The Role of Lysogenic Bacteriophage in Virulence and Survival of Streptococcus Pyogenes

This thesis investigates the interactions of lysogenic bacteriophage with Streptococcus pyogenes. We were specifically interested in elucidating novel ways in which the prophage influence GrAS virulence and survival either within the host cell or within the bacterial population. In turn, we also hoped to determine how the bacteria (and possibly other integrated phage) might influence prophage activity or gene expression. Our studies focused on two polylysogenized GrAS strains, a M6 serotype (MGAS10394) and a M1 serotype (SF370). In our M6 studies, we chose a streptococcal strain lysogenized by a chimeric bacteriophage element (Φ10394.4), which contains the erythromycin resistance gene mefA, in order to elucidate why genomes of certain erythromycin-resistant isolates of group A streptococci are resistant to SmaI endonuclease cleavage. In the work presented here, we identified a restriction modification system contained on the bacteriophage chimeric-element and successfully characterized the first methyltransferase (M.SpyI) encoded in S. pyogenes. In the M1 strain, allelic recombination techniques were used to begin to analyze the role and regulation of a bacteriophage-encoded potential superantigen, SpeH. Next, we designed a novel system to specifically select for the loss of integrated phages entirely from the genome. This technique allowed us to study the novel role that two SF370 phage play in regulating GrAS chromosomal genes involved in bacterial metabolism, DNA repair and mutagenesis. We were also able to begin to elucidate the effects that individual and multiple phage loss have on SF370 virulence and survival, as we successfully constructed the first strain of GrAS that is completely devoid of bacteriophages. In the course of these studies, we also manipulated lytic bacteriophage genes from two different Staphylococcus phage genomes to genetically engineer a novel chimeric endolysin (ClyS). We demonstrated the antimicrobial lytic activity of ClyS against methicillin-resistant Staphylococcus aureus (MRSA) in both in vivo colonization and septicemia models, and were also able to show its synergistic activity with oxacillin in in vitro and in vivo models. This work highlights the potential of ClyS as a novel therapeutic agent for the treatment of MRSA and other staphylococcal infections

Novel Algorithms Reveal Streptococcal Transcriptomes and Clues about Undefined Genes

Bacteria–host interactions are dynamic processes, and understanding transcriptional responses that directly or indirectly regulate the expression of genes involved in initial infection stages would illuminate the molecular events that result in host colonization. We used oligonucleotide microarrays to monitor (in vitro) differential gene expression in group A streptococci during pharyngeal cell adherence, the first overt infection stage. We present neighbor clustering, a new computational method for further analyzing bacterial microarray data that combines two informative characteristics of bacterial genes that share common function or regulation: (1) similar gene expression profiles (i.e., co-expression); and (2) physical proximity of genes on the chromosome. This method identifies statistically significant clusters of co-expressed gene neighbors that potentially share common function or regulation by coupling statistically analyzed gene expression profiles with the chromosomal position of genes. We applied this method to our own data and to those of others, and we show that it identified a greater number of differentially expressed genes, facilitating the reconstruction of more multimeric proteins and complete metabolic pathways than would have been possible without its application. We assessed the biological significance of two identified genes by assaying deletion mutants for adherence in vitro and show that neighbor clustering indeed provides biologically relevant data. Neighbor clustering provides a more comprehensive view of the molecular responses of streptococci during pharyngeal cell adherence

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

Elimination of Chromosomal Island SpyCIM1 from Streptococcus pyogenes Strain SF370 Reverses the Mutator Phenotype and Alters Global Transcription

This work was made possible by an Oklahoma Center for the Advancement of Science and Technology (OCAST) grant HR11-133 and by NIH Grant Number R15A1072718 to WMM and NIH Grant AI11822 to VAF.Streptococcus pyogenes chromosomal island M1 (SpyCIM1) integrates by site-specific recombination into the 5’ end of DNA mismatch repair (MMR) gene mutL in strain SF370SmR, blocking transcription of it and the downstream operon genes. During exponential growth, SpyCIM1 excises from the chromosome and replicates as an episome, restoring mutL transcription. This process is reversed in stationary phase with SpyCIM1 re-integrating into mutL, returning the cells to a mutator phenotype. Here we show that elimination of SpyCIM1 relieves this mutator phenotype. The downstream MMR operon genes, multidrug efflux pump lmrP, Holliday junction resolution helicase ruvA, and DNA base excision repair glycosylase tag, are also restored to constitutive expression by elimination of SpyCIM1. The presence of SpyCIM1 alters global transcription patterns in SF370SmR. RNA sequencing (RNA-Seq) demonstrated that loss of SpyCIM1 in the SpyCIM1 deletion mutant, CEM1Δ4, impacted the expression of over 100 genes involved in virulence and metabolism both in early exponential phase, when the SpyCIM1 is episomal, as well as at the onset of stationary phase, when SpyCIM1 has reintegrated into mutL. Among these changes, the up-regulation of the genes for the antiphagocytic M protein (emm1), streptolysin O (slo), capsule operon (hasABC), and streptococcal pyrogenic exotoxin (speB), are particularly notable. The expression pattern of the MMR operon confirmed our earlier observations that these genes are transcribed in early exponential phase but silenced as stationary phase is approached. Thus, the direct role of SpyCIM1 in causing the mutator phenotype is confirmed, and further, its influence upon the biology of S. pyogenes was found to impact multiple genes in addition to the MMR operon, which is a novel function for a mobile genetic element. We suggest that such chromosomal islands are a remarkable evolutionary adaptation to promote the survival of its S. pyogenes host cell in changing environments.Yeshttp://www.plosone.org/static/editorial#pee

M.SpyI, a DNA Methyltransferase Encoded on a mefA Chimeric Element, Modifies the Genome of Streptococcus pyogenes

While screening the clonality of Streptococcus pyogenes isolates from an outbreak of erythromycin-resistant pharyngitis in Pittsburgh, PA, we found a correlation between the presence of the chimeric element Φ10394.4 (carrying the macrolide efflux gene, mefA) and genomic DNA being resistant to cleavage by SmaI restriction endonuclease. A search of the open reading frames in Φ10394.4 identified a putative type II restriction-modification (R-M) cassette containing a cytosine methyltransferase gene (spyIM). Heterologous expression of the cloned spyIM gene, as well as allelic-replacement experiments, showed that the action of this methyltransferase (M.SpyI) was responsible for the inhibition of SmaI digestion of genomic DNA in the Φ10394.4-containing isolates. Analysis of the methylation patterns of streptococcal genomic DNA from spyIM-positive strains, a spyIM deletion mutant, and a spyIM-negative strain determined that M.SpyI specifically recognized and methylated the DNA sequence to generate 5′-C(m)CNGG. To our knowledge, this is the first methyltransferase gene from S. pyogenes to be cloned and to have its activity characterized. These results reveal why pulsed field gel electrophoresis analysis of SmaI-digested genomic DNA cannot be used to analyze the clonality of some streptococci containing Φ10394.4 and may explain the inability of previous epidemiological studies to use SmaI to analyze DNAs from macrolide-resistant streptococci. The presence of the SpyI R-M cassette in Φ10394.4 could impart a selective advantage to host strain survival and may provide another explanation for the observed increase in macrolide-resistant streptococci

Strains of bacterial species induce a greatly varied acute adaptive immune response: The contribution of the accessory genome

<div><p>A fundamental question in human susceptibility to bacterial infections is to what extent variability is a function of differences in the pathogen species or in individual humans. To focus on the pathogen species, we compared in the same individual the human adaptive T and B cell immune response to multiple strains of two major human pathogens, <i>Staphylococcus aureus</i> and <i>Streptococcus pyogenes</i>. We found wide variability in the acute adaptive immune response induced by various strains of a species, with a unique combination of activation within the two arms of the adaptive response. Further, this was also accompanied by a dramatic difference in the intensity of the specific protective T helper (Th) response. Importantly, the same immune response differences induced by the individual strains were maintained across multiple healthy human donors. A comparison of isogenic phage KO strains, demonstrated that of the pangenome, prophages were the major contributor to inter-strain immune heterogeneity, as the T cell response to the remaining “core genome” was noticeably blunted. Therefore, these findings extend and modify the notion of an adaptive response to a pathogenic bacterium, by implying that the adaptive immune response signature of a bacterial species should be defined either per strain or alternatively to the species’ ‘core genome’, common to all of its strains. Further, our results demonstrate that the acquired immune response variation is as wide among different strains within a single pathogenic species as it is among different humans, and therefore may explain in part the clinical heterogeneity observed in patients infected with the same species.</p></div

The inter-individual variability of the immune response to a strain vs the intra-individual variability of the immune response to various strains within a species.

<p><b>A, B, C, and D</b>. The percentage Coefficient of Variation (CV) in % CD3+CD4+ proliferation (A), %IFNγ expressing CD4+ cells (B), % B cell Proliferation (C), and % IgG expression by proliferating B cells (D), was calculated across bacterial strains vs donors. Expressed is the mean percentage CV±2*SEM.</p

Strains of a species may differ prominently in Th1/Th17 response intensity.

<p><b>A</b>. CFSE labeled PBMC from 10 donors were stimulated and re-stimulated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006726#ppat.1006726.g001" target="_blank">Fig 1A</a> (as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006726#ppat.1006726.g001" target="_blank">Fig 1</a>) with either Newman or USA600, stained and analyzed by FACS for percent CFSE dilution (%proliferation) in live CD3+CD4+ cells that were gated on Th subsets based on cell surface markers (CxCR3, CCR6, CCR4). <b>B</b>. Example of FACS plot of one of the donors in Figure A. <b>C</b>. Absolute number of proliferating cells in response to stimulation with either Newman or USA600, when gated on cell surface markers for Th subsets cells. <b>D</b>. Intracellular staining for IFNγ, IL17A expression in proliferating live CD3+CD4+ cells following stimulation with either Newman or USA600 as in A. <b>E</b>. As in D, but staining for transcription factor Tbet and RORγt. <b>F</b>. As in D after stimulation with <i>Streptococcus pyogenes</i> strains. <b>G</b>. The MANOVA test for difference between bivariate means of induced IFNγ+IL17A- vs IL17A+IFNγ- absolute (Abs) number of cells following stimulation with <i>Streptococcus pyogenes</i> strains among 10 donors, showed significant results (p<0.00001). The post-hoc pairwise comparisons identified the differences between strains to be significant (for M1 vs M3 and M1 vs M6, P<0.0001; for M3 vs M6 P<0.005).</p

The effect of phage KO on adaptive immune response.

<p><b>A. and C</b>. CFSE labeled PBMC from 10 donors were stimulated with strains Newman or TB4 (Newman KO of its 4 phages) as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006726#ppat.1006726.g001" target="_blank">Fig 1A</a>, stained and analyzed by FACS for % proliferation (CFSE dilution) of live CD3+CD4+ cells (A left), % IFNγ expression by proliferating live CD3+CD4+ cells (A right), % proliferation of B cell (live CD3-CD19+, C left), or % IgG expression by live B cells (C right). <b>B</b>. Example of FACS plot of one of the donors in Figure A. <b>D</b>. Intracellular staining for IFNγ, IL17A expression in proliferating live CD3+CD4+ cells following stimulation with either Newman or TB4. <b>E</b>. As in D, but staining for transcription factor Tbet and RORγt. <b>F. and G</b>. are as in A and C left, respectively, but after stimulation with wild type <i>Streptococcus pyogenes</i> M1 strain SF370 or its complete phage KO. <b>H</b>. Intracellular staining for IFNγ, IL17A expression in proliferating live CD3+CD4+ cells following stimulation with either wild type <i>Streptococcus pyogenes</i> M1 strain SF370 or its complete phage KO (CEM1ΔΦ). <b>I</b>. The MANOVA test for difference between bivariate means of % T cell proliferation and % B cell proliferation cells following stimulation with either TB4 or <i>Streptococcus pyogenes</i> M1 (SF370) complete phage KO (CEM1ΔΦ) strains.</p