Prophage-encoded methyltransferase drives adaptation of community-acquired methicillin-resistant Staphylococcus aureus
Robert J. Ulrich, Magdalena Podkowik, Rebecca Tierce, Irnov Irnov, Gregory Putzel, Nora M. Samhadaneh, Keenan A. Lacey, Daiane Boff, Sabrina M. Morales, Sohei Makita, Theodora K. Karagounis, Erin E. Zwack, Chunyi Zhou, Randie H. Kim, Karl Drlica, Alejandro Pironti, Harm van Bakel, Victor J. Torres, Bo Shopsin
Robert J. Ulrich, Magdalena Podkowik, Rebecca Tierce, Irnov Irnov, Gregory Putzel, Nora M. Samhadaneh, Keenan A. Lacey, Daiane Boff, Sabrina M. Morales, Sohei Makita, Theodora K. Karagounis, Erin E. Zwack, Chunyi Zhou, Randie H. Kim, Karl Drlica, Alejandro Pironti, Harm van Bakel, Victor J. Torres, Bo Shopsin
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Research Article Infectious disease Microbiology

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.

Authors

Robert J. Ulrich, Magdalena Podkowik, Rebecca Tierce, Irnov Irnov, Gregory Putzel, Nora M. Samhadaneh, Keenan A. Lacey, Daiane Boff, Sabrina M. Morales, Sohei Makita, Theodora K. Karagounis, Erin E. Zwack, Chunyi Zhou, Randie H. Kim, Karl Drlica, Alejandro Pironti, Harm van Bakel, Victor J. Torres, Bo Shopsin

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Figure 4

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

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The pamA-mediated skin abscess phenotype depends on the methylase activi...
(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.