This application proposes the continuation of a molecular genetic analysis of the mobile pathogenicity islands, SaPIs, encoding SEB, TSST-1, and other superantigens and pathogenicity factors. Our laboratory has discovered and characterized many of these elements in S. aureus, and we have recently demonstrated natural SaPI transfer to Listeria monocytogenes, as well as to other staphylococcal species. This transfer may generate Listeria derivatives with enhanced virulence. The SaPIs are discrete 15-20 kb DNA segments that occupy specific chromosomal sites. They are induced to excise and replicate by certain staphylococcal phages and are efficiently packaged into infectious particles for transmission.
Specific Aims are 1. To elucidate the internal regulatory circuitry of the SaPI genome Under this aim, we will analyze SaPI gene expression and replication dynamics during the ERP cycle. It is hypothesized that following induction, SaPI genes are expressed in an explicit temporal sequence that varies according to induction scenario. We propose to test this hypothesis by analyzing the temporal pattern of SaPI gene expression (transcription pattern) under 4 different paradigms: SaPI induction by superinfecting or SOS-induced phage;incoming SaPI along with or in the absence of helper phage. 2. To analyze the SaPI-phage interface. The SaPI interacts with its inducing phage in two basic ways: it uses just those phage products that are necessary to enable the formation of infective SaPI particles, and it interferes with phage development ensuring that there is a very low probability that any potential recipient cell will be infected by an active phage particle as well as by a SaPI particle. Firstly, the SaPI uses one or more phage functions to inactivate its repressor, resulting in excision and replication;it remodels the phage capsid proteins to form its specific small particles;it diverts the phage packaging system to promote packaging of SaPI genomes at the expense of phage genomes. Secondly, the SaPI directly interferes with phage maturation and it ensures that the phage DNA is packaged mostly into small SaPI capsids, resulting in defective phage particles. Under this aim, we address both parts of this SaPI strategy. 3. To investigate the role of SaPIs in the microbiosphere. Under this aim, we will analyze the behavior of SaPI DNA during its replication cycle in S. aureus, evaluate the consequences of regulatory mutations, and analyze the phenomenon of displacement of a resident by an incoming SaPI. We will also study the interactions between co-resident SaPIs. Also under this aim, we will investigate SaPI biology in L. monocytogenes and other organisms to which it may be transferred, and we will determine the prevalence of SaPIs among different bacterial species in addition to clinical S. aureus isolates.
Staphylococci are extremely difficult to control owing to their remarkable ability to acquire and transmit genes for antibiotic resistance and for virulence. Our project is focused on genetic units that carry toxin genes and are transferred at very high frequencies, even to other species. By understanding the biology of these gene transfer systems, it is our hope to be able to control staphylococcal gene transfer and so reduce the danger of staphylococcal disease.
|Chen, John; Ram, Geeta; Penadés, José R et al. (2015) Pathogenicity island-directed transfer of unlinked chromosomal virulence genes. Mol Cell 57:138-49|
|Ram, Geeta; Chen, John; Ross, Hope F et al. (2014) Precisely modulated pathogenicity island interference with late phage gene transcription. Proc Natl Acad Sci U S A 111:14536-41|
|Quiles-Puchalt, Nuria; Carpena, Nuria; Alonso, Juan C et al. (2014) Staphylococcal pathogenicity island DNA packaging system involving cos-site packaging and phage-encoded HNH endonucleases. Proc Natl Acad Sci U S A 111:6016-21|
|Chen, John; Yoong, Pauline; Ram, Geeta et al. (2014) Single-copy vectors for integration at the SaPI1 attachment site for Staphylococcus aureus. Plasmid 76C:1-7|
|Quiles-Puchalt, Nuria; Tormo-Mas, Maria Angeles; Campoy, Susana et al. (2013) A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria. Nucleic Acids Res 41:7260-75|
|Ubeda, Carles; Tormo-Mas, Maria Angeles; Penades, Jose R et al. (2012) Structure-function analysis of the SaPIbov1 replication origin in Staphylococcus aureus. Plasmid 67:183-90|
|Alonzo 3rd, Francis; Benson, Meredith A; Chen, John et al. (2012) Staphylococcus aureus leucocidin ED contributes to systemic infection by targeting neutrophils and promoting bacterial growth in vivo. Mol Microbiol 83:423-35|
|Ferrer, Maria Desamparados; Quiles-Puchalt, Nuria; Harwich, Michael D et al. (2011) RinA controls phage-mediated packaging and transfer of virulence genes in Gram-positive bacteria. Nucleic Acids Res 39:5866-78|
|Tormo-Mas, Maria Angeles; Mir, Ignacio; Shrestha, Archana et al. (2010) Moonlighting bacteriophage proteins derepress staphylococcal pathogenicity islands. Nature 465:779-82|
|Christie, G E; Matthews, A M; King, D G et al. (2010) The complete genomes of Staphylococcus aureus bacteriophages 80 and 80*--implications for the specificity of SaPI mobilization. Virology 407:381-90|
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