Comparative mitogenomic analyses provide evolutionary insights into the retrolateral tibial apophysis clade (Araneae: Entelegynae).

The retrolateral tibial apophysis (RTA) clade is the largest spider lineage within Araneae. To better understand the diversity and evolution, we newly determined mitogenomes of ten RTA species from six families and performed a comparative mitogenomics analysis by combining them with 40 sequenced RTA mitogenomes available on GenBank. The ten mitogenomes encoded 37 typical mitochondrial genes and included a large non-coding region (putative control region). Nucleotide composition and codon usage were well conserved within the RTA clade, whereas diversity in sequence length and structural features was observed in control region. A reversal of strand asymmetry in nucleotide composition, i.e., negative AT-skews and positive GC-skews, was observed in each RTA species, likely resulting from mitochondrial gene rearrangements. All protein-coding genes were evolving under purifying selection, except for atp8 whose Ka/Ks was larger than 1, possibly due to positive selection or selection relaxation. Both mutation pressure and natural selection might contribute to codon usage bias of 13 protein-coding genes in the RTA lineage. Phylogenetic analyses based on mitogenomic data recovered a family-level phylogeny within the RTA; {[(Oval calamistrum clade, Dionycha), Marronoid clade], Sparassidae}. This study characterized RTA mitogenomes and provided some new insights into the phylogeny and evolution of the RTA clade.

Mitochondrial phylogenomics provides insights into the phylogeny and evolution of spiders (Arthropoda: Araneae).

Spiders are among the most varied terrestrial predators, with highly diverse morphology, ecology, and behavior. Morphological and molecular data have greatly contributed to advances in the phylogeny and evolutionary dynamics of spiders. Here, we performed comprehensive mitochondrial phylogenomics analysis on 78 mitochondrial genomes (mitogenomes) representing 29 families; of these, 23 species from eight families were newly generated. Mesothelae retained the same gene arrangement as the arthropod ancestor ( Limulus polyphemus), while Opisthothelae showed extensive rearrangement, with 12 rearrangement types in transfer RNAs (tRNAs) and control region. Most spider tRNAs were extremely truncated and lacked typical dihydrouridine or TΨC arms, showing high tRNA structural diversity; in particular, trnS1 exhibited anticodon diversity across the phylogeny. The evolutionary rates of mitochondrial genes were potentially associated with gene rearrangement or truncated tRNAs. Both mitogenomic sequences and rearrangements possessed phylogenetic characteristics, providing a robust backbone for spider phylogeny, as previously reported. The monophyly of suborder, infraorder, retrolateral tibial apophysis clade, and families (except for Pisauridae) was separately supported, and high-level relationships were resolved as (Mesothelae, (Mygalomorphae, (Entelegynae, (Synspermiata, Hypochilidae)))). The phylogenetic positions of several families were also resolved (e.g., Eresidae, Oecobiidae and Titanoecidae). Two reconstructions of ancestral web type obtained almost identical results, indicating that the common ancestor of spiders likely foraged using a silk-lined burrow. This study, the largest mitochondrial phylogenomics analysis of spiders to date, highlights the usefulness of mitogenomic data not only for providing efficient phylogenetic signals for spider phylogeny, but also for characterizing trait diversification in spider evolution.

Lactobacillus Modulates Chlamydia Infectivity and Genital Tract Pathology in vitro and in vivo.

Since we previously reported that women infected with chlamydia had a significant overall reduction in Lactobacillus in the vagina microbiota as compared to those uninfected individuals; the interactions between the altered Lactobacillus and Chlamydia trachomatis, on the other hand, need to be elucidated. Here, we employed both in vitro and in vivo models to evaluate the effects of this changed Lactobacillus on Chlamydia infection. We found that L. iners, L. crispatus, L. jensenii, L. salivarius, L. gasseri, L. mucosae, and L. reuteri all significantly reduced C. trachomatis infection in a dose- and time-dependent manner. The strongest anti-Chlamydia effects were found in L. crispatus (90 percent reduction), whereas the poorest was found in L. iners (50 percent reduction). D (-) lactic acid was the key component in Lactobacillus cell-free supernatants (CFS) to inactivate Chlamydia EBs, showing a positive correlation with the anti-Chlamydia activity. The effects of D (-) lactic acid were substantially attenuated by neutralizing the pH value to 7.0. In vivo, mice intravaginally inoculated with Lactobacillus mixtures (L. crispatus, L. reuteri, and L. iners at a ratio of 1:1:1), but not single Lactobacillus, after genital Chlamydia infection, significantly attenuated the levels of Chlamydia live organism shedding in both the lower genital tract and the intestinal tract, reduced cytokines production (TNF-α, IFN-γ, and IL-1ß) in the vagina, and lessened upper genital tract inflammation and pathogenicity. Taken together, these data demonstrate that Lactobacillus inhibits Chlamydia infectivity both in vivo and in vitro, providing useful information for the development of Lactobacillus as adjunctive treatment in Chlamydia infection.

LncRNA ZEB1-AS1/miR-1224-5p / MAP4K4 axis regulates mitochondria-mediated HeLa cell apoptosis in persistent Chlamydia trachomatis infection.

Persistent infection of Chlamydia trachomatis is thought to be responsible for the debilitating sequelae of blinding trachoma and infertility. Inhibition of host cell apoptosis is a persistent C. trachomatis infection mechanism. ZEB1-AS1 is a long non-coding RNA (lncRNA), which was up-regulated in persistent C. trachomatis infection in our previous work. In this study, we investigated the role of ZEB1-AS1 in persistent infection and the potential mechanisms. The results showed that ZEB1-AS1 was involved in the regulation of apoptosis, and targeted silencing of ZEB1-AS1 could increase the apoptosis rate of persistently infected cells. Mechanically, interference ZEB1-AS1 caused an apparent down-regulation of the Bcl-2/Bax ratio and the repression of the mitochondrial membrane potential with the remarkable release of cytochrome c, resulting in the significant elevation level of caspase-3 activation. Meanwhile, the luciferase reporter assay confirmed that ZEB1-AS1 acted as a sponge for miR-1224-5p to target MAP4K4. The regulatory effect of miR-1224-5p/MAP4K4 on persistent infection-induced antiapoptosis was regulated by ZEB1-AS1. In addition, ZEB1-AS1 inhibited the apoptosis of Chlamydia-infected cells by activating the MAPK/ERK pathway. In conclusion, we found a new molecular mechanism that the ZEB1-AS1/miR-1224-5p/MAP4K4 axis contributes to apoptosis resistance in persistent C. trachomatis infection. This work may help understand the pathogenic mechanisms of persistent C. trachomatis infection and reveal a potential therapeutic strategy for its treatment.

Chlamydia trachomatis induces lncRNA MIAT upregulation to regulate mitochondria-mediated host cell apoptosis and chlamydial development.

Chlamydia trachomatis persistent infection is the leading cause of male prostatitis and female genital tract diseases. Inhibition of host cell apoptosis is the key to maintaining Chlamydia survival in vivo, and long noncoding RNAs (lncRNAs) play important roles in its developmental cycle and pathogenesis. However, it is not clear how lncRNAs regulate persistent Chlamydia infection. Here, using a microarray method, we identified 1718 lncRNAs and 1741 mRNAs differentially expressed in IFN-γ-induced persistent C. trachomatis infection. Subsequently, 10 upregulated and 5 downregulated differentially expressed lncRNAs were verified by qRT-PCR to confirm the reliability of the chip data. The GO and KEGG analyses revealed that differentially regulated transcripts were predominantly involved in various signalling pathways related to host immunity and apoptosis response. Targeted silencing of three lncRNAs (MIAT, ZEB1-AS1 and IRF1) resulted in increased apoptosis rates. Furthermore, interference with lncRNA MIAT caused not only an obvious downregulation of the Bcl-2/Bax ratio but also a marked release of cytochrome c, resulting in a significantly elevated level of caspase-3 activation. Meanwhile, MIAT was involved in the regulation of chlamydial development during the persistent infection. Collectively, these observations shed light on the enormous complex lncRNA regulatory networks involved in mitochondria-mediated host cell apoptosis and the growth and development of C. trachomatis.

Long Non-Coding RNA FGD5-AS1 Induced by Chlamydia trachomatis Infection Inhibits Apoptosis via Wnt/ß-Catenin Signaling Pathway.

Background: Chlamydia trachomatis (Ct) is one of the most common bacterial sexually transmitted infection (STI) pathogens in the world, but the exact pathogenic mechanism still needs to be further elucidated. Long non-coding RNAs (lncRNAs) have become vital regulators in many biological processes. Their role in the interaction between Ct and host cells has not been reported. Methods: Microarrays were used to study the expression profiles of lncRNAs and mRNAs in HeLa cells at 12, 24, and 40 h post-infection (hpi). Differentially expressed lncRNAs and mRNAs were verified by RT-qPCR. Coding-non-coding (CNC) network analysis showed co-expression molecules of selected lncRNA. Western blot, flow cytometry, and indirect immunofluorescence were used to detect the effect of lncRNA FGD5-AS1 on apoptosis during Ct infection. Results: Compared with the uninfected group, the number of differential lncRNAs were 2,130, 1,081, and 1,101 at 12, 24, and 40 hpi, and the number of differential mRNAs was 1,998, 1,129, and 1,330, respectively. Ct induced differential expression of large amounts of lncRNAs and mRNAs in HeLa cells, indicating that lncRNAs may play roles in the pathogenesis of Ct. RT-qPCR verified six differential lncRNAs and six differential mRNAs, confirming the reliability of the microarray. Among these molecules, lncRNA FGD5-AS1 was found to be upregulated at 12 and 24 hpi. Coding-non-coding (CNC) network analysis showed that co-expressed differential molecules of FGD5-AS1 at 12 and 24 hpi were enriched in the DNA replication and Wnt signaling pathway. The downregulation of FGD5-AS1 decreased the expression of ß-catenin and inhibited the translocation of ß-catenin and the DNA replication, while it promoted apoptosis of the host cells. Conclusions: DNA replication and apoptosis of host cells were affected by upregulating FGD5-AS1 via Wnt/ß-catenin pathway during Ct infection. This study provides evidence that lncRNAs are involved in the coaction between Ct and hosts, and provides new insights into the study of lncRNAs that regulate chlamydial infection.

Chlamydia trachomatis Pgp3 protein regulates oxidative stress via activation of the Nrf2/NQO1 signal pathway.

AIM: Chlamydia trachomatis has evolved various strategies to alleviate oxidative stress of host cells to maintain their intracellular survival. However, the exact mechanism of anti-oxidative stress of C. trachomatis is still unclear. The activation of nuclear factor erythroid 2-related factor 2/quinone oxidoreductase (Nrf2/NQO1) signal pathway has been identified as an efficient antioxidant defensive mechanism used by host cells to counteract oxidative stress. Pgp3 is a pivotal virulence factor of C. trachomatis involved in intracellular survival. The aim of this study is to explore the role of Pgp3 on Nrf2/NQO1 signal pathway against oxidative stress. MAIN METHODS: After HeLa cells were stimulated with Pgp3 protein, Nrf2 location and the inclusion bodies of C. trachomatis were detected by indirect immunofluorescence, western blotting and Oxidative stress assay kits were used to separately determine the protein expression and the content of malondialdehyde (MDA), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) before and after the interference of Nrf-2 and NQO1. KEY FINDINGS: Pgp3 promoted the nuclear translocation of Nrf2 to increase NQO1 expression and reduced oxidative stress induced by LPS to contribute to the survival of C. trachomatis. Inhibition of Nrf2/NQO1 signal pathway with Nrf2 inhibitor and down-regulation of NQO1 with siRNA-NQO1 suppressed oxidative stress resistance induced by Pgp3. SIGNIFICANCE: Here we found that Pgp3 alleviated oxidative stress to promote the infectivity of C. trachomatis through activation of Nrf2/NQO1 signal pathway, which provided a novel understanding of the effects of Pgp3 in the pathogenesis of C. trachomatis.

Roles of long noncoding RNAs in bacterial infection.

Bacteria can induce significant alteration in the cell transcriptome and develop many strategies to modify immune signaling for its survival. In recent years, a new class of regulatory RNAs, long noncoding RNAs (lncRNAs), has been demonstrated to play an essential role in host gene expression. Growing literature indicate that lncRNAs function as positive or negative effectors on antibacterial immunity. On the one hand, the host regulates immune-related genes at epigenetic, transcriptional, and post-transcriptional levels by lncRNAs, thereby protecting itself from pathogen invasion. On the other hand, bacteria can manipulate the host signaling pathways by regulating the host lncRNAs to escape immune clearance. In addition, some bacteria even produce lncRNAs, which are involved in the pathogenic process of pathogens. Some dysregulated lncRNAs during bacterial infections can be used as a potential diagnostic marker for infection. Understanding of gene expression regulation through lncRNAs helps illustrate bacterial pathogenesis. Here, we summarize the functions of lncRNAs and current advances of lncRNAs in different bacterial infections and look forward to the future research orientation.

Roles of long non-coding RNAs in cervical cancer.

Cervical cancer (CC) is regarded as the second serious threat to women's health worldwide; it's associated with certain viruses that are transmitted through sexual intercourse. Therefore, the pathogenesis of CC remains to be studied. The identified long non-coding RNAs (lncRNAs) as a key genomic product were found to be commonly dysregulated in CC and to exert significant effects in the initiation, migration, invasion and therapeutic response of CC. Therefore lncRNAs may be used as tumor suppressor genes or oncogenes to interact with DNA, RNA or proteins for the regulation of gene expression and cell signaling pathways. The relationship between single lncRNA and CC has been discovered. However, full-scale reviews on the lncRNAs function in CC are deficiency. In this review, we describe the recent reports on the dysregulated patterns regulation of lncRNAs in CC. We also conclude the recent advances on biologic functions and molecular regulation mechanism and potential clinical application of lncRNAs in CC.

Chlamydia trachomatis plasmid-encoded protein pORF5 activates unfolded protein response to induce autophagy via MAPK/ERK signaling pathway.

Chlamydia trachomatis (C. trachomatis) is an obligate intracellular organism that depends on nutrients from the host cell for their replication and proliferation. Therefore, the interaction between this pathogen and host induces sustained endoplasmic reticulum (ER) stress in the infected cells. Unfolded protein response (UPR) has been demonstrated to be activated by chlamydial secreted effectors, allowing host cells to recover from the stressful state. In this study, we attempted to explore the role of the only secreted plasmid-encoded protein pORF5 of C. trachomatis between UPR and autophagy induction. The results showed that three branches of UPR (PERK, IRE1, and ATF6) were activated by pORF5. pORF5-induced autophagy was repressed by UPR inhibitors GSK2606414 and 4µ8C, while the autophagy inhibition was failed to influence pORF5-induced UPR significantly. MAPK/ERK inhibitor PD98059 partially suppressed the pORF5-induced autophagy, but had little effect on UPR, indicating that pORF5 actives UPR to induce autophagy via the MAPK/ERK signaling pathway. These observations provide clues on how the host maintains the cellular homeostasis during C. trachomatis infection.

Chlamydia trachomatis Plasmid Protein pORF5 Up-Regulates ZFAS1 to Promote Host Cell Survival via MAPK/p38 Pathway.

Long non-coding RNAs (lncRNAs) have been demonstrated to play essential roles in many diseases. However, few studies have shown that lncRNAs take part in the pathogenesis of Chlamydia trachomatis (C. trachomatis). Here, we used a lncRNA microarray to detect the global lncRNA expression profiles in HeLa cells transfected with pORF5 plasmid protein, an important virulence factor for C. trachomatis. The differentially expressed lncRNAs and mRNAs screened by microarray were selected for validation by quantitative real-time PCR. The up-regulated lncRNA zinc finger antisense 1 (ZFAS1) was presumed to involved in MAPK pathways by bioinformatics analysis. Inhibition of ZFAS1 decreased the apoptotic rate of pORF5 and reduced the infectivity of C. trachomatis, and MAPK/p38 pathway was involved in anti-apoptotic effect induced by ZFAS1. Therefore, the present study confirmed that pORF5 up-regulates ZFAS1 to promote host cell survival via MAPK/p38 pathway and influences the infectivity of C. trachomatis.

The Complete Mitogenome of Pyrrhocoris tibialis (Hemiptera: Pyrrhocoridae) and Phylogenetic Implications.

We determined the complete mitogenome of Pyrrhocoris tibialis (Hemiptera: Heteroptera: Pyrrhocoridae) to better understand the diversity and phylogeny within Pentatomomorpha, which is the second largest infra-order of Heteroptera. Gene content, gene arrangement, nucleotide composition, codon usage, ribosomal RNA (rRNA) structures, and sequences of the mitochondrial transcription termination factor were well conserved in Pyrrhocoroidea. Different protein-coding genes have been subject to different evolutionary rates correlated with the G + C content. The size of control regions (CRs) was highly variable among mitogenomes of three sequenced Pyrrhocoroidea species, with the P. tibialis CR being the largest. All the transfer RNA genes found in Pyrrhocoroidea had the typical clover leaf secondary structure, except for trnS1 (AGN), which lacked the dihydrouridine arm and possessed an unusual anticodon stem (9 bp vs. the normal 5 bp). A total of three different phylogenetic relationships among the five super-families of Pentatomomorpha were obtained using three analytical methods (MrBayes and RAxML under site-homogeneous models and PhyloBayes under a site-heterogeneous CAT + GTR model) and two mitogenomic datasets (nucleotides and amino acids). The tree topology test using seven methods statistically supported a phylogeny of (Aradoidea + (Pentatomoidea + (Lygaeoidea + (Pyrrhocoroidea + Coreoidea)))) as the best topology, as recognized by both RAxML and MrBayes based on the two datasets.

Antiapoptotic activity of Chlamydia trachomatis Pgp3 protein involves activation of the ERK1/2 pathway mediated by upregulation of DJ-1 protein.

Chlamydia trachomatis has evolved strategies to prevent host cell apoptosis to evade the host immune defense. However, the precise mechanisms of antiapoptotic activity of C. trachomatis still need to be clarified. Pgp3, one of eight plasmid proteins of C. trachomatis, has been identified to be closely associated with chlamydial virulence. In this study, we attempted to explore the effects and the mechanisms of Pgp3 protein on apoptosis in HeLa cells; the results showed that Pgp3 increased Bcl-2/Bax ratio and prevented caspase-3 activation to suppress apoptosis induced by TNF-α and cycloheximide (CHX) through ERK1/2 pathway activation. Downregulation of DJ-1 with siRNA-DJ-1(si-DJ-1) reduced ERK1/2 phosphorylation and elevated apoptotic rate significantly in Pgp3-HeLa cells. However, inhibition of ERK1/2 signal pathway with ERK inhibitor PD98059 had little effect on DJ-1 expression. These findings confirm that plasmid protein Pgp3 contributes to apoptosis resistance through ERK1/2 signal pathway mediated by upregulation of DJ-1 expression. Therefore, the present study provided novel insights into the role of Pgp3 in apoptosis and suggested that manipulation of the host apoptosis response could be a new approach for the prevention and treatment of C. trachomatis infection.