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1.
Protein Sci ; 30(12): 2474-2481, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34676610

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPRs)-CRISPR-associated protein systems are bacterial and archaeal defense mechanisms against invading elements such as phages and viruses. To overcome these defense systems, phages and viruses have developed inhibitors called anti-CRISPRs (Acrs) that are capable of inhibiting the host CRISPR-Cas system via different mechanisms. Although the inhibitory mechanisms of AcrIIC1, AcrIIC2, and AcrIIC3 have been revealed, the inhibitory mechanisms of AcrIIC4 and AcrIIC5 have not been fully understood and structural data are unavailable. In this study, we elucidated the crystal structure of Type IIC anti-CRISPR protein, AcrIIC4. Our structural analysis revealed that AcrIIC4 exhibited a helical bundle fold comprising four helixes. Further biochemical and biophysical analyses showed that AcrIIC4 formed a monomer in solution, and monomeric AcrIIC4 directly interacted with Cas9 and Cas9/sgRNA complex. Discovery of the structure of AcrIIC4 and their interaction mode on Cas9 will help us elucidate the diversity in the inhibitory mechanisms of the Acr protein family.


Assuntos
Antibiose/genética , Proteínas de Bactérias/química , Proteína 9 Associada à CRISPR/química , Sistemas CRISPR-Cas , Haemophilus parainfluenzae/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Proteína 9 Associada à CRISPR/antagonistas & inibidores , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Clonagem Molecular , Cristalografia por Raios X , DNA/química , DNA/genética , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Edição de Genes , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Haemophilus parainfluenzae/metabolismo , Modelos Moleculares , Neisseria meningitidis/genética , Neisseria meningitidis/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
3.
Microb Drug Resist ; 22(4): 247-52, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26683319

RESUMO

AIMS: To investigate the relative contributions of naturally occurring penicillin-binding protein 3 (PBP3) substitutions, and TEM-1, TEM-15, and ROB-1 beta-lactamases on resistance to a third-generation cephalosporin in Haemophilus influenzae and Haemophilus parainfluenzae. RESULTS: The minimum inhibitory concentration (MIC) of cefotaxime (CTX) was assessed after transformation with PCR-amplified ftsI genes expressing altered PBP3 and/or small plasmids encoding beta-lactamases into an isogenic environment of H. influenzae and H. parainfluenzae. Group III PBP3, comprising substitutions N526K, S385T, and L389F, conferred CTX resistance to H. influenzae according to EUCAST interpretative criteria. Group III-like PBP3, comprising substitutions N526H and S385T, increased the CTX MIC of H. parainfluenzae ninefold, but the level did not transgress the resistance breakpoint. Production of TEM-15 beta-lactamase conferred CTX resistance on both H. influenzae and H. parainfluenzae. A nitrocefin hydrolysis assay showed TEM-15 to be a less efficient enzyme compared to TEM-1. CONCLUSIONS: TEM-15 and PBP3 substitutions impose an additive effect on resistance to third-generation cephalosporins in both H. influenzae and H. parainfluenzae. The effect of PBP3 substitutions on beta-lactam resistance in H. parainfluenzae can be addressed by transfer of ftsI genes in vitro.


Assuntos
Haemophilus influenzae/genética , Haemophilus parainfluenzae/genética , Proteínas de Ligação às Penicilinas/genética , Resistência beta-Lactâmica/genética , beta-Lactamases/genética , Substituição de Aminoácidos , Antibacterianos/farmacologia , Cefotaxima/farmacologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/metabolismo , Haemophilus parainfluenzae/efeitos dos fármacos , Haemophilus parainfluenzae/metabolismo , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/genética , Peptidoglicano Glicosiltransferase/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Transformação Bacteriana , beta-Lactamases/metabolismo
4.
Int J Med Microbiol ; 303(8): 603-17, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24035104

RESUMO

Lipopolysaccharide O-antigens are the basis of serotyping schemes for Gram negative bacteria and help to determine the nature of host-bacterial interactions. Haemophilus parainfluenzae is a normal commensal of humans but is also an occasional pathogen. The prevalence, diversity and biosynthesis of O-antigens were investigated in this species for the first time. 18/18 commensal H. parainfluenzae isolates contain a O-antigen biosynthesis gene cluster flanked by glnA and pepB, the same position as the hmg locus for tetrasaccharide biosynthesis in Haemophilus influenzae. The O-antigen loci show diverse restriction digest patterns but fall into two main groups: (1) those encoding enzymes for the synthesis and transfer of FucNAc4N in addition to the Wzy-dependent mechanism of O-antigen synthesis and transport and (2) those encoding galactofuranose synthesis/transfer enzymes and an ABC transporter. The other glycosyltransferase genes differ between isolates. Three H. parainfluenzae isolates fell outside these groups and are predicted to synthesise O-antigens containing ribitol phosphate or deoxytalose. Isolates using the ABC transporter system encode a putative O-antigen ligase, required for the synthesis of O-antigen-containing LPS glycoforms, at a separate genomic location. The presence of an O-antigen contributes significantly to H. parainfluenzae resistance to the killing effect of human serum in vitro. The discovery of O-antigens in H. parainfluenzae is striking, as its close relative H. influenzae lacks this cell surface component.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Glicosiltransferases/metabolismo , Haemophilus parainfluenzae/metabolismo , Lipopolissacarídeos/metabolismo , Antígenos O/metabolismo , Vias Biossintéticas/genética , Criança , DNA Bacteriano/química , DNA Bacteriano/genética , Genes Bacterianos , Haemophilus parainfluenzae/genética , Haemophilus parainfluenzae/isolamento & purificação , Humanos , Dados de Sequência Molecular , Família Multigênica , Análise de Sequência de DNA
5.
Glycoconj J ; 30(6): 561-76, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23093380

RESUMO

Cell surface lipopolysaccharide (LPS) is a well characterized virulence determinant for the human pathogen Haemophilus influenzae, so an investigation of LPS in the less pathogenic Haemophilus parainfluenzae could yield important insights. Using a panel of 18 commensal H. parainfluenzae isolates we demonstrate that the set of genes for inner core LPS biosynthesis largely resembles that of H. influenzae, with an additional heptosyltransferase I gene similar to waaC from Pasteurella multocida. Inner core LPS structure is therefore likely to be largely conserved across the two Haemophilus species. Outer core LPS biosynthetic genes are much less prevalent in H. parainfluenzae, although homologues of the H. influenzae LPS genes lpsB, non-phase variable lic2A and lgtC, and losA1, losB1 and lic2C are found in certain isolates. Immunoblotting using antibodies directed against selected LPS epitopes was consistent with these data. We found no evidence for tetranucleotide repeat-mediated phase variation in H. parainfluenzae. Phosphocholine, a phase variable H. influenzae LPS epitope that has been implicated in disease, was absent in H. parainfluenzae LPS as were the respective (lic1) biosynthetic genes. The introduction of the lic1 genes into H. parainfluenzae led to the phase variable incorporation of phosphocholine into its LPS. Differences in LPS structure between Haemophilus species could affect interactions at the bacterial-host interface and therefore the pathogenic potential of these bacteria.


Assuntos
Haemophilus parainfluenzae/metabolismo , Lipopolissacarídeos/biossíntese , Repetições de Microssatélites , Sequência de Aminoácidos , Epitopos/química , Epitopos/genética , Genes Bacterianos , Haemophilus parainfluenzae/química , Haemophilus parainfluenzae/genética , Lipopolissacarídeos/química , Lipopolissacarídeos/genética , Dados de Sequência Molecular
6.
J Mol Biol ; 386(2): 406-18, 2009 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-19103206

RESUMO

The Cu,Zn superoxide dismutase from Haemophilus ducreyi is characterized by the unique ability to bind heme at its dimer interface. Here we report the high-resolution crystal structures of this protein in the heme-loaded (holo) and heme-free (apo) forms. Heme is asymmetrically bound between the two enzyme subunits, where heme iron is coordinated by two histidine residues, His64 and His 124, provided by the two subunits. Moreover, the binding of heme to the protein is ensured by stabilizing contacts between the prosthetic group and a limited number of other residues, most of which are not present in other bacterial enzyme variants. We show that the introduction of only three mutations at the dimer interface of the enzyme from Haemophilus parainfluenzae, a closely related bacterial species, is sufficient to induce heme-binding ability by this enzyme variant. Heme binding does not alter protein activity. Moreover, the binding of the prosthetic group does not induce any significant structural perturbation at the subunit level and requires only limited local structural rearrangements that widen the cleft at the dimer interface and cause a limited shift in the relative orientation between the subunits. The presence of a preformed heme-binding pocket and the significant solvent exposure of the cofactor to the solvent are compatible with the suggested protective role of the enzyme against heme toxicity or with its involvement in heme trafficking in the periplasmic space.


Assuntos
Proteínas de Bactérias/química , Haemophilus ducreyi/química , Heme/metabolismo , Superóxido Dismutase/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Haemophilus parainfluenzae/genética , Haemophilus parainfluenzae/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação Puntual , Estrutura Quaternária de Proteína , Alinhamento de Sequência , Superóxido Dismutase/metabolismo
7.
J Clin Microbiol ; 45(12): 4051-3, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17942653

RESUMO

The biotypes of Haemophilus influenzae and Haemophilus parainfluenzae isolates were determined with three commercially available biochemical test kits: the IDS RapID NH system, the Neisseria-Haemophilus identification test (NHI card), and the API NH strip. The API NH strip performed best, correctly classifying the biotypes of 371 of 380 (97.6%) different challenge strains.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Haemophilus influenzae/classificação , Haemophilus influenzae/isolamento & purificação , Haemophilus parainfluenzae/classificação , Haemophilus parainfluenzae/isolamento & purificação , Infecções por Haemophilus/diagnóstico , Haemophilus influenzae/metabolismo , Haemophilus parainfluenzae/metabolismo , Humanos , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade
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