RESUMO
We have identified and partially characterized a putative HD domain hydrolase, LMOf2365_2464, which is highly expressed during listerial intracellular replication. LMOf2365_2464 is annotated as a putative HD domain-containing hydrolase. The ability of an isogenic mutant strain, F2365Δ2464, to adhere, invade and replicate in intestinal epithelial cells (Caco-2) was significantly lower than parent strain F2365. Colonization of mouse liver and spleen by L. monocytogenes F2365 was significantly higher than it was for the mutant. The recombinant protein showed phosphodiesterase activity in the presence of divalent metal ions, indicating its role in nucleotide metabolism. It has activity against several cyclic nucleotides and cyclic dinucleotides, but its strongest activity is against cyclic di-AMP and cyclic AMP. Based on this enzymatic activity, we designated LMOf2365_2464 phosphodiesterase E (PdeE).
Assuntos
Hidrólise , Listeria monocytogenes/enzimologia , Listeria monocytogenes/patogenicidade , Nucleotídeos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Virulência , Animais , Aderência Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células CACO-2 , AMP Cíclico/metabolismo , DNA Bacteriano , Modelos Animais de Doenças , Ensaios Enzimáticos , Feminino , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Humanos , Concentração de Íons de Hidrogênio , Listeria monocytogenes/genética , Listeria monocytogenes/crescimento & desenvolvimento , Listeriose/microbiologia , Fígado/microbiologia , Manganês/metabolismo , Camundongos , Mutagênese , Mutação , Diester Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Recombinantes , Baço/microbiologia , Temperatura , Virulência/genéticaRESUMO
The structure of Archaeal chromatin or nucleoid is believed to have characteristics similar to that found in both eukaryotes and bacteria. Recent comparative studies have suggested that DNA compaction in Archaea requires a bridging protein (e.g., Alba) along with either a wrapping protein (e.g., a histone) or a bending protein such as Sac7d. While X-ray crystal structures demonstrate that Sac7d binds as a monomer to create a significant kink in duplex DNA, the structure of a multiprotein-DNA complex has not been established. Using cross-linked dimers of Sac7d with a defined orientation, we present evidence that indicates that Sac7d is able to largely coat duplex DNA in vivo by binding in alternating head-to-head and tail-to-tail orientations. Although each Sac7d monomer promotes a significant kink of nearly 70°, coated DNA is expected to be largely extended because of compensation of repetitive kinks with helical symmetry.
Assuntos
Proteínas Arqueais/metabolismo , Cromatina/metabolismo , DNA Arqueal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sulfolobus acidocaldarius/metabolismo , Algoritmos , Proteínas Arqueais/química , Proteínas Arqueais/genética , Cromatina/química , Cromatina/genética , Dicroísmo Circular , Cristalografia por Raios X , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , DNA Arqueal/química , DNA Arqueal/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Eletroforese em Gel de Poliacrilamida , Engenharia Genética , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Estrutura Secundária de Proteína , Sulfolobus acidocaldarius/genéticaRESUMO
Aeromonas hydrophila is a reemerging pathogen of channel catfish (Ictalurus punctatus); recent outbreaks from 2009 to 2014 have caused the loss of more than 12 million pounds of market size catfish in Alabama and Mississippi. Genome sequencing revealed a clonal group of A. hydrophila isolates with unique genetic and phenotypic features that is highly pathogenic in channel catfish. Comparison of the genome sequence of a representative catfish isolate (ML09-119) from this virulent clonal group with lower virulence A. hydrophila isolates revealed four fimbrial proteins unique to strain ML09-119. In this work, we expressed and purified four A. hydrophila fimbrial proteins (FimA, Fim, MrfG, and FimOM) and assessed their ability to protect and stimulate protective immunity in channel catfish fingerlings against A. hydrophila ML09-119 infection for vaccine development. Our results showed catfish immunized with FimA, Fim, FimMrfG, and FimOM exhibited 59.83%, 95.41%, 85.72%, and 75.01% relative percent survival, respectively, after challenge with A. hydrophila strain ML09-119. Bacterial concentrations in liver, spleen, and anterior kidney were significantly (p<0.05) lower in vaccinated fish compared to the non-vaccinated sham groups at 48h post-infection. However, only the Fim immunized group showed a significantly higher antibody titer in comparison to the non-vaccinated treatment group (p<0.05) at 21days post-vaccination. Altogether, Fim and FimMrfG recombinant proteins have potential for vaccine development against virulent A. hydrophila infection.