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Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes.

Le Breton Y, Belew AT, Valdes KM, Islam E, Curry P, Tettelin H, Shirtliff ME, El-Sayed NM, McIver KS - Sci Rep (2015)

Bottom Line: A large proportion of GAS essential genes corresponded to key cellular processes and metabolic pathways, and 177 were found conserved within the GAS core genome established from 20 available GAS genomes.Selected essential genes were validated using conditional-expression mutants.Finally, comparison to previous essentiality analyses in S. sanguinis and S. pneumoniae revealed significant overlaps, providing valuable insights for the development of new antimicrobials to treat infections by GAS and other pathogenic streptococci.

View Article: PubMed Central - PubMed

Affiliation: 1] [2].

ABSTRACT
Streptococcus pyogenes (Group A Streptococcus, GAS) remains a major public health burden worldwide, infecting over 750 million people leading to over 500,000 deaths annually. GAS pathogenesis is complex, involving genetically distinct GAS strains and multiple infection sites. To overcome fastidious genetic manipulations and accelerate pathogenesis investigations in GAS, we developed a mariner-based system (Krmit) for en masse monitoring of complex mutant pools by transposon sequencing (Tn-seq). Highly saturated transposant libraries (Krmit insertions in ca. every 25 nucleotides) were generated in two distinct GAS clinical isolates, a serotype M1T1 invasive strain 5448 and a nephritogenic serotype M49 strain NZ131, and analyzed using a Bayesian statistical model to predict GAS essential genes, identifying sets of 227 and 241 of those genes in 5448 and NZ131, respectively. A large proportion of GAS essential genes corresponded to key cellular processes and metabolic pathways, and 177 were found conserved within the GAS core genome established from 20 available GAS genomes. Selected essential genes were validated using conditional-expression mutants. Finally, comparison to previous essentiality analyses in S. sanguinis and S. pneumoniae revealed significant overlaps, providing valuable insights for the development of new antimicrobials to treat infections by GAS and other pathogenic streptococci.

No MeSH data available.


Related in: MedlinePlus

Experimental validation of selected essential genes.(A) Schematic showing construction of GAS conditional expression mutants controlled by riboswitch E. The 5’-end of the selected gene was cloned along with the Psag promoter and a theophylline-inducible riboswitch into the new pSinS vector for stable chromosomal integration in GAS. Merodiploid integrants have target gene expression under the control of the riboswitch, while the wild-type promoter controls the expression of a truncated target gene. (B to C) Validation of vicR (B to C) and murE (D to E) as an essential gene in GAS 5448 (B and D) and NZ131 (C and D). The top section of each panel represents the distribution of Krmit TIS in the each locus. The bottom section shows the growth of an inducible mutant in the presence (black squares) or absence (grey circles) of theophylline.
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f5: Experimental validation of selected essential genes.(A) Schematic showing construction of GAS conditional expression mutants controlled by riboswitch E. The 5’-end of the selected gene was cloned along with the Psag promoter and a theophylline-inducible riboswitch into the new pSinS vector for stable chromosomal integration in GAS. Merodiploid integrants have target gene expression under the control of the riboswitch, while the wild-type promoter controls the expression of a truncated target gene. (B to C) Validation of vicR (B to C) and murE (D to E) as an essential gene in GAS 5448 (B and D) and NZ131 (C and D). The top section of each panel represents the distribution of Krmit TIS in the each locus. The bottom section shows the growth of an inducible mutant in the presence (black squares) or absence (grey circles) of theophylline.

Mentions: To confirm that conserved genes identified in our screen are required for in vitro growth of GAS, we utilized a conditionally lethal approach that took advantage of a theophylline-sensitive synthetic riboswitch functional in GAS41. A suicide/helper system pSin-pHlp was created for GAS to allow for stable insertional gene inactivation (see Materials and Methods). Stable plasmid integration was generated in the GAS chromosome resulting in a merodiploid strain containing the heterologous Psag promoter followed by the theophylline-inducible riboswitch E controlling expression of the full length targeted gene, while the wild type promoter of the targeted gene transcribed a truncated non-functional allele (Fig. 5A).


Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes.

Le Breton Y, Belew AT, Valdes KM, Islam E, Curry P, Tettelin H, Shirtliff ME, El-Sayed NM, McIver KS - Sci Rep (2015)

Experimental validation of selected essential genes.(A) Schematic showing construction of GAS conditional expression mutants controlled by riboswitch E. The 5’-end of the selected gene was cloned along with the Psag promoter and a theophylline-inducible riboswitch into the new pSinS vector for stable chromosomal integration in GAS. Merodiploid integrants have target gene expression under the control of the riboswitch, while the wild-type promoter controls the expression of a truncated target gene. (B to C) Validation of vicR (B to C) and murE (D to E) as an essential gene in GAS 5448 (B and D) and NZ131 (C and D). The top section of each panel represents the distribution of Krmit TIS in the each locus. The bottom section shows the growth of an inducible mutant in the presence (black squares) or absence (grey circles) of theophylline.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440532&req=5

f5: Experimental validation of selected essential genes.(A) Schematic showing construction of GAS conditional expression mutants controlled by riboswitch E. The 5’-end of the selected gene was cloned along with the Psag promoter and a theophylline-inducible riboswitch into the new pSinS vector for stable chromosomal integration in GAS. Merodiploid integrants have target gene expression under the control of the riboswitch, while the wild-type promoter controls the expression of a truncated target gene. (B to C) Validation of vicR (B to C) and murE (D to E) as an essential gene in GAS 5448 (B and D) and NZ131 (C and D). The top section of each panel represents the distribution of Krmit TIS in the each locus. The bottom section shows the growth of an inducible mutant in the presence (black squares) or absence (grey circles) of theophylline.
Mentions: To confirm that conserved genes identified in our screen are required for in vitro growth of GAS, we utilized a conditionally lethal approach that took advantage of a theophylline-sensitive synthetic riboswitch functional in GAS41. A suicide/helper system pSin-pHlp was created for GAS to allow for stable insertional gene inactivation (see Materials and Methods). Stable plasmid integration was generated in the GAS chromosome resulting in a merodiploid strain containing the heterologous Psag promoter followed by the theophylline-inducible riboswitch E controlling expression of the full length targeted gene, while the wild type promoter of the targeted gene transcribed a truncated non-functional allele (Fig. 5A).

Bottom Line: A large proportion of GAS essential genes corresponded to key cellular processes and metabolic pathways, and 177 were found conserved within the GAS core genome established from 20 available GAS genomes.Selected essential genes were validated using conditional-expression mutants.Finally, comparison to previous essentiality analyses in S. sanguinis and S. pneumoniae revealed significant overlaps, providing valuable insights for the development of new antimicrobials to treat infections by GAS and other pathogenic streptococci.

View Article: PubMed Central - PubMed

Affiliation: 1] [2].

ABSTRACT
Streptococcus pyogenes (Group A Streptococcus, GAS) remains a major public health burden worldwide, infecting over 750 million people leading to over 500,000 deaths annually. GAS pathogenesis is complex, involving genetically distinct GAS strains and multiple infection sites. To overcome fastidious genetic manipulations and accelerate pathogenesis investigations in GAS, we developed a mariner-based system (Krmit) for en masse monitoring of complex mutant pools by transposon sequencing (Tn-seq). Highly saturated transposant libraries (Krmit insertions in ca. every 25 nucleotides) were generated in two distinct GAS clinical isolates, a serotype M1T1 invasive strain 5448 and a nephritogenic serotype M49 strain NZ131, and analyzed using a Bayesian statistical model to predict GAS essential genes, identifying sets of 227 and 241 of those genes in 5448 and NZ131, respectively. A large proportion of GAS essential genes corresponded to key cellular processes and metabolic pathways, and 177 were found conserved within the GAS core genome established from 20 available GAS genomes. Selected essential genes were validated using conditional-expression mutants. Finally, comparison to previous essentiality analyses in S. sanguinis and S. pneumoniae revealed significant overlaps, providing valuable insights for the development of new antimicrobials to treat infections by GAS and other pathogenic streptococci.

No MeSH data available.


Related in: MedlinePlus