Complete Genome and Transcriptomes of Streptococcus parasanguinis FW213: Phylogenic Relations and Potential Virulence Mechanisms

Streptococcus parasanguinis, a primary colonizer of the tooth surface, is also an opportunistic pathogen for subacute endocarditis. The complete genome of strain FW213 was determined using the traditional shotgun sequencing approach and further refined by the transcriptomes of cells in early exponential and early stationary growth phases in this study. The transcriptomes also discovered 10 transcripts encoding known hypothetical proteins, one pseudogene, five transcripts matched to the Rfam and additional 87 putative small RNAs within the intergenic regions defined by the GLIMMER analysis. The genome contains five acquired genomic islands (GIs) encoding proteins which potentially contribute to the overall pathogenic capacity and fitness of this microbe. The differential expression of the GIs and various open reading frames outside the GIs at the two growth phases suggested that FW213 possess a range of mechanisms to avoid host immune clearance, to colonize host tissues, to survive within oral biofilms and to overcome various environmental insults. Furthermore, the comparative genome analysis of five S. parasanguinis strains indicates that albeit S. parasanguinis strains are highly conserved, variations in the genome content exist. These variations may reflect differences in pathogenic potential between the strains

Enhancement of polar phases in PVDF by forming PVDF/SiC nanowire composite

Different contents of silicon carbide (SiC) nanowires were mixed with Poly(vinylidene fluoride) (PVDF) to facilitate the polar phase crystallization. It was shown that the annealing temperature and SiC content affected on the phase and crystalline structures of PVDF/SiC samples. Furthermore, the addition of SiC nanowire enhanced the transformation of non-polar α phase to polar phases and increased the relative fraction of β phase in PVDF. Due to the nucleating agent mechanism of SiC nanowires, the ion-dipole interaction between the negatively charged surface of SiC nanowires and the positive CH2 groups in PVDF facilitated the formation of polar phases in PVDF

Pyk2 activates the NLRP3 inflammasome by directly phosphorylating ASC and contributes to inflammasome-dependent peritonitis

The inflammasome adaptor protein, ASC, contributes to both innate immune responses and inflammatory diseases via self-oligomerization, which leads to the activation of the protease, caspase-1. Here, we report that the cytosolic tyrosine kinases, FAK and Pyk2, are differentially involved in NLRP3 and AIM2 inflammasome activation. The inhibition of FAK and Pyk2 with RNA interference or chemical inhibitors dramatically abolished ASC oligomerization, caspase-1 activation, and IL-1β secretion in response to NLRP3 or AIM2 stimulation. Pyk2 is phosphorylated by the kinase Syk and relocalizes to the ASC specks upon NLRP3 inflammasome activation. Pyk2, but not FAK, could directly phosphorylate ASC at Tyr146, and only the phosphorylated ASC could participate in speck formation and trigger IL-1β secretion. Moreover, the clinical-trial-tested Pyk2/FAK dual inhibitor PF-562271 reduced monosodium urate-mediated peritonitis, a disease model used for studying the consequences of NLRP3 activation. Our results suggest that although Pyk2 and FAK are involved in inflammasome activation, only Pyk2 directly phosphorylates ASC and brings ASC into an oligomerization-competent state by allowing Tyr146 phosphorylation to participate ASC speck formation and subsequent NLRP3 inflammation

The Expression of the <i>fim</i> Operon Is Crucial for the Survival of <i>Streptococcus parasanguinis</i> FW213 within Macrophages but Not Acid Tolerance

<div><p>The acquisition of transition metal ions is essential for the viability and in some cases the expression of virulence genes in bacteria. The <i>fimCBA</i> operon of <i>Streptococcus parasanguinis</i> FW213 encodes a Mn²⁺/Fe²⁺-specific ATP-binding cassette transporter. FimA, a lipoprotein in the system, is essential for the development of endocarditis, presumably by binding to fibrin monolayers on the damaged heart tissue. Recent sequence analysis revealed that Spaf_0344 was homologous to <i>Streptococcus gordonii scaR</i>, encoding a metalloregulatory protein for the Sca Mn²⁺-specific transporter. Based on the homology, Spaf_0344 was designated <i>fimR</i>. By using various <i>fim</i> promoter (p<i>_fim</i>) derivatives fused with a promoterless chloramphenicol acetyltransferase gene, the functions of the <i>cis</i>-elements of p<i>_fim</i> were analyzed in the wild-type and <i>fimR</i>-deficient hosts. The result indicated that FimR represses the expression of p<i>_fim</i> and the palindromic sequences 5′ to <i>fimC</i> are involved in repression of p<i>_fim</i>. A direct interaction between FimR and the palindromic sequences was further confirmed by <i>in vitro</i> electrophoresis gel mobility shift assay and <i>in vivo</i> chromatin immunoprecipitation assay (ChIP)-quantitative real-time PCR (qPCR). The result of the ChIP-qPCR analysis also indicated that FimR is activated by Mn²⁺ and, to a lesser degree, Fe²⁺. Functional analysis indicated that the expression of FimA in <i>S. parasanguinis</i> was critical for wild-type levels of survival against oxidative stress and within phagocytes, but not for acid tolerance. Taken together, in addition to acting as an adhesin (FimA), the expression of the <i>fim</i> operon is critical for the pathogenic capacity of <i>S. parasanguinis</i>.</p></div

EMSA demonstrating the interaction between FimR and p<b><i>_fim</i></b><b>.</b>

<p>Lanes 1 to 4 are reactions containing 0, 20, 40, and 80 µM His-FimR, respectively; lane 5 is reaction containing 80 µM His-FimR and unlabeled <i>tcrB</i>. The positions of the FimR-probe complexes are indicated by triangles.</p

ChIP-qPCR demonstrating the relative quantity of p<i>_fim</i> bound by FimR.

<p>Cells were grown under 0.01 µM MnCl₂ and 0.1 µM FeSO₄ (I), 0.01 µM MnCl₂ and 50 µM FeSO₄ (II), 50 µM MnCl₂ and 0.1 µM FeSO₄ (III), and 50 µM MnCl₂ and 50 µM FeSO₄ (IV). The ΔCq of the sample from III was used as the reference. Significant differences between samples were determined using one-way ANOVA. A significant difference (<i>P</i><0.05) was detected between all pairs of comparison.</p

Growth kinetics of the wild-type <i>S.</i><i>parasanguinis</i>, VT930, Δ<i>fimR</i>, and VT930_Δ<i>fimR</i> grown in TH (A), TH containing 2 mM (B) and 4 mM (C) paraquat.

<p>A representative figure of at least three experiments under each condition is shown.</p

The regulation of FimR on p<i>_fim</i> expression.

<p>(A) The nt sequence of the 5′ flanking region of <i>fimC</i>. The <i>pepO</i> and <i>fimC</i> are transcribed from the opposite DNA strands, thus the sequence of <i>pepO</i> presented here is the noncoding strand, and the sequence of <i>fimC</i> is the coding strand. The transcription initiation sites (+1) of <i>fimC</i> and <i>pepO</i> are shown by a solid triangle above the sequence, and two open triangles below the sequence, respectively. The putative −10 and −35 sequences of p<i>_fim</i> are shaded. The potential Per box is underlined. The inverted repeat sequences are shown by horizontal arrows above the sequence. The sequence of the probe used in EMSA is boxed. The limits of the deletion derivatives are indicated by the numbers. (B) The CAT activities in wild-type FW213 and Δ<i>fimR</i> harboring various p<i>_fim</i>-<i>cat</i> fusions. All strains were grown in TH. Values shown are means and standard deviations of three independent experiments. All experiments were done in triplicate reactions and negative controls were reactions carried out in the absence of Cm.</p

Effect of Mn²⁺ and Fe²⁺ on p<i>_fim</i> expression.

<p>Wild-type FW213 and Δ<i>fimR</i> harboring p<i>_fim</i>(445 b)-<i>cat</i> were grown in FMC containing 0.01 µM MnCl₂ and 0.1 µM FeSO₄ (I), 0.01 µM MnCl₂ and 50 µM FeSO₄ (II), 50 µM MnCl₂ and 0.1 µM FeSO₄ (III), 50 µM MnCl₂ and 50 µM FeSO₄ (IV). All cultures were supplemented with 1 mM MgSO₄ and 1 mM CaCl₂. Values are means and standard deviations of three independent experiments. Significant differences between samples were determined by two-way ANOVA using SPSS Statistic 17.0. The <i>P</i> values between the wild-type strain and Δ<i>fimR</i> under all four conditions are less than 0.01. <i>P</i> values between condition I and II are indicated in the figure. *, <i>P</i><0.05; **, <i>P</i><0.01.</p