Prophage-encoded methyltransferase drives adaptation of community-acquired methicillin-resistant Staphylococcus aureus

Abstract

We recently described the evolution of a community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 variant responsible for an outbreak of skin and soft tissue infections. Acquisition of a mosaic version of the Φ11 prophage (mΦ11) that increases skin abscess size was an early step in CA-MRSA adaptation that primed the successful spread of the clone. The present study shows how prophage mΦ11 exerts its effect on virulence for skin infection without encoding known toxin or fitness genes. Abscess size and skin inflammation were associated with DNA methylase activity of an mΦ11-encoded adenine methyltransferase (designated pamA). pamA increased expression of fibronectin-binding protein A (fnbA; FnBPA), and inactivation of fnbA eliminated the effect of pamA on abscess virulence without affecting strains lacking pamA. Thus, fnbA is a pamA-specific virulence factor. Mechanistically, pamA was shown to promote biofilm formation in vivo in skin abscesses, a phenotype linked to FnBPA's role in biofilm formation. Collectively, these data reveal a critical mechanism - epigenetic regulation of staphylococcal gene expression - by which phage can regulate virulence to drive adaptive leaps by S. aureus.

Keywords: Bacterial infections; Epigenetics; Infectious disease; Microbiology; Molecular biology.

Figure 1. Effect of en bloc deletions on the mΦ11-mediated skin abscess phenotype.

(A) Skin infection workflow. Created with BioRender.com. (B) Map of mΦ11 in strain USA300-BKV, adapted with permission from Copin et al. (5), with en bloc deletion locations. Arrows indicate predicted ORFs and the direction of the transcription of genes within the unique mΦ11 modules. Homologous (red) and nonhomologous (blue) ORFs are shown, compared with prototypical Φ11. Black arrow indicates pamA. Black bars beneath the gene map correspond to the gene blocks deleted from the indicated strain. (C) Representative images of skin abscesses 72 hours after subcutaneous infection with the indicated strains. Scale bar (black): 1 cm. (D) Skin abscess infections with en bloc deletion mutants. Skin abscess area at the indicated times after infection with approximately 10⁷ bacterial CFU of LAC* lysogens containing mΦ11 (blue, n = 20, strain BS989), mΦ11Δ32–43 (purple, n = 16–18, strain RU47), mΦ11Δ44–57 (salmon, n = 20, strain RU108), or mΦ11Δ32–64 (cyan, n = 18–20, strain RU42). Data are pooled from 2 independent experiments and represent mean ± SD. Statistical significance was determined by Kruskal-Wallis and Dunn’s tests, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Figure 2. mΦ11 phage adenine methyltransferase increases skin abscess size without affecting tissue bacterial burden.

(A) Effect of pamA on the mΦ11 skin abscess phenotype. Abscess area at the indicated times after infection with approximately 1.5 × 10⁷ bacterial CFU of LAC* containing Φ11 (orange, n = 50 abscesses, strain BS990), mΦ11 (blue, n = 48–50 abscesses, strain BS989), or mΦ11ΔpamA (green, n = 50 abscesses, strain RU39). Data are pooled from 4 independent experiments and represent mean ± SD. Statistical significance determined by Kruskal-Wallis and Dunn’s tests, ***P ≤ 0.001, ****P ≤ 0.0001. (B) pamA skin abscess bacterial burden. Skin abscesses from A with LAC* containing Φ11 (orange, n = 30 abscesses, strain BS990), mΦ11 (blue, n = 30 abscesses, strain BS989), or mΦ11ΔpamA (green, n = 30 abscesses, strain RU39) were harvested at 72 hours and abscess CFU enumerated. Data represent mean ± SD. Statistical significance determined by Kruskal-Wallis and Dunn’s tests. (C) Effect of pamA complementation on abscess size. Abscess area at the indicated times after infection with approximately 1 × 10⁷ bacterial CFU of LAC* containing mΦ11:EV (blue, n = 36 abscesses, strain RU138), mΦ11ΔpamA:EV (green, n = 36 abscesses, strain RU128), or mΦ11ΔpamA:pamA (red, n = 34–36 abscesses, strain RU131). EV, empty vector. Data are pooled from 4 independent experiments and represent mean ± SD. Statistical significance determined by Kruskal-Wallis and Dunn’s tests, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. (D) Bacterial burden in abscesses. Skin abscesses from C of LAC* containing mΦ11:EV (blue, n = 22 abscesses, strain RU138), mΦ11ΔpamA:EV (green, n = 22 abscesses, strain RU128), and mΦ11ΔpamA:pamA (red, n = 20 abscesses, strain RU131) were harvested at 72 hours and CFU enumerated. CFU/abscess is shown due to missing abscess weights during one of the replicate experiments. With the available weight-adjusted data, we found no significant differences between strains (not shown). Data represent mean ± SD. Statistical significance determined by Kruskal-Wallis and Dunn’s tests.

Figure 3. pamA increases skin abscess size and inflammation in the absence of mΦ11.

(A) Effect of pamA on skin abscess size. Abscess area at the indicated times after infection with approximately 1 × 10⁷ bacterial CFU of LAC* with empty vector (EV) (maroon, n = 50 abscesses, strain RU129) or constitutively expressed pamA (purple, n = 50 abscesses, strain RU121) integrated into the chromosome in single copy. Data are pooled from 4 independent experiments and represent mean ± SD. Statistical significance was determined by Mann-Whitney test, ****P ≤ 0.0001. (B) Effect of pamA on CFU recovered from skin abscesses. Skin abscesses (n = 25 abscesses per strain) from 2 independent infections in A were harvested at 72 hours and CFU enumerated. Data represent mean ± SD. Statistical significance determined by Mann-Whitney test. (C) Effect of pamA on skin inflammation. Biopsies of skin abscess (n = 15 per strain, pooled from 2 independent experiments) 72 hours after infection with LAC* containing pamA (strain RU121) or EV control (strain RU129) were H&E stained and inflammatory burden was graded by a blinded dermatopathologist. Statistical significance determined by χ² test (P = 0.0014). (D) Representative images of skin abscess biopsies from C. One image from each strain is presented according to dermatopathologist architecture classification as nodular (above) or diffuse (below). (E) Effect of pamA on local proinflammatory and vascular proliferation cytokines. Skin abscess biopsies from 3 independent experiments of LAC* with pamA (n = 24 abscesses, strain RU121) or EV control (n = 22 abscesses, strain RU129) were homogenized, and levels of the indicated cytokines were measured. Data represent mean ± SD. Statistical significance was determined by Mann-Whitney test, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Figure 4. The pamA-mediated skin abscess phenotype depends on the methylase activity of PamA.

(A) Predicted structure of mΦ11 PamA. Amino acid backbone represented in green, with N-terminus (M1), C-terminus (Q141), and putative active site (N64, P65, P66, Y67) highlighted. Generated by AlphaFold, visualized using PyMol Molecular Graphics System, version 2.5.2 (Schrödinger, LLC). (B) Effect of PamA point mutants on methylase activity. Genomic DNA was isolated from LAC* strains containing the indicated pamA alleles and digested with DpnI (DpnI+) or PBS control (DpnI–), then visualized on a 1% agarose gel. The analysis confirms that PamA methylates at the predicted GATC site and that PamA point mutants lack methylation activity. EV, empty vector. (C) Skin abscess size. Abscess area of LAC* with EV (maroon, n = 20 abscesses, strain RU129), pamA (purple, n = 20 abscesses, strain RU121), and pamAP65T (cyan, n = 20 abscesses, strain RU162) at the indicated time points after skin infection with approximately 1 × 10⁷ CFU of bacteria per abscess. Data are pooled from 2 independent experiments and represent mean ± SD. Statistical significance was determined by Kruskal-Wallis and Dunn’s tests, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. (D) Bacterial burden in abscesses. Skin abscesses from infections in panel C (n = 9–11 abscesses per strain) were harvested at 72 hours and CFU enumerated. Data represent mean ± SD. Statistical significance was determined by Kruskal-Wallis and Dunn’s tests.

Figure 5. pamA induces widespread transcriptional changes, including a large increase in the expression of fibronectin-binding protein A (fnbA; FnBPA).

(A) Whole genome transcriptome. Volcano plot of RNA-Seq data comparing LAC* strains containing pamA (n = 3 biological replicates, strain RU121) or empty vector (EV) control (n = 2 biological replicates, strain RU129) after 5 hours of growth in RPMI media. Data points to the right of 0 (green arrow) represent upregulated genes in LAC*:pamA and data points to the left of 0 (red arrow) represent downregulated genes in LAC*:pamA; pamA and fnbA are highlighted. Blue data points represent genes that achieved statistical significance (P ≤ 0.05); pink data points indicate genes that did not. (B) Effect of pamA on fnbA expression. Quantitative real-time PCR of fnbA expression in LAC* strains containing pamA or EV control. Strains were grown and prepared in the same manner as A. Data represent mean SD of 3 biological replicates.

Figure 6. pamA methylase increases biofilm production through fnbA (FnBPA).

(A) Effect of pamA methylase on biofilm production. In vitro biofilm quantified by OD after static growth for 24 hours by LAC* strains with the indicated pamA alleles integrated into the chromosome. EV, empty vector. Data represent mean ± SD and are pooled from 2 independent experiments. Statistical significance was determined by ANOVA with Tukey’s test, ****P ≤ 0.0001. (B) Effect of pamA on abscess biofilm. Representative images of skin abscess tissue stained with DAPI (blue) and 5-methylcytosine (5-mC, green) 72 hours after infection with approximately 1 × 10⁷ CFU of LAC* containing pamA (strain RU121) or EV (strain RU129). Scale bar: 200 μm. (C) Biofilm area of LAC* containing pamA (n = 12 abscesses, strain RU121) or EV (n = 11 abscesses, strain RU129) quantified as the difference between DAPI (total extracellular DNA) and 5-mC (eukaryotic host extracellular DNA) (48). Red data points correspond to representative images in B. Data are pooled from 2 independent experiments; individual results are shown in Supplemental Figure 9A. Statistical significance was determined by Mann-Whitney test, **P ≤ 0.01. The difference remained significant (P = 0.007) after removal of the pamA strain outlier (Supplemental Figure 9B). (D) Cell wall proteins. Cell wall–associated proteins from biofilms of LAC* strains containing pamA or EV (3 biological replicates each) were separated by SDS-PAGE. Gel image represents 2 independent experiments. Yellow star = band of interest. (E) Identification of FnBPA bands. Western blot of cell wall–associated protein bands from D using polyclonal anti-FnBPA. (F) Biofilm production. In vitro biofilms from LAC* strains containing the indicated genetic changes quantified by OD. Data represent mean ± SD of 6 biological replicates per strain, pooled from 2 independent experiments. (G) FnBPA production. Western blot of cell wall–associated proteins during in vitro biofilm production by the indicated strains.

Figure 7. fnbA deficiency decreases pamA-mediated skin abscess size and biofilm production in vivo.

(A) Effect of fnbA on the pamA-mediated abscesses. Abscess area at the indicated times after skin infection with approximately 1 × 10⁷ bacterial CFU of LAC* with EV (maroon, n = 20 abscesses, strain RU129), EV+fnbA:bursa (green, n = 18–20 abscesses, strain RU170), pamA (purple, n = 20 abscesses, strain RU121), or pamA+fnbA:bursa (orange, n = 18 abscesses, strain RU169). Data are pooled from 2 independent experiments and represent mean ± SD. (B) Bacterial burden in abscesses. Abscesses from A (n = 14–15 per strain) were harvested and CFU enumerated 72 hours after infection. Data represent mean ± SD. (C) Effect of fnbA on the mΦ11-mediated abscesses. Abscess area at the indicated times after infection with approximately 1 × 10⁷ bacterial CFU of LAC* containing mΦ11 (blue, n = 40 abscesses, strain BS989) or mΦ11+fnbA:bursa (pink, n = 40 abscesses, strain RU171). Data are pooled from 4 independent experiments and represent mean ± SD. (D) Skin abscesses from C were harvested 72 hours after infection and CFU enumerated. Data represent mean ± SD. (E) Representative images 72 hours after infection with the indicated strains; abscess area circled. (F) Effect of fnbA on biofilm formation in mΦ11-mediated skin abscesses. Representative images of abscess tissue stained for DAPI (blue) and 5-methylcytosine (5-mC, green) 72 hours after infection with approximately 1 × 10⁷ CFU of LAC* containing mΦ11 (strain BS989, left image) or mΦ11 with fnbA:bursa (strain RU171, right image). The corner inset is magnified abscess area. Scale bars: 1,000 μm. (G) Biofilm area of the strains from F (n = 10 abscesses each) quantified as the difference between DAPI and 5-mC (48). Red data points correspond to representative images in F. Statistical significance for A and B was determined by Kruskal-Wallis and Dunn’s tests. Statistical significance in the remaining panels was determined by Mann-Whitney test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.