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Epigenetic Switch Driven by DNA Inversions Dictates Phase Variation in <i>Streptococcus pneumoniae</i>

<div><p>DNA methylation is an important epigenetic mechanism for phenotypic diversification in all forms of life. We previously described remarkable cell-to-cell heterogeneity in epigenetic pattern within a clonal population of <i>Streptococcus pneumoniae</i>, a leading human pathogen. We here report that the epigenetic diversity is caused by extensive DNA inversions among <i>hsdS</i>_<i>A</i>, <i>hsdS</i>_<i>B</i>, and <i>hsdS</i>_<i>C</i>, three methyltransferase <i>hsdS</i> genes in the Spn556II type-I restriction modification (R-M) locus. Because <i>hsdS</i>_<i>A</i> encodes the sequence recognition subunit of this type-I R-M DNA methyltransferase, these site-specific recombinations generate pneumococcal cells with variable HsdS_A alleles and thereby diverse genome methylation patterns. Most importantly, the DNA methylation pattern specified by the HsdS_A1 allele leads to the formation of opaque colonies, whereas the pneumococci lacking HsdS_A1 produce transparent colonies. Furthermore, this HsdS_A1-dependent phase variation requires intact DNA methylase activity encoded by <i>hsdM</i> in the Spn556II (renamed <u>c</u>olony <u>o</u>pacity <u>d</u>eterminant or <i>cod</i>) locus. Thus, the DNA inversion-driven ON/OFF switch of the <i>hsdS</i>_<i>A1</i> allele in the <i>cod</i> locus and resulting epigenetic switch dictate the phase variation between the opaque and transparent phenotypes. Phase variation has been well documented for its importance in pneumococcal carriage and invasive infection, but its molecular basis remains unclear. Our work has discovered a novel epigenetic cause for this significant pathobiology phenomenon in <i>S</i>. <i>pneumoniae</i>. Lastly, our findings broadly represents a significant advancement in our understanding of bacterial R-M systems and their potential in shaping epigenetic and phenotypic diversity of the prokaryotic organisms because similar site-specific recombination systems widely exist in many archaeal and bacterial species.</p></div

Significant impact of epigenetic-mediated phase variation on pneumococcal adhesion to host epithelial cells.

<p>The <i>hsdS</i>_<i>A1</i>, <i>hsdS</i>_<i>A2</i>, or <i>hsdS</i>_<i>A3</i> allele-carrying derivatives of strains ST556 (panels A and B), P384 (panels C and D), and ST877 (panels E and F) were cultured on the TSA plates supplemented with catalase as represented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005762#ppat.1005762.g006" target="_blank">Fig 6</a>, and used to determine adhesion to human lung (A549 line) and nasopharyngeal (Detroit 562 line) cells in 24-well plates by counting CFU of adhering bacteria after extensive washing of the cell monolayers. The pneumococci carrying the <i>hsdS</i>_<i>A1</i> (A1) (producing opaque colonies) are significantly less adherent than those carrying the <i>hsdS</i>_<i>A2</i> (A2) or <i>hsdS</i>_<i>A3</i> (A3).</p

Detection of DNA rearrangements in the Spn556II locus by PCR.

<p><b>A.</b> Positions of the primers used for PCR amplification in the Spn556II locus of ST556. The predicted rho-independent transcription terminator is indicated by a hairpin. The primers used in (<b>B</b>) and (<b>C</b>) are indicated by small arrows. The JC-replaced region in TH6501 is marked with dashed lines. <b>B.</b> Amplification of the Spn556II locus in ST556 and isogenic mutant TH5792 lacking the entire Spn556II locus with primers P1 and P11. The PCR mixtures were processed by DNA electrophoresis and stained by the Goldview dye (Yeasen, Beijing, China). The PCR products that were absent in the mutant strains are marked with asterisks (*). The sizes of the DNA markers are indicated in kilobases. <b>C.</b> Detection of DNA rearrangements in the <i>hsdS</i> regions of the Spn556II locus. PCR reactions were performed with the genomic DNA of ST556 using the same set of primer pairs indicated at the top of each lane, and marked as in (<b>B</b>). <b>D</b>. Same as in (C) except for using the genomic DNA from the ST556 derivative lacking <i>hsdS</i>_<i>A</i> strain (TH6501).</p

Significant impact of epigenetic-mediated phase variation on nasopharyngeal colonization of the pneumococci in the mouse co-carriage model.

<p>The pneumococcal derivatives of strains ST556 (A), P384 (B), and ST877 (C) each carrying the <i>hsdS</i>_<i>A1</i>, <i>hsdS</i>_<i>A2</i>, or <i>hsdS</i>_<i>A3</i> allele were grown on the TSA plates supplemented with catalase as represented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005762#ppat.1005762.g006" target="_blank">Fig 6</a>. Two of the three unique <i>hsdS</i>_<i>A</i> allelic derivatives (A1, A2, and A3) from each strain background were mixed at a 1:1 ratio before being used to inoculate intranasally C57BL/6 mice. The colonizing pneumococci were recovered from each mouse by washing the nasal cavity 7 days post inoculation. The output ratio of the two the <i>hsdS</i>_<i>A</i> allele-specific variants co-infecting the same mouse was determined with the nasal lavage sample by PCR with the <i>hsdS</i>_<i>A</i> allele-specific primers. The <i>hsdS</i>_<i>A1</i>-specific variant derived from each of three different strain backgrounds (A1) (forming opaque colonies) was less fit than the counterpart carrying <i>hsdS</i>_<i>A2</i> (A2) or <i>hsdS</i>_<i>A3</i> (A3) (forming transparent colonies) in the nasopharynx.</p

Genetic arrangement in the Spn556II locus.

<p><b>A.</b> The three functional DNA methylation motifs recognized by the three R-M systems in strain ST556 according to our previous study [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005762#ppat.1005762.ref035" target="_blank">35</a>]. The methylated bases are highlighted with red characters. <b>B.</b> The gene order and other features in the Spn556 locus of three pneumococcal strains. The orientations of the coding sequences are indicated by arrowheads. Each <i>hsdS</i> segment with identical or nearly identical sequences between the two of three strains (ST556, TIGR4 and D39) is indicated with a dashed line. Drawing is not to scale.</p

Colony morphology of six <i>S</i>. <i>pneumoniae</i> strains and their derivatives each carrying an invariable <i>hsdS</i>_<i>A</i> allele.

<p>Pneumococcal strains ST556 (19F), P384 (6A), TH2901 (6B), TH2835 (14), TH2886 (23F), and ST877 (35B) were grown on TSA plates supplemented with catalase; the colonies photographed under a dissection microscope as described in reference [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005762#ppat.1005762.ref033" target="_blank">33</a>]. The Spn556II <i>hsdS</i>_<i>A</i> genotype and corresponding profile of chromosomal methylation in each strain are marked at the top of each column. Strain designation is indicated at the bottom of each photograph. The representative colonies with opaque and transparent appearance in the wild types are highlighted with blue and red arrowheads, respectively.</p

Allele A1 of <i>hsdS</i>_<i>A</i> dictates the opaque colony phenotype of <i>S</i>. <i>pneumoniae</i>.

<p>Chromosomal co-expression of <i>hsdS</i>_<i>A</i> alleles A1-A3 in the <i>bgaA</i> locus of the ST556 derivatives carrying the locked <i>hsdS</i>_<i>A</i> allele A2 (<b>A</b>), A3 (<b>B</b>) or A1 (<b>C</b>). A modified Janus cassette (JC1) was used to replace partially the coding sequence of <i>bgaA</i> in each parent strain. JC1 in the resulting strains were subsequently replaced by the fusion PCR product of the A1, A2, or A3 allele of <i>hsdS</i>_<i>A</i> by counter selection, which consisted of the <i>hsdRMS</i> promoter and full coding sequence of each allele. Colonies of each strain were prepared, photographed, and marked as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005762#ppat.1005762.g006" target="_blank">Fig 6</a>. The genotype and name of each strain are marked at the top and bottom of each photograph. All of the ST556 derivatives carrying allele A1 produced uniformly opaque colonies. In (<b>D</b>), the relationship of the <i>hsdS</i>_<i>A</i> allelic variations by DNA inversions and the resulting epigenetic and phenotypic switch is diagrammatically illustrated; the methylated and unmethylated adenine nucleotides in the DNA motif by the Hsd_A1-associated methyltransferase is highlighted with red and blue characters, respectively. R = A or G, Y = T or C.</p

DNA configurations generated by inversions and excisions in the <i>hsdS</i> genes of the Spn556II locus.

<p>DNA configurations derived by three inversions from form S1 (<b>A</b>), S3 (<b>B</b>), or S4 (<b>C</b>). Each gene and its orientation are indicated with a large arrow. The inverted repeats (IRs) in the <i>hsdS</i> genes are represented by yellow (IR1), black (IR2), and white (IR3) arrowheads. The inversion sites are indicated by dashed lines. Each DNA configuration is assigned with an S number. DNA configurations generated by excisions between <i>hsdS</i>_<i>A</i> and <i>hsdS</i>_<i>C</i> demarcated by direct repeat sets 1 (DR1, red arrows) and (DR2, purple arrows) (<b>D</b>). Excisions mediated by the DR1 and DR2 yields <i>hsdS</i>_<i>A</i> variant S9 and S7. Further inversion in S7 generates S8. S7 may also generate variant S9 by further DNA excision between DR1.1 and DR1.2.</p

Colony morphology of strain D39 and its derivatives each carrying one of the 6 invariable <i>hsdS</i>_<i>A</i> alleles.

<p>The <i>hsdS</i>_<i>A</i> allele-locked derivatives of strain D39 (WT, type 2) and isogenic capsule switch variant producing a type-19F capsule (D39^cps19F) were grown for 16 or 24 hours and processed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005762#ppat.1005762.g006" target="_blank">Fig 6</a>. The name of each strain is listed at the bottom of each photograph; the <i>hsdS</i>_<i>A</i> allele genotype marked at the bottom and left side of each row. The representative colonies with opaque and transparent appearance in the parental strains are indicated with blue and red arrowheads, respectively.</p