Genetic Manipulation of Group A Streptococcus—Gene Deletion by Allelic Replacement

Barnett, Timothy C.; Daw, Jessica; Walker, Mark J.; Brouwer, Stephan

doi:10.1007/978-1-0716-0467-0_5

“…A markerless HKU16Δ gshT isogenic mutant strain was generated using a highly efficient plasmid (pLZts) as previously described ( 73 ). PCR primers were designed to keep the first and last 24 nucleotides (8 amino acids), respectively, of the gshT coding sequence intact to reduce the possibility of downstream effects.…”

Section: Methodsmentioning

confidence: 99%

Streptococcus pyogenes Hijacks Host Glutathione for Growth and Innate Immune Evasion

Brouwer

¹

,

Jespersen

²

,

Ong

³

et al. 2022

mBio

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“…Plasmids were purified using the Monarch Plasmid Miniprep kit (New England Biolabs T1010L), and sequences verified by whole-plasmid sequencing (PlasmidSeq, Genomics WA). Plasmids were transformed into electrocompetent GAS strains using the following settings: voltage = 1.75 kV, resistance 400 Ω, capacitance = 25 µF 41 . Transformants were selected by plating on THYA containing 100 µg/ml spectinomycin (THYA + Sp100).…”

Section: Methodsmentioning

confidence: 99%

“…Insertion of thfT into the genome of NS5347 was performed using the temperature-sensitive plasmid pLZts 41 (Addgene # 128799). The thfT gene and ~2.5 kb flanking DNA was amplified by PCR from TB08 DNA using primers pLZ-thfT-S (5’-CAT AAC CTG AAG GAA GAT CTA AAA CCA TAA TTT CCT TTC G-3’) and pLZ-thfT-A (5’-GTC GTC AGA CTG ATG GGC CCA GTA AAA CTC AAT GAG AAA ATA TTT TTT TAG-3’), and cloned into pLZts amplified by PCR with primers pLZts-S (5’-AGA TCT TCC TTC AGG TTA TG-3’) and pLZts-A (5’-GGG CCC ATC AGT CTG ACG-3’).…”

Section: Methodsmentioning

confidence: 99%

“…The resulting plasmid was transformed into electrocompetent NS5347 GAS cells as described above and selected by plating on THYA + Sp100 at 28 °C. Insertion of thfT into the genome of NS5347 was accomplished by homologous recombination, following plasmid integration at 37.5 °C and plasmid excision at 28 °C 41 . Colonies containing the desired allelic exchange were screened for the presence of thfT by PCR, using primers thfT+222 S (5’-GCC AGT GGG TCA GGT AAT TT-3’) and thfT+524 A (5’-CTT TGA AGG GCT GGC ACT AAT A-3’).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Host-dependent resistance of Group A Streptococcus to sulfamethoxazole mediated by a horizontally-acquired reduced folate transporter

Rodrigo

¹

,

Saiganesh

²

,

Hayes

³

et al. 2022

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Described antimicrobial resistance mechanisms enable bacteria to avoid the direct effects of antibiotics and can be monitored by in vitro susceptibility testing and genetic methods. Here we describe a mechanism of sulfamethoxazole resistance that requires a host metabolite for activity. Using a combination of in vitro evolution and metabolic rescue experiments, we identify an energy-coupling factor (ECF) transporter S component gene (thfT) that enables Group A Streptococcus to acquire extracellular reduced folate compounds. ThfT likely expands the substrate specificity of an endogenous ECF transporter to acquire reduced folate compounds directly from the host, thereby bypassing the inhibition of folate biosynthesis by sulfamethoxazole. As such, ThfT is a functional equivalent of eukaryotic folate uptake pathways that confers very high levels of resistance to sulfamethoxazole, yet remains undetectable when Group A Streptococcus is grown in the absence of reduced folates. Our study highlights the need to understand how antibiotic susceptibility of pathogens might function during infections to identify additional mechanisms of resistance and reduce ineffective antibiotic use and treatment failures, which in turn further contribute to the spread of antimicrobial resistance genes amongst bacterial pathogens.

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“…Plasmids were purified using the Monarch Plasmid Miniprep kit (New England Biolabs), and sequences verified by whole-plasmid sequencing (PlasmidSeq, Genomics WA). Plasmids were transformed into electrocompetent GAS strains as described 29 .…”

Section: Metabolic Rescue Assaymentioning

confidence: 99%

Host-dependent resistance of Group A Streptococcus to sulfamethoxazole mediated by a horizontally-acquired ECF transporter S component

Rodrigo

¹

,

Saiganesh

²

,

Hayes

³

et al. 2022

Preprint

0

View full text Add to dashboard Cite

Described antimicrobial resistance mechanisms enable bacteria to avoid the direct effects of antibiotics and can be monitored by in vitro susceptibility testing and genetic methods. We have identified a new mechanism of sulfamethoxazole resistance that requires a host metabolite for activity. Using a combination of in vitro evolution and metabolic rescue experiments, we identified an energy-coupling factor (ECF) transporter S component gene (thfT) that enables Group A Streptococcus to acquire extracellular tetrahydrofolate and related compounds. ThfT likely modifies the substrate specificity of an ECF transporter to acquire the end products of folate biosynthesis. As ThfT-mediated resistance is undetectable by routine surveillance methods, our study highlights the need to understand antibiotic susceptibility during infection to reduce inappropriate antibiotic use and treatment failures.

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Genetic Manipulation of Group A Streptococcus—Gene Deletion by Allelic Replacement

Cited by 14 publications

References 13 publications

Streptococcus pyogenes Hijacks Host Glutathione for Growth and Innate Immune Evasion

Streptococcus pyogenes Hijacks Host Glutathione for Growth and Innate Immune Evasion

Host-dependent resistance of Group A Streptococcus to sulfamethoxazole mediated by a horizontally-acquired reduced folate transporter

Host-dependent resistance of Group A Streptococcus to sulfamethoxazole mediated by a horizontally-acquired ECF transporter S component

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