The Dynamic Streptococcus Pyogenes Transcriptome in the Host Cell Environment and Contributions by Phage

This thesis investigates the transcriptional responses of Streptococcus pyogenes in the pharyngeal environment and characterizes two transcriptional regulators involved in the adaptive response to the host. Furthermore, this thesis explores the potential role of integrated prophage on the streptococcal transcriptome. We are specifically interested in global regulatory systems in group A streptococci and how they impact virulence regulation. With this work, we hoped to identify new regulatory elements involved in the infection process. The first objective of this thesis was to determine the transcriptional shift induced in streptococci upon introduction into the in vitro host environment. Using three time points, we examined the dynamic transcriptome remodeling program that streptococci undergo following exposure to cell-free pharyngeal culture supernatants or during co-culture with intact pharyngeal monolayers. These studies highlighted that streptococci modulate expression of virulence factors in the host environment using a combination of stand-alone regulators (most of which are uncharacterized) and two-component regulatory systems. The next phase of this work involved characterizing two of the stand-alone regulators identified in our transcriptome screen. The first spy1215 encodes a sirtuin-like deacetylase that is homologous to a repressor of virulence in the malarial parasite Plasmodium falciparum. Our work presents the first evidence of a direct link between a bacterial sirtuin (Spy1215) and virulence regulation. Interestingly, Spy1215-mediated virulence repression was determined to be dependent on signals from pharyngeal cells in S. pyogenes. The second regulator spy1755 was found to be an activator of fatty acid biosynthesis in group A streptococci and required for normal growth rates in laboratory media. Finally, we endeavored to elucidate the role of integrated prophage in the regulation of streptococcal gene expression. Using the first S. pyogenes strain cured of all phage, we explored the effect of phage deletion on the streptococcal transcriptome during early and late exponential growth in laboratory media. We found a limited effect on gene expression in the absence of integrated phage. The genes whose expression was most affected by phage deletion were found to lie downstream of phage insertion sites. Overall, this work supports observations thus far that the streptococcal strain cured of all phage displays a limited phenotype via numerous measures in vitro and in vivo

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

Strains described in this study.

<p>Strains described in this study.</p

Confirmed phage <i>attB</i> sequences in <i>S</i>. <i>pyogenes</i> SF370 prophage KO mutants.

<p>Confirmed phage <i>attB</i> sequences in <i>S</i>. <i>pyogenes</i> SF370 prophage KO mutants.</p

Lytic potential of phage KO mutants.

<p>Lytic potential of phage KO mutants.</p

Location of the major bacteriophage elements in the chromosome of SF370.

<p>Triangles represent the location of the four main bacteriophage-like elements in the circular genome of <i>S</i>. <i>pyogenes</i> strain SF370. Numbers in triangles represent the corresponding phage elements with their size and encoded virulence factors identified in the table below. Derived from GenBank nucleotide accession number: AE004092.</p

Secreted DNase activity of SF370SmR and CEM1ΔΦ.

<p>Both strains were cultured on the same DNase Test Agar plate with Methyl Green for 17 h. A, SF370SmR WT colonies; B, CEM1ΔΦ full phage KO. Clearing zones around the colonies resulted from hydrolysis of the DNA-methyl green substrate, and signify DNase activity.</p

Pulse field gel electrophoresis (PFGE) analysis of <i>S</i>. <i>pyogenes</i> SF370SmR and single phage deletion mutants.

<p>PFGE patterns of SmaI digested genomic DNA from A, the phage wild type (WT) strain SF370SmR and the single phage KOs CEM1Δ(1–4), and B, the quadruple full phage KO CEM1ΔΦ compared to WT strain SF370. Labeled arrows indicate DNA fragments that contain the corresponding integrated phage or phage-like elements, based on the genome sequence of SF370. A loss or drop of these DNA fragments represents a phage or SpyCIM1 deletion. Numbers at top indicate which phages have been deleted: 1, Φ370.1 (40.9kb), strain CEM1Δ1; 2, Φ370.2 (42.5 kb), strain CEM1Δ2; 3, Φ370.3 (33.5Kb), strain CEM1Δ3; 4, Φ370.4/SpyCIM1 (13.5kb), strain CEM1Δ4; (1,2,3,4), all 4 phage elements deleted to create strain CEM1ΔΦ. All lanes in A and B originated from the same PFGE gel as shown in (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146408#pone.0146408.s002" target="_blank">S2 Fig</a>). λ, Lambda ladder, New England Biolabs PFG Marker with DNA fragment sizes on adjacent to gel images.</p