Dataset Information

Modeling the Architecture of Depolymerase-Containing Receptor Binding Proteins in Klebsiella Phages.

ABSTRACT: Klebsiella pneumoniae carries a thick polysaccharide capsule. This highly variable chemical structure plays an important role in its virulence. Many Klebsiella bacteriophages recognize this capsule with a receptor binding protein (RBP) that contains a depolymerase domain. This domain degrades the capsule to initiate phage infection. RBPs are highly specific and thus largely determine the host spectrum of the phage. A majority of known Klebsiella phages have only one or two RBPs, but phages with up to 11 RBPs with depolymerase activity and a broad host spectrum have been identified. A detailed bioinformatic analysis shows that similar RBP domains repeatedly occur in K. pneumoniae phages with structural RBP domains for attachment of an RBP to the phage tail (anchor domain) or for branching of RBPs (T4gp10-like domain). Structural domains determining the RBP architecture are located at the N-terminus, while the depolymerase is located in the center of protein. Occasionally, the RBP is complemented with an autocleavable chaperone domain at the distal end serving for folding and multimerization. The enzymatic domain is subjected to an intense horizontal transfer to rapidly shift the phage host spectrum without affecting the RBP architecture. These analyses allowed to model a set of conserved RBP architectures, indicating evolutionary linkages.

SUBMITTER: Latka A

PROVIDER: S-EPMC6872550 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Modeling the Architecture of Depolymerase-Containing Receptor Binding Proteins in Klebsiella Phages.

Latka Agnieszka A Leiman Petr G PG Drulis-Kawa Zuzanna Z Briers Yves Y

Frontiers in microbiology 20191115

Klebsiella pneumoniae carries a thick polysaccharide capsule. This highly variable chemical structure plays an important role in its virulence. Many Klebsiella bacteriophages recognize this capsule with a receptor binding protein (RBP) that contains a depolymerase domain. This domain degrades the capsule to initiate phage infection. RBPs are highly specific and thus largely determine the host spectrum of the phage. A majority of known Klebsiella phages have only one or two R ...[more]

PMID: 31803168

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Project description:The highly pathogenic bacterium Yersinia pestis is the causative agent of plague, a notorious infectious zoonotic disease. When transmitted from person to person as pneumonic plague via droplets, Y. pestis is highly contagious and in most cases is fatal if left untreated. Thus, when plague is suspected, rapid diagnosis is crucial, as a serious course of the infection is only averted by early antibiotic therapy. The bacterium is easy to cultivate, accessible and has a high potential for nefarious use such as bioterrorism. Highly specific, rapid and easy-to-use confirmatory diagnostic methods are required to reliably identify the pathogen independently from PCR-based methods or F1 antigen-based immunological detection. Yersinia pestis specific phages such as L-413C and ?A1122 are already used for detection of Y. pestis in bacterial plaque or biosensor assays. Here, we made use of the host specificities conferred by phage receptor binding (or tail fiber/spike) proteins (RBP) for developing a specific, fast and simple fluorescence-microscopy-based detection method for Y. pestis. Genes of putative RBP of phages L-413C (gpH) and ?A1122 (gp17) were fused with those of fluorescent proteins and recombinant receptor-reporter fusion proteins were produced heterologously in Escherichia coli. When first tested on attenuated Y. pestis strain EV76, RBP-reporters bound to the bacterial cell surface. This assay could be completed within a few minutes using live or formaldehyde-inactivated cells. Specificity tests using cultures of closely related Yersinia species and several inactivated fully virulent Y. pestis strains exhibited high specificities of the RBP-reporters against Y. pestis. The L-413C RBP proved to be especially specific, as it only detected Y. pestis at all temperatures tested, whereas the RBP of ?A1122 also bound to Y. pseudotuberculosis strains at 37 °C (but not at 28, 20 or 6 °C). Finally, the Y. pestis-specific capsule, produced when grown at 37 °C, significantly reduced binding of phage ?A1122 RBP, whereas the capsule only slightly diminished binding of L-413C RBP.

Project description:BACKGROUND: Presence of capsule enhances the virulence of bacteria that cause pneumonia, meningitis, cystic fibrosis, dental caries, periodontitis. Capsule is an important virulence factor for Klebsiella pneumoniae and infections due to this pathogen have been associated with high mortality rates. In the present study, use of an Aeromonas punctata derived capsule depolymerase against K. pneumoniae, to reinstate the efficacy of gentamicin during pneumonia and septicemia was investigated. METHODS: Depolymerase was administered in mice intraperitoneally (50 ?g) alone as well in combination with gentamicin (1.5 mg/kg), 24 h post infection during acute lung infection and 6 h later during septicemia. Bacterial load, neutrophil infiltration and cytokine levels were estimated. The immunogenicity of protein was also studied. RESULTS: In comparison to groups treated with gentamicin alone, combination treatment with depolymerase and gentamicin significantly reduced (P < 0.01) bacterial titer in the lungs, liver, kidney, spleen and blood of experimental animals. Highly significant reduction in neutrophil infiltration and levels of pro-inflammatory and anti-inflammatory cytokines was also observed. This indicated an efficient capsule removal by the enzyme, that improved gentamicin efficacy in vivo. Although the enzyme was found to be immunogenic, but no significant reduction in treatment efficacy was observed in the preimmunized as well as naïve mice. In addition, as confirmed through flow cytometry, the hyperimmune sera raised against the enzyme did not neutralize its activity. CONCLUSION: The results confirm that administration of enzyme 'depolymerase' along with gentamicin not only checked the virulence of K. pneumoniae in vivo but it also increased its susceptibility to gentamicin at a lower concentration. Such a strategy would help to avoid exposure to higher concentration of gentamicin. Moreover, since this decapsulating protein does not possess a lytic activity therefore there would be no chances of development of bacterial resistance against it. Therefore, it should be studied further for its successful inclusion in our prophylactic/therapeutic regimes.

Project description:Thanks to their wide host range and virulence, staphylococcal bacteriophages (phages) belonging to the genus Twortlikevirus (staphylococcal Twort-like phages) are regarded as ideal candidates for clinical application for Staphylococcus aureus infections due to the emergence of antibiotic-resistant bacteria of this species. To increase the usability of these phages, it is necessary to understand the mechanism underlying host recognition, especially the receptor-binding proteins (RBPs) that determine host range. In this study, we found that the staphylococcal Twort-like phage ?SA012 possesses at least two RBPs. Genomic analysis of five mutant phages of ?SA012 revealed point mutations in orf103, in a region unique to staphylococcal Twort-like phages. Phages harboring mutated ORF103 could not infect S. aureus strains in which wall teichoic acids (WTAs) are glycosylated with ?-N-acetylglucosamine (?-GlcNAc). A polyclonal antibody against ORF103 also inhibited infection by ?SA012 in the presence of ?-GlcNAc, suggesting that ORF103 binds to ?-GlcNAc. In contrast, a polyclonal antibody against ORF105, a short tail fiber component previously shown to be an RBP, inhibited phage infection irrespective of the presence of ?-GlcNAc. Immunoelectron microscopy indicated that ORF103 is a tail fiber component localized at the bottom of the baseplate. From these results, we conclude that ORF103 binds ?-GlcNAc in WTAs, whereas ORF105, the primary RBP, is likely to bind the WTA backbone. These findings provide insight into the infection mechanism of staphylococcal Twort-like phages.Staphylococcus phages belonging to the genus Twortlikevirus (called staphylococcal Twort-like phages) are considered promising agents for control of Staphylococcus aureus due to their wide host range and highly lytic capabilities. Although staphylococcal Twort-like phages have been studied widely for therapeutic purposes, the host recognition process of staphylococcal Twort-like phages remains unclear. This work provides new findings about the mechanisms of host recognition of the staphylococcal Twort-like phage ?SA012. The details of the host recognition mechanism of ?SA012 will allow us to analyze the mechanisms of infection and expand the utility of staphylococcal Twort-like phages for the control of S. aureus.

Project description: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.

Dataset Information

Modeling the Architecture of Depolymerase-Containing Receptor Binding Proteins in Klebsiella Phages.

Publications

Modeling the Architecture of Depolymerase-Containing Receptor Binding Proteins in <i>Klebsiella</i> Phages.

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