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 6

pamA methylase increases biofilm production through fnbA (FnBPA).

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pamA methylase increases biofilm production through fnbA (FnBPA). (A) E...
(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.