Staphylococcus aureus is an important human pathogen whose success is largely attributed to its vast arsenal of virulence factors that facilitate its invasion into, and survival within, the human host. The expression of these virulence factors is controlled by the quorum sensing accessory gene regulator (Agr) system. However, a large proportion of clinical S. aureus isolates are consistently found to have a mutationally inactivated Agr system. These mutants have a survival advantage in the host but are considered irreversible mutants. Here we show, for the first time, that a fraction of Agr-negative mutants can revert their Agr activity. By serially passaging Agr-negative strains and screening for phenotypic reversion of hemolysis and subsequent sequencing, we identified two mutational events responsible for reversion: a genetic duplication plus inversion event and a poly(A) tract alteration. Additionally, we demonstrate that one clinical Agr-negative methicillin-resistant S. aureus (MRSA) isolate could reproducibly generate Agr-revertant colonies with a poly(A) tract genetic mechanism. We also show that these revertants activate their Agr system upon phagocytosis. We propose a model in which a minor fraction of Agr-negative S. aureus strains are phase variants that can revert their Agr activity and may act as a cryptic insurance strategy against host-mediated stress. IMPORTANCE Staphylococcus aureus is responsible for a broad range of infections. This pathogen has a vast arsenal of virulence factors at its disposal, but avirulent strains are frequently isolated as the cause of clinical infections. These isolates have a mutated agr locus and have been believed to have no evolutionary future. Here we show that a fraction of Agr-negative strains can repair their mutated agr locus with mechanisms resembling phase variation. The agr revertants sustain an Agr OFF state as long as they exist as a minority but can activate their Agr system upon phagocytosis. These revertant cells might function as a cryptic insurance strategy to survive immune-mediated host stress that arises during infection.

Phase variation (PV) is a well-known phenomenon of high-frequency reversible gene-expression switching. PV arises from genetic and epigenetic mechanisms and confers a range of benefits to bacteria, constituting both an innate immune strategy to infection from bacteriophages as well as an adaptation strategy within an infected host. PV has been well-characterized in numerous bacterial species; however, there is limited direct evidence of PV in the human opportunistic pathogen Staphylococcus aureus. This review provides an overview of the mechanisms that generate PV and focuses on earlier and recent findings of PV in S. aureus, with a brief look at the future of the field.

The influenza virus RNA genome exists as a ribonucleoprotein (RNP) complex by interacting with NP, one of virus-encoded RNA binding proteins. It is proposed that trimeric NP is a functional form, but it is not clear how trimeric NP is formed and transferred to RNA. UAP56, a cellular splicing factor, functions as a molecular chaperone for NP and is required for the replication-coupled RNP formation of newly synthesized viral genome, but the details of NP transfer to viral RNA by UAP56 is unclear. Here we found that UAP56 is complexed with trimeric NP, but not monomeric NP. Gel filtration analysis and atomic force microscopy analysis indicated that the complex consists of two trimeric NP connected by UAP56. We also found that UAP56 stimulates trimeric NP formation from monomeric NP even at physiological salt concentrations. Thus, UAP56 facilitates the transfer of NP to viral RNA since trimeric NP has higher RNA binding activity than monomeric NP. Further, UAP56 represses the binding of excess amount of NP to RNA possibly by transferring trimeric NP. Collectively, we propose that UAP56 stimulates viral RNP formation through promotion of the assembly of trimeric NP and is important for the structural integrity of NP-RNA complex.The genome of influenza type A viruses is single-stranded RNAs of negative polarity. The viral genome (vRNA) exists as ribonucleoprotein (designated vRNP) complexes with heterotrimeric viral RNA-dependent RNA polymerases and nucleoprotein (NP) 1 . NP is one of the basic viral proteins and binds single-stranded RNA without sequence specificity. NP is essential to maintain the RNA template in an ordered conformation suitable for viral RNA syntheses 2-6 . Cryo-electron microscopy analysis revealed that the oligomerization of NP is important to form a double helical structure with anti-parallel strand of vRNP 7,8 . Oligomeric NP shows higher RNA binding activity than monomeric NP 9 . Although crystal structures of RNA-free NP showed that NP forms oligomers in the crystalline state, a broad size distribution was observed by gel filtration chromatography in solution at physiological salt concentrations [9][10][11][12] , suggesting that NP exists in an equilibrium between monomers and oligomers including trimers and tetramers 13 . Thus, an exact form of NP to be assembled into vRNP remains unknown. For efficient viral transcription and replication, not only viral factors but also host factors are required. It has been reported that RAF-2p48/NPI-5/UAP56, Tat-SF1, and Prp18 function as molecular chaperones for NP to recruit NP to the viral RNAs 4,14,15 . NP chaperones are also required to suppress the aggregation of NP. UAP56 is a cellular splicing factor belonging to the DExD-box family of ATP-dependent RNA helicase 16 . It is reported that the newly synthesized viral genome is co-replicationally assembled into RNP complex by UAP56 3 . UAP56 binds to NP free of RNA but not NP-RNA complexes 15 . It is also reported that UAP56 interacts with N-terminal region of NP, and NP interacts with C-terminal reg...

Staphylococcus aureus is a Gram-positive opportunistic pathogen that imposes a heavy burden on society. What sets this pathogen apart is the sheer spectrum of infections it can cause, which range from benign skin and soft tissue infections to lethal endocarditis and bacteraemia. The ability of S. aureus to cause this gamut of infections is conferred by its arsenal of virulence factors that are under the control of the Accessory Gene Regulator (Agr) system. However, a large proportion of clinical isolates have inactivating mutations in this important regulatory system. We previously showed that, contrary to the common dogma, not all these mutations are evolutionary ‘dead-ends’ and a fraction are phase variants which can revert to an Agr active state. Here we report that some Agr deficient isolates can revert a haemolytic phenotype without repairing their Agr system. We collected a series of 30 Agr negative primary patient samples in order to assess the significance of our previous findings on the existence of Agr phase variants. We used primary samples to avoid strains that had undergone multiple clonal expansions before being tested for reversibility. We assessed Agr reversibility by serially passaging strains and screening for phenotypic reversion of haemolysis. We show that two strains reverted haemolysis and one reverted alpha haemolysin activity without any genetic changes in agr (and hla for the alpha revertant). These results add further complexity to the phenomenon of Agr shutdown observed in the clinical setting and corroborate recent findings of compensatory mutations arising in Agr deficient clinical strains.

Staphylococcus aureus is an important human pathogen whose success is largely attributed to its vast arsenal of virulence factors that facilitate its invasion into, and survival within, the human host. The expression of these virulence factors is controlled by the quorum sensing Accessory Gene Regulator (Agr) system. However, a large proportion of clinical S. aureus isolates are consistently found to have a mutationally inactivated Agr system. These mutants have a survival advantage in the host but are considered irreversible mutants. Here we show, for the first time, that a fraction of Agr-negative mutants can revert their Agr activity. By serially passaging Agr negative strains and screening for phenotypic reversion of haemolysis and subsequent sequencing, we identified two mutational events responsible for reversion: a genetic duplication plus inversion event and a poly(A) tract alteration. Additionally, we demonstrate that one clinical Agr-negative MRSA isolate could reproducibly generate Agr-revertant colonies with a poly(A) tract genetic mechanism. We also show that these revertants activate their Agr system upon phagocytosis. To assess the significance of our findings we screened a series of primary clinical isolates, which had undergone minimal handling post-isolation, and successfully identified a fraction which were Agr phase variants. Taken together, we propose a model where some Agr-negative S. aureus strains are phase variants who can revert their Agr activity and may act as a cryptic insurance strategy against host-mediated stress.