Promiscuous signaling by a regulatory system unique to the pandemic PMEN1 pneumococcal lineage

Streptococcus pneumoniae (pneumococcus) is a leading cause of death and disease in children and elderly. Genetic variability among isolates from this species is high. These differences, often the product of gene loss or gene acquisition via horizontal gene transfer, can endow strains with new molecular pathways, diverse phenotypes, and ecological advantages. PMEN1 is a widespread and multidrug-resistant pneumococcal lineage. Using comparative genomics we have determined that a regulator-peptide signal transduction system, TprA2/PhrA2, was acquired by a PMEN1 ancestor and is encoded by the vast majority of strains in this lineage. We show that TprA2 is a negative regulator of a PMEN1-specific gene encoding a lanthionine-containing peptide (lcpA). The activity of TprA2 is modulated by its cognate peptide, PhrA2. Expression of phrA2 is density-dependent and its C-terminus relieves TprA2-mediated inhibition leading to expression of lcpA. In the pneumococcal mouse model with intranasal inoculation, TprA2 had no effect on nasopharyngeal colonization but was associated with decreased lung disease via its control of lcpA levels. Furthermore, the TprA2/PhrA2 system has integrated into the pneumococcal regulatory circuitry, as PhrA2 activates TprA/PhrA, a second regulator-peptide signal transduction system widespread among pneumococci. Extracellular PhrA2 can release TprA-mediated inhibition, activating expression of TprA-repressed genes in both PMEN1 cells as well as another pneumococcal lineage. Acquisition of TprA2/PhrA2 has provided PMEN1 isolates with a mechanism to promote commensalism over dissemination and control inter-strain gene regulation

Analysis of the TprA2 regulon by comparison of gene expression levels among WT, Δ<i>tprA2</i>, and Δ<i>tprA2</i>::<i>tprA2</i> strains.

<p>qRT-PCR measurements for genes <i>tprA2</i>, <i>phrA2</i>, <i>ABCATPase</i> and <i>lcpA</i>. X-axis represents genes that were tested for expression in strains WT, <i>ΔtprA2</i> and <i>ΔtprA2</i>::<i>tprA2</i>. Y-axis denotes starting concentration of mRNA in arbitrary fluorescence units as calculated from LinRegPCR. Data was normalized to the expression of 16S rRNA. Error bars represent standard deviation for biological replicates (n = 3).‘*’ significantly different expression relative to WT (<i>P</i>-value < 0.005), ‘+’ significantly different expression relative to Δ<i>tprA2</i> (<i>P</i>-value < 0.005).</p

Model for regulation of gene expression by TprA2-PhrA2.

<p>(A) In the OFF state, TprA2 inhibits gene expression. (B) In the ON state, PhrA2 releases TprA2-mediated gene inhibition. This effect of PhrA2 is observed from synthetic peptide added to the extracellular milieu and cell-free supernatant, suggesting that PhrA2 is exported, activated and re-imported before it modulates TprA2 activity, in both the producer PMEN1 cells and surrounding PMEN1 population. (C) PhrA2 secreted by PMEN1 cells activates gene expression of <i>tprA</i> and associated <i>lanA</i>, in both PMEN1 and non-PMEN1 cells. Red circular shape/TprA2, purple triangle/PhrA2, blue circular shape/TprA; blue triangle/PhrA.</p

Gene expression measured by qRT-PCR of QS-Lcp genes in WT strain PN4595-T23 upon treatments.

<p>Data was normalized to 16S rRNA expression. Y-axis displays fold change in gene expression upon exposure to a peptide treatment relative to untreated control. Error bars represent standard deviations for biological replicates (n = 3). On the left, dark bars display expression from cells exposed to the PhrA2 C-terminal heptapeptide (VDLGLAD); on the right side, stripped bars display expression from cells exposed to the scrambled control peptide (DAGVLDL). “**” Statistically significant difference in gene expression after PhrA2 treatment compared to scrambled peptide (<i>P</i>-value<0.01).</p

Density-dependent gene expression and extracellular secretion of <i>phrA2</i> during planktonic growth.

<p>qRT-PCR measurements of <i>phrA2</i> gene expression in PN4595-T23. The Y-axis displays expression levels as a ratio to expression in lag phase culture. The X-axis denotes culture conditions. Black bars displays density-dependent gene expression at lag phase (OD₆₀₀0.05), early-log phase (OD₆₀₀0.2), mid-log phase (OD₆₀₀0.6), and stationary phase (OD₆₀₀1.0). Striped bars display treatment by cell-free supernatants. The lag phase culture was divided into three tubes and grown for 1h in one of three ways in: original supernatant (lagWT+1hour), cell-free supernatant from a high density wild type culture (OD₆₀₀1.2), or cell-free supernatant from a high density <i>ΔphrA2-ABC</i> culture (OD₆₀₀1.2). 16SrRNA was used as normalization control. Error bars represent standard deviations from biological duplicate experiments. ‘**’ <i>P</i>-value<0.01 and ‘*’, <i>P</i>-value<0.05 as determined by Student’s <i>t</i>-test.</p

Genomic organization of TprA2/PhrA2 region in PMEN1 strains.

<p>Genomic organization of TprA2/PhrA2 region in PMEN1 strains.</p

Intra- and inter-species distribution of the TprA2/PhrA2 and TprA/PhrA genomic regions.

<p>Phylogenetic analyses displaying bootstrap values on the branches. <u>Left side</u>: Maximum likelihood tree of streptococcal genomes generated from the core genome. <u>Right side</u>: Gene distribution, where blue columns display the distribution of <i>tprA2</i>, <i>phrA2</i>, and associated <i>lcpAMT</i>, and purple columns display the distribution of <i>tprA</i>, <i>phrA</i>, and downstream lantibiotic genes (seven consecutive genes, including predicted <i>lanA</i> and <i>lanM</i> labeled as Lan). Presence of the gene is marked with the following symbols: ‘●’ gene present in one copy; ‘○’ low coverage of region; ‘□’multiple copies of the gene. Red box indicates isolates from the PMEN1 lineage.</p

Gene expression levels of the TprA2/PhrA2 system and associated <i>lcpAMT</i> locus in chinchilla middle ear effusions and planktonic cultures.

<p>nCounter nanoString technology was used to quantify mRNA transcripts from planktonic cultures (dotted bars, n = 2) and chinchilla middle ear effusions (black bars, n = 3). Data was normalized to the geometric mean of the expression of <i>gyrB</i> and <i>metG</i> using nSolver software. The X-axis denotes the test genes assayed for gene expression. The Y-axis displays the log₁₀ of the total number of transcripts for each gene averaged over biological replicates. Error bars represent the standard deviation. ‘*’ Significantly higher <i>in vivo</i> expression (<i>P</i>-value < 0.05), as determined by Student’s <i>t</i>-test.</p