Lecture 15: Antibiotic Resistance Modification & Destruction PDF

ANTIMICROBIAL RESISTANCE MODIFICATION & by Dr. Daniel Czyż DESTRUCTION Lecture 15 Today’s lecture will cover: Different mechanisms of enzymatic inactivation of antibiotics After completion of this lecture, students should be able to: Describe mechanisms o...

ANTIMICROBIAL RESISTANCE MODIFICATION & by Dr. Daniel Czyż DESTRUCTION Lecture 15 Today’s lecture will cover: Different mechanisms of enzymatic inactivation of antibiotics After completion of this lecture, students should be able to: Describe mechanisms of antibiotic inactivation Be familiar with different types of antibiotic inactivation Major mechanism of antibiotic resistance Antibiotic inactivation: enzymatic inactivation of antibiotic to confer drug resistance (Lecture 15) Antibiotic efflux: transport of antibiotics outside of the cell (Lecture 16) Reduced permeability to antibiotics: generally through reduced production or modification of porins (Lecture 16) Resistance by absence: deletion of a gene (usually a porin) (Lecture 16) Antibiotic target modification: mutational alteration or enzymatic modification of antibiotic target (Lecture 17) Antibiotic target replacement: replacement or substitution of antibiotic action target (Lecture 17) Antibiotic target protection: protection of antibiotic action target from antibiotic binding (Lecture 17) Card.mcmaster.ca/home Antibiotic inactivation: enzymatic modification of antibiotics Enzymatic degradation Hydrolysis: beta-lactamases Linearization: cutting Enzymatic modification Nucleotidylation: additional of nucleotides Phosphorylation: phosphate group addition Glycosylation: attachment of a carbohydrate Acylation: addition of the acyl (RCO-) group Hydroxylation: addition of hydroxy (-OH) group Antibiotic inactivation by sequestration Early indication of antibiotic resistance Edward Abraham Ernst Chain Abraham & Chain, 1940 Abraham & Chain, 1940 The unknown enzyme: Penicillinase Abraham & Chain, 1940 β-lactamases are enzymes that hydrolyze β-lactam antibiotics 1BSG Wikimedia Commons β-lactamases are enzymes that hydrolyze β-lactam antibiotics 1BSG KNOWLEDGE BOX Penicillinase (beta-lactamase) is an enzyme that facilitates hydrolysis of beta- lactams rendering them ineffective at killing bacteria Wikimedia Commons Currently, there are over 1000 β- lactamases identified Davies & Davies, 2010 Currently, there are over 1000 β- lactamases identified Pozzi and Ben-David, 2002 Bulky side groups protect the β-lactam ring from β-lactamases So bulky! Generation Antibiotic Class I II III IV Fischbach & Walsh (2009), Science β-lactamase classification Metalloenzymes Serine in the active site Ruppe et al. 2015 β-lactamase classification Ruppe et al. 2015 β-lactamase classification Ruppe et al. 2015 β-lactamase classification Ruppe et al. 2015  Mechanisms of β-lactamase induction: AmpG-AmpR-AmpC Gram (-) bacteria evolved two strategies to induce β-lactamases: AmpG-AmpR-AmpC pathway Two-component regulatory system (TCRS) Zeng & Lin (2013) Front. Microbiol.  Mechanisms of β-lactamase induction: AmpG-AmpR-AmpC Gram (-) bacteria evolved two strategies to induce β-lactamases: AmpG-AmpR-AmpC pathway Two-component regulatory system (TCRS) Upon β-lactam antibiotic treatment muropeptides (fragments of peptidoglycans) are transported into the cytoplasm by AmpG transporter In the cytoplasm, NagZ enzyme removes NAG sugar group and the remaining NAM- oligopeptide interacts with AmpR located upstream of ampC (β-lactamase gene) (ampR) (ampC) Zeng & Lin (2013) Front. Microbiol.  Mechanisms of β-lactamase induction: AmpG-AmpR-AmpC Gram (-) bacteria evolved two strategies to induce β-lactamases: AmpG-AmpR-AmpC pathway Two-component regulatory system (TCRS) Upon β-lactam antibiotic treatment muropeptides (fragments of peptidoglycans) are transported into the cytoplasm by AmpG transporter In the cytoplasm, NagZ enzyme removes NAG sugar group and the remaining NAM- oligopeptide interacts with AmpR located upstream of ampC (β-lactamase gene) (ampR) AmpR has other functions (i.e. in P. aeruginosa it also induces proteases, quorum sensing, and other virulence (ampC) factors) Zeng & Lin (2013) Front. Microbiol. Mechanisms of β-lactamase induction: Two-component regulatory system Two-component regulatory system (TCRS) allows the cell to sense the outside environment and respond accordingly through a signaling cascade KNOWLEDGE BOX Two-component regulatory system (TCRS): a signaling cascade that allows a cell to sense the outside environment and response accordingly Bretl et al. 2011 Mechanisms of β-lactamase induction: Two-component regulatory system, BlrAB The TCRS, BlrAB, is not well understood and most evidence from its involvement comes from genetic studies in Aeromonas: Overexpression of BlrA increased production of B-lactamase BlrA knockout attenuated expression of B- lactamase Studies of the promoter region revealed that deletion of the BlrB DNA binding site attenuated expression of B-lactamase Similar TCRS have been identified in other species. For example, Bretl et al. 2011 Mechanisms of β-lactamase induction: Two-component regulatory system, BlrAB The TCRS, BlrAB, is not well understood and most evidence from its involvement comes from genetic studies in Aeromonas: Overexpression of BlrA increased production of B-lactamase BlrA knockout attenuated expression of B- lactamase Studies of the promoter region revealed that deletion of the BlrB DNA binding site attenuated expression of B-lactamase Similar TCRS have been identified in other species. For example, E. coli Different Aeromonas spp. P. aeruginosa S. maltophilia Bretl et al. 2011 Mechanisms of β-lactamase induction: Two-component regulatory system, BlrAB The TCRS, BlrAB, is not well understood and most evidence from its involvement comes from genetic studies in Aeromonas: Overexpression of BlrA increased production of B-lactamase BlrA knockout attenuated expression of β- lactamase Studies of the promoter region revealed that deletion of the BlrB DNA binding site attenuated expression of B-lactamase Recent studies in V. parahaemolyticus identified the histidine kinase sensor to function directly as β-lactam receptor that translates the signal to a response regulator that controls the expression of a β-lactamase β-lactamase Li et al. (2016), PNAS TCRSs are involved in sensing and inactivation of many antibiotics Chancey et al. 2012 TCRS controlling β-lactamase synthesis Staphylococcus resistance to beta-lactams is mediated by blaZ β-lactamase Upon exposure to β-lactams, BlaR1 cleaves itself and becomes an active protease The protease, BlaR2, cleaves and inactivates the BlaI repressor leading to activation of blaZ expression Lowy, 2003 Mechanisms of β-lactamase induction: Potential new targets to combat resistance Understanding the mechanisms of β- lactamase induction provides additional therapeutic targets to combat antibiotic resistance Bulgecin A, a compound that selectively binds to and inhibits bacterial lytic transglycosylase increases efficacy of β- lactam antibiotics Williams et al. (2017), Antibiotics Zeng & Lin (2013) Front. Microbiol. Mechanisms of β-lactamase induction: Potential new targets to combat resistance Additional small molecules that target LT, NagZ, AmpG, and AmpR were identified and were shown to increase the efficiency of β-lactam antibiotics by inhibiting levels β-lactamases Zeng & Lin (2013) Front. Microbiol. Small molecule inhibitors of β-lactamases Clavulanate Sulbactam Tazobactam Avibactam (non-b-lactam) Vaborbactam (non-b-lactam)  Bacteria can produce enzymes to enzymatically inactivate antibiotics (other than β-lactams) Tetracyclines destructases are enzymes that degrade tetracycline antibiotics Antibiotic inactivation: enzymatic modification of antibiotics Enzymatic degradation Hydrolysis: beta-lactamases Linearization: cutting Enzymatic modification Nucleotidylation: additional of nucleotides Phosphorylation: phosphate group addition Glycosylation: attachment of a carbohydrate Acylation: addition of the acyl (RCO-) group Hydroxylation: addition of hydroxy (-OH) group Antibiotic inactivation by sequestration Enzymatic linearization Enzymatic linearization of antibiotics: Rifamycin monooxygenase (ROX) enzyme linearizes rifampicin Common again cyclic antibiotics: lipopeptides ansamycins streptogramins Koteva et al. 2018 Antibiotic inactivation: enzymatic modification of antibiotics Enzymatic degradation Hydrolysis: beta-lactamases Linearization: cutting Enzymatic modification Nucleotidylation: additional of nucleotides Phosphorylation: phosphate group addition Glycosylation: attachment of a carbohydrate Acylation: addition of the acyl (RCO-) group Hydroxylation: addition of hydroxy (-OH) group Antibiotic inactivation by sequestration Antibiotic inactivation: enzymatic modification of antibiotics Enzymatic degradation Hydrolysis: beta-lactamases Linearization: cutting KNOWLEDGE BOX Enzymatic modification: biochemical Enzymatic modification modification of antibiotics that increases Nucleotidylation: additional steric hinderance of nucleotides as a result the antibiotic Phosphorylation: looses its ability to recognize phosphate group targets and addition the MIC increases Glycosylation: attachment of a carbohydrate Acylation: addition of the acyl (RCO-) group Hydroxylation: addition of hydroxy (-OH) group Antibiotic inactivation by sequestration Antibiotic inactivation: enzymatic modification of antibiotics Enzymatic degradation Hydrolysis: beta-lactamases Linearization: cutting Enzymatic modification Nucleotidylation: additional of nucleotides Phosphorylation: phosphate group addition Glycosylation: attachment of a carbohydrate Acylation: addition of the acyl (RCO-) group Hydroxylation: addition of hydroxy (-OH) group Antibiotic inactivation by sequestration Antibiotic inactivation by sequestration Sabnis et al. 2018 Question? 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