The Relative Importance of Cytotoxins Produced by Methicillin-Resistant Staphylococcus aureus Strain USA300 for Causing Human PMN Destruction
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacteria Strains and Culture Conditions
2.2. Human PMN Purification
2.3. Cytotoxicity Assays
2.4. Phagocytosis Assays
3. Results
3.1. Bicomponent Leukocidins Are the Primary Extracellular Cytotoxic Component Produced by USA300 against Human PMNs
3.2. PVL Is the Prominent Cytotoxic Extracellular Factor Produced by USA300 That Causes Human PMN Destruction
3.3. Lysis of Human PMNs Following Phagocytosis of USA300 Is Primarily Mediated by Bicomponent Leukocidins
3.4. LukGH Is the Primary Initial Cause of Human PMN Destruction Following Phagocytosis of USA300
4. Discussion
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Strain | Genes Deleted; Complemented | Toxin Genes Present (↓ = Downregulated) |
---|---|---|
USA300 strain LAC | none | hlgABC, lukED, lukGH, pvl, hla, and psmα [54] |
USA300ΔagrABCD | agrABCD | ↓hlgABC, ↓lukED, ↓lukGH, ↓pvl, ↓hla, and ↓psm [11,13,16] |
USA300ΔsaePQRS | saePQRS | ↓hlgABC, ↓lukED, ↓lukGH, ↓pvl, ↓hla, and psmα [13,59] |
USA300ΔarlRS | arlRS | ↓hlgABC, lukED, ↓lukGH, ↓pvl, hla, and psmα [62] |
USA300ΔhlgABC | hlgABC | lukED, lukGH, pvl, hla, and psmα |
USA300ΔlukGH | lukGH | hlgABC, lukED, pvl, hla, and psmα |
USA300ΔlukED | lukED | hlgABC, lukGH, pvl, hla, and psmα |
USA300Δpvl | pvl | hlgABC, lukED, lukGH, hla, and psmα |
USA300Δpsm-aΔhla | psm-a and hla | hlgABC, lukED, lukGH, and pvl |
USA300ΔhlgABCΔlukGHΔpvl | hlgABC, lukGH, and PVL | lukED, hla, and psmα |
USA300ΔhlgABCΔlukGHΔlukED | hlgABC, lukGH, and lukED | pvl, hla, and psmα |
USA300ΔpvlΔlukGHΔlukED | pvl, lukGH, and lukED | hlgABC, hla, and psmα |
USA300ΔpvlΔhlgABCΔlukED | pvl, hlgABC, and lukED | lukGH, hla, and psmα |
USA300ΔhlgABCΔlukGHΔpvlΔlukED | hlgABC, lukGH, PVL, and lukED | hla and psmα |
USA300ΔhlgABCΔlukGHΔpvlΔlukEDΔhlaΔpsm | hlgABC, lukAB, pvl, lukED, hla and psm-a | none |
USA300ΔhlgABCΔlukGHΔpvlΔlukED pRB473-pvl-comp | hlgABC, lukGH, pvl, lukED; pvl complemented | pvl, hla, and psmα |
USA300ΔhlgABCΔlukGHΔpvlΔlukED pRB473-hlgABC-comp | hlgABC, lukGH, pvl, lukED; hlgABC complemented | hlgABC, hla, and psmα |
USA300ΔhlgABCΔlukGHΔpvlΔlukED pRB473-lukED-comp | hlgABC, lukGH, pvl, lukED; lukED complemented | lukED, hla, and psmα |
USA300ΔhlgABCΔlukABΔpvlΔlukED pRB473-lukGH-comp | hlgABC, lukGH, pvl, lukED; lukGH complemented | lukGH, hla, and psmα |
Primer | Sequence |
---|---|
agrABCD-Top_fwd | 5′ - GGG GAC AAG TTT GTA CAA AAA AGC AGG CGA AGC GCC CGA AAT AAT ATT TAA CAC - 3′ |
agrABCD-SphI-Top_rvs | 5′ - GGT GGT GCA TGC CTC CTC ACT GTC ATT ATA CGA TTT AG - 3′ |
agrABCD-SphI-Bot_fwd | 5′ - GGT GGT GCA TGC GTC AGT TAA CGG CGT ATT CAA TTG - 3′ |
agrABCD-Bot_rvs | 5′ - GGG GAC CAC TTT GTA CAA GAA AGC TGG GTG TAA GCC CTC TGC TGA TAT G - 3′ |
SaePQRS-Top_Fwd | 5′ - GGG GAC AAG TTT GTA CAA AAA AGC AGG CGA AGG GGA AGT CAT TAC ACA AAC - 3′ |
SaePQRS-SphI-Top_Rvs | 5′ - GGT GGT GCA TGC CTC CCA TTA ATG AGG GCT TC - 3′ |
saePQRS-SphI-Bot_fwd | 5′ - GGT GGT GCA TGC CTC GGA GAG ATT GCA ATT GG - 3′ |
saePQRS-Bot_Rvs | 5′ - GGG GAC AAG TTT GTA CAA AAA AGC AGG CGT CAT ATG GCC GTT AAA CCA CA - 3′ |
arlRS-SalI-Top_fwd | 5′ - TGT CGA CCT CAT ATT ACG ACT TTT TC - 3′ |
arlRS-PstI-Top_rvs | 5′ - CTG CAG TAA ACC TAA AGT GTC GTA AG - 3′ |
arlRS-SacI-Bot_fwd | 5′ - TCA CTA TTG AGC TCT TTG TTA AAG TAG - 3′ |
arlRS-BamHI-Bot_rvs | 5′ - AAA TGG ATC CTA TCA TAA AAT TAG TCG AAG - 3′ |
hlgABC-SphI-Top_Fwd | 5′ - GGG GAC AAG TTT GTA CAA AAA AGC AGG CGT TCG TCA TGA TGA GCG TG - 3′ |
hlgABC-SphI-Top_rvs | 5′ - GGT GGT GCA TGC GGT CGC AGG CGT TTA TAT AG - 3′ |
hlgABC-SphI-Bot_Fwd | 5′ - GGT GGT GCA TGC GTG ACG ACC GTG - 3′ |
hlgABC-SphI-Bot_rvs | 5′ - GGG GAC CAC TTT GTA CAA GAA AGC TGG GTG CGC TAA ATC AAG GGA TG - 3′ |
lukGH-SphI-Top_fwd | 5′ - GGG GAC AAG TTT GTA CAA AAA AGC AGG CCA ATC AGG GTG GGA CAA AAC - 3′ |
lukGH-SphI-Top_rvs | 5′ - GGG GGT GGT GCA TGC GAC GTG CAG TGT ATG AAT CTT G - 3′ |
lukGH-SphI-Bot_fwd | 5′ - GGT GGT GCA TGC GAT TGA TAT TTG TTG ATA TGT ATC GAC ATG TG - 3′ |
lukGH-SphI-Bot_rvs | 5′ - GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC AAT GAT TTG AAC ATA GGC GCA AC - 3′ |
lukED-SphI-Top_fwd | 5′ - GGG GAC AAG TTT GTA CAA AAA AGC AGG CGA AGT TAA GGC CTA CTT CAA TTG TC - 3′ |
lukED-SphI-Top_rvs | 5′ - GGT GGT GCA TGC GAA ACT AAT CCT GGA GTA TAA CTG TTA G - 3′ |
lukED-SphI-Bot_fwd | 5′ - GGT GGT GCA TGC CTA CTG ACA AAG TTG CAG CTA AC - 3′ |
lukED-SphI-Bot_rvs | 5′ - GGG GAC CAC TTT GTA CAA GAA AGC TGG GTG TGC TCG TCG TCA AGA C - 3′ |
PVL-SphI-Top_fwd | 5′ - GGG GAC AAG TTT GTA CAA AAA AGC AGG CCT CAT ATC ATC GCC TTT GTC C - 3′ |
PVL-SphI-Top_rvs | 5′ - GGT GGT GCA TGC GGA ATC AAC TTC ACT GGA TAG G - 3′ |
PVL-SphI-Bot_fwd | 5′ - GGT GGT GCA TGC CTA ACG ACA ATG TTG CAG CTA ATA G - 3′ |
PVL-SphI-Bot_rvs | 5′ - GGG GAC CAC TTT GTA CAA GAA AGC TGG GTG AGA AAG CGC AAG TGG TG - 3′ |
PSMa-Top_fwd | 5’′ - GGG GAC AAG TTT GTA CAA AAA AGC AGG CGT CGT CTA CCT TTC CAT GC - 3′ |
PSMa-SphI-Top_rvs | 5′ - GGT GGT GCA TGC CTC AGG CCA CTA TAC CAA TAG - 3′ |
PSMa-SphI-Bot_fwd | 5′ - GGT GGT GCA TGC CAG CGA TGA TAC CCA TTA AGA TTA CC - 3′ |
PSMa-Bot_rvs | 5′ - GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC GAA TGC AAG CCA ACC AC - 3′ |
hla-Top_fwd | 5′ - GGG GAC AAG TTT GTA CAA AAA AGC AGG CGA AGT CCA TAC AAA ATC CGC ATC - 3′ |
hla-BamHI-Top_rvs | 5′ - GGT GGT GGA TCC CTA TCT ACT TGA TTT GCT TTC CTG AC - 3′ |
hla-BamHI-Bot_fwd | 5′ - GGT GGT GGA TCC CAA TTT CGA GGG TTA GTC AAA GTT G - 3′ |
hla-Bot_rvs | 5′ - GGG GAC CAC TTT GTA CAA GAA AGC TGG GTG CAA TAC TTT ATT GTC CCA TGA TTA GTG - 3′ |
pvl-EcoRI-comp_fwd | 5′ - AGG AGG GAA TTC GTT TGG TAA TGA ACG GGT TTT TTT CG - 3′ |
pvl-BamHI-comp_rvs | 5′ - GGT GGT GGA TCC CAA TTA AGA CGT GGT TAC CCT AAT ATA G - 3′ |
hlgABC-SacI-comp_fwd | 5′ - GGT GGT GAG CTC CAG TTA ATT CGA AAA CGC TTA CAA ATG G - 3′ |
hlgABC-BamHI-comp_rvs | 5′ - GGT GGT GGA TCC CTG TTG GCG ACC GTG - 3′ |
lukED-SacI-Comp_fwd | 5′ - GGT GGT GAG CTC CCA TGA GAG TAG AAG CTT CAG - 3′ |
lukED-BamHI-Comp_rvs | 5′ - GGT GGT GGA TCC GAA GTT AAG ACC CAC TTC AAT TGT C - 3′ |
lukGH-EcoRI-comp_fwd | 5′ - GGT GGT GAA TTC GTA TCA ACG ATC TTA TTA ACG CTG - 3′ |
lukGH-BamHI-comp_rvs | 5′ - GGT GGT GGA TCC CTA CAT TCT ATG TAG CAG GCA AC - 3′ |
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Nygaard, T.K.; Borgogna, T.R.; Pallister, K.B.; Predtechenskaya, M.; Burroughs, O.S.; Gao, A.; Lubick, E.G.; Voyich, J.M. The Relative Importance of Cytotoxins Produced by Methicillin-Resistant Staphylococcus aureus Strain USA300 for Causing Human PMN Destruction. Microorganisms 2024, 12, 1782. https://doi.org/10.3390/microorganisms12091782
Nygaard TK, Borgogna TR, Pallister KB, Predtechenskaya M, Burroughs OS, Gao A, Lubick EG, Voyich JM. The Relative Importance of Cytotoxins Produced by Methicillin-Resistant Staphylococcus aureus Strain USA300 for Causing Human PMN Destruction. Microorganisms. 2024; 12(9):1782. https://doi.org/10.3390/microorganisms12091782
Chicago/Turabian StyleNygaard, Tyler K., Timothy R. Borgogna, Kyler B. Pallister, Maria Predtechenskaya, Owen S. Burroughs, Annika Gao, Evan G. Lubick, and Jovanka M. Voyich. 2024. "The Relative Importance of Cytotoxins Produced by Methicillin-Resistant Staphylococcus aureus Strain USA300 for Causing Human PMN Destruction" Microorganisms 12, no. 9: 1782. https://doi.org/10.3390/microorganisms12091782