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

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.

Specific N-terminal cleavage of ribosomal protein L27 in Staphylococcus aureus and related bacteria.

Ribosomal protein L27 is a component of the eubacterial large ribosomal subunit that has been shown to play a critical role in substrate stabilization during protein synthesis. This function is mediated by the L27 N-terminus, which protrudes into the peptidyl transferase center. In this report, we demonstrate that L27 in Staphylococcus aureus and other Firmicutes is encoded with an N-terminal extension that is not present in most Gram-negative organisms and is absent from mature ribosomes. We have identified a cysteine protease, conserved among bacteria containing the L27 N-terminal extension, which performs post-translational cleavage of L27. Ribosomal biology in eubacteria has largely been studied in the Gram-negative bacterium Escherichia coli; our findings indicate that there are aspects of the basic biology of the ribosome in S. aureus and other related bacteria that differ substantially from that of the E. coli ribosome. This research lays the foundation for the development of new therapeutic approaches that target this novel pathway.

The gp44 Ejection Protein of Staphylococcus aureus Bacteriophage 80α Binds to the Ends of the Genome and Protects It from Degradation.

Bacteriophage 80α is a representative of a class of temperate phages that infect Staphylococcus aureus and other Gram-positive bacteria. Many of these phages carry genes encoding toxins and other virulence factors. This phage, 80α, is also involved in high-frequency mobilization of S. aureus pathogenicity islands (SaPIs), mobile genetic elements that carry virulence factor genes. Bacteriophage 80α encodes a minor capsid protein, gp44, between the genes for the portal protein and major capsid protein. Gp44 is essential for a productive infection by 80α but not for transduction of SaPIs or plasmids. We previously demonstrated that gp44 is an ejection protein that acts to promote progression to the lytic cycle upon infection and suggested that the protein might act as an anti-repressor of CI in the lytic-lysogenic switch. However, an 80α Δ44 mutant also exhibited a reduced rate of lysogeny. Here, we show that gp44 is a non-specific DNA binding protein with affinity for the blunt ends of linear DNA. Our data suggest a model in which gp44 promotes circularization of the genome after injection into the host cell, a key initial step both for lytic growth and for the establishment of lysogeny.

A novel ejection protein from bacteriophage 80α that promotes lytic growth.

Many staphylococcal bacteriophages encode a minor capsid protein between the genes for the portal and scaffolding proteins. In Staphylococcus aureus bacteriophage 80α, this protein, called gp44, is essential for the production of viable phage, but dispensable for the phage-mediated mobilization of S. aureus pathogenicity islands. We show here that gp44 is not required for capsid assembly, DNA packaging or ejection of the DNA, nor for generalized transduction of plasmids. An 80α Δ44 mutant could be complemented in trans by gp44 expressed from a plasmid, indicating that gp44 plays a post-injection role in the host. Our results show that gp44 is an ejection (pilot) protein that is involved in deciding the fate of the phage DNA after injection. Our data are consistent with a model in which gp44 acts as a regulatory protein that promotes progression to the lytic cycle.

Competing scaffolding proteins determine capsid size during mobilization of Staphylococcus aureus pathogenicity islands.

Staphylococcus aureus pathogenicity islands (SaPIs), such as SaPI1, exploit specific helper bacteriophages, like 80α, for their high frequency mobilization, a process termed 'molecular piracy'. SaPI1 redirects the helper's assembly pathway to form small capsids that can only accommodate the smaller SaPI1 genome, but not a complete phage genome. SaPI1 encodes two proteins, CpmA and CpmB, that are responsible for this size redirection. We have determined the structures of the 80α and SaPI1 procapsids to near-atomic resolution by cryo-electron microscopy, and show that CpmB competes with the 80α scaffolding protein (SP) for a binding site on the capsid protein (CP), and works by altering the angle between capsomers. We probed these interactions genetically and identified second-site suppressors of lethal mutations in SP. Our structures show, for the first time, the detailed interactions between SP and CP in a bacteriophage, providing unique insights into macromolecular assembly processes.

Cleavage and Structural Transitions during Maturation of Staphylococcus aureus Bacteriophage 80α and SaPI1 Capsids.

In the tailed bacteriophages, DNA is packaged into spherical procapsids, leading to expansion into angular, thin-walled mature capsids. In many cases, this maturation is accompanied by cleavage of the major capsid protein (CP) and other capsid-associated proteins, including the scaffolding protein (SP) that serves as a chaperone for the assembly process. Staphylococcus aureus bacteriophage 80α is capable of high frequency mobilization of mobile genetic elements called S. aureus pathogenicity islands (SaPIs), such as SaPI1. SaPI1 redirects the assembly pathway of 80α to form capsids that are smaller than those normally made by the phage alone. Both CP and SP of 80α are N-terminally processed by a host-encoded protease, Prp. We have analyzed phage mutants that express pre-cleaved or uncleavable versions of CP or SP, and show that the N-terminal sequence in SP is absolutely required for assembly, but does not need to be cleaved in order to produce viable capsids. Mutants with pre-cleaved or uncleavable CP display normal viability. We have used cryo-EM to solve the structures of mature capsids from an 80α mutant expressing uncleavable CP, and from wildtype SaPI1. Comparisons with structures of 80α and SaPI1 procapsids show that capsid maturation involves major conformational changes in CP, consistent with a release of the CP N-arm by SP. The hexamers reorganize during maturation to accommodate the different environments in the 80α and SaPI1 capsids.

Convergent evolution of pathogenicity islands in helper cos phage interference.

Staphylococcus aureus pathogenicity islands (SaPIs) are phage satellites that exploit the life cycle of their helper phages for their own benefit. Most SaPIs are packaged by their helper phages using a headful (pac) packaging mechanism. These SaPIs interfere with pac phage reproduction through a variety of strategies, including the redirection of phage capsid assembly to form small capsids, a process that depends on the expression of the SaPI-encoded cpmA and cpmB genes. Another SaPI subfamily is induced and packaged by cos-type phages, and although these cos SaPIs also block the life cycle of their inducing phages, the basis for this mechanism of interference remains to be deciphered. Here we have identified and characterized one mechanism by which the SaPIs interfere with cos phage reproduction. This mechanism depends on a SaPI-encoded gene, ccm, which encodes a protein involved in the production of small isometric capsids, compared with the prolate helper phage capsids. As the Ccm and CpmAB proteins are completely unrelated in sequence, this strategy represents a fascinating example of convergent evolution. Moreover, this result also indicates that the production of SaPI-sized particles is a widespread strategy of phage interference conserved during SaPI evolution.This article is part of the themed issue 'The new bacteriology'.