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1.
Microbiol Spectr ; 9(1): e0102321, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34431721

RESUMO

The production of capsular polysaccharides by Klebsiella pneumoniae protects the bacterial cell from harmful environmental factors such as antimicrobial compounds and infection by bacteriophages (phages). To bypass this protective barrier, some phages encode polysaccharide-degrading enzymes referred to as depolymerases to provide access to cell surface receptors. Here, we characterized the phage RAD2, which infects K. pneumoniae strains that produce the widespread, hypervirulence-associated K2-type capsular polysaccharide. Using transposon-directed insertion sequencing, we have shown that the production of capsule is an absolute requirement for efficient RAD2 infection by serving as a first-stage receptor. We have identified the depolymerase responsible for recognition and degradation of the capsule, determined that the depolymerase forms globular appendages on the phage virion tail tip, and present the cryo-electron microscopy structure of the RAD2 capsule depolymerase at 2.7-Å resolution. A putative active site for the enzyme was identified, comprising clustered negatively charged residues that could facilitate the hydrolysis of target polysaccharides. Enzymatic assays coupled with mass spectrometric analyses of digested oligosaccharide products provided further mechanistic insight into the hydrolase activity of the enzyme, which, when incubated with K. pneumoniae, removes the capsule and sensitizes the cells to serum-induced killing. Overall, these findings expand our understanding of how phages target the Klebsiella capsule for infection, providing a framework for the use of depolymerases as antivirulence agents against this medically important pathogen. IMPORTANCE Klebsiella pneumoniae is a medically important pathogen that produces a thick protective capsule that is essential for pathogenicity. Phages are natural predators of bacteria, and many encode diverse "capsule depolymerases" which specifically degrade the capsule of their hosts, an exploitable trait for potential therapies. We have determined the first structure of a depolymerase that targets the clinically relevant K2 capsule and have identified its putative active site, providing hints to its mechanism of action. We also show that Klebsiella cells treated with a recombinant form of the depolymerase are stripped of capsule, inhibiting their ability to grow in the presence of serum, demonstrating the anti-infective potential of these robust and readily producible enzymes against encapsulated bacterial pathogens such as K. pneumoniae.


Assuntos
Cápsulas Bacterianas/virologia , Bacteriófagos/enzimologia , Klebsiella pneumoniae/virologia , Polissacarídeo-Liases/metabolismo , Proteínas Virais/metabolismo , Cápsulas Bacterianas/metabolismo , Cápsulas Bacterianas/ultraestrutura , Bacteriófagos/genética , Bacteriófagos/fisiologia , Microscopia Crioeletrônica , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Klebsiella pneumoniae/ultraestrutura , Polissacarídeo-Liases/genética , Proteínas Virais/genética
2.
Nat Microbiol ; 6(2): 157-161, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33432151

RESUMO

We characterized two bacteriophages, ΦFG02 and ΦCO01, against clinical isolates of Acinetobacter baumannii and established that the bacterial capsule is the receptor for these phages. Phage-resistant mutants harboured loss-of-function mutations in genes responsible for capsule biosynthesis, resulting in capsule loss and disruption of phage adsorption. The phage-resistant strains were resensitized to human complement, beta-lactam antibiotics and alternative phages and exhibited diminished fitness in vivo. Using a mouse model of A. baumannii infection, we showed that phage therapy was effective.


Assuntos
Infecções por Acinetobacter/microbiologia , Infecções por Acinetobacter/terapia , Acinetobacter baumannii/virologia , Antibacterianos/farmacologia , Bacteriófagos/fisiologia , Terapia por Fagos , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Animais , Cápsulas Bacterianas/virologia , Proteínas do Sistema Complemento/farmacologia , Modelos Animais de Doenças , Farmacorresistência Bacteriana , Feminino , Humanos , Mutação com Perda de Função , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Inibidores de beta-Lactamases/farmacologia
3.
PLoS One ; 14(6): e0218461, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31220143

RESUMO

Acinetobacter baumannii isolate NIPH 329 carries a novel capsular polysaccharide (CPS) gene cluster, designated KL46, that is closely related to the KL5 locus in A. baumannii isolate SDF but includes genes for synthesis of 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic (di-N-acetylpseudaminic) acid (Pse5Ac7Ac) instead of the corresponding D-glycero-D-galacto isomer (di-N-acetyllegionaminic acid) (Leg5Ac7Ac). In agreement with the genetic content of KL46, chemical studies of the K46 CPS produced by NIPH 329 revealed a branched tetrasaccharide repeat (K unit) with an overall structure the same as K5 from SDF but with â-Pse5Ac7Ac replacing α-Leg5Ac7Ac. As for K5, the K46 unit begins with d-GalpNAc and includes α-d-GlcpNAc-(1→3)-d-GalpNAc and α-d-Galp-(1→6)-d-GlcpNAc linkages, formed by Gtr14 and Gtr15 glycosyltransferases, respectively. The Gtr94K46 glycosyltransferase, which is related to Gtr13K5, links Pse5Ac7Ac to d-Galp in the growing K unit via a â-(2→6) linkage. Nearly identical Wzy enzymes connect the K46 and K5 units via a α-D-GalpNAc-(1→3)-α-D-Galp linkage to form closely related CPSs. Both Pse5Ac7Ac in K46 and Leg5Ac7Ac in K5 are acetylated at O4 but no acetyltransferase gene is present in KL46 or KL5. Related acetyltransferases were found encoded in the NIPH 329 and SDF genomes, but not in other strains carrying an unacetylated Pse or Leg derivative in the CPS. The genes encoding the acetyltransferases were in different putative phage genomes. However, related acetyltransferases were rare among the >3000 publically available genome sequences.


Assuntos
Acetiltransferases/genética , Cápsulas Bacterianas/química , Bacteriófagos/genética , Ácidos Siálicos/química , Acetilação , Acetiltransferases/química , Acinetobacter baumannii/química , Acinetobacter baumannii/genética , Acinetobacter baumannii/virologia , Cápsulas Bacterianas/metabolismo , Cápsulas Bacterianas/virologia , Bacteriófagos/enzimologia , Genoma Viral/genética , Espectroscopia de Ressonância Magnética , Ácidos Siálicos/genética
4.
J Virol ; 93(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30463964

RESUMO

Acinetobacter baumannii is an important pathogen causative of health care-associated infections and is able to rapidly develop resistance to all known antibiotics, including colistin. As an alternative therapeutic agent, we have isolated a novel myovirus (vB_AbaM_B9) which specifically infects and makes lysis from without in strains of the K45 and K30 capsule types, respectively. Phage B9 has a genome of 93,641 bp and encodes 167 predicted proteins, of which 29 were identified by mass spectrometry. This phage holds a capsule depolymerase (B9gp69) able to digest extracted exopolysaccharides of both K30 and K45 strains and remains active in a wide range of pH values (5 to 9), ionic strengths (0 to 500 mM), and temperatures (20 to 80°C). B9gp69 was demonstrated to be nontoxic in a cell line model of the human lung and to make the K45 strain fully susceptible to serum killing in vitro Contrary to the case with phage, no resistance development was observed by bacteria targeted with the B9gp69. Therefore, capsular depolymerases may represent attractive antimicrobial agents against A. baumannii infections.IMPORTANCE Currently, phage therapy has revived interest for controlling hard-to-treat bacterial infections. Acinetobacter baumannii is an emerging Gram-negative pathogen able to cause a variety of nosocomial infections. Additionally, this species is becoming more resistant to several classes of antibiotics. Here we describe the isolation of a novel lytic myophage B9 and its recombinant depolymerase. While the phage can be a promising alternative antibacterial agent, its success in the market will ultimately depend on new regulatory frameworks and general public acceptance. We therefore characterized the phage-encoded depolymerase, which is a natural enzyme that can be more easily managed and used. To our knowledge, the therapeutic potential of phage depolymerase against A. baumannii is still unknown. We show for the first time that the K45 capsule type is an important virulence factor of A. baumannii and that capsule removal via the recombinant depolymerase activity helps the host immune system to combat the bacterial infection.


Assuntos
Glicosídeo Hidrolases/metabolismo , Myoviridae/genética , Myoviridae/metabolismo , Acinetobacter baumannii/virologia , Cápsulas Bacterianas/fisiologia , Cápsulas Bacterianas/virologia , Bacteriófagos/genética , DNA Viral/genética , Genoma Viral , Glicosídeo Hidrolases/genética , Humanos , Fases de Leitura Aberta/genética , Análise de Sequência de DNA/métodos , Proteínas Virais/metabolismo
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