BPEI-Induced Delocalization of PBP4 Potentiates β-Lactams against MRSA

Abstract

With its high morbidity rate and increasing resistance to treatment, methicillin-resistant Staphylococcus aureus (MRSA) is a grave concern in the medical field. In methicillin-susceptible strains, β-lactam antibiotics disable the penicillin binding proteins (PBPs) that cross-link the bacterial cell wall. However, methicillin-resistant strains have PBP2a and PBP4, which continue enzymatic activity in the presence of β-lactam antibiotics. The activity of PBP2a and PBP4 is linked to the presence of wall teichoic acid (WTA); thus, WTA has emerged as a target for antibiotic drug discovery. In this work, we disable WTA in situ using its anionic phosphodiester backbone to attract cationic branched polyethylenimine (BPEI). Data show that BPEI removes β-lactam resistance in common MRSA strains and clinical isolates. Fluorescence microscopy was used to investigate this mechanism of action. The results indicate that BPEI prevents the localization of PBP4 to the cell division septum, thereby changing the cellular morphology and inhibiting cell division. Although PBP4 is not required for septum formation, proper cell division and morphology require WTA; BPEI prevents this essential function. The combination of BPEI and β-lactams is bactericidal and synergistic. Because BPEI allows us to study the role of WTA in the cell wall without genetic mutation or altered translocation of biomolecules and/or their precursors, this approach can help revise existing paradigms regarding the role of WTA in prokaryotic biochemistry at every growth stage.

Figure 1.

Checkerboard assays show synergy between BPEI and β-lactams on MRSA USA300 and MRSA MW2. BPEI had synergy with (A and B) oxacillin, (C and D) ceftizoxime, and (E and F) imipenem against (A, C, and E) MRSA USA300 and (B, D, and F) MRSA MW2. Each assay was performed as three separate trials, and the presented data are shown as the average change in OD₆₀₀.

Figure 2.

Mechanism by which BPEI makes MRSA susceptible via WTA. BPEI and oxacillin have synergy against MRSA MW2. Synergy is lost against (A) MRSA MW2 ΔtarO and (B) MRSA MW2 with sublethal tunicamycin (0.250 μg/mL). Each assay was performed as three separate trials, and the presented data are shown as the average change in OD₆₀₀.

Figure 3.

(A and B) Fluorescence images of MRSA COL PBP4-YFP show that PBP4 primarily localizes at the division septum of dividing cells. (C and D) BPEI delocalizes PBP4 from the division septum. (E) Quantitative analysis of the fluorescence intensity at the division septum vs fluorescence intensity at peripheral cell wall regions was performed for untreated and BPEI-treated samples. Analysis was performed for >100 cells of each sample that showed a visible closed septum. The PBP4 protein in untreated cells (S/L = 2.95 ± 0.06) localized more at the division septum than in the BPEI-treated samples (S/L = 2.15 ± 0.05). The scale bar is 1 μm.

Figure 4.

SEM of MRSA USA300 shows multiple septa formations when treated with BPEI. (A) Untreated cells have a single septal formation (black arrows). (B) Some BPEI-treated cells have multiple septa formations (white arrows). The scale bar is 1 μm.

Figure 5.

TEM images of MRSA USA300 cells do not show drastic changes in division septa. Cells treated with (B and D) BPEI have thicker division septa in comparison to (A and C) those of the untreated cells. Black arrows indicate complete septa formation. White arrows indicate incomplete septa formation. The scale bar is 500 nm.

Figure 6.

Fluorescence images of MRSA JE2 bacteria showing that BPEI binds to the peripheral cell wall and the division septum of dividing cells. The intensity of the Nile Red dye is used to count the number of cells with no septum (denoted as P1), a partial septum (denoted as P2), or a complete septum (denoted as P3).