Fig 1.
Construction of a chromosomal pmecA fusion in S. aureus SH1000.
A) Schematic overview of genomic region of lysA in SH1000 and post integration of pVP01-pmecA resulting into lysA::pmecA (SJF4996). B) The lysA::pmecA (SJF4996) construct was produced by transduction from RN4220 lysA::pmecA (SJF4994). This led to single copy mecA at the lysA locus expressed mecA under its native promoter. C) Subsequently, spontaneous oxacillin resistant mutants were selected using a gradient plate containing either 0–5 or 0–20 μg/ml of methicillin. Strains with low-level resistance were further selected using an antibiotic gradient plate containing 0–20 μg/ml methicillin to select high-level resistance. D) Etest strips revealed high-level oxacillin resistance was lost by removal of pmecA and restored via subsequent reintroduction of pmecA at the lysA locus. Oxacillin MICs shown in brackets were measured using the Etest strip.
Fig 2.
Identification of rpoB/C mutations and reconstitution of 8325–4 and Newman expressing single copy mecA.
A) Schematic representation of the genomic region of the rpoB/C operon, and downstream genes. Amino acid substitutions identified in resistant derivatives of lysA::pmecA (SJF4996) are indicated with a red box representing highly resistant mutants with oxacillin MIC of ≥256 μg/ml and a grey box representing intermediate resistant mutants with oxacillin MIC of up to 16 μg/ml. B) Chromosomal integration of mecA at lysA locus in 8325–4 did not result in high-level oxacillin resistance. C) The introduction of lysA::pmecA and rpoB-H929Q kan of SJF5046 into Newman resulted in the development of high-level resistance. Oxacillin susceptibility of Newman lysA::pmecA rpoB-H929Q kan (SJF5050) and its parental strain Newman were compared using Etest.
Fig 3.
Location of rpoB/C mutations and the impact on transcription initiation.
A) (i) Abortive products produced on the T7A1 promoter by S. aureus RNAPs over 10 min at 37°C. (ii) Abortive initiation by SH1000, lysA::pmecA rpoB-H929Q (SJF5003) and lysA::pmecA rpoC-G740R (SJF5034) holoenzymes on the promoters of mecA, pbp2, asp23 and clfB. Abortive products were normalised to abortive synthesis on T7A1 promoter. Error bars show ±SD from three replicates. B) The three-dimensional structure of E. coli RNAP holoenzyme was used to map the rpoB and rpoC mutations [67]. The two subunits β (red) and β’ (blue) are shown as molecular surface. The mutated residues of rpoB/C (S. aureus residue numbering) are highlighted with dots (Zoomed in section). *, residue not conserved in E. coli. C) S. aureus rpoB mutations mapped on the structure of E. coli RpoB shows relative location of the mutations to the rifampicin binding pocket regions l and ll [47], shown as orange and cyan sticks, respectively. Figure generated from PDB 6CUX using PYMOL.
Fig 4.
Growth characteristics and PBP2A production of S. aureus strains.
A) Growth of representative strains, lysA::pmecA rpoB-H929Q (SJF5003), lysA::kan rpoB-H929Q (SJF5010), lysA::pmecA rpoC-G740R (SJF5034) were compared to the SH1000, COL and lysA::pmecA (SJF4996) in the absence (left) and presence (right) of oxacillin. Following one hour of incubation, 0.5 μg/ml of oxacillin was added to SH1000 and lysA::pmecA (SJF4996) cultures, and 20 μg/ml of oxacillin was added to COL, lysA::pmecA rpoB-H929Q (SJF5003) and lysA::pmecA rpoC-G740R (SJF5034) cultures. Bacterial cultures were prepared in triplicate and the error bars represent standard deviation of the mean (±SEM). B) Whole cell lysates (~10 μg of protein) of strains expressing chromosomal pmecA (single-copy; lysA::pmecA) and plasmid-borne pmecA (multi-copy; pRB474-pmecA) were used to determine the amounts of PBP2A (~76kDa) and were compared between these two set of strains. Plasmid-borne pmecA carrying strains include, pRB474-pmecA (SJF4981), TI11 pRB474-pmecA (SJF5194), pRB474-pmecA gdpP-R318L (SJF4991) and gdpP-R318L lysA::pmecA (SJF4997). Chromosomal pmecA expressing strains include, lysA::pmecA (SJF4996), TI2 lysA::pmecA (SJF4999), lysA::pmecA rpoB-H929Q (SJF5003) and lysA::pmecA rpoC-G740R (SJF5034). Anti-YmdA antibodies were used as an endogenous control producing ~58 kDa band. Relative levels of PBP2A were calculated using ImageLab (Bio-Rad) quantitation tool, selecting recombinant PBP2A as a point of reference. The results of relative concentrations of PBP2A are the average of three independent repeats where error bars represent standard deviation of the mean (±SEM). Oxacillin MICs are listed in brackets for all strains above the bars.
Fig 5.
Differential gene expression in SH1000 follows the acquisition of chromosomal mecA and the rpoB/C mutations.
Two-set proportional Venn diagram illustrating the total number of differentially expressed genes (DEGs) altered in A) lysA::pmecA (SJF4996) (grey) and lysA::pmecA rpoB-H929Q (SJF5003) (red), B) lysA::pmecA (SJF4996) (grey) and lysA::pmecA rpoC-G740R (SJF5034) (blue) and C) lysA::pmecA (SJF4996) (grey) and lysA::kan rpoB-H929Q (SJF5010) (green) compared to WT (white background). D) Three-set proportional Venn diagram displaying the shared DEGs among lysA::pmecA (SJF4996) (grey), lysA::pmecA rpoB-H929Q (SJF5003) (red) and lysA::pmecA rpoC-G740R (SJF5034) (blue) compared to WT (white background). 3 common DEGs (mecA, lysA, nirR) are marked by a circle showing reduced expression. E) The lysA gene (marked by a circle) was shared among four strains based on comparisons from c) and d) compared to WT (white background). The expression of lysA was reduced in all four strains compared WT, marked by a downward arrow.
Fig 6.
Identification and functional characterisation of overlapping genes and their differential expression in derived strains with a range of resistance properties.
Two-set proportional Venn diagrams displaying the total number of DEGs (listed in brackets), common (intersection) and unique DEGs altered in A) lysA::pmecA rpoB-H929Q (SJF5003) (red) and lysA::pmecA rpoC-G740R (SJF5034) (blue), B) lysA::pmecA rpoC-G740R (SJF5034) (blue) and lysA::kan rpoB-H929Q (SJF5010) (green) and C) lysA::pmecA rpoB-H929Q (SJF5003) (red) and lysA::kan rpoB-H929Q (SJF5010) compared to lysA::pmecA (SJF4996) (white background). D) Three-set proportional Venn diagram comparing the three rpo strains relative to the lysA::pmecA (SJF4996) illustrated the 26 common DEGs among all three rpo strains and 95 overlapping DEGs exclusively common between lysA::pmecA rpoB-H929Q (SJF5003) (red) and lysA::pmecA rpoC-G740R (SJF5034) (blue). Chord plots illustrating the differential expression (DE) of 26 common genes (i) from lysA::pmecA (SJF4996) compared to WT. 24 out of 26 common genes (ii) were differentially expressed in the three rpo strains compared to lysA::pmecA (SJF4996). mecA was not included as lysA::kan rpoB-H929Q (SJF5010) showed an obvious reduced mecA expression. Also, icaB was not included because it was upregulated only lysA::pmecA rpoB-H929Q (SJF5003). The DE and functional categorisation of 95 common genes (iii) between lysA::pmecA rpoB-H929Q (SJF5003) and lysA::pmecA rpoC-G740R (SJF5034) compared to lysA::pmecA (SJF4996) strain. Each segment on the left represents the average log2FoldChange of individual genes from the three strains (SJF5003, SJF5010 and SJF5034) which connects to their functional categories on the right.
Fig 7.
Functional classification and transcriptional regulation of common genes.
A) COG (Clusters of Orthologous Groups) classification of 121 overlapping DEGs. Colours illustrate COG functional categories. C, Energy production and conversion; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport, and catabolism; M, cell wall/membrane/envelope biogenesis; O, post-translational modification, protein turnover, and chaperones; T, signal transduction mechanisms; V, defence mechanisms; J, translation, ribosomal structure and biogenesis; K, transcription; L, replication, recombination and repair; R and S are function prediction only or function unknown categories. Total number of genes associated with each category are listed outside the bar. B) The transcriptional regulators were predicted for 24 out of 26 and 95 common DEGs identified in the three rpo strains compared to their parent (Fig 6D). C) Chromosomal region of the genes involved in nitrite and nitrate reduction in S. aureus. A membrane bound nitrate reductase system (narGHJI) reduces nitrate to nitrite. Subsequently, nitrite is reduced to ammonia by NADH-dependent nitrite reductase system (nirBD) [53]. NreABC has been identified as an oxygen sensing system which regulates the expression of nitrate and nitrite reductase system as well as narK [68].
Fig 8.
Oxygen consumption rates of S. aureus strains.
Cellular respiration was measured for SH1000, lysA::pmecA (SJF4996), lysA::pmecA rpoB-H929Q (SJF5003), lysA::kan rpoB-H929Q (SJF5010) and lysA::pmecA rpoC-G740R (SJF5034) strains using Clark-type oxygen electrode. Data shown here reflect mean ±SEM of the three biological replicates. The data were compared and analysed against WT and lysA::pmecA (SJF4996) using unpaired t-test. * p <0.05.
Fig 9.
Virulence of S. aureus strains in a murine sepsis model.
Mice were injected with 1 x 107 CFU per mouse with SH1000, lysA::pmecA (SJF4996), lysA::pmecA rpoB-H929Q (SJF5003), lysA::kan rpoB-H929Q (SJF5010), lysA::pmecA rpoC-G740R (SJF5034), COL, COL rpoB+ (SJF5049) and animals (n = 10 mice per group) were euthanised at 72 hpi. A) Weight analysis. Colony forming units (CFUs) per organ B) Kidneys and C) Livers were recovered from animals infected with strains listed above. Statistical significance determined by Mann-Whitney two-tailed test; ns, p >0.05; * p <0.05; **p <0.005.
Fig 10.
Model of adaptations associated with the development of high-level β-lactam resistance.
Strain lysA::pmecA (SJF4996) (left) expresses chromosomal mecA, with PBP2A protein (red hour glass) incorporated into the cell envelope. This perturbs cell membrane (CM) function (⊥), resulting in decreased flux through the aerobic electron transport chain (ETC). The resulting increased NADH:NAD+ ratio (indicated by font size) inhibits DNA-binding by the transcription factor Rex (green hexagon) and the anaerobic respiratory and fermentative genes (anaerobic genes) are transcribed (red filled arrow) by the native RNA polymerase (RNAP). This results in a low-level resistance phenotype. Evolved strains have missense mutations in the rpoB/C (RNAP*) that permit enhanced expression of 11 genes, including mecA and two genes associated with the Type VII secretion system (SAHOUHSC_00270 and 00271). Incorporation of some of the gene products (e.g. SAHOUHSC_00270 and 00271; brown ellipses) into the cell membrane allows integration of PBP2A activity into ‘normal’ membrane function. This results in lower NADH:NAD+ ratios, permitting Rex to repress expression of anaerobic genes (blue rectangle), the restoration of aerobic respiratory growth and high level β-lactam resistance.