RESUMO
Mycobacterium tuberculosis (Mtb) possesses five type VII secretion systems (T7SS), virulence determinants that include the secretion apparatus and associated secretion substrates. Mtb strains deleted for the genes encoding substrates of the ESX-3 T7SS, esxG or esxH, require iron supplementation for in vitro growth and are highly attenuated in vivo. In a subset of infected mice, suppressor mutants of esxG or esxH deletions were isolated, which enabled growth to high titers or restored virulence. Suppression was conferred by mechanisms that cause overexpression of an ESX-3 paralogous region that lacks genes for the secretion apparatus but encodes EsxR and EsxS, apparent ESX-3 orphan substrates that functionally compensate for the lack of EsxG or EsxH. The mechanisms include the disruption of a transcriptional repressor and a massive 38- to 60-fold gene amplification. These data identify an iron acquisition regulon, provide insight into T7SS, and reveal a mechanism of Mtb chromosome evolution involving "accordion-type" amplification.
Assuntos
Mycobacterium tuberculosis/genética , Sistemas de Secreção Tipo VII/genética , Animais , Sistemas de Secreção Bacterianos/genética , Evolução Biológica , Evolução Molecular , Amplificação de Genes/genética , Camundongos , Mycobacterium tuberculosis/metabolismo , Sistemas de Secreção Tipo VII/fisiologia , Virulência , Fatores de Virulência/genéticaRESUMO
The increase of antimicrobial resistance (AMR), and lack of new classes of licensed antimicrobials, have made alternative treatment options for AMR pathogens increasingly attractive. Recent studies have demonstrated anti-bacterial efficacy of a humanised monoclonal antibody (mAb) targeting the O25b O-antigen of Escherichia coli ST131. To evaluate the phenotypic effects of antibody binding to diverse clinical E. coli ST131 O25b bacterial isolates in high-throughput, we designed a novel mAb screening method using high-content imaging (HCI) and image-based morphological profiling to screen a mAb targeting the O25b O-antigen. Screening the antibody against a panel of 86 clinical E. coli ST131 O25:H4 isolates revealed 4 binding phenotypes: no binding (18.60%), weak binding (4.65%), strong binding (69.77%) and strong agglutinating binding (6.98%). Impaired antibody binding could be explained by the presence of insertion sequences or mutations in O-antigen or lipopolysaccharide core biosynthesis genes, affecting the amount, structure or chain length of the O-antigen. The agglutinating binding phenotype was linked with lower O-antigen density, enhanced antibody-mediated phagocytosis and increased serum susceptibly. This study highlights the need to screen candidate mAbs against large panels of clinically relevant isolates, and that HCI can be used to evaluate mAb binding affinity and potential functional efficacy against AMR bacteria.
Assuntos
Antibacterianos/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Infecções por Escherichia coli/tratamento farmacológico , Escherichia coli/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Antibacterianos/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/ultraestrutura , Infecções por Escherichia coli/microbiologia , Estudos de Viabilidade , Humanos , Sequências Repetitivas Dispersas/genética , Testes de Sensibilidade Microbiana , Microscopia Eletrônica , Antígenos O/genética , Antígenos O/imunologia , Filogenia , Polimorfismo de Nucleotídeo Único , Virulência/imunologiaRESUMO
The interplay between bacterial antimicrobial susceptibility, phylogenetics and patient outcome is poorly understood. During a typhoid clinical treatment trial in Nepal, we observed several treatment failures and isolated highly fluoroquinolone-resistant Salmonella Typhi (S. Typhi). Seventy-eight S. Typhi isolates were genome sequenced and clinical observations, treatment failures and fever clearance times (FCTs) were stratified by lineage. Most fluoroquinolone-resistant S. Typhi belonged to a specific H58 subclade. Treatment failure with S. Typhi-H58 was significantly less frequent with ceftriaxone (3/31; 9.7%) than gatifloxacin (15/34; 44.1%)(Hazard Ratio 0.19, p=0.002). Further, for gatifloxacin-treated patients, those infected with fluoroquinolone-resistant organisms had significantly higher median FCTs (8.2 days) than those infected with susceptible (2.96) or intermediately resistant organisms (4.01)(pS. Typhi clade internationally, but there are no data regarding disease outcome with this organism. We report an emergent new subclade of S. Typhi-H58 that is associated with fluoroquinolone treatment failure.
Assuntos
Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Ciprofloxacina/farmacologia , Fluoroquinolonas/uso terapêutico , Genótipo , Salmonella typhi/efeitos dos fármacos , Febre Tifoide/tratamento farmacológico , Técnicas de Tipagem Bacteriana , Ceftriaxona/uso terapêutico , Gatifloxacina , Humanos , Nepal , Salmonella typhi/classificação , Salmonella typhi/isolamento & purificação , Análise de Sequência de DNA , Falha de Tratamento , Febre Tifoide/microbiologiaAssuntos
Antibacterianos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Escherichia coli Êntero-Hemorrágica/efeitos dos fármacos , Proteínas de Escherichia coli/metabolismo , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Animais , Antibacterianos/uso terapêutico , Descoberta de Drogas/história , Escherichia coli Êntero-Hemorrágica/metabolismo , Escherichia coli Êntero-Hemorrágica/patogenicidade , Francisella tularensis/efeitos dos fármacos , História do Século XX , Infecções por Salmonella/tratamento farmacológico , Salmonella typhimurium/efeitos dos fármacos , Tularemia/tratamento farmacológico , Fatores de VirulênciaRESUMO
Immunisation of BALB/c mice with a vaccine containing Vi polysaccharide conjugated to the Klebsiella pneumoniae outer membrane 40 kDa protein (rP40), in combination with Escherichia coli heat-labile toxin adjuvant (LT), elicited anti-Vi IgG antibodies after administration using different routes. Testing of the immune serum in opsonisation assays demonstrated the specific enhancement of Vi-positive bacterial uptake by cultured murine bone marrow derived macrophages. Intra-peritoneal challenge of mice immunised with the Vi-based vaccine elicited a degree of protection against virulent Vi+ Salmonella enterica serovar typhimurium (S. typhimurium). In contrast, Vi vaccination did not confer protection against oral challenge with virulent Vi-positive S. typhimurium or S. dublin.
Assuntos
Vacinas Bacterianas/imunologia , Modelos Animais de Doenças , Polissacarídeos Bacterianos/imunologia , Infecções por Salmonella/prevenção & controle , Animais , Avaliação Pré-Clínica de Medicamentos , Ensaio de Imunoadsorção Enzimática , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Salmonella/imunologiaRESUMO
Fragment C (TetC) is a non-toxic 47 kDa polypeptide fragment of tetanus toxin that can be used as a subunit vaccine against tetanus. Expression of TetC in Escherichia coli and yeast was dependent on the availability of synthetic genes that were required to improve translation efficiency and stabilize the mRNA. To explore the feasibility of producing TetC in tobacco leaves, we attempted expression of both the bacterial high-AT (72.3% AT) and the synthetic higher-GC genes (52.5% AT) in tobacco chloroplasts. We report here that the bacterial high-AT mRNA is stable in tobacco chloroplasts. Significant TetC accumulation was obtained from both genes, 25 and 10% of total soluble cellular protein, respectively, proving the versatility of plastids for expression of unmodified high-AT and high-GC genes. Mucosal immunization of mice with the plastid- produced TetC induced protective levels of TetC antibodies. Thus, expression of TetC in chloroplasts provides a potential route towards the development of a safe, plant-based tetanus vaccine for nasal and oral applications.