The Staphylococcus aureus Chaperone PrsA Is a New Auxiliary Factor of Oxacillin Resistance Affecting Penicillin-Binding Protein 2A

Effect of prsA on PBP profile. (A) Detection of PBP1, PBP2, PBP3, and PBP4 in membrane preparations of wild-type and ΔprsA strains. Equal amounts (20 μg) of Bocillin-FL-labeled membrane proteins were separated on a 10% SDS-PAGE gel. Fluorescently labeled PBPs are indicated by arrows. (B) Western blot analysis of PBP2 in S. aureus protein membrane extracts. PBP2 protein (80 kDa) was detected using rabbit-polyclonal anti-PBP2 antibodies. A total of 20 μg of membrane protein extract was loaded on a 10% SDS-PAGE gel. (C) Western blot analysis of PBP2A in S. aureus membrane protein extracts. PBP2A protein (70 kDa) was detected using rabbit-polyclonal anti-PBP2A antibodies. The lower panel corresponds to FtsZ antibody probing on the same PVDF membrane, used as a loading control. A total of 20 μg of membrane protein extract was loaded on a 10% SDS-PAGE gel.

Effect of prsA deletion on oxacillin resistance in different SCCmec strain backgrounds and on mecA gene transcription. (A) Oxacillin spot plating PAPs of COL (ΔblaI ΔblaR1 ΔblaZ ΔmecR1 ΔmecI ΔmecR2), MW2 (blaI⁺ blaR1⁺ blaZ⁺ ΔmecR1 ΔmecI ΔmecR2), and Mu3 (ΔblaI ΔblaR1 ΔblaZ mecR1 mecI mecR2⁺) and their corresponding derivatives. The upper panels correspond to MHB agar without oxacillin. The lower panels correspond to MHB agar containing-oxacillin, and the oxacillin concentration is indicated at the left margin. Spot serial dilutions are indicated at the right margin. The first spot, 10 μl, corresponds to 10⁵ CFU. (B) Steady-state levels of mecA transcript of wild-type COL strain compared to COLΔprsA and COLΔprsA-C strains, determined by qRT-PCR and normalized to 16S rRNA. Values represent the mean C_T values ± the standard deviations of three independent experiments.

Expression of PrsA and truncated PrsA derivatives. (A) Schematic representations of PrsA domain derivatives used in this study, according to Heikkinen et al. (19). Each PrsA domain derivative was introduced into to the prsA deletion strain by chromosomal insertion in the geh locus. The white box represents the first 140 N-terminal residues of PrsA containing the cysteine membrane-anchored amino acid, the gray box represents the 105 amino acid residues of the PrsA PPIase domain, and the darker gray box corresponds to the 75 amino acid residues of the C-terminal domain. The corresponding detection (+) or absence (−) of prsA mRNA or PrsA protein of each genetic construct is shown. (B) PrsA Western blot of total protein extracts from strains COL, COLΔprsA, and COLΔprsA complemented in the geh locus with wild-type prsA (COLΔprsA-C), with N- and C-terminal domains (COLΔprsA-Nter-Cter) or with PPIase domain (COLΔprsA-PPIase). Total protein extracts were run in a 15% SDS gel, and Western blotting was done with a PrsA-specific antibody (see Materials and Methods). For the detection of the PPIase domain, a longer exposure time was used.

Effect of PrsA domains on PBP2A protein levels and oxacillin resistance. (A) The upper panel shows a PBP2A Western blot of the total protein extracts from strains COL, COLΔprsA, and COLΔprsA complemented in the geh locus with wild-type prsA (COLΔprsA-C), with N- and C-terminal domains (COLΔprsA_NterCter), or with PPIase domain (COLΔprsA_PPIase). Total protein extracts were run on a 10% SDS gel, and the membrane was probed with PBP2A- and FtsZ-specific antibodies (see Materials and Methods). The lower panel shows a quantification of three different Western blot membranes detecting PBP2A and FtsZ proteins, performed as described above. Relative abundance (AU) of PBP2A was measured by normalizing to FtsZ protein levels used as internal controls of total protein loading. An asterisk (*) denotes PBP2A protein levels significantly (P < 0.05) different from wild-type levels. (B) Oxacillin spot plating PAPs of COL, COLΔprsA, COLΔprsA-C, COLΔprsA_NterCter, and COLΔprsA_PPIase. Spot serial dilutions are indicated in the right margin. The first spot, 10 μl, corresponds to 10⁵ CFU.