A Novel
            <i>Staphylococcus</i>
            Podophage Encodes a Unique Lysin with Unusual Modular Design

Cater, Katie; Dandu, Vidya Sree; Bari, S. M. Nayeemul; Lackey, Kim; Everett, Gabriel F. Kuty; Hatoum-Aslan, Asma

doi:10.1128/msphere.00040-17

“…Therefore, we first tested CRISPR-Cas10 immunity against representatives from both virulent staphylococcal phage families: Podoviridae phage Andhra 43 and Myoviridae phage ISP. 12 Since the S. epidermidis CRISPR-Cas10 system lacks natural spacers targeting these phages, the system had to be reprogrammed to target Andhra and ISP.…”

Section: Resultsmentioning

confidence: 99%

“…Media were supplemented with the following antibiotics as needed: 10 μ g/mL chloramphenicol (for selection of p crispr and p crispr-cas based plasmids) and 15 μ g/mL neomycin (for selection of S. epidermidis ). Phage Andhra was discovered in-house, 43 and phage ISP was a generous gift from Luciano Marraffini. For phage propagation, S. epidermidis was grown in BHI plus 5 mM CaCl 2 to an early logarithmic phase at 37 °C with shaking.…”

Section: Methodsmentioning

confidence: 99%

Strategies for Editing Virulent Staphylococcal Phages Using CRISPR-Cas10

Bari

¹

,

Walker

²

,

Cater

³

et al. 2017

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Staphylococci are prevalent skin-dwelling bacteria that are also leading causes of antibiotic-resistant infections. Viruses that infect and lyse these organisms (virulent staphylococcal phages) can be used as alternatives to conventional antibiotics and represent promising tools to eliminate or manipulate specific species in the microbiome. However, since over half their genes have unknown functions, virulent staphylococcal phages carry inherent risk to cause unknown downstream side effects. Further, their swift and destructive reproductive cycle make them intractable by current genetic engineering techniques. CRISPR-Cas10 is an elaborate prokaryotic immune system that employs small RNAs and a multisubunit protein complex to detect and destroy phages and other foreign nucleic acids. Some staphylococci naturally possess CRISPR-Cas10 systems, thus providing an attractive tool already installed in the host chromosome to harness for phage genome engineering. However, the efficiency of CRISPR-Cas10 immunity against virulent staphylococcal phages and corresponding utility as a tool to facilitate their genome editing has not been explored. Here, we show that the CRISPR-Cas10 system native to Staphylococcus epidermidis exhibits robust immunity against diverse virulent staphylococcal phages. On the basis of this activity, a general two-step approach was developed to edit these phages that relies upon homologous recombination machinery encoded in the host. Variations of this approach to edit toxic phage genes and access phages that infect CRISPR-less staphylococci are also presented. This versatile set of genetic tools enables the systematic study of phage genes of unknown functions and the design of genetically defined phage-based antimicrobials that can eliminate or manipulate specific Staphylococcus species.

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“…The distantly related S. aureus-infecting podovirus P68 has a "tail fiber" protein (gp17) that is remarkably similar to the 80α RBP [37] (25% sequence identity), suggesting that this structure is conserved among phages that infect S. aureus. In contrast, phage Andhra, which is structurally very similar to P68, but infects S. epidermidis [40], has a completely different predicted RBP structure based on a β-helical architecture. Similarly, S. aureus phage ϕ187, which infects the ST395 lineage of S. aureus, lacks an RBP-like protein, but has a tail fiber protein (gp24) that, by HHpred analysis, resembles parts of FibL, including an N-terminal Ig-like domain, a coiled-coil domain, a tower domain, and a C-terminal domain similar to the phage K tail fiber protein, gp68 (S6 Fig).…”

Section: Discussionmentioning

confidence: 99%

Structure of the host cell recognition and penetration machinery of a Staphylococcus aureus bacteriophage

Kizziah

¹

,

Manning

²

,

Dearborn

³

et al. 2020

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Staphylococcus aureus is a common cause of infections in humans. The emergence of virulent, antibiotic-resistant strains of S. aureus is a significant public health concern. Most virulence and resistance factors in S. aureus are encoded by mobile genetic elements, and transduction by bacteriophages represents the main mechanism for horizontal gene transfer. The baseplate is a specialized structure at the tip of bacteriophage tails that plays key roles in host recognition, cell wall penetration, and DNA ejection. We have used high-resolution cryo-electron microscopy to determine the structure of the S. aureus bacteriophage 80α baseplate at 3.75 Å resolution, allowing atomic models to be built for most of the major tail and baseplate proteins, including two tail fibers, the receptor binding protein, and part of the tape measure protein. Our structure provides a structural basis for understanding host recognition, cell wall penetration and DNA ejection in viruses infecting Gram-positive bacteria. Comparison to other phages demonstrates the modular design of baseplate proteins, and the adaptations to the host that take place during the evolution of staphylococci and other pathogens. Author summaryThe emergence of virulent strains of Staphylococcus aureus that are resistant to most antibiotics has become a major public health concern. Virulence and resistance determinants in S. aureus are usually carried on mobile genetic elements (MGEs). Transduction by bacteriophages provides the main means by which MGEs are disseminated horizontally through the bacterial population, and is therefore essential to the evolution of pathogenicity of S. aureus and other pathogens. The baseplate is a complex structure at the tip of bacteriophage tails that serves multiple roles, including host recognition and binding, cell wall penetration, and ejection of the phage DNA. We have determined the structure of the baseplate from bacteriophage 80α, a representative of phages involved in host pathogenicity and in the mobilization of MGEs in S. aureus. Our structure provides a basis for understanding host recognition and infection by phages infecting Gram-positive hosts, and the PLOS Pathogens | https://doi.

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“…The distantly related S. aureus-infecting podovirus P68 has a "tail fiber" protein (gp17) that is remarkably similar to the 80α RBP (36) (25% sequence identity), suggesting that this structure is conserved among phages that infect S. aureus. In contrast, phage Andhra, which is structurally very similar to P68, but infects Staphylococcus epidermidis (39), has a completely different predicted RBP structure based on a β-helical architecture. S. epidermidis, a CoNS species, has a different type of WTA from S. aureus, which could explain this difference.…”

Section: Discussionmentioning

confidence: 99%

Structure of the host cell recognition and penetration machinery of a Staphylococcus aureus bacteriophage

Kizziah

¹

,

Manning

²

,

Dearborn

³

et al. 2019

Preprint

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Staphylococcus aureus is a common cause of infections in humans. The emergence of virulent, antibiotic-resistant strains of S. aureus is a significant public health concern. Most virulence and resistance factors in S. aureus are encoded by mobile genetic elements, and transduction by bacteriophages represents the main mechanism for horizontal gene transfer. The baseplate is a specialized structure at the tip of bacteriophage tails that plays key roles in host recognition, cell wall penetration, and DNA ejection. We have used high-resolution cryo-electron microscopy to determine the structure of the S. aureus bacteriophage 80α baseplate at 3.7 Å resolution, allowing atomic models to be built for most of the major tail and baseplate proteins, including two tail fibers, a trimeric receptor binding protein, and part of the tape measure protein. Our structure provides a structural basis for understanding host recognition, cell wall penetration and DNA ejection in viruses infecting Gram-positive bacteria. Comparison to other phages demonstrate the modular design of baseplate proteins, and the adaptations to the host that take place during the evolution of staphylococci and other pathogens.

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A Novel Staphylococcus Podophage Encodes a Unique Lysin with Unusual Modular Design

Cited by 37 publications

References 26 publications

Strategies for Editing Virulent Staphylococcal Phages Using CRISPR-Cas10

Strategies for Editing Virulent Staphylococcal Phages Using CRISPR-Cas10

Structure of the host cell recognition and penetration machinery of a Staphylococcus aureus bacteriophage

Structure of the host cell recognition and penetration machinery of a Staphylococcus aureus bacteriophage

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