Bacterial Resistance Strategies

• Learning objectives include elaborating on bacterial resistance strategies and comparing methods to identify antibiotic mechanisms

Bacterial Resistance

• An antibiotic needs to reach high enough concentrations to engage its target and modulate survival pathways
• Bacteria can be resistant to antibiotics in these main ways:
  • Preventing antibiotic entry (modulating uptake through the cell envelope)
  • Actively pumping the antibiotic out (efflux pumps)
  • Altering the antibiotic target to inhibit binding
  • Modifying the antibiotic to inhibit binding
  • Bypassing a blocked pathway via alternative enzymes

Resistance via Alternative Enzymes

• This mechanism depends on the specific pathway and its essentiality
• MRSA often contains the mecA gene, encoding PBP2A, which mediates transpeptidase activity during cell wall synthesis, and resists β-lactam antibiotics

Resistance by Preventing Entry

• Gram-negative bacteria modulate antibiotic uptake by altering porins or reducing porin expression

Resistance via Efflux Pumps

• Bacteria use efflux pumps with broad substrate scope to export many antibiotics
• Antibiotic levels are kept low, preventing an effect
• Efflux pumps are prevalent in Gram-negative bacteria

Resistance via Target Modification

• A strategy involves changing the antibiotic target to prevent binding
• Point mutations are simple examples: gyrase for fluoroquinolones, dihydropteroate synthase for sulfonamides
• Modified enzymes stay active but antibiotics bind less well

Target Modulation for Vancomycin

• Glycopeptides (like vancomycin) bind the D-Ala-D-Ala end of peptidoglycan precursors, blocking transpeptidase
• Vancomycin binds D-Ala-D-Ala through multiple hydrogen bonds
• Resistant bacteria may re-engineer the precursor to contain D-Ala-D-Lac
• Re-engineering causes a lost hydrogen bond, making Vancomycin much less active
• Gene clusters VanA or VanB cause this
• There are three genes:
  • A dehydrogenase (VanH) reduces pyruvate to D-Lac
  • A ligase (VanA or VanB) synthesizes D-Ala-D-Lac
  • A D,D-dipeptidase (VanX) hydrolyzes D-Ala-D-Ala
• D-Ala-D-Lac is high to support synthesis, while D-Ala-D-Ala is low to not compete

Strategies to Overcome Vancomycin Resistance

• E. faecalis (VanA) has D-Ala-D-Lac in the cell wall
• D-Ala-D-Lac binds natural Vancomycin 5 (Y=O) weaker (>1000x) and has no activity against E. faecalis (VanA)
• An amidine analog (Y=NH) of Vancomycin binds both D-Ala-D-Ala and D-Ala-D-Lac almost equally well, restoring activity against E. faecalis (VanA)

Resistance by Inactivating the Antibiotic

• Chemical modification to the antibiotic leads to resistance

Chloramphenicol Acetyltransferase

• Acetylated Chloramphenicol binds much less well to the ribosome

Modifying Aminoglycosides

• Enzymatic modifications and can inactivate aminoglycoside scaffolds

β-lactam Resistance

• The acyl-enzyme complex of Penicillin-binding proteins (PBPs) has a long half-life for long-term inhibition
• β-lactamases (BLAs) follow the mechanism, but the acyl-enzyme complex is very short-lived

Classes of β-lactamases

• β-lactamases are classified in the Ambler classification
• Classes A, C, and D follow the serine-based mechanism
• Class B uses a metal to hydrolyze the β-lactam
• Class B is the most problematic, resistant to normal serine-directed strategies

Strategies to Overcome β-lactamases

• It was possible to develop β-lactams that bind PBPs, but not β-lactamases
• Over time, the abundance and broad spectrums of β-lactamases have made the task more challenging
• β-lactamase inhibitors are becoming more prevalent
• Inhibitors lack an MIC, but protect β-lactams via combination therapy

β-lactam-based BLIs

• They go through the BLA mechanism, but the acyl-enzyme complex is more stable on BLAs
• This is often caused by further fragmentation of the initial adduct

Non-ß-Lactam-Based BLIs

• Inhibit BLAs through reversible covalent action to avoid hydrolysis
• Boronate of vaborbactam complex binds serine of BLAs. Giving reversible covalent inhibition that is not prone to destroy vaborbactam
• In avibactam complexes, carbamate formed is more stable than normal ester on acyl-enzyme-complex with hydrolysis being extremely slow. Mechanism is reversibly covalent.

Strategies for Resistance

• Target alteration and alternative enzymes depend on a specific target
• Others all depend on the specific compounds used
• Even with a resistance-free target, bacteria can still develop resistance through compound-directed approaches

Studying Mechanisms and Targets of Antibiotics

• Figuring out how it works is an important step when an antibiotic is proving promising
• Many assays can be used to determine the general pathway/specific target that is affected
• A combination of different methods would be required to fully understand the mechanism

Macromolecule Synthesis Assays

• Testing if the synthesis of one of them is blocked is an early step, as many antibiotics target the synthesis of macromolecules
• Radioactive substrates monitor biosynthesis of major macromolecules

Reporter Strains

• Lab strains have the reporter LacZ under control of stress promotors in B. subtilis
• Blue color will appear if that specific pathway is triggered by the compound

Bacterial Cytological Profiling (BCP)

• It is possible to stain In bacterial cell wall (red) and DNA (blue) and see phenotype changes when antibiotics are introduced

Bacterial Membrane Integrity

• The use of Propidium Iodide can be adopted to study membrane integrity. Due to it's polar nature it doesn't get into the cell. If membrane is permeabilized it will get in and form a fluorescent complex with DNA.

Outer Membrane Permeabilization in Gram-Negatives

• Hydrophobic dyes cannot enter the outer membrane of these bacteria due to the presence of LPS
• If the LPS layer is damaged, said dyes can reach the phospholipid bilayer and give off fluorescence
• This allows measurement of the intactness of the LPS in these outer membranes
• 1-N-phenylnaphthylamine is used (NPN)

Bacterial Membrane Potential

• This can be quantified using DiSC3(5)
• Certain compounds may not disrupt the membrane but cause effects regardless
• It binds to the membrane dependent on the potential and is quenched
• Should a compound disrupt the membrane potential, fluorescence increases

Proton Motive Force (PMF)

• Bacteria need PMF across the membrane to stay alive, and active transport/ATP synthesis are necessary for PMF Membrane potential helps keep PMF up should the bacteria need to increase the difference

Case Study: Xanthocillin X

• Xanthocillin X has activity against gram-negative pathogens, but its mechanism was largely unknown
• Resistance mutant sequencing showed involvement of heme biosynthesis and its mechanism (hemB)

Global Proteomics and Transcriptomics

• Bacteria can react by adjusting transcription or the translation of certain genes. This will make them be less affected by a compound
• Using these levels can help to identify the target when looking at affected pathways
• Can compare antibiotic-treated to non-treated (can be resistant vs non-resistant) to gain insights

Chemical Proteomics for Target Identification

• Chemical proteomics can highlight direct targets
• Fully functionalized probes with diazirine and alkyne can be used and bind to a target

Chemical Proteomics for PK150

• SpsB and MenG were validated as PK150 functional targets
• Double target strategy may explain the low frequency of resistance

Competitive Residue-Specific Proteomics

• This can be used for the purposes of indentifying targets for antibiotics
• Strengths of the method include target engagement, residue-specific information, and global ligandability