RESUMO
The human gastric pathogen Helicobacter pylori occurs in two basic variants, either exhibiting a functional cagPAI-encoded type-4-secretion-system (T4SS) or not. Only a few cagPAI-positive strains have been successfully adapted for long-term infection of mice, including the pre-mouse Sydney strain 1 (PMSS1). Here we confirm that PMSS1 induces gastric inflammation and neutrophil infiltration in mice, progressing to intestinal metaplasia. Complete genome analysis of PMSS1 revealed 1,423 coding sequences, encompassing the cagPAI gene cluster and, unusually, the location of the cytotoxin-associated gene A (cagA) approximately 15 kb downstream of the island. PMSS1 harbours three genetically exchangeable loci that are occupied by the hopQ coding sequences. HopQ represents a critical co-factor required for the translocation of CagA into the host cell and activation of NF-κB via the T4SS. Long-term colonisation of mice led to an impairment of cagPAI functionality. One of the bacterial clones re-isolated at four months post-infection revealed a mutation in the cagPAI gene cagW, resulting in a frame shift mutation, which prevented CagA translocation, possibly due to an impairment of T4SS function. Rescue of the mutant cagW re-established CagA translocation. Our data reveal intriguing insights into the adaptive abilities of PMSS1, suggesting functional modulation of the H. pylori cagPAI virulence attribute.
Assuntos
Proteínas de Bactérias/genética , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Helicobacter pylori/patogenicidade , Sistemas de Secreção Tipo IV/genética , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Família Multigênica , Virulência , Sequenciamento Completo do Genoma/métodosRESUMO
P. acnes is a skin commensal that is frequently associated with inflammatory diseases such as acne vulgaris. Despite the availability of the genome sequence functional studies on P. acnes are scarce due to a lack of methods for genetic manipulation of this bacterium. Here we present an insertional mutagenesis approach for the inactivation of specific P. acnes genes. The gene of interest can be disrupted and replaced with an erythromycin-resistance cassette by employing homologous recombination. We used this method to generate knock-out mutants of camp2 (PPA0687) and camp4 (PPA1231), encoding CAMP factor homologs with predicted co-hemolytic activities. The successful inactivation of the two genes was confirmed by PCR and Western blotting experiments using specific anti-CAMP2/CAMP4 sera. The Δcamp2 but not the Δcamp4 mutant exhibited reduced hemolytic activity in the CAMP reaction with sheep erythrocytes, indicating that CAMP2 is the major active co-hemolytic factor of P. acnes. The biological relevance of the CAMP factors remains unclear as disruption of camp2 or camp4 did not significantly alter the transcriptome response of HaCaT cells to P. acnes. The here presented insertional mutagenesis approach will facilitate future studies on P. acnes.
Assuntos
Proteínas de Bactérias/genética , Técnicas de Inativação de Genes/métodos , Proteínas Hemolisinas/genética , Mutagênese Insercional/métodos , Propionibacterium acnes/genética , Animais , Antibacterianos/farmacologia , Western Blotting , Eritrócitos/efeitos dos fármacos , Eritromicina/farmacologia , Hemólise , Camundongos , Reação em Cadeia da Polimerase , Recombinação Genética , Seleção Genética , OvinosRESUMO
We constructed an oral live vaccine based on the attenuated aroA mutant Salmonella enterica serovar Typhimurium strain SL3261 expressing outer membrane proteins F and I (OprF-OprI) from Pseudomonas aeruginosa and investigated it in a mouse model. Strains with in vivo inducible protein expression with the PpacC promoter showed good infection rates and immunogenicity but failed to engender detectable antibodies in the lung. However, a systemic booster vaccination following an oral primary immunization yielded high immunoglobulin A (IgA) and IgG antibody levels in both upper and lower airways superior to conventional systemic or mucosal booster vaccination alone. In addition, the proportion of IgG1 and IgG2a antibodies suggested that the systemic booster does not alter the more TH1-like type of response induced by the oral Salmonella primary vaccination. We conclude that an oral primary systemic booster vaccination strategy with an appropriate mucosal vector may be advantageous in diseases with the risk of P. aeruginosa airway infection, such as cystic fibrosis.
Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Lipoproteínas/imunologia , Porinas/imunologia , Pseudomonas aeruginosa/imunologia , Mucosa Respiratória/imunologia , Salmonella typhimurium/genética , Administração Oral , Animais , Anticorpos Antibacterianos/análise , Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/genética , Vacinas Bacterianas/genética , Humanos , Imunidade nas Mucosas , Lipoproteínas/administração & dosagem , Lipoproteínas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Porinas/administração & dosagem , Porinas/genética , Infecções por Pseudomonas/prevenção & controle , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Salmonella typhimurium/imunologia , Vacinação , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/imunologiaRESUMO
Vaccines effective against intracellular pathogens could save the lives of millions of people every year, but vaccine development has been hampered by the slow largely empirical search for protective antigens. In vivo highly expressed antigens might represent a small attractive antigen subset that could be rapidly evaluated, but experimental evidence supporting this rationale, as well as practical strategies for its application, is largely lacking because of technical difficulties. Here, we used Salmonella strains expressing differential amounts of a fluorescent model antigen during infection to show that, in a mouse typhoid fever model, CD4 T cells preferentially recognize abundant Salmonella antigens. To identify a large number of natural Salmonella antigens with high expression levels during infection, we used a quantitative in vivo screening strategy. Immunization studies with five particularly attractive candidates revealed two highly protective antigens that might permit the development of an improved typhoid fever vaccine. In conclusion, we have established a rationale and an experimental strategy that will substantially facilitate vaccine development for Salmonella and possibly other intracellular pathogens.
Assuntos
Antígenos de Bactérias/genética , Vacinas Bacterianas/imunologia , Salmonella typhimurium/genética , Salmonella typhimurium/imunologia , Animais , Vacinas Bacterianas/genética , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , DNA Bacteriano/genética , Feminino , Expressão Gênica , Genes Bacterianos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Salmonelose Animal/imunologia , Salmonelose Animal/microbiologia , Salmonelose Animal/prevenção & controle , Febre Tifoide/imunologia , Febre Tifoide/microbiologia , Febre Tifoide/prevenção & controleRESUMO
A rapidly maturing variant of the red fluorescent protein DsRed was optimized for bacterial expression by random mutagenesis. The brightest variant contains six mutations, two of which (S4T and a silent mutation in codon 2) explain most of the fluorescence enhancement. The novel variants are expressed at 9-60-fold higher levels in Escherichia coli compared to DsRed.T3, but are not superior fluorophores on a per molecule basis. In contrast to previously available DsRed variants, DsRed.T3_S4T is sufficiently bright to monitor Salmonella gene expression in infected animals using flow cytometry. However, no fluorescence enhancement was observed in Leishmania or HeLa cells, indicating that these novel variants are specifically useful for bacteria.