Properties of a novel PBP2A protein homolog from Staphylococcus aureus strain LGA251 and its contribution to the β-lactam-resistant phenotype

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) strains show strain-to-strain variation in resistance level, in genetic background, and also in the structure of the chromosomal cassette (SCCmec) that carries the resistance gene mecA. In contrast, strain-to-strain variation in the sequence of the mecA determinant was found to be much more limited among MRSA isolates examined so far. The first exception to this came with the recent identification of MRSA strain LGA251, which carries a new homolog of this gene together with regulatory elements mecI/mecR that also have novel, highly divergent structures. After cloning and purification in Escherichia coli, PBP2A(LGA), the protein product of the new mecA homolog, showed aberrant mobility in SDS-PAGE, structural instability and loss of activity at 37 °C, and a higher relative affinity for oxacillin as compared with cefoxitin. The mecA homolog free of its regulatory elements was cloned into a plasmid and introduced into the background of the β-lactam-susceptible S. aureus strain COL-S. In this background, the mecA homolog expressed a high-level resistance to cefoxitin (MIC = 400 μg/ml) and a somewhat lower resistance to oxacillin (minimal inhibitory concentration = 200 μg/ml). Similar to PBP2A, the protein homolog PBP2A(LGA) was able to replace the essential function of the S. aureus PBP2 for growth. In contrast to PBP2A, PBP2A(LGA) did not depend on the transglycosylase activity of the native PBP2 for expression of high level resistance to oxacillin, suggesting that the PBP2A homolog may preferentially cooperate with a monofunctional transglycosylase as the alternative source of transglycosylase activity.

FIGURE 1.

SDS-PAGE and Western blotting of PBP2A and PBP2A_LGA. A, the purity of the purified His-tagged proteins was checked by loading indicated amount on the 10% gel for SDS-PAGE and staining with Coomassie Blue R-250. B, the size of each protein was confirmed by immunoblotting with anti-6× His antibody and anti-PBP2A antibody. The upper panels and lower panels in B represent the intensity of the proteins developed by antibodies and Coomassie Blue, respectively. The chemiluminescence of PBP2A_LGA was much weaker than that of PBP2A for both antibodies. M represents a size marker.

FIGURE 2.

Optimal temperatures for the activity and the stability of PBP2A and PBP2A_LGA. A, purified proteins were incubated at different temperatures (25 °C, 30 °C and 37 °C) for various time periods, after which the activity of the preparations was determined by Bocillin FL binding assay. Lanes I and II represent the fluorescence of Bocillin FL-bound proteins and the amount of loaded proteins, respectively. B, the activity of PBP2A_LGA was plotted as a function of preincubation time. The activity of each protein was normalized by dividing the fluorescent intensity of each lane by the corresponding protein amount. The value at 0 min was set as 100%. PBP2A_LGA dramatically lost its activity at 37 °C. The experiment was independently performed in triplicate.

FIGURE 3.

Measurement of the affinities of PBP2A and PBP2A_LGA for two structurally different β-lactam antibiotics. The two proteins were preincubated with different concentrations of oxacillin and/or cefoxitin for 15 min, after which the fraction of the proteins that remained non-acylated was determined by Bocillin FL binding assay. A, the decreasing fluorescence by β-lactam antibiotics on SDS-PAGE. B, the plot for the percentage of unbound proteins as a function of oxacillin concentration. C, the plot for the percentage of unbound proteins as a function of cefoxitin concentration. IC₅₀ values of oxacillin and cefoxitin were calculated from the plots in B and C. The affinity was independently evaluated in triplicate.

FIGURE 4.

The far UV CD spectra for PBP2A and PBP2A_LGA at 25 °C and 37 °C. The proteins were preincubated for 20 min at the indicated temperatures prior to CD measurement. The conformation of PBP2A_LGA was disrupted at 37 °C, whereas PBP2A was adjusted to a more active form. The measurement was carried out three times independently.

FIGURE 5.

High-level antibiotic resistance produced in the β-lactam-susceptible S. aureus strain COL-S by introducing plasmid-borne copies of the mecA_LGA251. The mecA determinant of strain LGA251 was cloned free of its mecI/mecR1 regulatory elements into the cadmium-inducible plasmid pBCB8 and transduced into strain COL-S. The antibiotic resistance of the transductants was determined by population analysis. Colony-forming units (CFU) were calculated by counting colonies after 48 h of incubation on tryptic soy agar plates supplemented with 0.2 μm CdCl₂ at 37 °C and 30 °C. The closed symbols indicate the resistance of strain COL-S_LGAmecA to oxacillin (■) and cefoxitin (●) at 37 °C. The open symbols indicate the resistance of strain COL-S_LGAmecA to oxacillin (□) and cefoxitin (○) at 30 °C. The triangles represent the susceptibility profile of strain COL-S to oxacillin (△) and cefoxitin (▴).

FIGURE 6.

Measurement of the affinities of PBP2A and PBP2A_LGA from membrane preparations for two structurally different β-lactam antibiotics. PBP2A and PBP2A_LGA were produced in S. aureus COL and transductants of S. aureus COL-S, respectively. Membrane proteins (100 μg) prepared from COL and the transductants were analyzed by SDS-PAGE. Prior to the analysis, membrane extracts, except the ones labeled NC, were exposed to clavulanate (1.0 mg/ml) to make all PBPs, except for PBP2A and PBPB2A_LGA “invisible,” and then incubated with various concentrations of oxacillin (A) or cefoxitin (B). Gels were developed both by the Bocillin FL binding assay and by Coomassie Blue staining. The figure also shows the corresponding PBP patterns for the isogenic MRSA strain COL.

FIGURE 7.

The mecA_LGA251 homolog can replace the transpeptidase function of PBP2. COL-S_spac::pbpB represents the conditional mutant of S. aureus strain of COL-S in which the transcription of pbpB was under the control of an IPTG-inducible promoter. COL-S_spac::pbpB_/SAmecA and COL-S_spac::pbpB_/LGAmecA) indicate the strains to which the mecA gene and the mecA homolog were introduced by transduction, respectively. Growth of strains was followed by determination of A at 30 °C temperature, at which the temperature-sensitive plasmid pSTSW2C (19) carrying the mecA gene was stable in S. aureus.

FIGURE 8.

The mecALGA251 homolog does not require the TGase function of PBP2 for optimal expression of oxacillin resistance. The number of bacteria capable of growing in the presence of different concentrations of oxacillin was determined by population analysis at 30 °C.