A Triad of Lys12, Lys41, Arg78 Spatial Domain, a Novel Identified Heparin Binding Site on Tat Protein, Facilitates Tat-Driven Cell Adhesion

Tat protein, released by HIV-infected cells, has a battery of important biological effects leading to distinct AIDS-associated pathologies. Cell surface heparan sulfate protoglycans (HSPGs) have been accepted as endogenous Tat receptors, and the Tat basic domain has been identified as the heparin binding site. However, findings that deletion or substitution of the basic domain inhibits but does not completely eliminate Tat–heparin interactions suggest that the basic domain is not the sole Tat heparin binding site. In the current study, an approach integrating computational modeling, mutagenesis, biophysical and cell-based assays was used to elucidate a novel, high affinity heparin-binding site: a Lys12, Lys41, Arg78 (KKR) spatial domain. This domain was also found to facilitate Tat-driven β1 integrin activation, producing subsequent SLK cell adhesion in an HSPG-dependent manner, but was not involved in Tat internalization. The identification of this new heparin binding site may foster further insight into the nature of Tat-heparin interactions and subsequent biological functions, facilitating the rational design of new therapeutics against Tat-mediated pathological events

Targeted Curing of All Lysogenic Bacteriophage from Streptococcus pyogenes Using a Novel Counter-selection Technique

We thank the members of the Laboratory of Microbial Pathogenesis and Immunology, especially Annette Nelkenbaum and Ben Winer for their technical assistance. We also thank Estee Colleen Cervantes and Sutapa Banerjee from Hunter College for their technical contribution to this project. We are grateful to Joseph Ferretti for S. pyogenes strain SF370.Streptococcus pyogenes is a human commensal and a bacterial pathogen responsible for a wide variety of human diseases differing in symptoms, severity, and tissue tropism. The completed genome sequences of >37 strains of S. pyogenes, representing diverse disease-causing serotypes, have been published. The greatest genetic variation among these strains is attributed to numerous integrated prophage and prophage-like elements, encoding several virulence factors. A comparison of isogenic strains, differing in prophage content, would reveal the effects of these elements on streptococcal pathogenesis. However, curing strains of prophage is often difficult and sometimes unattainable. We have applied a novel counter-selection approach to identify rare S. pyogenes mutants spontaneously cured of select prophage. To accomplish this, we first inserted a two-gene cassette containing a gene for kanamycin resistance (KanR) and the rpsL wild-type gene, responsible for dominant streptomycin sensitivity (SmS), into a targeted prophage on the chromosome of a streptomycin resistant (SmR) mutant of S. pyogenes strain SF370. We then applied antibiotic counter-selection for the re-establishment of the KanS/SmR phenotype to select for isolates cured of targeted prophage. This methodology allowed for the precise selection of spontaneous phage loss and restoration of the natural phage attB attachment sites for all four prophage-like elements in this S. pyogenes chromosome. Overall, 15 mutants were constructed that encompassed every permutation of phage knockout as well as a mutant strain, named CEM1ΔΦ, completely cured of all bacteriophage elements (a ~10% loss of the genome); the only reported S. pyogenes strain free of prophage-like elements. We compared CEM1ΔΦ to the WT strain by analyzing differences in secreted DNase activity, as well as lytic and lysogenic potential. These mutant strains should allow for the direct examination of bacteriophage relationships within S. pyogenes and further elucidate how the presence of prophage may affect overall streptococcal survival, pathogenicity, and evolution.Yeshttp://www.plosone.org/static/editorial#pee

HIV-1 proteins gp120 and tat induce the epithelial–mesenchymal transition in oral and genital mucosal epithelial cells

The oral, cervical, and genital mucosa, covered by stratified squamous epithelia with polarized organization and strong tight and adherens junctions, play a critical role in preventing transmission of viral pathogens, including human immunodeficiency virus (HIV). HIV-1 interaction with mucosal epithelial cells may depolarize epithelia and disrupt their tight and adherens junctions; however, the molecular mechanism of HIV-induced epithelial disruption has not been completely understood. We showed that prolonged interaction of cell-free HIV-1 virions, and viral envelope and transactivator proteins gp120 and tat, respectively, with tonsil, cervical, and foreskin epithelial cells induces an epithelial-mesenchymal transition (EMT). EMT is an epigenetic process leading to the disruption of mucosal epithelia and allowing the paracellular spread of viral and other pathogens. Interaction of cell-free virions and gp120 and tat proteins with epithelial cells substantially reduced E-cadherin expression and activated vimentin and N-cadherin expression, which are well-known mesenchymal markers. HIV gp120- and tat-induced EMT was mediated by SMAD2 phosphorylation and activation of transcription factors Slug, Snail, Twist1 and ZEB1. Activation of TGF-β and MAPK signaling by gp120, tat, and cell-free HIV virions revealed the critical roles of these signaling pathways in EMT induction. gp120- and tat-induced EMT cells were highly migratory via collagen-coated membranes, which is one of the main features of mesenchymal cells. Inhibitors of TGF-β1 and MAPK signaling reduced HIV-induced EMT, suggesting that inactivation of these signaling pathways may restore the normal barrier function of mucosal epithelia