Table_1_A Novel Iron Transporter SPD_1590 in Streptococcus pneumoniae Contributing to Bacterial Virulence Properties.DOCX

<p>Streptococcus pneumoniae, a Gram-positive human pathogen, has evolved three main transporters for iron acquisition from the host: PiaABC, PiuABC, and PitABC. Our previous study had shown that the mRNA and protein levels of SPD_1590 are significantly upregulated in the ΔpiuA/ΔpiaA/ΔpitA triple mutant, suggesting that SPD_1590 might be a novel iron transporter in S. pneumoniae. In the present study, using spd1590-knockout, -complemented, and -overexpressing strains and the purified SPD_1590 protein, we show that SPD_1590 can bind hemin, probably supplementing the function of PiuABC, to provide the iron necessary for the bacterium. Furthermore, the results of iTRAQ quantitative proteomics and cell-infection studies demonstrate that, similarly to other metal-ion uptake proteins, SPD_1590 is important for bacterial virulence properties. Overall, these results provide a better understanding of the biology of this clinically important bacterium.</p

Intra-Genomic Heterogeneity in 16S rRNA Genes in Strictly Anaerobic Clinical Isolates from Periodontal Abscesses

<div><p>Background</p><p>Members of the genera <i>Prevotella</i>, <i>Veillonella</i> and <i>Fusobacterium</i> are the predominant culturable obligate anaerobic bacteria isolated from periodontal abscesses. When determining the cumulative number of clinical anaerobic isolates from periodontal abscesses, ambiguous or overlapping signals were frequently encountered in 16S rRNA gene sequencing chromatograms, resulting in ambiguous identifications. With the exception of the genus <i>Veillonella</i>, the high intra-chromosomal heterogeneity of <i>rrs</i> genes has not been reported.</p><p>Methods</p><p>The 16S rRNA genes of 138 clinical, strictly anaerobic isolates and one reference strain were directly sequenced, and the chromatograms were carefully examined. Gene cloning was performed for 22 typical isolates with doublet sequencing signals for the 16S rRNA genes, and four copies of the <i>rrs-ITS</i> genes of 9 <i>Prevotella intermedia</i> isolates were separately amplified by PCR, sequenced and compared. Five conserved housekeeping genes, <i>hsp60</i>, <i>recA</i>, <i>dnaJ</i>, <i>gyrB1</i> and <i>rpoB</i> from 89 clinical isolates of <i>Prevotella</i> were also amplified by PCR and sequenced for identification and phylogenetic analysis along with 18 <i>Prevotella</i> reference strains.</p><p>Results</p><p>Heterogeneity of 16S rRNA genes was apparent in clinical, strictly anaerobic oral bacteria, particularly in the genera <i>Prevotella</i> and <i>Veillonella</i>. One hundred out of 138 anaerobic strains (72%) had intragenomic nucleotide polymorphisms (SNPs) in multiple locations, and 13 strains (9.4%) had intragenomic insertions or deletions in the 16S rRNA gene. In the genera <i>Prevotella</i> and <i>Veillonella</i>, 75% (67/89) and 100% (19/19) of the strains had SNPs in the 16S rRNA gene, respectively. Gene cloning and separate amplifications of four copies of the <i>rrs-ITS</i> genes confirmed that 2 to 4 heterogeneous 16S rRNA copies existed.</p><p>Conclusion</p><p>Sequence alignment of five housekeeping genes revealed that intra-species nucleotide similarities were very high in the genera <i>Prevotella</i>, ranging from 94.3–100%. However, the inter-species similarities were relatively low, ranging from 68.7–97.9%. The housekeeping genes <i>rpoB</i> and <i>gyrB1</i> were demonstrated to be alternative classification markers to the species level based on intra- and inter-species comparisons, whereas based on phylogenetic tree <i>rpoB</i> proved to be reliable phylogenetic marker for the genus <i>Prevotella</i>.</p></div

Phylogenetic trees based on the concatenated sequences of the <i>rpoB</i> gene.

<p>Detailed phylogenetic trees of <i>P</i>. <i>melaninogenica</i> strains are shown in Figure (B). The trees were constructed by the neighbour-joining (NJ) method. The numbers at nodes indicate the percentage bootstrap values of 1000 replicates (>70%). Bars indicate the expected nucleotide substitutions per site. * represents reference strains.</p

16S rRNA gene cloning of 22 <i>Prevotella spp</i>. clinical isolates and four copies of <i>rrs</i> sequencing of 9 <i>Prev</i>otella intermedia <i>clinical</i> isolates.

<p>16S rRNA gene cloning of 22 <i>Prevotella spp</i>. clinical isolates and four copies of <i>rrs</i> sequencing of 9 <i>Prev</i>otella intermedia <i>clinical</i> isolates.</p

Characteristics of intragenomic heterogeneous 16S rRNA genes in clinical anaerobic strains.

<p><b>^a</b><i>Alloprevotella rava</i> was previously classified as <i>Prevotella oral taxon 302</i>. Although it has been separated from the genus <i>Prevotella</i>, this strain was analysed together with the other <i>Prevotella spp</i>. in this study.</p><p>Characteristics of intragenomic heterogeneous 16S rRNA genes in clinical anaerobic strains.</p

Analysis of 106 <i>hsp60</i>, <i>recA</i>, <i>dnaJ</i>, <i>gyrB1</i>, <i>rpoB</i> and 16S rRNA gene sequences from <i>Prevotella</i>.

<p>*Synonymous substitutions per site (dS) and non-synonymous substitutions per site (dN) (means±SEM) were determined by the Nei–Gojobori method using the Jukes–Cantor distance. NA, not applicable.</p><p>Analysis of 106 <i>hsp60</i>, <i>recA</i>, <i>dnaJ</i>, <i>gyrB1</i>, <i>rpoB</i> and 16S rRNA gene sequences from <i>Prevotella</i>.</p

16S rRNA sequence trace data for the isolates <i>Prevotella denticola</i> HJX050 and <i>Prevotella nigrescens</i> HJX046, showing comparison sequence data for heterogeneous clones and direct sequencing with single ambiguous bases (A) or continuous ambiguous bases (B).

<p><i>Prevotella nigrescens</i> HJX046 clone A had a deletion at nucleotide position 229.</p

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

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