Pseudomonas aeruginosa outer membrane vesicle-packed sRNAs can enter host cells and regulate innate immune responses.

Bacterial outer membrane vesicles (OMVs) can package and deliver virulence factors into host cells, which is an important mechanism mediating host-pathogen interactions. It has been reported that small RNAs (sRNAs) can be packed into OMVs with varying relative abundance, which might affect the function and/or stability of host mRNAs. In this study, we used OptiPrep density gradient ultra-high-speed centrifugation to purify OMVs from Pseudomonas aeruginosa. Next, the sequences and abundance of sRNAs were detected by using Small RNA-Seq. In particular, sRNA4518698, sRNA2316613 and sRNA809738 were the three most abundant sRNAs in OMVs, which are all fragments of P. aeruginosa non-coding RNAs. sRNAs were shielded within the interior of OMVs and remained resistant to external RNase cleavage. The miRanda and RNAhybrid analysis demonstrated that those sRNAs could target a large number of host mRNAs, which were enriched in host immune responses by the functions of GO and KEGG enrichment. Experimentally, we demonstrated that the transfection of synthetic sRNA4518698, sRNA2316613, or sRNA809738 could reduce the expression of innate immune response genes in RAW264.7 cells. Together, we demonstrated that P. aeruginosa OMVs sRNAs can regulate innate immune responses. This study uncovered a mechanism in which the OMVs regulate host responses by transferring bacterial sRNAs.

A core and pan gene map of Leptospira genus and its interactions with human host.

Leptospira species are the etiological agent of an emerging zoonotic disease known as "Leptospirosis" that substantially affects both human health and economy across the globe. Despite the global importance of the disease, pathogenetic features, host-adaptation and proper diagnosis of this bacteria remains lacking. To accomplish these gaps, pan-genome of Leptospira genus was explored in the present study. The pan-genome of Leptospira genus was comprised of core (692) and accessory parts (softcore:1804, shell:6432, cloud:16,600). The functional analysis revealed the abundancy of "Translation, ribosomal structure and biogenesis" COG class in core-genes; whereas in accessory parts, genes involved in signal transduction was the most abundant. Furthermore, pathogen-host interaction (PHI) analysis of core and accessory proteins with human proteins showed the presence of a total of 599 and 510 interactions, respectively. There were eight hubs in core PHI network and five hubs in PHI network of accessory proteins. The human's proteins involved in these interactions were found functionally enriched in metabolic processes, responses to stimulus and immune system processes. Further, pan-genome based phylogeny separated the Leptospira genus in three major clades (belonging to P1, P2 and S) which relates with their pathogenicity level. Additionally, pathogenic and saprophytic clade specific genes of Leptospira have also been identified and functionally annotated for COG, KEGG and virulence factors. The results revealed the presence of 102 pathogenic and 215 saprophytic group specific gene clusters. The COG functional annotation of pathogen specific genes showed that defence mechanism followed by signal transduction mechanisms category were most significantly enriched COG categories; whereas in saprophytic group, signal transduction mechanisms was the most abundant COG, suggesting their role in adaptation and hence important for microbe's evolution and survival. In conclusion, this study provides a new insight of genomic features of Leptospira genus which may further be implemented for development of better control actions of the disease.

ZBP1: A Powerful Innate Immune Sensor and Double-Edged Sword in Host Immunity.

Z-conformation nucleic acid binding protein 1 (ZBP1), a powerful innate immune sensor, has been identified as the important signaling initiation factor in innate immune response and the multiple inflammatory cell death known as PANoptosis. The initiation of ZBP1 signaling requires recognition of left-handed double-helix Z-nucleic acid (includes Z-DNA and Z-RNA) and subsequent signaling transduction depends on the interaction between ZBP1 and its adapter proteins, such as TANK-binding kinase 1 (TBK1), interferon regulatory factor 3 (IRF3), receptor-interacting serine/threonine-protein kinase 1 (RIPK1), and RIPK3. ZBP1 activated innate immunity, including type-I interferon (IFN-I) response and NF-κB signaling, constitutes an important line of defense against pathogenic infection. In addition, ZBP1-mediated PANoptosis is a double-edged sword in anti-infection, auto-inflammatory diseases, and tumor immunity. ZBP1-mediated PANoptosis is beneficial for eliminating infected cells and tumor cells, but abnormal or excessive PANoptosis can lead to a strong inflammatory response that is harmful to the host. Thus, pathogens and host have each developed multiplex tactics targeting ZBP1 signaling to maintain strong virulence or immune homeostasis. In this paper, we reviewed the mechanisms of ZBP1 signaling, the effects of ZBP1 signaling on host immunity and pathogen infection, and various antagonistic strategies of host and pathogen against ZBP1. We also discuss existent gaps regarding ZBP1 signaling and forecast potential directions for future research.

The contributions of fliG gene to the pathogenicity of Pseudomonas plecoglossicida and pathogen-host interactions with Epinephelus coioides.

Pseudomonas plecoglossicida is a Gram-negative aerobic rod-shaped bacterium with polar flagella. It is the causative agent of visceral white spot disease in cultured fish, resulting in serious economic losses. In our previous study, RNA sequencing showed that the expression of the fliG gene in P. plecoglossicida is significantly up-regulated during infection of orange-spotted grouper (Epinephelus coioides). In this study, four P. plecoglossicida RNA interference (RNAi) mutants were successfully constructed by linking four short hairpin RNAs (shRNAs), which target different sites of the fliG gene, to pCM130/tac, respectively. The mRNA expression levels of the fliG gene in P. plecoglossicida were significantly decreased in four mutants. The shRNA-335 mutant (fliG-RNAi strain) showed the best silencing efficiency (88.2%) and was thus chosen for further analysis. Electron microscopy indicated that the flagella of the fliG-RNAi strain of P. plecoglossicida were shorter and finer than those of the wild type strain. The fliG-RNAi strain also showed significantly decreased mobility, chemotaxis, adhesion, and biofilm formation. Furthermore, compared with wild type strain infection, E. coioides infected with the fliG-RNAi strain exhibited a 0.5-d delay in the time of first death and 55% reduction in accumulated mortality, as well as milder splenic symptoms. RNAi of the fliG gene significantly affected the transcriptomes of both pathogen and host in the infected spleens of E. coioides. KEGG analysis revealed that the flagellar assembly pathway, bacterial chemotaxis pathway, and starch and sucrose metabolism pathway were significantly enriched in the pathogen at 3 days post infection (dpi). In contrast, the complement and coagulation cascade pathway and antigen processing and presentation pathway were significantly enriched in the host at 3 dpi. More immune-related pathways were enriched at 5 dpi and more differentially expressed genes were found in the complement and coagulation cascade and antigen processing and presentation pathways. Cytokine-cytokine receptor interaction, hematopoietic cell lineage, and IgA-producing intestinal immune network pathways were significantly enriched in the host at 5 dpi. These results indicate that fliG is an important virulence gene of P. plecoglossicida and contributes to the pathogenicity of P. plecoglossicida as well as pathogen-host interactions with E. coioides.

AAA+ Molecular Chaperone ClpB in Leptospira interrogans: Its Role and Significance in Leptospiral Virulence and Pathogenesis of Leptospirosis.

Bacterial ClpB is an ATP-dependent disaggregase that belongs to the Hsp100/Clp subfamily of the AAA+ ATPases and cooperates with the DnaK chaperone system in the reactivation of aggregated proteins, as well as promotes bacterial survival under adverse environmental conditions, including thermal and oxidative stresses. In addition, extensive evidence indicates that ClpB supports the virulence of numerous bacteria, including pathogenic spirochaete Leptospira interrogans responsible for leptospirosis in animals and humans. However, the specific function of ClpB in leptospiral virulence still remains to be fully elucidated. Interestingly, ClpB was predicted as one of the L. interrogans hub proteins interacting with human proteins, and pathogen-host protein interactions are fundamental for successful invasion of the host immune system by bacteria. The aim of this review is to discuss the most important aspects of ClpB's function in L. interrogans, including contribution of ClpB to leptospiral virulence and pathogenesis of leptospirosis, a zoonotic disease with a significant impact on public health worldwide.

Tracking Pathogen Infections by Time-Resolved Chemical Proteomics.

Studying the dynamic interaction between host cells and pathogen is vital but remains technically challenging. We describe herein a time-resolved chemical proteomics strategy enabling host and pathogen temporal interaction profiling (HAPTIP) for tracking the entry of a pathogen into the host cell. A novel multifunctional chemical proteomics probe was introduced to label living bacteria followed by in vivo crosslinking of bacteria proteins to their interacting host-cell proteins at different time points initiated by UV for label-free quantitative proteomics analysis. We observed over 400 specific interacting proteins crosslinked with the probe during the formation of Salmonella-containing vacuole (SCV). This novel chemical proteomics approach provides a temporal interaction profile of host and pathogen in high throughput and would facilitate better understanding of the infection process at the molecular level.

RNA-Seq based transcriptome analysis during bovine viral diarrhoea virus (BVDV) infection.

BACKGROUND: Bovine viral diarrhoea virus (BVDV) is the member of the genus Pestivirus within the Flaviviridae family and responsible for severe economic losses in the cattle industry. BVDV can employ 'infect-and-persist' strategy and 'hit-and-run' strategy to remain associated with hosts and thus contributes to BVDV circulation in cattle herds. BVDV have also evolved various strategies to evade the innate immunity of host. To further understand the mechanisms by which BVDV overcomes the host cell innate immune response and provide more clues for further understanding the BVDV-host interaction, in this descriptive study, we conducted a investigation of differentially expressed genes (DEGs) of the host during BVDV infection by RNA-Seq analysis. RESULTS: Our analysis identified 1297, 1732, 3072, and 1877 DEGs in the comparison groups mock vs. MDBK cells infected with BVDV post 2 h (MBV2h), mock vs. MBV6h, mock vs. MBV12h, and mock vs. MBV24h, respectively. The reproducibility and repeatability of the results were validated by RT-qPCR. Enrichment analyses of GO annotations and KEGG pathways revealed the host DEGs that are potentially induced by BVDV infection and may participate in BVDV-host interactions. Protein-protein interaction (PPI) network analyses identified the potential interactions among the DEGs. Our findings suggested that BVDV infection induced the upregulation of genes involved in lipid metabolism. The expression of genes that have antiviral roles, including ISG15, Mx1, OSA1Y, were found to be downregulated and are thus potentially associated with the inhibition of host innate immune system during BVDV infection. The expression levels of F3, C1R, KNG1, CLU, C3, FB, SERPINA5, SERPINE1, C1S, F2RL2, and C2, which belong to the complement and coagulation signalling cascades, were downregulated during BVDV infection, which suggested that the complement system might play a crucial role during BVDV infection. CONCLUSION: In this descriptive study, our findings revealed the changes in the host transcriptome expression profile during BVDV infection and suggested that BVDV-infection induced altering the host's metabolic network, the inhibition of the expression of antiviral proteins and genes within the complement system might be contributed to BVDV proliferation. The above findings provided unique insights for further studies on the mechanisms underlying BVDV-host interactions.

Targeting host mitochondria: A role for the Trypanosoma cruzi amastigote flagellum.

Trypanosoma cruzi is the kinetoplastid protozoan parasite that causes human Chagas disease, a chronic disease with complex outcomes including severe cardiomyopathy and sudden death. In mammalian hosts, T. cruzi colonises a wide range of tissues and cell types where it replicates within the host cell cytoplasm. Like all intracellular pathogens, T. cruzi amastigotes must interact with its immediate host cell environment in a manner that facilitates access to nutrients and promotes a suitable niche for replication and survival. Although potentially exploitable to devise strategies for pathogen control, fundamental knowledge of the host pathways co-opted by T. cruzi during infection is currently lacking. Here, we report that intracellular T. cruzi amastigotes establish close contact with host mitochondria via their single flagellum. Given the key bioenergetic and homeostatic roles of mitochondria, this striking finding suggests a functional role for host mitochondria in the infection process and points to the T. cruzi amastigote flagellum as an active participant in pathogenesis. Our study establishes the basis for future investigation of the molecular and functional consequences of this intriguing host-parasite interaction.

Pathogen receptor discovery with a microfluidic human membrane protein array.

The discovery of how a pathogen invades a cell requires one to determine which host cell receptors are exploited. This determination is a challenging problem because the receptor is invariably a membrane protein, which represents an Achilles heel in proteomics. We have developed a universal platform for high-throughput expression and interaction studies of membrane proteins by creating a microfluidic-based comprehensive human membrane protein array (MPA). The MPA is, to our knowledge, the first of its kind and offers a powerful alternative to conventional proteomics by enabling the simultaneous study of 2,100 membrane proteins. We characterized direct interactions of a whole nonenveloped virus (simian virus 40), as well as those of the hepatitis delta enveloped virus large form antigen, with candidate host receptors expressed on the MPA. Selected newly discovered membrane protein-pathogen interactions were validated by conventional methods, demonstrating that the MPA is an important tool for cellular receptor discovery and for understanding pathogen tropism.

Chronic Wasting Disease Prion Strain Emergence and Host Range Expansion.

Human and mouse prion proteins share a structural motif that regulates resistance to common chronic wasting disease (CWD) prion strains. Successful transmission of an emergent strain of CWD prion, H95+, into mice resulted in infection. Thus, emergent CWD prion strains may have higher zoonotic potential than common strains.

Insights from protein-protein interaction studies on bacterial pathogenesis.

INTRODUCTION: The threat bacterial pathogens pose to human health is increasing with the number and distribution of antibiotic-resistant bacteria, while the rate of discovery of new antimicrobials dwindles. Proteomics is playing key roles in understanding the molecular mechanisms of bacterial pathogenesis, and in identifying disease outcome determinants. The physical associations identified by proteomics can provide the means to develop pathogen-specific treatment methods that reduce the spread of antibiotic resistance and alleviate the negative effects of broad-spectrum antibiotics on beneficial bacteria. Areas covered: This review discusses recent trends in proteomics and introduces new and developing approaches that can be applied to the study of protein-protein interactions (PPIs) underlying bacterial pathogenesis. The approaches examined encompass options for mapping proteomes as well as stable and transient interactions in vivo and in vitro. We also explored the coverage of bacterial and human-bacterial PPIs, knowledge gaps in this area, and how they can be filled. Expert commentary: Identifying potential antimicrobial candidates is confounded by the complex molecular biology of bacterial pathogenesis and the lack of knowledge about PPIs underlying this process. Proteomics approaches can offer new perspectives for mechanistic insights and identify essential targets for guiding the discovery of next generation antimicrobials.

Zombie soldier beetles: Epizootics in the goldenrod soldier beetle, Chauliognathus pensylvanicus (Coleoptera: Cantharidae) caused by Eryniopsis lampyridarum (Entomophthoromycotina: Entomophthoraceae).

Adult goldenrod soldier beetles, Chauliognathus pensylvanicus, were found infected by the fungus Eryniopsis lampyridarum (Entomophthoromycotina) in Arkansas during September - October (1996, 2001, 2015 and 2016). Living and dead infected beetles were found on flowering frost aster, Symphyotrichum pilosum, common boneset, Eupatorium perfoliatum, and Canada goldenrod, Solidago canadensis. Live and dead beetles (n=446) were collected in 1996 from S. pilosum flowers and held individually in the laboratory for determination of fungal prevalence. Of the beetles collected, 281 (63%) were males and 165 (37%) were females. A total of 90 beetles were infected with E. lampyridarum, an overall prevalence of 20.2%. Prevalence in males was 19.6% (n=55 infected/281 males total) and prevalence in females was 21.2% (n=35 infected /165 females total). Conidia were produced from 57% of the infected beetles, 23% of the infected beetles produced resting spores, and 20% contained the hyphal body stage. Infected beetles produced either conidia or resting spores but never both in the same host. Post-mortem morphological changes in the hosts due to E. lampyridarum were observed periodically for 24h. Shortly before death, by unknown mechanisms, dying infected beetles tightly clamped their mandibles into flower heads and ca. 15-22h later (between 2400 and 0700h) the fungus caused dead beetles to raise their elytra and expand their metathoracic wings.

Enzymatic Engineering of Live Bacterial Cell Surfaces Using Butelase†1.

Butelase-mediated ligation (BML) can be used to modify live bacterial cell surfaces with diverse cargo molecules. Surface-displayed butelase recognition motif NHV was first introduced at the C-terminal end of the anchoring protein OmpA on E. coli cells. This then served as a handle of BML for the functionalization of E. coli cell surfaces with fluorescein and biotin tags, a tumor-associated monoglycosylated peptide, and mCherry protein. The cell-surface ligation reaction was achieved at low concentrations of butelase and the labeling substrates. Furthermore, the fluorescein-labeled bacterial cells were used to show the interactions with cultured HeLa cells and with macrophages in live transgenic zebrafish, capturing the latter's powerful phagocytic effect in action. Together these results highlight the usefulness of butelase 1 in live bacterial cell surface engineering for novel applications.

Genome sequence and comparative analysis of clavicipitaceous insect-pathogenic fungus Aschersonia badia with Metarhizium spp.

BACKGROUND: Aschersonia badia [(Ab) Teleomorph: Hypocrella siamensis] is an entomopathogenic fungus that specifically infects scale insects and whiteflies. We present the whole genome sequence of Ab and its comparison with two clavicipitaceous fungi Metarhizium robertsii (MR: generalist entomopathogen) and M. acridum (MAC: acridid-specific entomopathogen) that exhibit variable host preferences. Here, through comparative analysis of pathogen-host interacting genes, carbohydrate active enzymes, secondary metabolite biosynthesis genes, and sexuality genes, we explore the proteins with possible virulence functions in clavicipitaceous fungi. Comprehensive overview of GH18 family chitinases has been provided to decipher the role of chitinases in claviceptaceous fungi that are either host specific or generalists. RESULTS: We report the 28.8 Mb draft genome of Ab and its comparative genome analysis with MR and MAC. The comparative analyses suggests expansion in pathogen-host interacting gene families and carbohydrate active enzyme families in MR, whilst their contraction in Ab and MAC genomes. The multi-modular NRPS gene (dtxS1) responsible for biosynthesis of the secondary metabolite destruxin in MR is not conserved in Ab, similar to the specialist pathogen MAC. An additional siderophore biosynthetic gene responsible for acquisition of iron was identified in MR. Further, the domain survey of chitinases suggest that the CBM50 (LysM) domains, which participate in chitin-binding functions, were not observed in MAC, but were present in Ab and MR. However, apparent differences in frequency of CBM50 domains associated with chitinases of Ab and MR was identified, where MR chitinases displayed a higher proportion of associated CBM50 domains than Ab chitinases. CONCLUSIONS: This study suggests differences in distribution of dtxS1 and chitinases in specialists (Ab and MAC) and generalists (MR) fungi. Our analysis also suggests the presence of a siderophore biosynthetic gene in the MR genome which perhaps aids in enhanced virulence potential and host range. The variation in association of CBMs, being higher in generalists (MR) and lower in specialists (Ab and MAC) fungi may further be responsible for the differences in host affiliation.

Resolution pharmacology and the treatment of infectious diseases.

Inflammation is elicited by the host in response to microbes, and is believed to be essential for protection against infection. However, we have previously hypothesized that excessive or misplaced inflammation may be a major contributor to tissue dysfunction and death associated with viral and bacterial infections. The resolutive phase of inflammation is a necessary condition to achieve homeostasis after acute inflammation. It is possible that targeting inflammation resolution may be beneficial for the host during infection. In this review, we summarize the evidence demonstrating the expression, roles and effects of the best described pro-resolving molecules in the context of bacterial and viral infections. Pro-resolving molecules play a pivotal role in modulating a spectrum of pathways associated with tissue inflammation and damage during both viral and bacterial infections. These molecules offer a blend of anti-inflammatory, pro-resolving and sometimes anti-infective benefits, all the while circumventing the undesired and immune-suppressive unwanted effects associated with glucocorticoids. Whether these beneficial effects will translate into benefits to patients clearly deserve further investigation.

A framework for community curation of interspecies interactions literature.

The quantity and complexity of data being generated and published in biology has increased substantially, but few methods exist for capturing knowledge about phenotypes derived from molecular interactions between diverse groups of species, in such a way that is amenable to data-driven biology and research. To improve access to this knowledge, we have constructed a framework for the curation of the scientific literature studying interspecies interactions, using data curated for the Pathogen-Host Interactions database (PHI-base) as a case study. The framework provides a curation tool, phenotype ontology, and controlled vocabularies to curate pathogen-host interaction data, at the level of the host, pathogen, strain, gene, and genotype. The concept of a multispecies genotype, the 'metagenotype,' is introduced to facilitate capturing changes in the disease-causing abilities of pathogens, and host resistance or susceptibility, observed by gene alterations. We report on this framework and describe PHI-Canto, a community curation tool for use by publication authors.

The increasingly vast amount of data being produced in research communities can be difficult to manage, making it challenging for both humans and computers to organise and connect information from different sources. Currently, software tools that allow authors to curate peer-reviewed life science publications are designed solely for single species, or closely related species that do not interact. Although most research communities are striving to make their data FAIR (Findable, Accessible, Interoperable and Reusable), it is particularly difficult to curate detailed information based on interactions between two or more species (interspecies), such as pathogen-host interactions. As a result, there was a lack of tools to support multi-species interaction databases, leading to a reliance on labour-intensive curation methods. To address this problem, Cuzick et al. used the Pathogen-Host Interactions database (PHI-base), which curates knowledge from the text, tables and figures published in over 200 journals, as a case study. A framework was developed that could capture the many observable traits (phenotype annotations) for interactions and link them directly to the combination of genotypes involved in those interactions across multiple scales ­ ranging from microscopic to macroscopic. This demonstrated that it was possible to build a framework of software tools to enable curation of interactions between species in more detail than had been done before. Cuzick et al. developed an online tool called PHI-Canto that allows any researcher to curate published pathogen-host interactions between almost any known species. An ontology ­ a collection of concepts and their relations ­ was created to describe the outcomes of pathogen-host interactions in a standardised way. Additionally, a new concept called the 'metagenotype' was developed which represents the combination of a pathogen and a host genotype and can be easily annotated with the phenotypes arising from each interaction. The newly curated multi-species FAIR data on pathogen-host interactions will enable researchers in different disciplines to compare and contrast interactions across species and scales. Ultimately, this will assist the development of new approaches to reduce the impact of pathogens on humans, livestock, crops and ecosystems with the aim of decreasing disease while increasing food security and biodiversity. The framework is potentially adoptable by any research community investigating interactions between species and could be adapted to explore other harmful and beneficial interspecies interactions.

α-Hydroxyketone synthesis and sensing by Legionella and Vibrio.

Bacteria synthesize and sense low molecular weight signaling molecules, termed autoinducers, to measure their population density and community complexity. One class of autoinducers, the α-hydroxyketones (AHKs), is produced and detected by the water-borne opportunistic pathogens Legionella pneumophila and Vibrio cholerae, which cause Legionnaires' disease and cholera, respectively. The "Legionella quorum sensing" (lqs) or "cholera quorum sensing" (cqs) genes encode enzymes that produce and sense the AHK molecules "Legionella autoinducer-1" (LAI-1; 3-hydroxypentadecane-4-one) or cholera autoinducer-1 (CAI-1; 3-hydroxytridecane-4-one). AHK signaling regulates the virulence of L. pneumophila and V. cholerae, pathogen-host cell interactions, formation of biofilms or extracellular filaments, expression of a genomic "fitness island" and competence. Here, we outline the processes, wherein AHK signaling plays a role, and review recent insights into the function of proteins encoded by the lqs and cqs gene clusters. To this end, we will focus on the autoinducer synthases catalysing the biosynthesis of AHKs, on the cognate trans-membrane sensor kinases detecting the signals, and on components of the down-stream phosphorelay cascade that promote the transmission and integration of signaling events regulating gene expression.

Hepatitis B Virus Integration into Transcriptionally Active Loci and HBV-Associated Hepatocellular Carcinoma.

Hepatitis B Virus (HBV) DNA integrations into the human genome are considered major causative factors to HBV-associated hepatocellular carcinoma development. In the present study, we investigated whether HBV preferentially integrates parts of its genome in specific genes and evaluated the contribution of the integrations in HCC development per gene. We applied dedicated in-house developed pipelines on all of the available HBV DNA integration data and performed a statistical analysis to identify genes that could be characterized as hotspots of integrations, along with the evaluation of their association with HBV-HCC. Our results suggest that 15 genes are recurrently affected by HBV integrations and they are significantly associated with HBV-HCC. Further studies that focus on HBV integrations disrupting these genes are mandatory in order to understand the role of HBV integrations in clonal advantage gain and oncogenesis promotion, as well as to determine whether inhibition of the HBV-disrupted genes can provide a therapy strategy for HBV-HCC.

A Secondary Metabolism Pathway Involved in the Production of a Putative Toxin Is Expressed at Early Stage of Monilinia laxa Infection.

The necrotrophic pathogenic fungus Monilinia laxa causes brown rot disease on stone fruit generating significant yield losses. So far, a limited number of pathogenesis-related virulence factors, such as cell wall degrading enzymes and potential phytotoxins, have been described in Monilinia spp. Using RNA-sequencing data from highly virulent M. laxa ML8L strain at early stages of the infection process (6, 14, 24, and 48 h post-inoculation, hpi) on nectarine and the Pathogen-Host-Interactions (PHI) database, we selected a number of genes for further study and ranked them according to their transcription levels. We identified a class of genes highly expressed at 6 hpi and that their expression decreased to almost undetectable levels at 14 to 48 hpi. Among these genes we found Monilinia__061040 encoding a non-ribosomal peptide synthase (NRPS). Monilinia__061040 together with other five co-regulated genes, forms a secondary metabolism cluster potentially involved in the production of epipolythiodioxopiperazine (ETP) toxin. Quantitative-PCR data confirmed previous RNA sequencing results from the virulent ML8L strain. Interestingly, in a less virulent M. laxa ML5L strain the expression levels of this pathway were reduced compared to the ML8L strain during nectarine infection. In vitro experiments showed that liquid medium containing peach extract mimicked the results observed using nectarines. In fact, upregulation of the NRPS coding gene was also observed in minimal medium suggesting the existence of a fruit-independent mechanism of regulation for this putative toxin biosynthetic pathway that is also downregulated in the less virulent strain. These results emphasize the role of this secondary metabolism pathway during the early stage of brown rot disease development and show alternative models to study the induction of virulence genes in this fungus.

Comparative transcriptome analysis of MDBK cells reveals that BoIFN-γ augmented host immune responses to bovine herpesvirus 1 infection.

Bovine herpesvirus 1 (BoHV-1) is an alphaherpesvirus that causes infectious bovine rhinotracheitis and infectious pustular vulvovaginitis in cattle. Ιnterferon-gamma (IFN-γ) is a pleiotropic cytokine with antiviral activity that modulates the innate and adaptive immune responses. In this study, we prepared high-purity bovine interferon gamma (BoIFN-γ) dimer protein using prokaryotic expression system and affinity chromatography. We subsequently investigated the effect of BoIFN-γ on BoHV-1 infection in Madin-Darby bovine kidney (MDBK) cells. The results showed that BoIFN-γ pre-treament not only decreased the production of BoHV-1 but also reduced the cytopathic effect of the virus. Differential gene expression profiles of BoHV-1 infected MDBK cells were then analysed through high-throughput RNA sequencing. The data showed that BoIFN-γ pre-treatment reduced lipid metabolism disorder and DNA damage caused by BoHV-1 infection. Furthermore, BoIFN-γ treatment upregulated the transcription of interferon regulatory transcription factors (IRF1 and GBP5) and interferon-stimulated genes (ISGs) of MDBK cells. Additionally, BoIFN-γ promotes expression of cellular protein involved in complement activation and coagulation cascades response as well as antigen processing and presentation process, while BoHV-1 infection dramatically downregulates transcription of these immune components including C3, C1r, C1s, PLAT, ITGB2, PROCR, BoLA, CD74, B2M, PA28, BoLA-DRA, and TAPBP. Collectively, our findings revealed that BoIFN-γ pre-treatment can improve host resistance to BoHV-1 infection and regulate transcription or expression of host protein associated with cellular metabolism and innate immune response. This provides insights into the development of prophylactic agents for prevention and control of BoHV-1 infection.