The Hippo kinases control inflammatory Hippo signaling and restrict bacterial infection in phagocytes.

The Hippo kinases MST1 and MST2 initiate a highly conserved signaling cascade called the Hippo pathway that limits organ size and tumor formation in animals. Intriguingly, pathogens hijack this host pathway during infection, but the role of MST1/2 in innate immune cells against pathogens is unclear. In this report, we generated Mst1/2 knockout macrophages to investigate the regulatory activities of the Hippo kinases in immunity. Transcriptomic analyses identified differentially expressed genes (DEGs) regulated by MST1/2 that are enriched in biological pathways, such as systemic lupus erythematosus, tuberculosis, and apoptosis. Surprisingly, pharmacological inhibition of the downstream components LATS1/2 in the canonical Hippo pathway did not affect the expression of a set of immune DEGs, suggesting that MST1/2 control these genes via alternative inflammatory Hippo signaling. Moreover, MST1/2 may affect immune communication by influencing the release of cytokines, including TNFα, CXCL10, and IL-1ra. Comparative analyses of the single- and double-knockout macrophages revealed that MST1 and MST2 differentially regulate TNFα release and expression of the immune transcription factor MAF, indicating that the two homologous Hippo kinases individually play a unique role in innate immunity. Notably, both MST1 and MST2 can promote apoptotic cell death in macrophages upon stimulation. Lastly, we demonstrate that the Hippo kinases are critical factors in mammalian macrophages and single-cell amoebae to restrict infection by Legionella pneumophila, Escherichia coli, and Pseudomonas aeruginosa. Together, these results uncover non-canonical inflammatory Hippo signaling in macrophages and the evolutionarily conserved role of the Hippo kinases in the anti-microbial defense of eukaryotic hosts. IMPORTANCE: Identifying host factors involved in susceptibility to infection is fundamental for understanding host-pathogen interactions. Clinically, individuals with mutations in the MST1 gene which encodes one of the Hippo kinases experience recurrent infection. However, the impact of the Hippo kinases on innate immunity remains largely undetermined. This study uses mammalian macrophages and free-living amoebae with single- and double-knockout in the Hippo kinase genes and reveals that the Hippo kinases are the evolutionarily conserved determinants of host defense against microbes. In macrophages, the Hippo kinases MST1 and MST2 control immune activities at multiple levels, including gene expression, immune cell communication, and programmed cell death. Importantly, these activities controlled by MST1 and MST2 in macrophages are independent of the canonical Hippo cascade that is known to limit tissue growth and tumor formation. Together, these findings unveil a unique inflammatory Hippo signaling pathway that plays an essential role in innate immunity.

Exogenous application of nanocarrier-mediated double-stranded RNA manipulates physiological traits and defence response against bacterial diseases.

Stability and delivery are major challenges associated with exogenous double-stranded RNA (dsRNA) application into plants. We report the encapsulation and delivery of dsRNA in cationic poly-aspartic acid-derived polymer (CPP6) into plant cells. CPP6 stabilizes the dsRNAs during long exposure at varied temperatures and pH, and protects against RNase A degradation. CPP6 helps dsRNA uptake through roots or foliar spray and facilitates systemic movement to induce endogenous gene silencing. The fluorescence of Arabidopsis GFP-overexpressing transgenic plants was significantly reduced after infiltration with gfp-dsRNA-CPP6 by silencing of the transgene compared to plants treated only with gfp-dsRNA. The plant endogenous genes flowering locus T (FT) and phytochrome interacting factor 4 (PIF4) were downregulated by a foliar spray of ft-dsRNA-CPP6 and pif4-dsRNA-CPP6 in Arabidopsis, with delayed flowering and enhanced biomass. The rice PDS gene targeted by pds-dsRNA-CPP6 through root uptake was effectively silenced and plants showed a dwarf and albino phenotype. The NaCl-induced OsbZIP23 was targeted through root uptake of bzip23-dsRNA-CPP6 and showed reduced transcripts and seedling growth compared to treatment with naked dsRNA. The negative regulators of plant defence SDIR1 and SWEET14 were targeted through foliar spray to provide durable resistance against bacterial leaf blight disease caused by Xanthomonas oryzae pv. oryzae (Xoo). Overall, the study demonstrates that transient silencing of plant endogenous genes using polymer-encapsulated dsRNA provides prolonged and durable resistance against Xoo, which could be a promising tool for crop protection and for sustaining productivity.

Host-response transcriptional biomarkers accurately discriminate bacterial and viral infections of global relevance.

Diagnostic limitations challenge management of clinically indistinguishable acute infectious illness globally. Gene expression classification models show great promise distinguishing causes of fever. We generated transcriptional data for a 294-participant (USA, Sri Lanka) discovery cohort with adjudicated viral or bacterial infections of diverse etiology or non-infectious disease mimics. We then derived and cross-validated gene expression classifiers including: 1) a single model to distinguish bacterial vs. viral (Global Fever-Bacterial/Viral [GF-B/V]) and 2) a two-model system to discriminate bacterial and viral in the context of noninfection (Global Fever-Bacterial/Viral/Non-infectious [GF-B/V/N]). We then translated to a multiplex RT-PCR assay and independent validation involved 101 participants (USA, Sri Lanka, Australia, Cambodia, Tanzania). The GF-B/V model discriminated bacterial from viral infection in the discovery cohort an area under the receiver operator curve (AUROC) of 0.93. Validation in an independent cohort demonstrated the GF-B/V model had an AUROC of 0.84 (95% CI 0.76-0.90) with overall accuracy of 81.6% (95% CI 72.7-88.5). Performance did not vary with age, demographics, or site. Host transcriptional response diagnostics distinguish bacterial and viral illness across global sites with diverse endemic pathogens.

The evolution of NLRC3 subfamily genes in Sebastidae teleost fishes.

BACKGROUND: With more than 36,000 valid fish species, teleost fishes constitute the most species-rich vertebrate clade and exhibit extensive genetic and phenotypic variation, including diverse immune defense strategies. NLRC3 subfamily genes, which are specific to fishes, play vital roles in the immune system of teleosts. The evolution of teleosts has been impacted by several whole-genome duplication (WGD) events, which might be a key reason for the expansions of the NLRC3 subfamily, but detailed knowledge of NLRC3 subfamily evolution in the family Sebastidae is still limited. RESULTS: Phylogenetic inference of NLRC3 subfamily protein sequences were conducted to evaluate the orthology of NLRC3 subfamily genes in black rockfish (Sebastes schlegilii), 13 other fish species from the families Sebastidae, Serranidae, Gasterosteidae and Cyclopteridae, and three species of high vertebrates (bird, reptile and amphibian). WGD analyses were used to estimate expansions and contractions of the NLRC3 subfamily, and patterns of expression of NLRC3 subfamily genes in black rockfish following bacterial infections were used to investigate the functional roles of these genes in the traditional and mucosal immune system of the Sebastidae. Different patterns of gene expansions and contractions were observed in 17 fish and other species examined, and one and two whole-genome duplication events were observed in two members of family Sebastidae (black rockfish and honeycomb rockfish, Sebastes umbrosus), respectively. Subsequently, 179 copy numbers of NLRC3 genes were found in black rockfish and 166 in honeycomb rockfish. Phylogenetic analyses corroborated the conservation and evolution of NLRC3 orthologues between Sebastidae and other fish species. Finally, differential expression analyses provided evidence of the immune roles of NLRC3 genes in black rockfish during bacterial infections and gene ontology analysis also indicated other functional roles. CONCLUSIONS: We hypothesize that NLRC3 genes have evolved a variety of different functions, in addition to their role in the immune response, as a result of whole genome duplication events during teleost diversification. Importantly, this study had underscored the importance of sampling across taxonomic groups, to better understand the evolutionary patterns of the innate immunity system on which complex immunological novelties arose. Moreover, the results in this study could extend current knowledge of the plasticity of the immune system.

A Mendelian Randomization-Based Causal Investigation on Bacterial Infection-Related Genes and Neurodegenerative Diseases.

Neurodegenerative diseases remain the most prevalent and unsolved health problems in human society, especially Alzheimer's disease (AD) and Parkinson's disease (PD). The pathogenesis, pathology, and potential clinical treatments of neurodegenerative diseases still require in-depth research. In the wake of the association between pandemics and a growing number of neurodegeneration patients, there has been growing speculation that infections are linked to AD and PD. The Aß peptide is an important causal-related biomarker of AD and is reported to share structural and functional similarities with certain antimicrobial peptides, suggesting that it has a role in eliciting an immune response against microbes. But how neurodegeneration is related to bacterial chronic infection has not been thoroughly investigated. Using the data from genome-wide association studies (GWAS), we performed Mendelian Randomization (MR) and map 7 genes in multiple bacterial infection pathways as exposure, which show a significant association with the outcome of AD or PD. As co-verification, we perform Gene Set Enrichment Analysis (GSEA) on selected genetic variants incorporating their perturb-seq gene list (combining single-cell RNA-seq and CRISPR-based perturbations). We observed clustering of the differentially expressed genes (DEGs) in the upstream and downstream of AD and PD-related KEGG pathways, hence confirming their causal association with AD and PD and providing new perspectives on the true cause of neurodegeneration.

RVFScan predicts virulence factor genes and hypervirulence of the clinical metagenome.

Bacterial infections often involve virulence factors that play a crucial role in the pathogenicity of bacteria. Accurate detection of virulence factor genes (VFGs) is essential for precise treatment and prognostic management of hypervirulent bacterial infections. However, there is a lack of rapid and accurate methods for VFG identification from the metagenomic data of clinical samples. Here, we developed a Reads-based Virulence Factors Scanner (RVFScan), an innovative user-friendly online tool that integrates a comprehensive VFG database with similarity matrix-based criteria for VFG prediction and annotation using metagenomic data without the need for assembly. RVFScan demonstrated superior performance compared to previous assembly-based and read-based VFG predictors, achieving a sensitivity of 97%, specificity of 98% and accuracy of 98%. We also conducted a large-scale analysis of 2425 clinical metagenomic datasets to investigate the utility of RVFScan, the species-specific VFG profiles and associations between VFGs and virulence phenotypes for 24 important pathogens were analyzed. By combining genomic comparisons and network analysis, we identified 53 VFGs with significantly higher abundances in hypervirulent Klebsiella pneumoniae (hvKp) than in classical K. pneumoniae. Furthermore, a cohort of 1256 samples suspected of K. pneumoniae infection demonstrated that RVFScan could identify hvKp with a sensitivity of 90%, specificity of 100% and accuracy of 98.73%, with 90% of hvKp samples consistent with clinical diagnosis (Cohen's kappa, 0.94). RVFScan has the potential to detect VFGs in low-biomass and high-complexity clinical samples using metagenomic reads without assembly. This capability facilitates the rapid identification and targeted treatment of hvKp infections and holds promise for application to other hypervirulent pathogens.

Deciphering the molecular and cellular atlas of immune cells in septic patients with different bacterial infections.

BACKGROUND: Sepsis is a life-threatening organ dysfunction caused by abnormal immune responses to various, predominantly bacterial, infections. Different bacterial infections lead to substantial variation in disease manifestation and therapeutic strategies. However, the underlying cellular heterogeneity and mechanisms involved remain poorly understood. METHODS: Multiple bulk transcriptome datasets from septic patients with 12 types of bacterial infections were integrated to identify signature genes for each infection. Signature genes were mapped onto an integrated large single-cell RNA (scRNA) dataset from septic patients, to identify subsets of cells associated with different sepsis types, and multiple omics datasets were combined to reveal the underlying molecular mechanisms. In addition, an scRNA dataset and spatial transcriptome data were used to identify signaling pathways in sepsis-related cells. Finally, molecular screening, optimization, and de novo design were conducted to identify potential targeted drugs and compounds. RESULTS: We elucidated the cellular heterogeneity among septic patients with different bacterial infections. In Escherichia coli (E. coli) sepsis, 19 signature genes involved in epigenetic regulation and metabolism were identified, of which DRAM1 was demonstrated to promote autophagy and glycolysis in response to E. coli infection. DRAM1 upregulation was confirmed in an independent sepsis cohort. Further, we showed that DRAM1 could maintain survival of a pro-inflammatory monocyte subset, C10_ULK1, which induces systemic inflammation by interacting with other cell subsets via resistin and integrin signaling pathways in blood and kidney tissue, respectively. Finally, retapamulin was identified and optimized as a potential drug for treatment of E. coli sepsis targeting the signature gene, DRAM1, and inhibiting E. coli protein synthesis. Several other targeted drugs were also identified in other types of sepsis, including nystatin targeting C1QA in Neisseria sepsis and dalfopristin targeting CTSD in Streptococcus viridans sepsis. CONCLUSION: Our study provides a comprehensive overview of the cellular heterogeneity and underlying mechanisms in septic patients with various bacterial infections, providing insights to inform development of stratified targeted therapies for sepsis.

To target or not to target? The role of DNA and histone methylation in bacterial infections.

Epigenetics describes chemical modifications of the genome that do not alter DNA sequence but participate in the regulation of gene expression and cellular processes such as proliferation, division, and differentiation of eukaryotic cell. Disruption of the epigenome pattern in a human cell is associated with different diseases, including infectious diseases. During infection pathogens induce epigenetic modifications in the host cell. This can occur by controlling expression of genes involved in immune response. That enables bacterial survival and replication within the host and evasion of the immune response. Methylation is an example of epigenetic modification that occurs on DNA and histones. Reasoning that DNA and histone methylation of human host cells plays a crucial role during pathogenesis, these modifications are promising targets for the development of alternative treatment strategies in infectious diseases. Here, we discuss the role of DNA and histone methyltransferases in human host cell upon bacterial infections. We further hypothesize that compounds targeting methyltransferases are tools to study epigenetics in the context of host-pathogen interactions and can open new avenues for the treatment of bacterial infections.

MUC13 Cell Surface Mucin Limits Salmonella Typhimurium Infection by Protecting the Mucosal Epithelial Barrier.

BACKGROUND & AIMS: MUC13 cell surface mucin is highly expressed on the mucosal surface throughout the intestine, yet its role against bacterial infection is unknown. We investigated how MUC13 impacts Salmonella typhimurium (S Tm) infection and elucidated its mechanisms of action. METHODS: Muc13-/- and wild-type littermate mice were gavaged with 2 isogenic strains of S Tm after pre-conditioning with streptomycin. We assessed clinical parameters, cecal histology, local and systemic bacterial load, and proinflammatory cytokines after infection. Cecal enteroids and epithelial cell lines were used to evaluate the mechanism of MUC13 activity after infection. The interaction between bacterial SiiE and MUC13 was assessed by using siiE-deficient Salmonella. RESULTS: S Tm-infected Muc13-/- mice had increased disease activity, histologic damage, and higher local and systemic bacterial loads. Mechanistically, we found that S Tm binds to MUC13 through its giant SiiE adhesin and that MUC13 acts as a pathogen-binding decoy shed from the epithelial cell surface after pathogen engagement, limiting bacterial invasion. In addition, MUC13 reduces epithelial cell death and intestinal barrier breakdown by enhancing nuclear factor kappa B signaling during infection, independent of its decoy function. CONCLUSIONS: We show for the first time that MUC13 plays a critical role in antimicrobial defense against pathogenic S Tm at the intestinal mucosal surface by both acting as a releasable decoy limiting bacterial invasion and reducing pathogen-induced cell death. This further implicates the cell surface mucin family in mucosal defense from bacterial infection.

A review on circular RNAs and bacterial infections.

Bacterial infections and related diseases have been a major burden on social public health and economic stability around the world. However, the effective diagnostic methods and therapeutic approaches to treat bacterial infections are still limited. As a group of non-coding RNA, circular RNAs (circRNAs) that were expressed specifically in host cells and played a key regulatory role have the potential to be of diagnostic and therapeutic values. In this review, we systematically summarize the role of circRNAs in common bacterial infections and their potential roles as diagnostic markers and therapeutic targets.

The emerging roles of long non-coding RNA in host immune response and intracellular bacterial infections.

The long non-coding RNAs (lncRNAs) are evolutionarily conserved classes of non-coding regulatory transcripts of > 200 nucleotides in length. They modulate several transcriptional and post-transcriptional events in the organism. Depending on their cellular localization and interactions, they regulate chromatin function and assembly; and alter the stability and translation of cytoplasmic mRNAs. Although their proposed range of functionality remains controversial, there is increasing research evidence that lncRNAs play a regulatory role in the activation, differentiation and development of immune signaling cascades; microbiome development; and in diseases such as neuronal and cardiovascular disorders; cancer; and pathogenic infections. This review discusses the functional roles of different lncRNAs in regulation of host immune responses, signaling pathways during host-microbe interaction and infection caused by obligate intracellular bacterial pathogens. The study of lncRNAs is assuming significance as it could be exploited for development of alternative therapeutic strategies for the treatment of severe and chronic pathogenic infections caused by Mycobacterium, Chlamydia and Rickettsia infections, as well as commensal colonization. Finally, this review summarizes the translational potential of lncRNA research in development of diagnostic and prognostic tools for human diseases.

MicroRNAs reshape the immunity of insects in response to bacterial infection.

The interaction between bacteria and insects can significantly impact a wide range of different areas because bacteria and insects are widely distributed around the globe. The bacterial-insect interactions have the potential to directly affect human health since insects are vectors for disease transmission, and their interactions can also have economic consequences. In addition, they have been linked to high mortality rates in economically important insects, resulting in substantial economic losses. MicroRNAs (miRNAs) are types of non-coding RNAs involved in regulating gene expression post-transcriptionally. The length of miRNAs ranges from 19 to 22 nucleotides. MiRNAs, in addition to their ability to exhibit dynamic expression patterns, have a diverse range of targets. This enables them to govern various physiological activities in insects, like innate immune responses. Increasing evidence suggests that miRNAs have a crucial biological role in bacterial infection by influencing immune responses and other mechanisms for resistance. This review focuses on some of the most recent and exciting discoveries made in recent years, including the correlation between the dysregulation of miRNA expression in the context of bacterial infection and the progression of the infection. Furthermore, it describes how they profoundly impact the immune responses of the host by targeting the Toll, IMD, and JNK signaling pathways. It also emphasizes the biological function of miRNAs in regulating immune responses in insects. Finally, it also discusses current knowledge gaps about the function of miRNAs in insect immunity, in addition to areas that require more research in the future.

Comprehensive Characterization of a Novel Bacteriophage, vB_VhaS_MAG7 against a Fish Pathogenic Strain of Vibrio harveyi and Its In Vivo Efficacy in Phage Therapy Trials.

Vibrio harveyi, a significant opportunistic marine pathogen, has been a challenge to the aquaculture industry, leading to severe economical and production losses. Phage therapy has been an auspicious approach in controlling such bacterial infections in the era of antimicrobial resistance. In this study, we isolated and fully characterized a novel strain-specific phage, vB_VhaS_MAG7, which infects V. harveyi MM46, and tested its efficacy as a therapeutic agent in challenged gilthead seabream larvae. vB_VhaS_MAG7 is a tailed bacteriophage with a double-stranded DNA of 49,315 bp. No genes linked with virulence or antibiotic resistance were harbored in the genome. The phage had a remarkably large burst size of 1393 PFU cell-1 and showed strong lytic ability in in vitro assays. When applied in phage therapy trials in challenged gilthead seabream larvae, vB_VhaS_MAG7 was capable of improving the survival of the larvae up to 20%. Due to its distinct features and safety, vB_VhaS_MAG7 is considered a suitable candidate for applied phage therapy.

Host A-to-I RNA editing signatures in intracellular bacterial and single-strand RNA viral infections.

Background: Microbial infection is accompanied by remodeling of the host transcriptome. Involvement of A-to-I RNA editing has been reported during viral infection but remains to be elucidated during intracellular bacterial infections. Results: Herein we analyzed A-to-I RNA editing during intracellular bacterial infections based on 18 RNA-Seq datasets of 210 mouse samples involving 7 tissue types and 8 intracellular bacterial pathogens (IBPs), and identified a consensus signature of RNA editing for IBP infections, mainly involving neutrophil-mediated innate immunity and lipid metabolism. Further comparison of host RNA editing patterns revealed remarkable similarities between pneumonia caused by IBPs and single-strand RNA (ssRNA) viruses, such as altered editing enzyme expression, editing site numbers, and levels. In addition, functional enrichment analysis of genes with RNA editing highlighted that the Rab GTPase family played a common and vital role in the host immune response to IBP and ssRNA viral infections, which was indicated by the consistent up-regulated RNA editing of Ras-related protein Rab27a. Nevertheless, dramatic differences between IBP and viral infections were also observed, and clearly distinguished the two types of intracellular infections. Conclusion: Our study showed transcriptome-wide host A-to-I RNA editing alteration during IBP and ssRNA viral infections. By identifying and comparing consensus signatures of host A-to-I RNA editing, our analysis implicates the importance of host A-to-I RNA editing during these infections and provides new insights into the diagnosis and treatment of infectious diseases.

CRISPR-Cas system as a promising player against bacterial infection and antibiotic resistance.

The phenomenon of antibiotic resistance (AR) and its increasing global trends and destructive waves concerns patients and the healthcare system. In order to combat AR, it is necessary to explore new strategies when the current antibiotics fail to be effective. Thus, knowing the resistance mechanisms and appropriate diagnosis of bacterial infections may help enhance the sensitivity and specificity of novel strategies. On the other hand, resistance to antimicrobial compounds can spread from resistant populations to susceptible ones. Antimicrobial resistance genes (ARGs) significantly disseminate AR via horizontal and vertical gene transfer. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is a member of the bacterial immune system with the ability to remove the ARGs; therefore, it can be introduced as an effective and innovative strategy in the battle against AR. Here, we reviewed CRISPR-based bacterial diagnosis technologies. Moreover, the strategies to battle AR based on targeting bacterial chromosomes and resistance plasmids using the CRISPR-Cas system have been explained. Besides, we have presented the limitations of CRISPR delivery and potential solutions to help improve the future development of CRISPR-based platforms.

CRISPR/Cas9-Induced Knockout of Sting Increases Susceptibility of Zebrafish to Bacterial Infection.

Stimulator of interferon genes (STING) is an adapter protein that is activated when cyclic dinucleotides (CDNs) are present. CDNs originate from the cytosolic DNA of both pathogens and hosts. STING activation promotes efficient immune responses against viral infections; however, its impact in bacterial infections is unclear. In this study, we investigated the role of Sting in bacterial infections by successfully creating a sting-deficient (sting(-/-) with a 4-bp deletion) knockout zebrafish model using CRISPR/Cas9. The transcriptional modulation of genes downstream of cGAS (cyclic GMP-AMP synthase)-Sting pathway-related genes was analyzed in seven-day-old wild-type (WT) and sting(-/-) embryos, as well as in four-day-old LPS-stimulated embryos. The expression of downstream genes was higher in sting(-/-) than in healthy WT fish. The late response was observed in sting(-/-) larvae following LPS treatment, demonstrating the importance of Sting-induced immunity during bacterial infection by activating the cGAS-STING pathway. Furthermore, adult sting(-/-) fish had a high mortality rate and significantly downregulated cGAS-STING pathway-related genes during Edwardsiella piscicida (E. piscicida) infection. In addition, we assessed NF-κB pathway genes following E. piscicida infection. Our results show fluctuating patterns of interleukin-6 (il6) and tumor necrosis factor-α (tnfα) expression, which is likely due to the influence of other NF-κB pathway-related immune genes. In summary, this study demonstrates the important role of Sting against bacterial infection.

The Current Status of Antisense Gene Therapies for Bacteria-caused Diseases Challenges and Opportunities.

Bacteria-caused diseases continue to pose a serious threat to human health. The current situation of overused antibiotics against those diseases further spurs and exacerbates the ever-increasing drug resistance problems, which really leaves us very few options to combat those nasty bugs. Gene therapies based on the antisense oligonucleotide, though developed more than 40 years ago, did not reform the current treatments as originally expected. Along with the advances of new delivery technologies, this old field thrives again. In addition, newly evolving gene-editing tools based on the CRISPR-Cas system shed new light on this old field, bringing a breeze of hope to gene therapies for bacteria-caused diseases. As a fast-growing field, we strive to summarize in this review the recent progress in using gene therapies in those areas, analyze the potential challenges or problems from using antisense or gene-editing tools for targeting bacterial diseases and seek to explore any potential solutions to the current dilemmas. As a short review, we will focus our discussion mainly on antisense oligonucleotide-based gene therapies while briefly touching on the CRISPR-Cas based ones as the latter is just beginning to get more attention for application in the prokaryotic kingdom.

Nitric oxide synthase is required for the pea aphid's defence against bacterial infection.

Compared to other insects, the pea aphid Acyrthosiphon pisum has a reduced immune system with an absence of genes coding for a lot of immunity-related molecules. Notably, nitric oxide synthase (NOS), which catalyses the synthesis of nitric oxide (NO), is present in the pea aphid. However, the role of NO in the immune system of pea aphid remains unclear. In this study, we explored the role of NO in the defence of the pea aphid against bacterial infections and found that the NOS gene of the pea aphid responded to an immune challenge, with the expression of ApNOS observably upregulated after bacterial infections. Knockdown of ApNOS using RNA interference or inhibition of NOS activity increased the number of live bacterial cells in aphids and the mortality of aphids after bacterial infection. Conversely, the increase in NO level in aphids using NO donor inhibited the bacterial growth, increased the survival of bacteria-infected aphids, and upregulated immune genes, such as Toll pathway and phagocytosis related genes. Thus, NO promotes immune responses and plays an important role in the immune system of pea aphid.