mtb strain promoted highly differentiated KLRG1+CX3CR1+ CD4 T cells with limited lung-homing capacity. Importantly, this differentiated phenotype could be prevented by vaccination, and against the overexpressing strain, vaccination with MPT70 conferred protection similar to vaccination with ESAT-6. Together, our data indicate that high in vivo antigen expression drives T cells toward terminal differentiation and that targeted vaccination with adjuvanted protein can counteract this phenomenon by maintaining T cells in a protective less differentiated state. These observations shed new light on host-pathogen interactions and provide guidance on how future Mtb vaccines can be designed to tip the immune balance in favor of the host.IMPORTANCE Tuberculosis, caused by Mtb, constitutes a global health crisis of massive proportions and the impact of the current coronavirus disease 2019 (COVID-19) pandemic is expected to cause a rise in tuberculosis-related deaths. Improved vaccines are therefore needed more than ever, but a lack of knowledge on protective immunity hampers their development. The present study shows that constitutively expressed antigens with high availability drive highly differentiated CD4 T cells with diminished protective capacity, which could be a survival strategy by Mtb to evade T cell immunity against key antigens. We demonstrate that immunization with such antigens can counteract this phenomenon by maintaining antigen-specific T cells in a state of low differentiation. Future vaccine strategies should therefore explore combinations of multiple highly expressed antigens and we suggest that T cell differentiation could be used as a readily measurable parameter to identify these in both preclinical and clinical studies.


Assuntos
Antígenos de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/farmacologia , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/prevenção & controle , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/microbiologia , Diferenciação Celular/imunologia , Modelos Animais de Doenças , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Expressão Gênica , Genes Bacterianos , Humanos , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/genética , Vacinas contra a Tuberculose/genética , Vacinas contra a Tuberculose/imunologia , Tuberculose Pulmonar/microbiologia
7.
Int J Food Microbiol ; 347: 109188, 2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-33839439

RESUMO

Vibrio parahaemolyticus, which is commonly found in marine and estuarine environments worldwide and isolated from aquatic products, is one of the most important food-borne pathogens. Among the various typing methods, serotyping is widely accepted and utilized by infectious disease specialists and infection control agencies for the detection and epidemiological investigation of this pathogen. Thus far, 13 O serotypes and 71 K serotypes have been defined; however, untypeable strains are frequently isolated during routine detection, and some new O and/or K antigens have been identified and characterized. During a serotyping survey in Shandong province, China from 2016 to 2018, we collected 411 clinical V. parahaemolyticus strains and found that nine of them are untypeable K antigen strains. In this study, we identified three K serotypes of V. parahaemolyticus through in-depth genetic analysis of the K antigen gene cluster, serological tests, and the production of antisera. Among the nine strains, seven possess K untypeable 2 (KUT2) antigens, which have been reported recently by another group. However, two new O and K combinations (O3:KUT2 and O11:KUT2) were first characterized by us, with the remaining two each representing a novel K serotype. Moreover, through comparative genomic analysis, we showed that the Shandong KUT2 strains exhibit different virulence profiles compared to their identical K serotype partners from Zhejiang province, another Chinese coastal province; however, strains from these two regions are clustered into the same linage and may have evolved from a recent common ancestor. Additionally, one isolate, SD2016062, was phylogenetically similar to the strains associated with several local gastroenteritis outbreaks, with similar toxin patterns, suggesting its potential to cause sporadic occurrences of disease or even local pandemics. Finally, we developed a sero-specific PCR assay targeting the three novel K serotypes, which can monitor the V. parahaemolyticus spectrum for clinical and epidemiological purposes. Thus, we identified and characterized novel strains of V. parahaemolyticus and proposed a new technique for tracking the diversity of strains, which can help manage this food-borne pathogen.


Assuntos
Antígenos de Bactérias/genética , Antígenos de Superfície/genética , Vibrioses/epidemiologia , Vibrioses/microbiologia , Vibrio parahaemolyticus/genética , China/epidemiologia , Gastroenterite/epidemiologia , Gastroenterite/microbiologia , Humanos , Tipagem Molecular , Antígenos O/genética , Filogenia , Reação em Cadeia da Polimerase , Sorogrupo , Vibrio parahaemolyticus/classificação , Vibrio parahaemolyticus/patogenicidade , Virulência/genética
8.
mBio ; 12(2)2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33879592

RESUMO

New vaccines are urgently needed against Mycobacterium tuberculosis (Mtb), which kills more than 1.4 million people each year. CD4 T cell differentiation is a key determinant of protective immunity against Mtb, but it is not fully understood how host-pathogen interactions shape individual antigen-specific T cell populations and their protective capacity. Here, we investigated the immunodominant Mtb antigen, MPT70, which is upregulated in response to gamma interferon (IFN-γ) or nutrient/oxygen deprivation of in vitro-infected macrophages. Using a murine aerosol infection model, we compared the in vivo expression kinetics of MPT70 to a constitutively expressed antigen, ESAT-6, and analyzed their corresponding CD4 T cell phenotype and vaccine protection. For wild-type Mtb, we found that in vivo expression of MPT70 was delayed compared to ESAT-6. This delayed expression was associated with induction of less differentiated MPT70-specific CD4 T cells but, compared to ESAT-6, also reduced protection after vaccination. In contrast, infection with an MPT70-overexpressing Mtb strain promoted highly differentiated KLRG1+CX3CR1+ CD4 T cells with limited lung-homing capacity. Importantly, this differentiated phenotype could be prevented by vaccination, and against the overexpressing strain, vaccination with MPT70 conferred protection similar to vaccination with ESAT-6. Together, our data indicate that high in vivo antigen expression drives T cells toward terminal differentiation and that targeted vaccination with adjuvanted protein can counteract this phenomenon by maintaining T cells in a protective less differentiated state. These observations shed new light on host-pathogen interactions and provide guidance on how future Mtb vaccines can be designed to tip the immune balance in favor of the host.IMPORTANCE Tuberculosis, caused by Mtb, constitutes a global health crisis of massive proportions and the impact of the current coronavirus disease 2019 (COVID-19) pandemic is expected to cause a rise in tuberculosis-related deaths. Improved vaccines are therefore needed more than ever, but a lack of knowledge on protective immunity hampers their development. The present study shows that constitutively expressed antigens with high availability drive highly differentiated CD4 T cells with diminished protective capacity, which could be a survival strategy by Mtb to evade T cell immunity against key antigens. We demonstrate that immunization with such antigens can counteract this phenomenon by maintaining antigen-specific T cells in a state of low differentiation. Future vaccine strategies should therefore explore combinations of multiple highly expressed antigens and we suggest that T cell differentiation could be used as a readily measurable parameter to identify these in both preclinical and clinical studies.


Assuntos
Antígenos de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/farmacologia , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/prevenção & controle , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/microbiologia , Diferenciação Celular/imunologia , Modelos Animais de Doenças , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Expressão Gênica , Genes Bacterianos , Humanos , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/genética , Vacinas contra a Tuberculose/genética , Vacinas contra a Tuberculose/imunologia , Tuberculose Pulmonar/microbiologia
9.
Nat Commun ; 12(1): 2085, 2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33837194

RESUMO

Long-term infection of the stomach with Helicobacter pylori can cause gastric cancer. However, the mechanisms by which the bacteria adapt to the stomach environment are poorly understood. Here, we show that a small non-coding RNA of H. pylori (HPnc4160, also known as IsoB or NikS) regulates the pathogen's adaptation to the host environment as well as bacterial oncoprotein production. In a rodent model of H. pylori infection, the genomes of bacteria isolated from the stomach possess an increased number of T-repeats upstream of the HPnc4160-coding region, and this leads to reduced HPnc4160 expression. We use RNA-seq and iTRAQ analyses to identify eight targets of HPnc4160, including genes encoding outer membrane proteins and oncoprotein CagA. Mutant strains with HPnc4160 deficiency display increased colonization ability of the mouse stomach, in comparison with the wild-type strain. Furthermore, HPnc4160 expression is lower in clinical isolates from gastric cancer patients than in isolates derived from non-cancer patients, while the expression of HPnc4160's targets is higher in the isolates from gastric cancer patients. Therefore, the small RNA HPnc4160 regulates H. pylori adaptation to the host environment and, potentially, gastric carcinogenesis.


Assuntos
Adaptação Fisiológica/genética , Infecções por Helicobacter/patologia , Helicobacter pylori/fisiologia , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Neoplasias Gástricas/microbiologia , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Carcinogênese , Modelos Animais de Doenças , Mucosa Gástrica/microbiologia , Mucosa Gástrica/patologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Genoma Bacteriano/genética , Gerbillinae , Infecções por Helicobacter/microbiologia , Helicobacter pylori/isolamento & purificação , Helicobacter pylori/patogenicidade , Interações entre Hospedeiro e Microrganismos , Humanos , Masculino , Mutação , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , RNA-Seq , Neoplasias Gástricas/patologia
10.
Front Immunol ; 12: 615011, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33717102

RESUMO

Introduction: Borrelia burgdorferi sensu lato (sl) is the causative agent of Lyme borreliosis. Currently there is no human vaccine against Lyme borreliosis, and most research focuses on recombinant protein vaccines. DNA tattoo vaccination with B. afzelii strain PKo OspC in mice has proven to be fully protective against B. afzelii syringe challenge and induces a favorable humoral immunity compared to recombinant protein vaccination. Alternatively, several recombinant protein vaccines based on tick proteins have shown promising effect in tick-bite infection models. In this study, we evaluated the efficacy of DNA vaccines against Borrelia OspC or tick antigens in a tick-bite infection model. Method: We vaccinated C3H/HeN mice with OspC using a codon-optimized DNA vaccine or with recombinant protein. We challenged these mice with B. burgdorferi sensu stricto (ss)-infected Ixodes scapularis nymphs. Subsequently, we vaccinated C3H/HeN mice with DNA vaccines coding for tick proteins for which recombinant protein vaccines have previously resulted in interference with tick feeding and/or Borrelia transmission: Salp15, tHRF, TSLPI, and Tix-5. These mice were also challenged with B. burgdorferi ss infected Ixodes scapularis nymphs. Results: DNA tattoo and recombinant OspC vaccination both induced total IgG responses. Borrelia cultures and DNA loads of skin and bladder remained negative in the mice vaccinated with OspC DNA vaccination, except for one culture. DNA vaccines against tick antigens Salp15 and Tix-5 induced IgG responses, while those against tHRF and TSLPI barely induced any IgG response. In addition, Borrelia cultures, and DNA loads from mice tattooed with DNA vaccines against tick proteins TSLPI, Salp15, tHRF, and Tix-5 were all positive. Conclusion: A DNA tattoo vaccine against OspC induced high specific IgG titers and provided near total protection against B. burgdorferi ss infection by tick challenge. In contrast, DNA tattoo vaccines against tick proteins TSLPI, Salp15, tHRF, and Tix-5 induced low to moderate IgG titers and did not provide protection. Therefore, DNA tattoo vaccination does not seem a suitable vaccine strategy to identify, or screen for, tick antigens for anti-tick vaccines. However, DNA tattoo vaccination is a straightforward and effective vaccination platform to assess novel B. burgdorferi sl antigen candidates in a relevant tick challenge model.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Artrópodes/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Borrelia burgdorferi/imunologia , Ixodes/imunologia , Vacinas contra Doença de Lyme/imunologia , Doença de Lyme/prevenção & controle , Vacinas de DNA/imunologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Borrelia burgdorferi/genética , Feminino , Imunização , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Doença de Lyme/transmissão , Camundongos
11.
Microb Pathog ; 154: 104842, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33762199

RESUMO

BACKGROUND: Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tuberculosis), is one of the most common and dangerous infectious diseases in the world. Despite vaccination with BCG, it is still considered as a major health problem. Therefore, design and production of an effective novel vaccine against TB is necessary. Our aim was to evaluate immunogenicity of HspX/EsxS fusion protein of M. tuberculosis along with ISCOMATRIX, PLUSCOM nano-adjuvants and MPLA through the subcutaneous route in mice model. METHODS: HspX/EsxS fused protein of M. tuberculosis was cloned, expressed and purified in the prokaryotic system. ISCOMATRIX and PLUSCOM nano-adjuvants were prepared by film hydration method. Subcutaneous immunization of BALB/c mice was performed by different formulations. IFN-γ, IL-4, IL-17 and TGF-ß cytokines levels as well as serum IgG1, IgG2a. RESULTS: Our results showed that subcutaneous administration of mice with HspX/EsxS along with three adjuvants, ISCOMATRIX, PLUSCOM and MPLA increased immunogenicity of multi-stage fusion protein of M. tuberculosis. Additionally, HspX/EsxS protein + ISCOMATRIX or + PLUSCOM nano-adjuvants induced stronger Th1, IgG2a and IgG1 immune responses compared to MPLA adjuvant. Totally, HspX/EsxS/ISCOMATRIX/MPLA, HspX/EsxS/PLUSCOM/MPLA and two BCG booster groups could significantly induce higher Th1 and IgG2a immune responses. CONCLUSION: With regard to ability of ISCOMATRIX, PLUSCOM and MPLA adjuvants to increase immunogenicity of HspX/EsxS protein through induction of IFN-γ and IgG2a immune responses, it seems that these adjuvants and especially ISCOMATRIX and PLUSCOM, could also improve BCG efficacy as a BCG booster.


Assuntos
Mycobacterium tuberculosis , Vacinas contra a Tuberculose , Adjuvantes Imunológicos , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Colesterol , Modelos Animais de Doenças , Combinação de Medicamentos , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/genética , Fosfolipídeos , Saponinas
12.
Int J Med Microbiol ; 311(3): 151496, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33756191

RESUMO

Streptococcal toxic shock syndrome (STSS) is a severe invasive infection characterized by the sudden onset of shock, multi-organ failure, and puerperal sepsis and shows high mortality. Its primary cause is group A streptococcus (GAS, Streptococcus pyogenes). In this study, we genotyped the cell-surface M virulence protein gene (emm) from 621 GAS isolates obtained from patients with STSS in Japan in 2013-2018 and performed antimicrobial susceptibility testing using the broth microdilution method. The predominant emm type was found to be 1, followed by 89, 12, and 3, which were identified in more than 70 % of STSS isolates. The proportions of emm3 and emm89 increased from 2.4 % and 12.0 %, respectively, during 2010-2012 to 5.6 % and 23.3 % during 2013-2018. In contrast, the proportion of emm1 decreased from 60.6 % to 39.3 % during the same two periods. Some emm types showed increasing proportions and were not isolated from patients with STSS in 2010-2012. Among these, an emm76 type increased in prevalence and was not included in the 30-valent M protein-based vaccine. Continual investigation of changes in the epidemiology of GAS which causes STSS can provide useful monitoring information such as future vaccination strategies and the emergence status of antimicrobial-resistant bacteria.


Assuntos
Choque Séptico , Infecções Estreptocócicas , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Farmacorresistência Bacteriana , Humanos , Japão , Choque Séptico/tratamento farmacológico , Infecções Estreptocócicas/tratamento farmacológico , Infecções Estreptocócicas/epidemiologia , Streptococcus pyogenes/genética
13.
Nat Commun ; 12(1): 1546, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33750771

RESUMO

Many bacterial pathogens rely on virulent type III secretion systems (T3SSs) or injectisomes to translocate effector proteins in order to establish infection. The central component of the injectisome is the needle complex which assembles a continuous conduit crossing the bacterial envelope and the host cell membrane to mediate effector protein translocation. However, the molecular principles underlying type III secretion remain elusive. Here, we report a structure of an active Salmonella enterica serovar Typhimurium needle complex engaged with the effector protein SptP in two functional states, revealing the complete 800Å-long secretion conduit and unraveling the critical role of the export apparatus (EA) subcomplex in type III secretion. Unfolded substrates enter the EA through a hydrophilic constriction formed by SpaQ proteins, which enables side chain-independent substrate transport. Above, a methionine gasket formed by SpaP proteins functions as a gate that dilates to accommodate substrates while preventing leaky pore formation. Following gate penetration, a moveable SpaR loop first folds up to then support substrate transport. Together, these findings establish the molecular basis for substrate translocation through T3SSs and improve our understanding of bacterial pathogenicity and motility.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Transporte Proteico/fisiologia , Salmonella typhimurium/metabolismo , Sistemas de Secreção Tipo III/química , Sistemas de Secreção Tipo III/metabolismo , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Microscopia Crioeletrônica , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Salmonella enterica/metabolismo , Salmonella typhimurium/genética , Sistemas de Secreção Tipo III/genética
14.
Hum Vaccin Immunother ; 17(7): 2225-2231, 2021 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-33522380

RESUMO

The four-component meningococcal serogroup B vaccine (4CMenB) contains antigens present in the majority of meningococci causing invasive meningococcal disease (IMD) and may potentially offer protection against strains belonging to non-B serogroups.This study aimed to evaluate the ability of 4CMenB-induced antibodies to kill, in a human serum bactericidal assay (hSBA), non-B meningococci belonging to the main genotypes responsible for IMD in Italy.Meningococci, collected between 2015 and 2017, was characterized for PorA, FetA and sequence type, and for clonal complex. Twenty non-B isolates, representative of the most frequent genotypes, were molecularly characterized for 4CMenB antigens and tested in hSBA with sera from 4CMenB-vaccinated infants and adolescents.Among twenty isolates, eleven were serogroup C, five were Y, two W and two X. All isolates contained genes encoding for fHbp and NHBA antigens and four harbored the NadA full-length encoding gene. Positive hSBA titers were obtained against all serogroup W, X and Y isolates and against five serogroup C isolates.These data show that the 4CMenB vaccine can induce bactericidal antibodies against genetically representative meningococcal W, Y and X strains from Italy. For serogroup C, different susceptibilities to killing were observed for strains with similar antigenic repertoires.


Assuntos
Infecções Meningocócicas , Vacinas Meningocócicas , Neisseria meningitidis Sorogrupo B , Neisseria meningitidis , Adolescente , Antígenos de Bactérias/genética , Humanos , Lactente , Itália/epidemiologia , Infecções Meningocócicas/epidemiologia , Infecções Meningocócicas/prevenção & controle , Neisseria meningitidis/genética , Neisseria meningitidis Sorogrupo B/genética , Sorogrupo
15.
Mol Biol Rep ; 48(2): 1645-1649, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33550574

RESUMO

This study aimed to investigate the distribution of virulence factor genes in Shigella strains isolated from children with diarrhea in the southwest, Iran. In this cross-sectional study, 1530 diarrheal stool specimens were collected from children aged under 15 years. The Shigella strains were identified by biochemical methods and polymerase chain reaction (PCR). Subsequently, all Shigella isolates were evaluated by PCR for the presence of nine virulence genes ipaH (responsible for dissemination from cell to cell), ial (responsible for epithelial cell penetration), sat (displays cytopathic activity in several intestinal cell lines), sigA (toxic to epithelial cells), pic (associated with colonization), pet (cytotoxic for epithelial cells), sepA (contribute to intestinal inflammation and colonization), virF and invE (regulatory proteins). A total of 91 isolates including 47 S. flexneri, 36 S. sonnei, and 8 S. boydii were identified. All isolates were positive for the ipaH gene. The other genes include ial, virF, invE, sigA, sat, sepA, pic and pet found in 84.6%, 72.5%, 68.1%, 62.6%, 51.6%, 39.5%, 37.3% and 28.5% of the isolates, respectively. The results showed a high distribution of virulence genes among Shigella strains in our region. It seems that for different Shigella spp. different virulence factors contribute to pathogenesis. The current study provided insights into some baseline information about the distribution of some virulence genes of Shigella isolates in Southwest Iran.


Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Diarreia/genética , Shigella/genética , Adolescente , Idoso , Proteínas de Bactérias/classificação , Proteínas de Bactérias/isolamento & purificação , Criança , Pré-Escolar , Diarreia/epidemiologia , Diarreia/microbiologia , Diarreia/patologia , Fezes/microbiologia , Feminino , Humanos , Lactente , Irã (Geográfico)/epidemiologia , Shigella/classificação , Shigella/patogenicidade , Fatores de Virulência/genética
16.
Vaccine ; 39(11): 1621-1630, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33597116

RESUMO

Invasive meningococcal disease (IMD) is associated with high case fatality rates and long-term sequelae among survivors. Meningococci belonging to six serogroups (A, B, C, W, X, and Y) cause nearly all IMD worldwide, with serogroup B meningococci (MenB) the predominant cause in many European countries, including Greece (~80% of all IMD). In the absence of protein-conjugate polysaccharide MenB vaccines, two protein-based vaccines are available to prevent MenB IMD in Greece: 4CMenB (Bexsero™, GlaxoSmithKline), available since 2014; and MenB-FHbp, (Trumenba™, Pfizer), since 2018. This study investigated the potential coverage of MenB vaccines in Greece using 107 MenB specimens, collected from 2010 to 2017 (66 IMD isolates and 41 clinical samples identified solely by non-culture PCR), alongside 6 MenB isolates from a carriage study conducted during 2017-2018. All isolates were characterized by multilocus sequence typing (MLST), PorA, and FetA antigen typing. Whole Genome Sequencing (WGS) was performed on 66 isolates to define the sequences of vaccine components factor H-binding protein (fHbp), Neisserial Heparin Binding Antigen (NHBA), and Neisseria adhesin A (NadA). The expression of fHbp was investigated with flow cytometric meningococcal antigen surface expression (MEASURE) assay. The fHbp gene was present in-frame in all isolates tested by WGS and in 41 MenB clinical samples. All three variant families of fHbp peptides were present, with subfamily B peptides (variant 1) occurring in 69.2% and subfamily A in 30.8% of the samples respectively. Sixty three of 66 (95.5%) MenB isolates expressed sufficient fHbp to be susceptible to bactericidal killing by MenB-fHbp induced antibodies, highlighting its potential to protect against most IMD in Greece.


Assuntos
Infecções Meningocócicas , Vacinas Meningocócicas , Neisseria meningitidis Sorogrupo B , Antígenos de Bactérias/genética , Europa (Continente) , Grécia/epidemiologia , Humanos , Infecções Meningocócicas/epidemiologia , Infecções Meningocócicas/prevenção & controle , Tipagem de Sequências Multilocus , Neisseria meningitidis Sorogrupo B/genética , Estudos Retrospectivos , Sorogrupo
17.
Mol Immunol ; 133: 44-52, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33631554

RESUMO

Brucella is an intracellular zoonotic pathogen that can affect many hosts. Brucella melitensis Rev.1 is a live attenuated, is one of the most effective vaccine strain against brucellosis. It can be used safely in sheep, goats, and even cattle. Although many studies are available on this topic, there is no effective vaccine strain for sheep and goats that distinguishes the antibody titer produced between the field infections and vaccinations. Outer membrane protein 19 (Omp 19) is both virulent and a protective antigen found on the cell-wall of the Brucella strain. In this study, used the suicide plasmid pJQ200KS, which contained homologous region without Omp19 Open Reading Frame (ORF) that was transferred to B. melitensis Rev.1 and further transformed into spheroplasts along with penicillin, ampicillin, and glycine by electroporation. To obtain a mutant vector from Escherichia coli, we used the heat shock transformation method along with the blue-white colony screening using X-gal media, whereas for the gene transfer in Brucella, we used electroporation. A scanning electron microscope (S.E.M) was used to observe the spheroplast transformation while the mutant vector and deletion mutants were confirmed through PCR and sequence analysis. In the mouse model efficacy trials, three commercial vaccines were found to comply with the OIE standards. Although the deletion mutants 19 and 44/10 had similar efficiency as the commercial vaccines in terms of stimulation power, the ELISA test with Omp19 protein showed the same results as the negative control. The Rev.1 Omp19 deletion mutants obtained in this study contained sufficient residual virulence, and their protective immunity was similar to the commercial vaccines. The study showed that a vaccine prepared using a B. melitensis Rev.1 ΔOmp19 can act as a marker vaccine or differentiate infected from vaccinated animals (DIVA) through the ELISA test that detects the Omp19 protein.


Assuntos
Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Vacina contra Brucelose/imunologia , Brucella melitensis/genética , Brucella melitensis/imunologia , Brucelose/prevenção & controle , Lipoproteínas/genética , Animais , Brucella abortus/genética , Brucelose/microbiologia , Modelos Animais de Doenças , Eletroporação/métodos , Feminino , Camundongos , Plasmídeos/genética , Vacinação , Vacinas Atenuadas/imunologia , Virulência/genética , Virulência/imunologia
18.
J Infect ; 82(4): 28-36, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33610687

RESUMO

BACKGROUND: Surveillance of serogroup B Neisseria meningitidis (MenB) subcapsular antigen variant distribution in invasive disease (IMD) is fundamental for multicomponent vaccine coverage prediction. IMD incidence in Tuscany in 2018 was 0.37/100,000 inhabitants, with MenB representing 57% of cases. More than 50% of MenB responsible for IMD cannot be grown in culture, and molecular characterization of these cases is often lacking. The aim of the present study was to describe the distribution of MenB subcapsular antigens, comparing their distribution in culture-positive and culture-negative cases. METHODS: Molecular data regarding clonal complexes and subcapsular antigen variants of the 55 MenB-IMD occurring in Tuscany from 2007 to 2019 were made available, and their distribution between culture-positive and culture-negative cases was compared. Genetic-MATS and MenDeVAR prediction systems were used to assess multicomponent vaccine coverage predictions. RESULTS: Culture-positive and culture-negative cases presented a similar percentage representation of fHbp subfamilies. Clonal complex 162 was almost constantly associated with fHbp B231/v1.390, Neisserial-heparin-binding-antigen (NHBA) peptide 20, and PorinA P1.22,14 (BAST-3033): these were the most represented antigenic variants, both in culture-positive and culture-negative groups. Point-estimate 4CMenB coverage prediction was 88.5% (84.6%-92.3%). CONCLUSIONS: Our data demonstrate that non-cultivable meningococci, responsible for IMD, possess genetic variants of subcapsular antigens that are representative of what has been observed in culture. The vaccine-related antigenic epidemiology of MenB is thus similar in both groups. One of the first on-field applications of gMATS and MenDeVAR identifies their major advantage in their accessibility and in the possibility of dynamic data implementation that must be pursued continuously in the future.


Assuntos
Infecções Meningocócicas , Vacinas Meningocócicas , Neisseria meningitidis Sorogrupo B , Neisseria meningitidis , Antígenos de Bactérias/genética , Humanos , Infecções Meningocócicas/epidemiologia , Neisseria , Neisseria meningitidis/genética , Neisseria meningitidis Sorogrupo B/genética
19.
J Infect ; 82(4): 37-44, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33610688

RESUMO

Studies of meningococcal genetic population structure, including the potential associations between surface proteins variants and clonal complexes, are important to understand how new protein MenB vaccines might impact in specific scenarios. With the aim to analyze the diversity of Spanish invasive MenB strains, and genetic variability of the fHbp vaccine antigen, all MenB isolates received at National Reference Laboratory (NRL) from 2015 to 2018 were molecularly characterized. MATERIAL AND METHODS: 108, 103, 87 and 112 invasive MenB strains isolated during 2015-2018, respectively, were received at NRL. The strains were whole genome sequenced, and porA, fetA, MLST and fHbp variability was analyzed. Potential impact on MenB vaccines coverage was also assessed. RESULTS: A total of 42, 38 and 3 different FHbp subfamily A, B and A/B hybrid peptides, respectively, were found. FHbp subfamily A peptides were harboured by most of the strains (65.9%), being the most prevalent peptide 45 which was associated with genosubtype 22,14 and cc213. FHbp subfamily B peptides were harboured by 32.4% of the strains, and 6 strains harbouring subfamily A/B hybrid peptides were also found. The 64.15% of the strains showed FHbp variants "exact-match" or "cross-reactive" to the FHbp variants included in rLP2086 vaccine according to hSBA assays in the rLP2086 clinical development, and 15.85% showed FHbp peptides defined as predictors of FHbp-coverage for 4CMenB vaccine by gMATS. CONCLUSIONS: Due to invasive meningococcal strains temporal variability (eg prevalence of the cc213 increased from 3.6% in 2007 to 33% in 2018) affecting to the presence and distribution of the vaccine antigens, continuous detailed meningococcal surveillance and monitoring of the vaccine antigens is needed to determine the degree and durability of coverage provided by these protein vaccine.


Assuntos
Infecções Meningocócicas , Vacinas Meningocócicas , Neisseria meningitidis Sorogrupo B , Neisseria meningitidis , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Fator H do Complemento , Humanos , Infecções Meningocócicas/epidemiologia , Infecções Meningocócicas/prevenção & controle , Tipagem de Sequências Multilocus , Neisseria meningitidis/genética , Neisseria meningitidis Sorogrupo B/genética , Sorogrupo , Espanha/epidemiologia
20.
J Med Microbiol ; 70(3)2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33616518

RESUMO

Introduction. Streptococcus dysgalactiae subspecies equisimilis (SDSE) is becoming increasingly recognized as an important human pathogen. Recurrent bacteremia with SDSE has been described previously.Aim. The aims of the study were to establish the genetic relatedness of SDSE isolates with emm-type stG643 that had caused recurrent bacteraemia in three patients and to search for signs of horizontal gene transfer of the emm gene in a collection of SDSE stG643 genomes.Hypothesis. Recurring SDSE bacteremia is caused by the same clone in one patient.Methodology. Whole genome sequencing of 22 clinical SDSE stG643 isolates was performed, including three paired blood culture isolates and sixteen isolates from various sites. All assemblies were aligned to a reference assembly and SNPs were extracted. A total of 53 SDSE genomes were downloaded from GenBank. Two phylogenetic trees, including all 75 SDSE isolates, were created. One tree was based on the emm gene only and one tree was based on all variable positions in the genomes.Results. The genomes from the three pairs of SDSE isolates showed high sequence similarity (1-17 SNPs difference between the pairs), whereas the median SNP difference between the 22 isolates in our collection was 1694 (range 1-11257). The paired isolates were retrieved with 7-53 months between episodes. The 22 SDSE isolates from our collection formed a cluster in the phylogenetic tree based on the emm gene, while they were more scattered in the tree based on all variable positions.Conclusions. Our results show that the paired isolates were of the same clonal origin, which in turn supports carriage between bacteraemia episodes. The phylogenetic analysis indicates that horizontal gene transfer of the emm-gene between some of the SDSE isolates has occurred.


Assuntos
Bacteriemia/microbiologia , Reinfecção/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus/genética , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Transporte/genética , Genoma Bacteriano/genética , Humanos , Filogenia , Streptococcus/classificação , Streptococcus/isolamento & purificação
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