Unveiling the dance of evolution: Pla-mediated cleavage of Ymt modulates the virulence dynamics of Yersinia pestis.

Yersinia pestis has recently evolved into a highly lethal flea-borne pathogen through the pseudogenization of extensive genes and the acquisition of exogenous plasmids. Particularly noteworthy are the newly acquired pPCP1 and pMT1 plasmids, which encode the virulence determinants Pla and Yersinia murine toxin (Ymt), crucial for subcutaneous infection and survival within flea vector of Y. pestis, respectively. This study reveals that Pla can cleave Ymt at K299 both in vivo and in vitro. Y. pestis expressing YmtK299A displays enhanced in vitro biofilm formation and increased blood survival, indicating significant roles of Pla-mediated Ymt cleavage in these phenotypes. Intriguingly, although both the ancestral form of Pla and the prevalent Pla-I259T variant in modern Y. pestis strains are capable of cleaving Ymt at K299, the cleavage efficiency of Pla-I259T is only half that of the ancestral variant. In subcutaneous infection, mice infected with Δymt::ymt-K299A show significantly prolonged survival compared to those infected with Δymt::ymt. Similarly, infection with Δpla::pla-I259T also results in extended survival compared to Δpla::pla infection. These data demonstrate that the I259T substitution of Pla mitigates the enhanced virulence of Y. pestis in mice caused by Pla-mediated Ymt cleavage, thereby prolonging the survival period of infected animals and potentially conferring advantages on the transmission of Y. pestis to the next host. These findings deepen our understanding of the intricate interplay between two newly acquired plasmids and shed light on the positive selection of the Pla-I259T mutation, providing new insights into the virulence dynamics and transmission mechanisms of Y. pestis. IMPORTANCE: The emergence of Y. pestis as a highly lethal pathogen is driven by extensive gene pseudogenization and acquisition of exogenous plasmids pPCP1 and pMT1. However, the interplay between these two plasmids during evolution remains largely unexplored. Our study reveals intricate interactions between Ymt and Pla, two crucial virulence determinants encoded on these plasmids. Pla-mediated cleavage of Ymt significantly decreases Y. pestis survival in mouse blood and enhances its virulence in mice. The prevalent Pla-I259T variant in modern strains displays reduced Ymt cleavage, thereby extending the survival of infected animals and potentially increasing strain transmissibility. Our findings shed light on the nuanced evolution of Y. pestis, wherein reduced cleavage efficiency is a positive selection force, shaping the pathogen's natural trajectory.

Unveiling the impact of low-frequency electrical stimulation on network synchronization and learning behavior in cultured hippocampal neural networks.

Understanding the dynamics of neural networks and their response to external stimuli is crucial for unraveling the mechanisms associated with learning processes. In this study, we hypothesized that electrical stimulation (ES) would lead to significant alterations in the activity patterns of hippocampal neuronal networks and investigated the effects of low-frequency ES on hippocampal neuronal populations using the microelectrode arrays (MEAs). Our findings revealed significant alterations in the activity of hippocampal neuronal networks following low-frequency ES trainings. Post-stimulation, the neural activity exhibited an organized burst firing pattern characterized by increased spike and burst firings, increased synchronization, and enhanced learning behaviors. Analysis of peri-stimulus time histograms (PSTHs) further revealed that low-frequency ES (1Hz) significantly enhanced neural plasticity, thereby facilitating the learning process of cultured neurons, whereas high-frequency ES (>10Hz) impeded this process. Moreover, we observed a substantial increase in correlations and connectivity within neuronal networks following ES trainings. These alterations in network properties indicated enhanced synaptic plasticity and emphasized the positive impact of low-frequency ES on hippocampal neural activities, contributing to the brain's capacity for learning and memory.

A novel sORF gene mutant strain of Yersinia pestis vaccine EV76 offers enhanced safety and improved protection against plague.

We recently identified two virulence-associated small open reading frames (sORF) of Yersinia pestis, named yp1 and yp2, and null mutants of each individual genes were highly attenuated in virulence. Plague vaccine strain EV76 is known for strong reactogenicity, making it not suitable for use in humans. To improve the immune safety of EV76, three mutant strains of EV76, Δyp1, Δyp2, and Δyp1&yp2 were constructed and their virulence attenuation, immunogenicity, and protective efficacy in mice were evaluated. All mutant strains were attenuated by the subcutaneous (s.c.) route and exhibited more rapid clearance in tissues than the parental strain EV76. Under iron overload conditions, only the mice infected with EV76Δyp1 survived, accompanied by less draining lymph nodes damage than those infected by EV76. Analysis of cytokines secreted by splenocytes of immunized mice found that EV76Δyp2 induced higher secretion of multiple cytokines including TNF-α, IL-2, and IL-12p70 than EV76. On day 42, EV76Δyp2 or EV76Δyp1&yp2 immunized mice exhibited similar protective efficacy as EV76 when exposed to Y. pestis 201, both via s.c. or intranasal (i.n.) routes of administration. Moreover, when exposed to 200-400 LD50 Y. pestis strain 201Δcaf1 (non-encapsulated Y. pestis), EV76Δyp2 or EV76Δyp1&yp2 are able to afford about 50% protection to i.n. challenges, significantly better than the protection afforded by EV76. On 120 day, mice immunized with EV76Δyp2 or EV76Δyp1&yp2 cleared the i.n. challenge of Y. pestis 201-lux as quickly as those immunized with EV76, demonstrating 90-100% protection. Our results demonstrated that deletion of the yp2 gene is an effective strategy to attenuate virulence of Y. pestis EV76 while improving immunogenicity. Furthermore, EV76Δyp2 is a promising candidate for conferring protection against the pneumonic and bubonic forms of plague.

Comparative Lysine Acetylome Analysis of Y. pestis YfiQ/CobB Mutants Reveals that Acetylation of SlyA Lys73 Significantly Promotes Biofilm Formation of Y. pestis.

Increasing evidence shows that protein lysine acetylation is involved in almost every aspect of cellular physiology in bacteria. Yersinia pestis is a flea-borne pathogen responsible for millions of human deaths in three global pandemics. However, the functional role of lysine acetylation in this pathogen remains unclear. Here, we found more acetylated proteins and a higher degree of acetylation in Y. pestis grown under mammalian host (Mh) conditions than under flea vector (Fv) conditions, suggesting that protein acetylation could significantly change during fleabite transmission. Comparative acetylome analysis of mutants of YfiQ and CobB, the major acetyltransferase and deacetylase of Y. pestis, respectively, identified 23 YfiQ-dependent and 315 CobB-dependent acetylated proteins. Further results demonstrated that acetylation of Lys73 of the SlyA protein, a MarR-family transcriptional regulator, inhibits its binding to the promoter of target genes, including hmsT that encodes diguanylate cyclase responsible for the synthesis of c-di-GMP, and significantly enhances biofilm formation of Y. pestis. Our study presents the first extensive acetylome data of Y. pestis and a critical resource for the functional study of lysine acetylation in this pathogen. IMPORTANCE Yersinia pestis is the etiological agent of plague, historically responsible for three global pandemics. The 2017 plague epidemic in Madagascar was a reminder that Y. pestis remains a real threat in many parts of the world. Plague is a zoonotic disease that primarily infects rodents via fleabite, and transmission of Y. pestis from infected fleas to mammals requires rapid adaptive responses to adverse host environments to establish infection. Our study provides the first global profiling of lysine acetylation derived from mass spectrometry analysis in Y. pestis. Our data set can serve as a critical resource for the functional study of lysine acetylation in Y. pestis and provides new molecular insight into the physiological role of lysine acetylation in proteins. More importantly, we found that acetylation of Lys73 of SlyA significantly promotes biofilm formation of Y. pestis, indicating that bacteria can use lysine acetylation to fine-tune the expression of genes to improve adaptation.

Single-cell transcriptomics of immune cells in lymph nodes reveals their composition and alterations in functional dynamics during the early stages of bubonic plague.

Bubonic plague caused by Yersinia pestis is highly infectious and often fatal. Characterization of the host immune response and its subsequent suppression by Y. pestis is critical to understanding the pathogenesis of Y. pestis. Here, we utilized single-cell RNA sequencing to systematically profile the transcriptomes of immune cells in draining lymph nodes (dLNs) during the early stage of Y. pestis infection. Dendritic cells responded to Y. pestis within 2 h post-infection (hpi), followed by the activation of macrophages/monocytes (Mφs/Mons) and recruitment of polymorphonuclear neutrophils (PMNs) to dLNs at 24 hpi. Analysis of cell-to-cell communication suggests that PMNs may be recruited to lymph nodes following the secretion of CCL9 by Mφs/Mons stimulated through CCR1-CCL9 interaction. Significant functional suppression of all the three innate immune cell types occurred during the early stage of infection. In summary, we present a dynamic immune landscape, at single-cell resolution, of murine dLNs involved in the response to Y. pestis infection, which may facilitate the understanding of the plague pathogenesis of during the early stage of infection.

Subversion of GBP-mediated host defense by E3 ligases acquired during Yersinia pestis evolution.

Plague has caused three worldwide pandemics in history, including the Black Death in medieval ages. Yersinia pestis, the etiological agent of plague, has evolved a powerful arsenal to disrupt host immune defenses during evolution from enteropathogenic Y. pseudotuberculosis. Here, we find that two functionally redundant E3 ligase of Y. pestis, YspE1 and YspE2, can be delivered via type III secretion injectisome into host cytosol where they ubiquitinate multiple guanylate-binding proteins (GBPs) for proteasomal degradation. However, Y. pseudotuberculosis has no such capability due to lacking functional YspE1/2 homologs. YspE1/2-mediated GBP degradations significantly promote the survival of Y. pestis in macrophages and strongly inhibit inflammasome activation. By contrast, Gbpchr3-/-, chr5-/- macrophages exhibit much lowered inflammasome activation independent of YspE1/2, accompanied with an enhanced replication of Y. pestis. Accordingly, Gbpchr3-/-, chr5-/- mice are more susceptible to Y. pestis. We demonstrate that Y. pestis utilizes E3 ligases to subvert GBP-mediated host defense, which appears to be newly acquired by Y. pestis during evolution.

Yersinia pestis-Induced Mitophagy That Balances Mitochondrial Homeostasis and mROS-Mediated Bactericidal Activity.

Manipulating mitochondrial homeostasis is essential for host defense against infection and pathogen survival in cells. This study reports for the first time that Y. pestis infection caused mitochondria damage that subsequently leads to the activation of Pink1/Parkin-independent mitophagy in macrophage, and the effector YopH from the type III secretion system was required for these effects. The generation of mitochondrial reactive oxygen species (mROS) by damaged mitochondria enhances the antibacterial activity of macrophages against Y. pestis and promotes apoptosis of the infected cells. Therefore, Y. pestis-induced mitophagy was employed to eliminate dysfunctional mitochondria and relieve the mROS accumulation. This study reveals a novel role for YopH of Y. pestis in damaging host macrophage mitochondria during plague infection and underlines the vital role of mitophagy in maintaining mitochondrial homeostasis by clearing bacteria-damaged mitochondria. The results show that mitophagy or mitochondrial fission manipulation could be used as a new strategy to treat plague. IMPORTANCE Y. pestis, the pathogen of plague, also known as the "Black Death," has caused millions of deaths throughout history. This study reports that Y. pestis infection induces mitochondrial fragmentation and abnormal mROS accumulation, and releases mitochondrial contents into the cytoplasm in macrophages. mROS promotes the antibacterial activity of macrophages against Y. pestis and increases apoptosis of the infected cells. PINK-Parkin-independent mitophagy is activated to balance mitochondrial homeostasis and mROS-induced bactericidal activity in Y. pestis-infected macrophages. These findings deepen the understanding of Y. pestis pathogenesis on mitochondria damage to disturb the host cellular immune elimination. Manipulating mitophagic activity or mitochondrial fission may be a novel therapeutic approach to treat plague.

Bmal1 inhibits phenotypic transformation of hepatic stellate cells in liver fibrosis via IDH1/α-KG-mediated glycolysis.

Hepatic stellate cells (HSCs) play an important role in the initiation and development of liver fibrogenesis, and abnormal glucose metabolism is increasingly being considered a crucial factor controlling phenotypic transformation in HSCs. However, the role of the factors affecting glycolysis in HSCs in the experimental models of liver fibrosis has not been completely elucidated. In this study, we showed that glycolysis was significantly enhanced, while the expression of brain and muscle arnt-like protein-1 (Bmal1) was downregulated in fibrotic liver tissues of mice, primary HSCs, and transforming growth factor-ß1 (TGF-ß1)-induced LX2 cells. Overexpression of Bmal1 in TGF-ß1-induced LX2 cells blocked glycolysis and inhibited the proliferation and phenotypic transformation of activated HSCs. We further confirmed the protective effect of Bmal1 in liver fibrosis by overexpressing Bmal1 from hepatic adeno-associated virus 8 in mice. In addition, we also showed that the regulation of glycolysis by Bmal1 is mediated by the isocitrate dehydrogenase 1/α-ketoglutarate (IDH1/α-KG) pathway. Collectively, our results indicated that a novel Bmal1-IDH1/α-KG axis may be involved in regulating glycolysis of activated HSCs and might hence be used as a therapeutic target for alleviating liver fibrosis.

Proteogenomic discovery of sORF-encoded peptides associated with bacterial virulence in Yersinia pestis.

Plague caused by Yersinia pestis is one of the deadliest diseases. However, many molecular mechanisms of bacterial virulence remain unclear. This study engaged in the discovery of small open reading frame (sORF)-encoded peptides (SEPs) in Y. pestis. An integrated proteogenomic pipeline was established, and an atlas containing 76 SEPs was described. Bioinformatic analysis indicated that 20% of these SEPs were secreted or localized to the transmembrane and that 33% contained functional domains. Two SEPs, named SEPs-yp1 and -yp2 and encoded in noncoding regions, were selected by comparative peptidomics analysis under host-specific environments and high-salinity stress. They displayed important roles in the regulation of antiphagocytic capability in a thorough functional assay. Remarkable attenuation of virulence in mice was observed in the SEP-deleted mutants. Further global proteomic analysis indicated that SEPs-yp1 and -yp2 affected the bacterial metabolic pathways, and SEP-yp1 was associated with the bacterial virulence by modulating the expression of key virulence factors of the Yersinia type III secretion system. Our study provides a rich resource for research on Y. pestis and plague, and the findings on SEP-yp1 and SEP-yp2 shed light on the molecular mechanism of bacterial virulence.

Highly Specific and Sensitive Detection of Yersinia pestis by Portable Cas12a-UPTLFA Platform.

The recent discovery of collateral cleavage activity of class-II clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas) makes CRISPR-based diagnosis a potential high-accuracy nucleic acid detection method. Colloidal gold-based lateral flow immunochromatographic assay (LFA), which has been combined with CRISPR/Cas-based nucleic detection, usually associates with drawbacks of relative high background and the subjectivity in naked-eye read-out of the results. Here, we developed a novel system composed of Cas12a-based nucleic acid detection and up-converting phosphor technology (UPT)-based LFA (UPT-LFA), termed Cas12a-UPTLFA. We further demonstrated the utility of this platform in highly sensitive and specific detection of Yersinia pestis, the causative agent of the deadly plague. Due to high infectivity and mortality, as well as the potential to be misused as bioterrorism agent, a culture-free, ultrasensitive, specific, and rapid detection method for Y. pestis has long been desired. By incorporating isothermal recombinase polymerase amplification, the Cas12a-UPTLFA we established can successfully detect genomic DNA of Y. pestis as low as 3 attomolar (aM) and exhibited high sensitivity (93.75%) and specificity (90.63%) for detection of spiked blood samples with a detection limit of 102 colony-forming unit per 100 µl of mouse blood. With a portable biosensor, Cas12a-UPTLFA assay can be operated easily by non-professional personnel. Taken together, we have developed a novel Cas12a-UPTLFA platform for rapid detection of Y. pestis with high sensitivity and specificity, which is portable, not expensive, and easy to operate as a point-of-care method. This detection system can easily be extended to detect other pathogens and holds great promise for on-site detection of emerging infectious pathogens.

Obtaining Specific Sequence Tags for Yersinia pestis and Visually Detecting Them Using the CRISPR-Cas12a System.

Three worldwide historical plague pandemics resulted in millions of deaths. Yersinia pestis, the etiologic agent of plague, is also a potential bioterrorist weapon. Simple, rapid, and specific detection of Y. pestis is important to prevent and control plague. However, the high similarity between Y. pestis and its sister species within the same genus makes detection work problematic. Here, the genome sequence from the Y. pestis CO92 strain was electronically separated into millions of fragments. These fragments were analyzed and compared with the genome sequences of 539 Y. pestis strains and 572 strains of 20 species within the Yersinia genus. Altogether, 97 Y. pestis-specific tags containing two or more single nucleotide polymorphism sites were screened out. These 97 tags efficiently distinguished Y. pestis from all other closely related species. We chose four of these tags to design a Cas12a-based detection system. PCR-fluorescence methodology was used to test the specificity of these tags, and the results showed that the fluorescence intensity produced by Y. pestis was significantly higher than that of non-Y. pestis (p < 0.0001). We then employed recombinase polymerase amplification and lateral flow dipsticks to visualize the results. Our newly developed plasmid-independent, species-specific library of tags completely and effectively screened chromosomal sequences. The detection limit of our four-tag Cas12a system reached picogram levels.

Secretome and Comparative Proteomics of Yersinia pestis Identify Two Novel E3 Ubiquitin Ligases That Contribute to Plague Virulence.

Plague is a zoonotic disease that primarily infects rodents via fleabite. Transmission from flea to host niches requires rapid adaption of Yersinia pestis to the outer environments to establish infection. Here, quantitative proteome and secretome analyses of Y. pestis grown under conditions mimicking the two typical niches, i.e., the mammalian host (Mh) and the flea vector (Fv), were performed to understand the adaption strategies of this deadly pathogen. A secretome of Y. pestis containing 308 proteins has been identified using TMT-labeling mass spectrometry analysis. Although some proteins are known to be secreted, such as the type III secretion substrates, PsaA and F1 antigen, most of them were found to be secretory proteins for the first time. Comparative proteomic analysis showed that membrane proteins, chaperonins and stress response proteins are significantly upregulated under the Mh condition, among which the previously uncharacterized proteins YP_3416∼YP_3418 are remarkable because they cannot only be secreted but also translocated into HeLa cells by Y. pestis. We further demonstrated that the purified YP_3416 and YP_3418 exhibited E3 ubiquitin ligase activity in in vitro ubiquitination assay and yp_3416∼3418 deletion mutant of Y. pestis showed significant virulence attenuation in mice. Taken together, our results represent the first Y. pestis secretome, which will promote the better understanding of Y. pestis pathogenesis, as well as the development of new strategies for treatment and prevention of plague.

Reversible Gene Expression Control in Yersinia pestis by Using an Optimized CRISPR Interference System.

Many genes in the bacterial pathogen Yersinia pestis, the causative agent of three plague pandemics, remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been shown to be an effective tool for gene knockdown in model bacteria. In this system, a catalytically dead Cas9 (dCas9) and a small guide RNA (sgRNA) form a complex, binding to the specific DNA target through base pairing, thereby impeding RNA polymerase binding and causing target gene repression. Here, we introduce an optimized CRISPRi system using Streptococcus pyogenes Cas9-derived dCas9 for gene knockdown in Y. pestis Multiple genes harbored on either the chromosome or plasmids of Y. pestis were efficiently knocked down (up to 380-fold) in a strictly anhydrotetracycline-inducible manner using this CRISPRi approach. Knockdown of hmsH (responsible for biofilm formation) or cspB (encoding a cold shock protein) resulted in greatly decreased biofilm formation or impaired cold tolerance in in vitro phenotypic assays. Furthermore, silencing of the virulence-associated genes yscB or ail using this CRISPRi system resulted in attenuation of virulence in HeLa cells and mice similar to that previously reported for yscB and ail null mutants. Taken together, our results confirm that this optimized CRISPRi system can reversibly and efficiently repress the expression of target genes in Y. pestis, providing an alternative to conventional gene knockdown techniques, as well as a strategy for high-throughput phenotypic screening of Y. pestis genes with unknown functions.IMPORTANCEYersiniapestis is a lethal pathogen responsible for millions of human deaths in history. It has also attracted much attention for potential uses as a bioweapon or bioterrorism agent, against which new vaccines are desperately needed. However, many Y. pestis genes remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been successfully used in a variety of bacteria in functional genomic studies, but no such genetic tool has been reported in Y. pestis Here, we systematically optimized the CRISPRi approach for use in Y. pestis, which ultimately repressed target gene expression with high efficiency in a reversible manner. Knockdown of functional genes using this method produced phenotypes that were readily detected by in vitro assays, cell infection assays, and mouse infection experiments. This is a report of a CRISPRi approach in Y. pestis and highlights the potential use of this approach in high-throughput functional genomics studies of this pathogen.

Interferon-stimulated gene 15 enters posttranslational modifications of p53.

The tumor suppressor protein p53 is a central governor of various cellular signals. It is well accepted that ubiquitination as well as ubiquitin-like (UBL) modifications of p53 protein is critical in the control of its activity. Interferon-stimulated gene 15 (ISG15) is a well-known UBL protein with pleiotropic functions, serving both as a free intracellular molecule and as a modifier by conjugating to target proteins. Initially, attentions have historically focused on the antiviral effects of ISG15 pathway. Remarkably, a significant role in the processes of autophagy, DNA repair, and protein translation provided considerable insight into the new functions of ISG15 pathway. Despite the deterministic revelation of the relation between ISG15 and p53, the functional consequence of p53 ISGylation appears somewhat confused. More important, more recent studies have hinted p53 ubiquitination or other UBL modifications that might interconnect with its ISGylation. Here, we aim to summarize the current knowledge of p53 ISGylation and the differences in other significant modifications, which would be beneficial for the development of p53-based cancer therapy.

Protein Acetylation Mediated by YfiQ and CobB Is Involved in the Virulence and Stress Response of Yersinia pestis.

Recent studies revealed that acetylation is a widely used protein modification in prokaryotic organisms. The major protein acetylation acetyltransferase YfiQ and the sirtuin-like deacetylase CobB have been found to be involved in basic physiological processes, such as primary metabolism, chemotaxis, and stress responses, in Escherichia coli and Salmonella However, little is known about protein acetylation modifications in Yersinia pestis, a lethal pathogen responsible for millions of human deaths in three worldwide pandemics. Here we found that Yp_0659 and Yp_1760 of Y. pestis encode the major protein acetylation acetyltransferase YfiQ and the sirtuin-like deacetylase CobB, respectively, which can acetylate and deacetylate PhoP enzymatically in vitro Protein acetylation impairment in cobB and yfiQ mutants greatly decreased bacterial tolerance to cold, hot, high-salt, and acidic environments. Our comparative transcriptomic data revealed that the strongly decreased tolerance to stress stimuli was probably related to downregulation of the genes encoding the heat shock proteins (HtpG, HslV, HslR, and IbpA), cold shock proteins (CspC and CspA1), and acid resistance proteins (HdeB and AdiA). We found that the reversible acetylation mediated by CobB and YfiQ conferred attenuation of virulence, probably partially due to the decreased expression of the psaABCDEF operon, which encodes Psa fimbriae that play a key role in virulence of Y. pestis This is the first report, to our knowledge, on the roles of protein acetylation modification in stress responses, biofilm formation, and virulence of Y. pestis.

Yersinia pestis YopK Inhibits Bacterial Adhesion to Host Cells by Binding to the Extracellular Matrix Adaptor Protein Matrilin-2.

Pathogenic yersiniae harbor a type III secretion system (T3SS) that injects Yersinia outer protein (Yop) into host cells. YopK has been shown to control Yop translocation and prevent inflammasome recognition of the T3SS by the innate immune system. Here, we demonstrate that YopK inhibits bacterial adherence to host cells by binding to the extracellular matrix adaptor protein matrilin-2 (MATN2). YopK binds to MATN2, and deleting amino acids 91 to 124 disrupts binding of YopK to MATN2. A yopK null mutant exhibits a hyperadhesive phenotype, which could be responsible for the established Yop hypertranslocation phenotype of yopK mutants. Expression of YopK, but not YopKΔ91-124, in a yopK mutant restored the wild-type phenotypes of adhesion and Yop translocation, suggesting that binding to MATN2 might be essential for YopK to inhibit bacterial adhesion and negatively regulate Yop translocation. A green fluorescent protein (GFP)-YopK fusion specifically binds to the endogenous MATN2 on the surface of HeLa cells, whereas GFP-YopKΔ91-124 cannot. Addition of purified YopK protein during infection decreased adhesion of Y. pestis to HeLa cells, while YopKΔ91-124 protein showed no effect. Taking these results together, we propose a model that the T3SS-secreted YopK hinders bacterial adhesion to HeLa cells by binding to MATN2, which is ubiquitously exposed on eukaryotic cells.

An Interaction between the Inner Rod Protein YscI and the Needle Protein YscF Is Required to Assemble the Needle Structure of the Yersinia Type Three Secretion System.

The type III secretion system is a highly conserved virulence mechanism that is widely distributed in Gram-negative bacteria. It has a syringe-like structure composed of a multi-ring basal body that spans the bacterial envelope and a projecting needle that delivers virulence effectors into host cells. Here, we showed that the Yersinia inner rod protein YscI directly interacts with the needle protein YscF inside the bacterial cells and that this interaction depends on amino acid residues 83-102 in the carboxyl terminus of YscI. Alanine substitution of Trp-85 or Ser-86 abrogated the binding of YscI to YscF as well as needle assembly and the secretion of effectors (Yops) and the needle tip protein LcrV. However, yscI null mutants that were trans-complemented with YscI mutants that bind YscF still assembled the needle and secreted Yops, demonstrating that a direct interaction between YscF and YscI is critical for these processes. Consistently, YscI mutants that did not bind YscF resulted in greatly decreased HeLa cell cytotoxicity. Together, these results show that YscI participates in needle assembly by directly interacting with YscF.

Host transcriptomic responses to pneumonic plague reveal that Yersinia pestis inhibits both the initial adaptive and innate immune responses in mice.

Pneumonic plague is the most deadly form of infection caused by Yersinia pestis and can progress extremely fast. However, our understanding on the host transcriptomic response to pneumonic plague is insufficient. Here, we used RNA-sequencing technology to analyze transcriptomic responses in mice infected with fully virulent strain 201 or EV76, a live attenuated vaccine strain lacking the pigmentation locus. Approximately 600 differentially expressed genes (DEGs) were detected in lungs from both 201- and EV76-infected mice at 12h post-infection (hpi). DEGs in lungs of 201-infected mice exceeded 2000 at 48hpi, accompanied by sustained large numbers of DEGs in the liver and spleen; however, limited numbers of DEGs were detected in those organs of EV-infected mice. Remarkably, DEGs in lungs were significantly enriched in critical immune responses pathways in EV76-infected but not 201-infected mice, including antigen processing and presentation, T cell receptor signaling among others. Pathological and bacterial load analyses confirmed the rapid systemic dissemination of 201-infection and the confined EV76-infection in lungs. Our results suggest that fully virulent Y. pestis inhibits both the innate and adaptive immune responses that are substantially stimulated in a self-limited infection, which update our holistic views on the transcriptomic response to pneumonic plague.

MicroRNA-145 Increases the Apoptosis of Activated Hepatic Stellate Cells Induced by TRAIL through NF-κB Signaling Pathway.

During the liver fibrosis recovery stage tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can effectively induce apoptosis of activated hepatic stellate cells (HSCs). Normal hepatic stellate cells are resistant to TRAIL cytotoxicity. Therefore, enhancing the sensitivity of TRAIL-induced apoptosis of HSCs may be useful to treat hepatic fibrogenesis. Here, we demonstrated that miR-145 and TRAIL were down-regulated in both liver fibrosis tissue samples and transforming growth factor-ß1 induced HSCs, concomitant with increased the expression of ZEB2. In addition, we found that mimics-mediated over-expression of miR-145 led to resistance to the ZEB2 expression and up-regulation of the TRAIL-induced apoptosis after treatment of LX-2 cells with TRAIL. Furthermore, ZEB2-siRNA transfected LX-2 cells showed the increased sensitivity to TRAIL-induced apoptosis. Whereas, opposite results were obtained in miR-145-inhibitor group or ZEB2 plasmid group. Moreover, miR-145 regulated ZEB2 gene expression by specifically interacting with the 3'-UTR of ZEB2 mRNA to inhibit the expression of ZEB2. Further studies showed that the over-expression of ZEB2 could inhibit TRAIL-induced apoptosis via inhibiting nuclear factor-κB (NF-κB) signaling pathway in LX-2 cells. Collectively, our data suggest that up-regulation of miR-145 can down-regulate ZEB2 expression, consequently promoting TRAIL-induced apoptosis in LX-2 cells through NF-κB signaling pathway, which facilitates the resolution of liver fibrosis.