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 report shows how prophage mΦ11 exerts its effect on virulence for skin infection without encoding a 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 novel mechanism-epigenetic regulation of staphylococcal gene expression-by which phage can regulate virulence to drive adaptive leaps by S. aureus.

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

(A) Schematic of skin infection workflow. CFU, colony forming units. Created with BioRender.com. (B) Map of mΦ11 in strain USA300-BKV, adapted with permission from (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 non-homologous (blue) ORFs are shown, as compared to 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 h after subcutaneous infection with the indicated strains. Scale bar (black) is 1cm. (D) Skin abscess infections with en bloc deletion mutants. Skin abscess area 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) and mΦ11Δ32-64 (cyan, N=18-20, strain RU42) at 24, 48, and 72 hours after subcutaneous infection with ~10⁷ CFU of bacteria. Data are pooled from two independent experiments and represent mean ± SD. Statistical significance was determined with the Kruskall-Wallis test and Dunn’s multiple comparisons test, *P≤.05, ** P≤.01, ***P≤.001.

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

(A) Effect of pamA on skin abscess size. Abscess area of LAC* lysogens containing Φ11 (orange, N=50 abscesses, strain BS990), mΦ11 (blue, N=48-50 abscesses, strain BS989), and mΦ11ΔpamA (green, N=50 abscesses, strain RU39) at 24, 48, and 72 h after infection with ~1.5x10⁷ CFU of bacteria per abscess. Results are pooled from four independent experiments. Data represent mean ± SD. Statistical significance was determined with the Kruskall-Wallis test and Dunn’s multiple comparisons test, ***P≤.001, ****P≤.0001. (B) pamA skin abscess phenotype and bacterial burden. Skin abscesses infections with Φ11 (orange, N=30 abscesses, strain BS990), mΦ11 (blue, N=30 abscesses, strain BS989), or mΦ11ΔpamA (green, N=30 abscesses, strain RU39) lysogens in LAC*. CFU were enumerated at 72 h. Data represent mean ± SD. Statistical significance was determined with the Kruskall-Wallis test and Dunn’s multiple comparisons test. (C) Effect of pamA complementation on abscess size. Abscess area of LAC* containing mΦ11::EV (blue, N=36 abscesses, strain RU138), mΦ11ΔpamA::EV (green, N=36 abscesses, strain RU128), mΦ11ΔpamA::pamA (red, N=34-36 abscesses, strain RU131) after infection with ~1x10⁷ CFU of bacteria for the indicated times. EV, empty vector. Results are pooled from four independent experiments. Data represent mean ± SD. Statistical significance was determined with the Kruskall-Wallis test and Dunn’s multiple comparisons test, ***P≤.001, ****P≤.0001. (D) Bacterial burden in abscesses. Skin abscesses 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 h and CFU enumerated. Of note, 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 (data not shown). Data represent mean ± SD. Statistical significance was determined with the Kruskall-Wallis test and Dunn’s multiple comparisons test.

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

(A) Effect of pamA on skin abscess size. Abscess area 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 after infection in with ~1x10⁷ CFU of bacteria per abscess for the indicated times. Data are pooled from four independent experiments and represent mean ± SD. Statistical significance was determined with the Mann-Whitney test, ****P≤.0001. (B) Effect of pamA on CFU recovered from skin abscesses. Skin abscesses (N=25 abscesses per strain) from two independent infections in panel A were harvested at 72h. Data represent mean ± SD of CFU recovered. Statistical significance was determined with the Mann-Whitney test. (C) Effect of pamA on skin inflammation. Biopsies of skin abscess (N=15 per strain, pooled from two independent experiments) from LAC* containing EV (strain RU129) or pamA (strain RU121) were stained with H&E and inflammatory burden graded by a blinded dermatopathologist. Statistical significance was determined with chi-square test (P = 0.0014). (D) Representative images of skin abscess biopsies from panel C. One representative image from each strain is presented according to dermatopathologist classification as nodular (above) or diffuse (below) architecture. (E) Effect of pamA on local proinflammatory and vascular proliferation cytokines. Biopsy of skin abscesses from three independent experiments of LAC* with EV control (N=22 abscesses, strain RU129) or pamA (N=24 abscesses, strain RU121) were homogenized and the indicated cytokine levels measured. Data represent mean ± SD. Statistical significance was determined with the Mann-Whitney test, ** P≤.01, ***P≤.001, ****P≤.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 ~1x10⁷ CFU of bacteria per abscess. Data are pooled from two independent experiments and represent mean ± SD. Statistical significance was determined with the Kruskall-Wallis test and Dunn’s multiple comparisons test, *P≤.05, **P≤.01, ***P≤.001, ****P<.0001. (D) Bacterial burden in abscesses. Skin abscesses from infections in panel C (N=9-11 abscesses per strain) were harvested at 72h and CFU enumerated. Data represent mean ± SD. Statistical significance was determined with the Kruskall-Wallis test and Dunn’s multiple comparisons test.

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-sequencing 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 zero (green arrow) represent upregulated genes in LAC*::pamA and data points to the left of zero (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 in LAC* strains containing pamA or EV control. Strains were grown and prepared in the same manner as panel A. Data represent mean ± SD of three biological replicates.

Figure 6.. The methylase activity of pamA acts to increase biofilm production through fibronectin-binding protein A (fnbA; FnBPA).

(A) Effect of pamA methylase activity on biofilm production. In vitro biofilm production by LAC* strains with the indicated pamA alleles integrated into the chromosome, quantified by optical density after static growth for 24 h (OD). EV, empty vector control. Data represent mean ± SD of six biological replicates per strain, pooled from two independent experiments. (B) Effect of pamA on biofilm formation in abscesses. Representative images of skin abscess tissue stained for DAPI (blue) and 5-methylcytosine (5mc, green) 72 h after infection with ~1x10⁷ CFU of LAC* containing pamA (strain RU121) or EV (strain RU129). Scale bar (white) is 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 and 5mC staining (48). Red data points correspond to representative images in panel B. Data was pooled from two independent experiments. Statistical significance was determined with the Mann-Whitney test, **P≤.01. (D) Cell wall proteins. Cell wall-associated proteins from biofilms of LAC* strains containing pamA or EV (three biological replicates each) were separated by SDS-PAGE and stained with Coomassie blue. Gel image is representative of two independent experiments. Yellow star corresponds to the band of interest. (E) Identification of FnBPA bands. Western blot of cell wall associated protein bands from panel D using polyclonal anti-FnBPA, focusing on high molecular weight protein band area. (F) Biofilm production. In vitro biofilms from LAC* strains containing the indicated genetic changes was quantified by optical density (OD). Data represent mean ± SD of six biological replicates per strain, pooled from two 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 reverses the pamA-mediated skin abscess phenotype.

(A) Effect of fnbA on the pamA-mediated skin abscess phenotype. Abscess area of LAC* with EV control (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) after skin infection with ~1x10⁷ CFU of bacteria per abscess for the indicated times. Data are pooled from two independent experiments and represent mean ± SD. Statistical significance was determined with the Kruskall-Wallis test and Dunn’s multiple comparisons test, *P≤.05, **P≤.01, ***P≤.001, ****P≤.0001. (B) Bacterial burden in abscesses. Skin abscesses in panel A (N=14-15 abscesses per strain) were harvested for CFU enumeration 72 h post-infection. Data represent mean ± SD. Statistical significance was determined with the Kruskall-Wallis test and Dunn’s multiple comparisons test. (C) Effect of fnbA on the mΦ11-mediated skin abscess phenotype; transposon insertion in fnbA in mΦ11-containing strains confirms that fnbA is necessary for the skin abscess phenotype. Abscess area of LAC* containing prophage mΦ11 (blue, N=40 abscesses, strain BS989) and mΦ11+fnbA::bursa (pink, N=40 abscesses, strain RU171) after infection with ~1x10⁷ CFU of bacteria per abscess for the indicated times. Data are pooled from four independent experiments and represent mean ± SD. Statistical significance was determined with the Mann-Whitney test, *P≤.05, **P≤.01, ***P≤.001, ****P≤.0001. (D) Skin abscesses from infections in panel C were harvested at 72 h post-infection and CFU enumerated. Data represent mean ± SD. Statistical significance was determined with the Kruskall-Wallis test and Dunn’s multiple comparisons test. (E) Representative images at 72 hours post-infection of the indicated strains from panel E, abscess area circled in red.