RESUMO
A strategy for vaccine design involves identifying proteins that could be involved in pathogen-host interactions. The aim of this proteomic study was to determine how iron limitation affects the protein expression of Tenacibaculum dicentrarchi, with a primary focus on virulence factors and proteins associated with iron uptake. The proteomic analysis was carried out using two strains of T. dicentrarchi grown under normal (control) and iron-limited conditions, mimicking the host environment. Our findings revealed differences in the proteins expressed by the type strain CECT 7612T and the Chilean strain TdCh05 of T. dicentrarchi. Nonetheless, both share a common response to iron deprivation, with an increased expression of proteins associated with iron oxidation and reduction metabolism (e.g., SufA, YpmQ, SufD), siderophore transport (e.g., ExbD, TonB-dependent receptor, HbpA), heme compound biosynthesis, and iron transporters under iron limitation. Proteins involved in gliding motility, such as GldL and SprE, were also upregulated in both strains. A negative differential regulation of metabolic proteins, particularly those associated with amino acid biosynthesis, was observed under iron limitation, reflecting the impact of iron availability on bacterial metabolism. Additionally, the TdCh05 strain exhibited unique proteins associated with gliding motility machinery and phage infection control compared to the type strain. These groups of proteins have been identified as virulence factors within the Flavobacteriaceae family, including the genus Tenacibaculum. These results build upon our previous report on iron acquisition mechanisms and could lay the groundwork for future studies aimed at elucidating the role of some of the described proteins in the infectious process of tenacibaculosis, as well as in the development of potential vaccines.
Assuntos
Proteínas de Bactérias , Doenças dos Peixes , Infecções por Flavobacteriaceae , Ferro , Oxirredução , Proteômica , Tenacibaculum , Regulação para Cima , Ferro/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Infecções por Flavobacteriaceae/veterinária , Infecções por Flavobacteriaceae/microbiologia , Animais , Doenças dos Peixes/microbiologia , Tenacibaculum/genética , Tenacibaculum/metabolismo , Proteoma , Fatores de Virulência/metabolismo , Fatores de Virulência/genética , Bass/microbiologiaRESUMO
Gram-negative bacteria from the Bacteroidota phylum possess a type-IX secretion system (T9SS) for protein secretion, which requires cargoes to have a C-terminal domain (CTD). Structurally analysed CTDs are from Porphyromonas gingivalis proteins RgpB, HBP35, PorU and PorZ, which share a compact immunoglobulin-like antiparallel 3+4 ß-sandwich (ß1-ß7). This architecture is essential as a P. gingivalis strain with a single-point mutant of RgpB disrupting the interaction of the CTD with its preceding domain prevented secretion of the protein. Next, we identified the C-terminus ('motif C-t.') and the loop connecting strands ß3 and ß4 ('motif Lß3ß4') as conserved. We generated two strains with insertion and replacement mutants of PorU, as well as three strains with ablation and point mutants of RgpB, which revealed both motifs to be relevant for T9SS function. Furthermore, we determined the crystal structure of the CTD of mirolase, a cargo of the Tannerella forsythia T9SS, which shares the same general topology as in Porphyromonas CTDs. However, motif Lß3ß4 was not conserved. Consistently, P. gingivalis could not properly secrete a chimaeric protein with the CTD of peptidylarginine deiminase replaced with this foreign CTD. Thus, the incompatibility of the CTDs between these species prevents potential interference between their T9SSs.
Assuntos
Proteínas de Bactérias , Sistemas de Secreção Bacterianos , Porphyromonas gingivalis , Porphyromonas gingivalis/metabolismo , Porphyromonas gingivalis/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/metabolismo , Sistemas de Secreção Bacterianos/genética , Sistemas de Secreção Bacterianos/química , Modelos Moleculares , Cristalografia por Raios X , Sequência de Aminoácidos , Sinais Direcionadores de Proteínas , Domínios Proteicos , Bacteroidetes/metabolismo , Bacteroidetes/genética , Tannerella forsythia/metabolismo , Tannerella forsythia/genética , Tannerella forsythia/química , Relação Estrutura-Atividade , Conformação ProteicaRESUMO
Riemerella anatipestifer infection is characterized by meningitis with neurological symptoms in ducklings and has adversely affected the poultry industry. R. anatipestifer strains can invade the duck brain to cause meningitis and neurological symptoms, but the underlying mechanism remains unknown. In this study, we showed that obvious clinical symptoms, an increase in bloodâbrain barrier (BBB) permeability, and the accumulation of inflammatory cytokines occurred after intravenous infection with the Yb2 strain but not the mutant strain Yb2ΔsspA, indicating that Yb2 infection can lead to cerebrovascular dysfunction and that the type IX secretion system (T9SS) effector SspA plays a critical role in this pathological process. In addition, we showed that Yb2 infection led to rapid degradation of occludin (a tight junction protein) and collagen IV (a basement membrane protein), which contributed to endothelial barrier disruption. The interaction between SspA and occludin was confirmed by coimmunoprecipitation. Furthermore, we found that SspA was the main enzyme mediating occludin and collagen IV degradation. These data indicate that R. anatipestifer SspA mediates occludin and collagen IV degradation, which functions in BBB disruption in R. anatipestifer-infected ducks. These findings establish the molecular mechanisms by which R. anatipestifer targets duckling endothelial cell junctions and provide new perspectives for the treatment and prevention of R. anatipestifer infection.
Assuntos
Infecções por Flavobacteriaceae , Meningite , Doenças das Aves Domésticas , Riemerella , Animais , Barreira Hematoencefálica/metabolismo , Patos/metabolismo , Virulência , Fatores de Virulência/metabolismo , Ocludina/genética , Ocludina/metabolismo , Infecções por Flavobacteriaceae/veterinária , Riemerella/metabolismo , Meningite/veterinária , Colágeno/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
The type 9 secretion system (T9SS) is a recently discovered machinery that both transports cargo proteins across the Gram-negative bacterial outer membrane and attaches them to lipopolysaccharides on the extracellular surface. Outer membrane proteins (OMPs) are key components of the T9SS and are involved in both steps. In this chapter, we describe a method for the in silico modeling of T9SS OMPs and their complexes, and model validation. This is useful when the production of recombinant OMPs is difficult, and these protocols can also be applied to OMP complexes outside of the T9SS.