The mitochondrial carboxylase PCCA interacts with Listeria monocytogenes phospholipase PlcB to modulate bacterial survival.

Listeria monocytogenes, a prominent foodborne pathogen responsible for zoonotic infections, owes a significant portion of its virulence to the presence of the phospholipase PlcB. In this study, we performed an in-depth examination of the intricate relationship between L. monocytogenes PlcB and host cell mitochondria, unveiling a novel participant in bacterial survival: the mitochondrial carboxylase propionyl-coenzyme A carboxylase (PCCA). Our investigation uncovered previously unexplored levels of interaction and colocalization between PCCA and PlcB within host cells, with particular emphasis on the amino acids 504-508 of PCCA, which play a pivotal role in this partnership. To assess the effect of PCCA expression on L. monocytogenes proliferation, PCCA expression levels were manipulated by siRNA-si-PCCA or pCMV-N-HA-PCCA plasmid transfection. Our findings demonstrated a clear inverse correlation between PCCA expression levels and the proliferation of L. monocytogenes. Furthermore, the effect of L. monocytogenes infection on PCCA expression was investigated by assessing PCCA mRNA and protein expression in HeLa cells infected with L. monocytogenes. These results indicate that L. monocytogenes infection did not significantly alter PCCA expression. These findings led us to propose that PCCA represents a novel participant in L. monocytogenes survival, and its abundance has a detrimental impact on bacterial proliferation. This suggests that L. monocytogenes may employ PlcB-PCCA interactions to maintain stable PCCA expression, representing a unique pro-survival strategy distinct from that of other intracellular bacterial pathogens. IMPORTANCE: Mitochondria represent attractive targets for pathogenic bacteria seeking to modulate host cellular processes to promote their survival and replication. Our current study has uncovered mitochondrial carboxylase propionyl-coenzyme A carboxylase (PCCA) as a novel host cell protein that interacts with L. monocytogenes PlcB. The results demonstrate that PCCA plays a negative regulatory role in L. monocytogenes infection, as heightened PCCA levels are associated with reduced bacterial survival and persistence. However, L. monocytogenes may exploit the PlcB-PCCA interaction to maintain stable PCCA expression and establish a favorable intracellular milieu for bacterial infection. Our findings shed new light on the intricate interplay between bacterial pathogens and host cell mitochondria, while also highlighting the potential of mitochondrial metabolic enzymes as antimicrobial agents.

A multiplex digital PCR assay for detection and quantitation of porcine circovirus type 2 and type 3.

Porcine circovirus (PCV) has become a major pathogen, causing major economic losses in the global pig industry, and PCV type 2 (PCV2) and 3 (PCV3) are distributed worldwide. We designed specific primer and probe sequences targeting PCV2 Cap and PCV3 Rap and developed a multiplex crystal digital PCR (cdPCR) method after optimizing the primer concentration, probe concentration, and annealing temperature. The multiplex cdPCR assay permits precise and differential detection of PCV2 and PCV3, with a limit of detection of 1.39 × 101 and 1.27 × 101 copies/reaction, respectively, and no cross-reaction with other porcine viruses was observed. The intra-assay and interassay coefficients of variation (CVs) were less than 8.75%, indicating good repeatability and reproducibility. To evaluate the practical value of this assay, 40 tissue samples and 70 feed samples were tested for both PCV2 and PCV3 by cdPCR and quantitative PCR (qPCR). Using multiplex cdPCR, the rates of PCV2 infection, PCV3 infection, and coinfection were 28.45%, 1.72%, and 12.93%, respectively, and using multiplex qPCR, they were 25.00%, 0.86%, and 4.31%, respectively This highly specific and sensitive multiplex cdPCR thus allows accurate simultaneous detection of PCV2 and PCV3, and it is particularly well suited for applications that require the detection of small amounts of input nucleic acid or samples with intensive processing and complex matrices.

Epidemiology, pathogenesis, immune evasion mechanism and vaccine development of porcine Deltacoronavirus.

Coronaviruses have been identified as pathogens of gastrointestinal and respiratory diseases in humans and various animal species. In recent years, the global spread of new coronaviruses has had profound influences for global public health and economies worldwide. As highly pathogenic zoonotic viruses, coronaviruses have become the focus of current research. Porcine Deltacoronavirus (PDCoV), an enterovirus belonging to the family of coronaviruses, has emerged on a global scale in the past decade and significantly influenced the swine industry. Moreover, PDCoV infects not only pigs but also other species, including humans, chickens and cattles, exhibiting a broad host tropism. This emphasizes the need for in-depth studies on coronaviruses to mitigate their potential threats. In this review, we provided a comprehensive summary of the current studies on PDCoV. We first reviewed the epidemiological investigations on the global prevalence and distribution of PDCoV. Then, we delved into the studies on the pathogenesis of PDCoV to understand the mechanisms how the virus impacts its hosts. Furthermore, we also presented some exploration studies on the immune evasion mechanisms of the virus to enhance the understanding of host-virus interactions. Despite current limitations in vaccine development for PDCoV, we highlighted the inhibitory effects observed with certain substances, which offers a potential direction for future research endeavors. In conclusion, this review summarized the scientific findings in epidemiology, pathogenesis, immune evasion mechanisms and vaccine development of PDCoV. The ongoing exploration of potential vaccine candidates and the insights gained from inhibitory substances have provided a solid foundation for future vaccine development to prevent and control diseases associated with PDCoV.

Detection and differentiation of seven porcine respiratory pathogens using a multiplex ligation-dependent probe amplification assay.

Respiratory diseases due to viral or bacterial agents, either alone or in combination, cause substantial economic burdens to the swine industry worldwide. Rapid and reliable detection of causal pathogens is crucial for effective epidemiological surveillance and disease management. This research aimed to employ the multiplex ligation-dependent probe amplification (MLPA) assay for simultaneous detection of seven distinct pathogens causing respiratory problems in swine, porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), porcine respiratory coronavirus (PRCV), porcine circovirus type 2 (PCV2), Pasteurella multocida, Actinobacillus pleuropneumoniae, and Glässerella parasuis. The results indicated no probe cross-reactivity among the seven target agents with other swine pathogens. The detection limits ranged from 5 to 34 copies per assay for the target organisms. The MLPA assay was evaluated with 88 samples and compared to real-time or multiplex PCR for the target pathogens. The MLPA assay demonstrated high relative test sensitivities (100 %) and reasonable to good relative specificities at 62.5 %, 95.1 %, 86.8 %, and 97.6 % for PRRSV, P. multocida, G. parasuis, and PCV2, respectively, relative to comparator PCR assays. In 71 samples where MLPA and comparator PCR assays matched exactly, infections were detected in 64 samples (90.1 %), with PRRSV being the most commonly found virus and 50.7 % of the samples showing co-infection with two to five of the pathogens. This approach serves as a valuable tool for conducting differential diagnoses and epidemiological investigations of pathogen prevalence within swine populations.

The nucleocapsid protein facilitates p53 ubiquitination-dependent proteasomal degradation via recruiting host ubiquitin ligase COP1 in PEDV infection.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of the coronavirus family and caused severe economic losses to the global swine industry. Previous studies have established that p53 is a host restriction factor for PEDV infection, and p53 degradation occurs in PEDV-infected cells. However, the underlying molecular mechanisms through which PEDV viral proteins regulate p53 degradation remain unclear. In this study, we found that PEDV infection or expression of the nucleocapsid protein downregulates p53 through a post-translational mechanism: increasing the ubiquitination of p53 and preventing its nuclear translocation. We also show that the PEDV N protein functions by recruiting the E3 ubiquitin ligase COP1 and suppressing COP1 self-ubiquitination and protein degradation, thereby augmenting COP1-mediated degradation of p53. Additionally, COP1 knockdown compromises N-mediated p53 degradation. Functional mapping using truncation analysis showed that the N-terminal domains of N protein were responsible for interacting with COP1 and critical for COP1 stability and p53 degradation. The results presented here suggest the COP1-dependent mechanism for PEDV N protein to abolish p53 activity. This study significantly increases our understanding of PEDV in antagonizing the host antiviral factor p53 and will help initiate novel antiviral strategies against PEDV.

Application of Three-Dimension Printing Nano-Carbonated-Hydroxylapatite to the Repair of Defects in Rabbit Bone.

Introduction: Hydroxylapatite (HAp) is a biodegradable bone graft material with high biocompatibility. However, the clinical application of HAp has been limited due to the poor absorption rate in vivo. Methods: In this study, carbonated hydroxylapatite (CHAp) with a chemical composition similar to natural bone was synthesized. HAp and CHAp scaffolds were fabricated by 3D printing. Each material was designed by two types of scaffold model with a maximum width of 8 mm and a thickness of 2 mm, ie, structure I (round shape) and structure II (grid shape). Then, the HAp scaffolds were loaded with lutein. These scaffolds were implanted into the 8 mm bone defect on the top of the rabbit skull within 3 hours in the morning. The curative effects of the scaffolds were assessed two months after implantation. Results: The 3D printed scaffolds did not cause severe inflammation or rejection after implantation. It showed that the porous structures allow bone cells to enter into the scaffolds. Furthermore, CHAp scaffolds were more biocompatible than HAp scaffolds, and showed a higher level of degradation and new bone formation after implantation. Structure II scaffolds with a smaller mineral content degraded faster than structure I, while structure I had better osteoconductive properties than structure II. Besides, the addition of lutein significantly enhanced the rate of new bone formation. Discussion: The uniqueness of this study lies in the synthesis of 3D printed CHAp scaffolds and the implantation of CHAp in rabbit bone defects. The incorporation of suitable carbonate and lutein into HAp can enhance the osteoinductivity of the graft, and CHAp has a faster degradation rate in vivo, all of which provide a new reference for the research and application of apatite-based composites.

Molecular mechanism of bovine Gasdermin D-mediated pyroptosis.

Pyroptosis is a form of programmed cell death characterized by cell swelling, pore formation in the plasma membrane, lysis, and releases of cytoplasmic contents. To date, the molecular mechanism of human and murine Gasdermin D-mediated pyroptosis have been fully investigated. However, studies focusing on molecular mechanism of bovine Gasdermin D (bGSDMD)-mediated pyroptosis and its function against pathogenic infection were unclear. In the present study, we demonstrate that bovine caspase-1 (bCaspase-1) cleaves bGSDMD at amino acid residue D277 to produce an N-terminal fragment (bGSDMD-p30) which leads to pyroptosis. The amino acid residues T238 and F239 are critical for bGSDMD-p30-mediated pyroptosis. The loop aa 278-299, L293 and A380 are the key sites for autoinhibitory structure of the full length of bGSDMD. In addition, bCaspase-3 also cleaves bGSDMD at residue Asp86 without inducing cell death. Therefore, our study provides the first detailed elucidation of the mechanism of bovine GSDMD-mediated pyroptosis. The results will establish a significant foundation for future research on the role of pyroptosis in bovine infectious diseases.

Streptococcus suis subtilisin-like serine proteases SspA-1 and SspA-2 interplay with complement C3a and C5a to facilitate bacterial immune evasion and infection.

Streptococcus suis (S. suis), a significant zoonotic bacterial pathogen impacting swine and human, is associated with severe systemic diseases such as streptococcal toxic shock-like syndrome, meningitis, septicaemia, and abrupt fatality. The multifaceted roles of complement components C5a and C3a extend to orchestrating inflammatory cells recruitment, oxidative burst induction, and cytokines release. Despite the pivotal role of subtilisin-like serine proteases in S. suis pathogenicity, their involvement in immune evasion remains underexplored. In the present study, we identify two cell wall-anchored subtilisin-like serine proteases in S. suis, SspA-1 and SspA-2, as binding partners for C3a and C5a. Through Co-Immunoprecipitation, Enzyme-Linked Immunosorbent and Far-Western Blotting Assays, we validate their interactions with the aforementioned components. However, SspA-1 and SspA-2 have no cleavage activity against complement C3a and C5a performed by Cleavage assay. Chemotaxis assays reveal that recombinant SspA-1 and SspA-2 effectively attenuate monocyte chemotaxis towards C3a and C5a. Notably, the ΔsspA-1, ΔsspA-1, and ΔsspA-1/2 mutant strains exhibit compromised survival in blood, and resistance of opsonophagocytosis, alongside impaired survival in blood and in vivo colonization compared to the parental strain SC-19. Critical insights from the murine and Galleria mellonella larva infection models further underscore the significance of sspA-1 in altering mortality rates. Collectively, our findings indicate that SspA-1 and SspA-2 are novel binding proteins for C3a and C5a, thereby shedding light on their pivotal roles in S. suis immune evasion and the pathogenesis.

Mycobacterial PE12 protein promotes bacterial survival through inhibiting cell apoptosis.

Mycobacterial PE_PGRS family proteins play key roles in pathogen-host interaction. However, the function of most PE_PGRS proteins remains unknown. In this study, we characterized the role of PE12 of Mycobacterium bovis (M. bovis) on bacterial growth, bacterial survival, and host cell apoptosis. Transcriptome sequencing of infected THP-1 cells was also performed. Compared to Ms_Vec, we found that M. bovis PE12 did not alter the colony morphology of M. smegmatis. The survival of Ms_PE12 was obviously higher than that of Ms_Vec. Furthermore, PE12 significantly suppressed the apoptosis of THP-1 induced by M. smegmatis infection. Transcriptome analysis results showed that there were 70 downregulated genes in the Ms_PE12 infection group in comparison with the Ms_Vec infection group, and these differentially expressed genes were enriched in 240 downregulated GO terms and 6 KEGG pathways. The downregulated expression genes are involved in cell adhesion, phagocytosis, apoptosis, inflammatory response, glycolysis and transmembrane transporter activity. Taken together, our study reveals that PE12 can suppress apoptosis and inhibit proinflammatory cytokine response. We propose that PE12 is related to macrophage phagocytosis and apoptosis, providing useful information to the pathogenic mechanisms of M. bovis.

Canine distemper virus (CDV)-neutralizing activities of an anti-CDV canine-derived single-chain variable antibody fragment 4-15 (scFv 4-15) screened by phage display technology.

Canine distemper virus (CDV) is a highly contagious pathogen that causes severe diarrhea, fever and vomiting in domestic dogs, posing a serious threat to the dog breeding industry. Currently, there are no effective therapeutic agents for emergency treatment despite the availability of vaccines against CDV infection. Single-chain fragment variable (scFv) antibody has been demonstrated to effectively inhibit virus infections, suggesting a potential candidate as a therapeutic agent for canine distemper. In this study, a phage-displayed scFv library was constructed from the peripheral blood lymphocytes of dog immunized intramuscularly with live-attenuated CDV vaccine, and was subjected to four rounds of pannings against CDV. Subsequent indirect enzyme-linked immunosorbent assay screening revealed high-affinity scFv antibodies specific to CDV, and indirect immunofluorescence assay screening revealed CDV-neutralizing activity of scFv antibodies. Our results showed that a scFv antibody 4-15 (scFv 4-15) with high-affinity binding to CDV and neutralizing activity against CDV was obtained, which displayed effective therapeutic potential in vivo for dogs challenged with a lethal dose of CDV. Conclusively, the scFv 4-15 with high-affinity binding and neutralizing activity to CDV that was obtained by phage display technology provides a promising candidate for the therapeutic agents against CDV infection.

Comparative characterization of microRNA-71 of Echinococcus granulosus exosomes.

Cystic echinococcosis (CE) is a global zoonotic disease caused by Echinococcus granulosus, posing a great threat to human and animal health. MiRNAs are small regulatory noncoding RNA involved in the pathogenesis of parasitic diseases, possibly via exosomes. Egr-miR-71 has been identified as one of the miRNAs in the blood of CE patients, but its secretory characteristics and functions remains unclear. Herein, we studied the secretory and biological activity of exosomal egr-miR-71 and its immunoregulatory functions in sheep peripheral blood mononuclear cells (PBMCs). Our results showed that egr-miR-71 was enriched in the exosome secreted by protoscoleces with biological activity. These egr-miR-71-containing exosomes were easily internalized and then induced the dysregulation of cytokines (IL-10 and TNF-α), nitric oxide (NO) and key components (CD14 and IRF5) in the LPS/TLR4 pathway in the coincubated sheep PBMCs. Similarly, egr-miR-71 overexpression also altered the immune functions but exhibited obvious differences in regulation of the cytokines and key components, preferably inhibiting proinflammatory cytokines (IL-1α, IL-1ß and TNF-α). These results demonstrate that exosomal egr-miR-71 is bioactive and capacity of immunomodulation of PBMCs, potentially being involved in immune responses during E. granulosus infection.

Title: Caractérisation comparative du microARN-71 des exosomes d'Echinococcus granulosus. Abstract: L'échinococcose kystique (EK) est une maladie zoonotique mondiale causée par Echinococcus granulosus, représentant une grande menace pour la santé humaine et animale. Les miARN sont des petits ARN régulateurs non codants impliqués dans la pathogenèse des maladies parasitaires, éventuellement via les exosomes. Egr-miR-71 a été identifié comme l'un des miARN présents dans le sang des patients atteints d'EK, mais ses caractéristiques et fonctions sécrétoires restent floues. Ici, nous avons étudié l'activité sécrétoire et biologique du egr-miR-71 exosomal et ses fonctions immunorégulatrices dans les cellules mononucléées du sang périphérique (CMSP) de mouton. Nos résultats ont montré qu'egr-miR-71 était enrichi dans l'exosome sécrété par les protoscolex ayant une activité biologique. Ces exosomes contenant egr-miR-71 ont été facilement internalisés et ont ensuite induit la dérégulation des cytokines (IL-10 et TNF-α), de l'oxyde nitrique (NO) et des composants clés (CD14 et IRF5) de la voie LPS/TLR4 dans les CMSP de mouton co-incubées. De même, la surexpression d'egr-miR-71 a également modifié les fonctions immunitaires mais a montré des différences évidentes dans la régulation des cytokines et des composants clés, inhibant de préférence les cytokines pro-inflammatoires (IL-1α, IL-1ß et TNF-α). Ces résultats démontrent que l'egr-miR-71 exosomal est bioactif et possède une capacité d'immunomodulation des CMSP, potentiellement impliquée dans les réponses immunitaires lors d'une infection à E. granulosus.

DegU-mediated suppression of carbohydrate uptake in Listeria monocytogenes increases adaptation to oxidative stress.

The foodborne bacterial pathogen Listeria monocytogenes exhibits remarkable survival capabilities under challenging conditions, severely threatening food safety and human health. The orphan regulator DegU is a pleiotropic regulator required for bacterial environmental adaptation. However, the specific mechanism of how DegU participates in oxidative stress tolerance remains unknown in L. monocytogenes. In this study, we demonstrate that DegU suppresses carbohydrate uptake under stress conditions by altering global transcriptional profiles, particularly by modulating the transcription of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS)-related genes, such as ptsH, ptsI, and hprK. Specifically, in the absence of degU, the transcripts of ptsI are significantly upregulated and those of hprK are significantly downregulated in response to copper ion-induced stress. Overexpression of ptsI significantly increases bacterial growth in vitro, while overexpression of hprK leads to a decrease in growth. We further demonstrate that DegU directly senses oxidative stress, downregulates ptsI transcription, and upregulates hprK transcription. Additionally, through an electrophoretic mobility shift assay, we demonstrate that DegU directly regulates the transcription of ptsI and hprK by binding to specific regions within their respective promoter sequences. Notably, the putative pivotal DegU binding sequence for ptsI is located from 38 to 68 base pairs upstream of the ptsH transcription start site (TSS), whereas for hprK, it is mapped from 36 to 124 base pairs upstream of the hprK TSS. In summary, we elucidate that DegU plays a significant role in suppressing carbohydrate uptake in response to oxidative stress through the direct regulation of ptsI and hprK.ImportanceUnderstanding the adaptive mechanisms employed by Listeria monocytogenes in harsh environments is of great significance. This study focuses on investigating the role of DegU in response to oxidative stress by examining global transcriptional profiles. The results highlight the noteworthy involvement of DegU in this stress response. Specifically, DegU acts as a direct sensor of oxidative stress, leading to the modulation of gene transcription. It downregulates ptsI transcription while it upregulates hprK transcription through direct binding to their promoters. Consequently, these regulatory actions impede bacterial growth, providing a defense mechanism against stress-induced damage. These findings gained from this study may have broader implications, serving as a reference for studying adaptive mechanisms in other pathogenic bacteria and aiding in the development of targeted strategies to control L. monocytogenes and ensure food safety.

Characterization of a DsbA family protein reveals its crucial role in oxidative stress tolerance of Listeria monocytogenes.

IMPORTANCE: The adaption and tolerance to various environmental stresses are the fundamental factors for the widespread existence of Listeria monocytogenes. Anti-oxidative stress is the critical mechanism for the survival and pathogenesis of L. monocytogenes. The thioredoxin (Trx) and glutaredoxin (Grx) systems are known to contribute to the anti-oxidative stress of L. monocytogenes, but whether the Dsb system has similar roles remains unknown. This study demonstrated that the DsbA family protein Lmo1059 of L. monocytogenes participates in bacterial oxidative stress tolerance, with Cys36 as the key amino acid of its catalytic activity and anti-oxidative stress ability. It is worth noting that Lmo1059 was involved in the invading and cell-to-cell spread of L. monocytogenes. This study lays a foundation for further understanding the specific mechanisms of oxidative cysteine repair and antioxidant stress regulation of L. monocytogenes, which contributes to an in-depth understanding of the environmental adaptation mechanisms for foodborne bacterial pathogens.

Listeria monocytogenes GshF contributes to oxidative stress tolerance via regulation of the phosphoenolpyruvate-carbohydrate phosphotransferase system.

Glutathione (GSH) is an essential component of the glutaredoxin (Grx) system, and it is synthesized by the enzyme glutathione synthase GshF in Listeria monocytogenes. GSH plays a crucial role in regulating Listeria virulence by modifying the virulence factors LLO and PrfA. In this study, we investigated the involvement of L. monocytogenes GshF in oxidative tolerance and intracellular infection. Our findings revealed that the deletion of gshF resulted in a significant reduction in bacterial growth in vitro when exposed to diamide and copper ions stress. More importantly, this deletion also impaired the efficiency of invasion and proliferation in macrophages and mice organs. Furthermore, GshF influenced global transcriptional profiles, including carbohydrate and amino acid metabolism, particularly those related to the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) genes lmo1997-lmo2004, under oxidative stress conditions. In the wild-type strain, the transcription of lmo1997-lmo2004 was notably downregulated in response to copper ions and diamide stress compared to normal conditions. However, in the absence of gshF, the transcripts of lmo1997-lmo2004 were upregulated in response to these stress conditions. Notably, the deletion of iiBman (lmo2002) enhanced oxidative stress tolerance to copper ions, whereas overexpression of iiBman reduced this resistance. In conclusion, our study provides the first evidence that L. monocytogenes GshF plays a crucial role in bacterial antioxidation through the regulation of iiBman.IMPORTANCEListeria monocytogenes has developed various mechanisms to withstand oxidative stress, including the thioredoxin and glutaredoxin systems. However, the specific role of the glutathione synthase GshF, responsible for synthesizing GSH in L. monocytogenes, in oxidative tolerance remains unclear. This study aimed to elucidate the relationship between GshF and oxidative tolerance in L. monocytogenes by examining the efficiency of invasion and proliferation in macrophages and mice organs, as well as analyzing global transcriptional profiles under oxidative stress conditions. The results revealed that GshF plays a significant role in L. monocytogenes' response to oxidative stress. Notably, GshF acts to suppress the transcription of phosphoenolpyruvate-carbohydrate phosphotransferase system genes lmo1997-lmo2004, among which iiBman (lmo2002) was identified as the most critical gene for resisting oxidative stress. These findings enhance our understanding of how L. monocytogenes adapts to its environment and provide valuable insights for investigating the environmental adaptation mechanisms of other pathogenic bacteria.

Hypoxia-induced HIF-1α promotes Listeria monocytogenes invasion into tilapia.

HIF-1α is a nuclear transcription factor, and its activity is tightly regulated by the level of available oxygen in cells. Here, we investigated the roles of HIF-1α in the invasion of Listeria monocytogenes into tilapia under hypoxic environments. We found that the expression levels of HIF-1α in examined tissues of hypoxic tilapia were significantly upregulated, indicating that the tissue cells have been in hypoxic conditions. After 24-h infection with L. monocytogenes, we found that bacterial burden counts increased significantly in all examined tissues of hypoxic fish. To explore why the bacterial count increased significantly in the tissues of hypoxic fish, we modulated HIF-1α expression through RNAi technology. The results indicated that c-Met expression levels were positively related to HIF-1α expression. Since c-Met is the receptor of InlB that plays critical roles in the adhesion and invasion of L. monocytogenes, the ∆InlB strain was used to further explore the reason for the significant increase in bacterial counts in hypoxic fish. As expected, the decrease in the adhesion ability of ∆InlB suggested that InlB mediates L. monocytogenes infection in tilapia. After being infected with ∆InlB strain, we found that the bacterial counts in hypoxic fish were not affected by hypoxic conditions or HIF-1α expression levels. These findings indicate that HIF-1α may promote the internalization of InlB by upregulating c-Met expression and therefore contributes to the invasion of L. monocytogenes into hypoxic tilapia. IMPORTANCE Listeria monocytogenes is a zoonotic food-borne bacterial pathogen with a solid pathogenicity for humans. After ingestion of highly contaminated food, L. monocytogenes is able to cross the intestine invading phagocytic and nonphagocytic cells and causes listeriosis. China is the world's largest supplier of tilapia. The contamination rate of L. monocytogenes to tilapia products was as high as 2.81%, causing a severe threat to public health. This study revealed the underlying regulatory mechanisms of HIF-1α in the invasion of L. monocytogenes into tilapia under hypoxic environments. This study will be helpful for better understanding the molecular mechanisms of hypoxic environments in L. monocytogenes infection to tilapia. More importantly, our data will provide novel insights into the prevention and control of this pathogen in aquaculture.

Interplays of liver fibrosis-associated microRNAs: Molecular mechanisms and implications in diagnosis and therapy.

microRNAs (miRNAs) are a class of non-coding functional small RNA composed of 21-23 nucleotides, having multiple associations with liver fibrosis. Fibrosis-associated miRNAs are roughly classified into pro-fibrosis or anti-fibrosis types. The former is capable of activating hepatic stellate cells (HSCs) by modulating pro-fibrotic signaling pathways, mainly including TGF-ß/SMAD, WNT/ß-catenin, and Hedgehog; the latter is responsible for maintenance of the quiescent phenotype of normal HSCs, phenotypic reversion of activated HSCs (aHSCs), inhibition of HSCs proliferation and suppression of the extracellular matrix-associated gene expression. Moreover, several miRNAs are involved in regulation of liver fibrosis via alternative mechanisms, such as interacting between hepatocytes and other liver cells via exosomes and increasing autophagy of aHSCs. Thus, understanding the role of these miRNAs may provide new avenues for the development of novel interventions against hepatic fibrosis.

microRNAs in parasite-induced liver fibrosis: from mechanisms to diagnostics and therapeutics.

Chronic parasite infections in the liver pose a global threat to human and animal health, often occurring with liver fibrosis that leads to cirrhosis, liver failure, and even cancer. Hepatic fibrogenesis is a complex yet reversible process of tissue repair and is associated with various factors, including immune cells, microenvironment, gut microbiome, and interactions of the different liver cells. As a profibrogenic or antifibrogenic driver, microRNAs (miRNAs) are closely involved in parasite-induced hepatic fibrosis. This article updates the current understanding of the roles of miRNAs in hepatic fibrogenesis by parasite infections and discusses the strategies using miRNAs as candidates for diagnostics and therapeutics.

Stem-loop structures control mRNA processing of the cellulosomal cip-cel operon in Ruminiclostridium cellulolyticum.

BACKGROUND: Anaerobic, mesophilic, and cellulolytic Ruminiclostridium cellulolyticum produces an efficient cellulolytic extracellular complex named cellulosome, which consist of a non-catalytic multi-functional integrating subunit, organizing the various catalytic subunits into the complex. Main components of cellulosome were encoded by the cip-cel operon in R. cellulolyticum, and their stoichiometry is controlled by the mechanism of selective RNA processing and stabilization, which allows to confer each processed RNA portion from the cip-cel mRNA on different fates due to their stability and resolve the potential contradiction between the equimolar stoichiometry of transcripts with a within a transcription unit and the non-equimolar stoichiometry of subunits. RESULTS: In this work, RNA processing events were found to occur at six intergenic regions (IRs) harboring stem-loop structures in cip-cel operon. These stem-loops not only stabilize processed transcripts at their both ends, but also act as cleavage signals specifically recognized by endoribonucleases. We further demonstrated that cleavage sites were often located downstream or 3' end of their associated stem-loops that could be classified into two types, with distinct GC-rich stems being required for RNA cleavage. However, the cleavage site in IR4 was found to be located upstream of the stem-loop, as determined by the bottom AT-pair region of this stem-loop, together with its upstream structure. Thus, our findings reveal the structural requirements for processing of cip-cel transcripts, which can be potentially used to control the stoichiometry of gene expression in an operon. CONCLUSIONS: Our findings reveal that stem-loop structures acting as RNA cleavage signals not only can be recognized by endoribonucleases and determine the location of cleavage sites but also determine the stoichiometry of their flanking processed transcripts by controlling stability in cip-cel operon. These features represent a complexed regulation of cellulosome in the post-transcriptional level, which can be exploited for designing synthetic elements to control gene expression.

Antigen epitopes of animal coronaviruses: a mini-review.

Coronaviruses are widespread in nature and can infect mammals and poultry, making them a public health concern. Globally, prevention and control of emerging and re-emerging animal coronaviruses is a great challenge. The mechanisms of virus-mediated immune responses have important implications for research on virus prevention and control. The antigenic epitope is a chemical group capable of stimulating the production of antibodies or sensitized lymphocytes, playing an important role in antiviral immune responses. Thus, it can shed light on the development of diagnostic methods and novel vaccines. Here, we have reviewed advances in animal coronavirus antigenic epitope research, aiming to provide a reference for the prevention and control of animal and human coronaviruses. Supplementary Information: The online version contains supplementary material available at 10.1186/s44149-023-00080-0.

The effect of O-antigen length determinant wzz on the immunogenicity of Salmonella Typhimurium for Escherichia coli O2 O-polysaccharides delivery.

Attenuated Salmonella Typhimurium is a promising antigen delivery system for live vaccines such as polysaccharides. The length of polysaccharides is a well-known key factor in modulating the immune response induced by glycoconjugates. However, the relationship between the length of Lipopolysaccharide (LPS) O-antigen (OAg) and the immunogenicity of S. Typhimurium remains unclear. In this study, we assessed the effect of OAg length determined by wzzST on Salmonella colonization, cell membrane permeability, antimicrobial activity, and immunogenicity by comparing the S. Typhimurium wild-type ATCC14028 strain to those with various OAg lengths of the ΔwzzST mutant and ΔwzzST::wzzECO2. The analysis of the OAg length distribution revealed that, except for the very long OAg, the short OAg length of 2-7 repeat units (RUs) was obtained from the ΔwzzST mutant, the intermediate OAg length of 13-21 RUs was gained from ΔwzzST::wzzECO2, and the long OAg length of over 20 RUs was gained from the wild-type. In addition, we found that the OAg length affected Salmonella colonization, cell permeability, and antibiotic resistance. Immunization of mice revealed that shortening the OAg length by altering wzzST had an effect on serum bactericidal ability, complement deposition, and humoral immune response. S. Typhimurium mutant strain ΔwzzST::wzzECO2 possessed good immunogenicity and was the optimum option for delivering E. coli O2 O-polysaccharides. Furthermore, the attenuated strain ATCC14028 ΔasdΔcrpΔcyaΔrfbPΔwzzST::wzzECO2-delivered E. coli O2 OAg gene cluster outperforms the ATCC14028 ΔasdΔcrpΔcyaΔrfbP in terms of IgG eliciting, cytokine expression, and immune protection in chickens. This study sheds light on the role of OAg length in Salmonella characteristics, which may have a potential application in optimizing the efficacy of delivered polysaccharide vaccines.