Studying the molecular basis of antibiotic resistance by assessing penicillin interaction with PBP5 using fluorescence polarization

Introduction

Benzylpenicillin was first discovered back in the 1920s and since then thousands of new penicillin derivates have been discovered¹. Penicillin is part of a class of antibiotics called β-lactams². β-lactams are the most commonly used antibiotics. New classes of β-lactams were expanded to cover a larger range of bacterial species or combat antibiotic resistance mechanisms¹. β-lactams bind to, and target proteins found in the bacterial cell wall called penicillin binding proteins (PBPs)². PBPs are enzymes that are involved in the last step of the peptidoglycan cross-linking in the bacterial cell wall¹. β-lactams inhibit the cross-linking and disrupt the building of the bacterial cell wall. The bacterial cell wall is important for cell structure, osmosis, and protection of the organism. Once this process is disrupted, the bacteria can no longer maintain their integrity³.

Each species of bacteria contains their own particular set of PBP enzymes, and each species can have between three to eight PBP enzymes¹. Resistance to β-lactams is a growing problem and is mainly the result of β-lactamase enzymes produced by bacteria. β-lactamase enzymes hydrolyse the β-lactams ring which render the antibiotic inactive¹. It is important to study different variants of PBPs to understand their molecular basis and binding to β-lactams.   Enterococcus faecium is known to cause severe infections and has high resistance to β-lactams. Clinical-derived resistant PBP5 variants of E. faecium have been isolated⁴. Interestingly, these mutations do not impact the PBP5 protein structures². In this work, we used fluorescence polarization to determine the binding affinity of penicillin to these clinical-derived resistant PBP5 variants.  

Assay Principle

BOCILLIN FL is a fluorescent derivative penicillin (BODIPY-labelled penicillin V). As illustrated in figure 1, mixing of a PBP5 with BOCILLIN FL results in the increase in FP signal over time if a binding event occurs.

Materials & Methods

• Black, 384-well, polystyrene plates (Corning, #4511)
• BOCILLIN FL Penicillin (Thermo Scientific, #B13233)
• ThermalSeal RTS, silicone adhesive film (EXCEL Scientific, #TSS-RTQ-100)
• CLARIOstar^® Plus (BMG LABTECH)

Experimental Procedure
WT and variant PBP5 proteins were expressed and purified from isolated Enterococcus faecium variants as previously described in Hunashal, Y. et al.².

BOCILLIN FL was prepared fresh from a 1 mM stock in DMSO and used at 30 nM final concentration in the assay. For the assay it was diluted in assay buffer (100 mM sodium phosphate, pH 7.0; 0.01% Triton X-100) and transferred to a black 384-well plate with 5  µL/well. The assay was initiated by the addition of the 15 µL indicated PBP5 per well (at 3.6 µM final concentration) and sealed with the ThermalSeal. Reactions without PBP5 included only assay buffer and served as a negative control.

Instrument Settings

Results & Discussion

Figure 2 shows wildtype PBP5 (WT, defined as PBP5 T485) binding to BOCILLIN over time, visible through the increase in the FP value (green curve). As expected, the catalytically inactive PBP5 S422A variant shows no binding (blue curve), as β-lactams strictly require the active site serine for binding. 

One of the clinically most resistant PBP5 variants (T485A) binds to BOCILLIN at a much slower rate (red curve), nearly similar to the PBP5 S422A variant. Conversely, the least resistant PBP5 variant (T485M) binds to BOCILLIN at higher rate (orange curve). 

Conclusion

The fluorescence polarization data shows that β-lactams bind strictly to the PBP5 active site via S422. Clinical PBP5 variants show large change in fluorescence polarization, correlating with their clinical activity. The CLARIOstar Plus is a strong support in determining the interaction differences between the variants by monitoring the FP increase with very high sensitivity. The incubation function allows the optimum temperature to be set and maintained during the kinetic incubation study.

References

1. Bush, K., Bradford, P. β-Lactams and β-Lactamase Inhibitors: An Overview. Cold Spring Harb Perspect Med.(2016) Volume 6 (8)
2. Hunashal, Y. et al. Molecular basis of β-lactam antibiotic resistance of ESKAPE bacterium E. faecium Penicillin Binding Protein. Nat. Commun. (2023) 14: 4268
3. Yip, D., Gerriets, V., Penicillin. StatPearls Publishing. (2023)
4. Rice L.B., Bellais S., Carias L. L., Hutton-Thomas R., Bonomo R. A., Caspers P., Page M. G. P., Gutmann L. Impact of specific pbp5 mutations on expression of β-lactam resistance in Enterococcus faecium. Antimicrob. Agents Chemother, 48 (2004), pp. 3028-3032