Lysine acetylation regulates the AT-rich DNA possession ability of H-NS.

H-NS, the histone-like nucleoid-structuring protein in bacteria, regulates the stability of the bacterial genome by inhibiting the transcription of horizontally transferred genes, such as the type III and type VI secretion systems (T3/T6SS). While eukaryotic histone posttranslational modifications (PTMs) have been extensively studied, little is known about prokaryotic H-NS PTMs. Here, we report that the acetylation of H-NS attenuates its ability to silence horizontally transferred genes in response to amino acid nutrition and immune metabolites. Moreover, LC-MS/MS profiling showed that the acetyllysine sites of H-NS and K120 are indispensable for its DNA-binding ability. Acetylation of K120 leads to a low binding affinity for DNA and enhances T3/T6SS expression. Furthermore, acetylation of K120 impairs the AT-rich DNA recognition ability of H-NS. In addition, lysine acetylation in H-NS modulates in vivo bacterial virulence. These findings reveal the mechanism underlying H-NS PTMs and propose a novel mechanism by which bacteria counteract the xenogeneic silencing of H-NS.

NITR12+ NK Cells Release Perforin to Mediate IgMhi B Cell Killing in Turbot (Scophthalmus maximus).

B lymphocytes engaged in humoral immunity play a critical role in combating pathogenic infections; however, the mechanisms of NK cells in regulating the responses of B cells remain largely unknown. In the present study, we established an Edwardsiella piscicida infection model in turbot (Scophthalmus maximus) and found that the production of IgM was decreased. Meanwhile, through establishing the head kidney-derived lymphocyte infection model, we revealed that the impairment of IgMhi B cells was associated with bacterial infection-induced perforin production. Interestingly, we reveal that perforin production in NK cells is tightly regulated by an inhibitory novel immune-type receptor, NITR12. Moreover, we confirm that inhibiting NITR12 can result in elevated perforin production, engaging the impairment of IgMhi B cells. Taken together, these findings demonstrate an innovative strategy of NK cells in mediating B lymphocyte killing in turbot and suggest that relieving NK cells through NITR12 might be the target for the development of efficacious vaccines.

An aluminium adjuvant compound with ginseng stem leaf saponins enhances the potency of inactivated Pseudomonas plecoglossicida vaccine in large yellow croaker (Larimichthys crocea).

Large yellow croaker (Larimichthys crocea) farm industry in China suffered from huge economic loss caused by Pseudomonas plecoglossicida infection. Due to multi-antibiotic resistance, efficient vaccines are urgent to be developed to combat this pathogen. In this study, an inactivated vaccine was developed with an aluminium adjuvant (Alum) plus ginseng stem and leaf saponins (GSLS). As a result, the relative percentage survival (RPS) against P. plecoglossicida was up to 67.8 %. Comparatively, RPS of groups that vaccinated with only inactivated vaccine and vaccine containing Alum or Montanide™ 763A as adjuvant were 21.8 %, 32.2 % and 62.1 %, respectively. Assays for total serum protein and serum lysozyme activity in group vaccinated with inactivated vaccine plus Alum + GSLS adjuvant were significantly higher than that in control group. Moreover, specific antibody in serum elicited a rapid and persistent level. According to the expression of some immune related genes, inactivated vaccine plus Alum + GSLS adjuvant induced a stronger cellular immune response which was vital to defend against P. plecoglossicida. In conclusion, our study demonstrated that the compound Alum and GSLS adjuvant is a potential adjuvant system to develop LYC vaccine.

Poly(I:C) induces anti-inflammatory response against secondary LPS challenge in zebrafish larvae.

Poly(I:C) is known as an agonist of the TLR3 receptor which could prime inflammation and elicit the host immune response, which is widely applied as adjuvant or antivirus treatment. However, the negative effects of poly(I:C) on regulating immune response to protect the host from inflammatory diseases remain largely unknown. Here, we establish an in vivo model to pre-treat zebrafish larvae with poly(I:C) at 2 dpf, then challenge them with LPS at 6 dpf, and find that poly(I:C) training could significantly alleviate the LPS challenge-induced septic shock and inflammatory phenotypes. Moreover, the poly(I:C)-trained larvae exhibit decreased number of macrophages, but not neutrophils, after secondary LPS challenge. Furthermore, training the larvae with poly(I:C) could elevate the transcripts of mTOR signaling and heighten the H3K4me3-mediated epigenetic modifications. And interestingly, we find that inhibiting the H3K4me3 modification, rather than mTOR signaling, could recover the number of macrophages in poly(I:C)-trained larvae, which is consistent with the observations of inflammatory phenotypes. Taken together, these results suggest that poly(I:C) training could induce epigenetic rewiring to mediate the anti-inflammatory response against secondary LPS challenge-induced septic shock through decreasing macrophages' number in vivo, which might expand our understanding of poly(I:C) in regulating fish immune response.

A bacterial ghost vaccine against Aeromonas salmonicida infection in turbot (Scophthalmus maximus).

Aeromonas salmonicida is one of the most prevalent pathogens that causes huge economic losses to aquaculture. Effective vaccination is the first choice for preventing infection. Bacterial ghost (BG), an empty bacterial shell devoid of cytoplasm, is a promising vaccine antigen with distinct advantages. Herein, we established strategies for producing a substantial yield of A. salmonicida ghost (ASG) and investigated the immune-protective properties of it. As a result, 2.84 mg/ml NaOH was discovered to be capable of inducing considerable amounts of ASG. Furthermore, the ASG vaccine elicited adaptive immunity in turbots after rapid activation of innate immunity. Even though formalin-killed cells (FKC) produced a few more antibodies than ASG, ASG ultimately provided a much stronger immune protection effect because it strengthened cellular immunity, with a relative percentage survival (RPS) of 50.1 % compared to FKC. These findings demonstrated that ASG effectively activated cell-mediated immunity, which helped get rid of microorganisms inside cells. Therefore, this study presented novel perspectives for future research on furunculosis vaccine products based on ASG as an antigen.

Single-Cell Transcriptomic Analysis Reveals Neutrophil as Orchestrator during ß-Glucan-Induced Trained Immunity in a Teleost Fish.

Trained immunity defines long-term memory of innate immunity based on transcriptional, epigenetic, and metabolic modifications of myeloid cells, which are characterized by elevated proinflammatory responses toward homologous or heterologous secondary stimuli in mammals. However, the evidence of trained immunity-associated immune cells and its molecular mechanism in teleost fish remains largely unknown. In this study, we established a trained immunity activation model in turbot (Scophthalmus maximus) and found that administration with ß-glucan induces protection against a bacterial infection. Through single-cell RNA sequencing to annotate 14 clusters of innate and adaptive immune cells, as well as two clusters of blood cells, from head kidney and spleen, respectively, we characterized that neutrophil displays cardinal features of trained immunity by analyzing the expression abundance of trained immunity database-related genes at the single-cell level. Subsequently, through establishing an in vivo training and in vitro neutrophil challenge model, we found that the trained neutrophils exhibit a significant elevation of the IL-1R signaling pathway after Edwardsiella piscicida infection. Furthermore, inhibition of neutrophil's IL-1R signaling pathway through anakinra treatment impaired the heightened production of reactive oxygen, nitrogen species, lactate, as well as the neutrophil extracellular traps formation and bacterial killing ability. Taken together, these findings characterized neutrophil as the orchestrator to express features of trained immunity, and revealed that the IL-1R signaling pathway plays a critical role in induction of trained immunity for bacterial clearance in teleost fish.

Yeast transcriptional device libraries enable precise synthesis of value-added chemicals from methanol.

Natural methylotrophs are attractive methanol utilization hosts, but lack flexible expression tools. In this study, we developed yeast transcriptional device libraries for precise synthesis of value-added chemicals from methanol. We synthesized transcriptional devices by fusing bacterial DNA-binding proteins (DBPs) with yeast transactivation domains, and linking bacterial binding sequences (BSs) with the yeast core promoter. Three DBP-BS pairs showed good activity when working with transactivation domains and the core promoter of PAOX1 in the methylotrophic yeast, Pichia pastoris. Fine-tuning of the tandem BSs, spacers and differentiated input promoters further enabled a constitutive transcriptional device library (cTRDL) composed of 126 transcriptional devices with an expression strength of 16-520% and an inducible TRDL (iTRDL) composed of 162 methanol-inducible transcriptional devices with an expression strength of 30-500%, compared with PAOX1. Selected devices from iTRDL were adapted to the dihydromonacolin L biosynthetic pathway by orthogonal experimental design, reaching 5.5-fold the production from the PAOX1-driven pathway. The full factorial design of the selected devices from the cTRDL was adapted to the downstream pathway of dihydromonacolin L to monacolin J. Monacolin J production from methanol reached 3.0-fold the production from the PAOX1-driven pathway. Our engineered toolsets ensured multilevel pathway control of chemical synthesis in methylotrophic yeasts.

Xenogeneic nucleoid-associated EnrR thwarts H-NS silencing of bacterial virulence with unique DNA binding.

Type III and type VI secretion systems (T3/T6SS) are encoded in horizontally acquired genomic islands (GIs) that play crucial roles in evolution and virulence in bacterial pathogens. T3/T6SS expression is subjected to tight control by the host xenogeneic silencer H-NS, but how this mechanism is counteracted remains to be illuminated. Here, we report that xenogeneic nucleoid-associated protein EnrR encoded in a GI is essential for virulence in pathogenic bacteria Edwardsiella and Salmonella. We showed that EnrR plays critical roles in T3/T6SS expression in these bacteria. Various biochemical and genetic analyses demonstrated that EnrR binds and derepresses the promoter of esrB, the critical regulator of T3/T6SS, to promote their expression by competing with H-NS. Additionally, EnrR targets AT-rich regions, globally modulates the expression of ∼363 genes and is involved in various cellular processes. Crystal structures of EnrR in complex with a specific AT-rich palindromic DNA revealed a new DNA-binding mode that involves conserved HTH-mediated interactions with the major groove and contacts of its N-terminal extension to the minor groove in the symmetry-related duplex. Collectively, these data demonstrate that EnrR is a virulence activator that can antagonize H-NS, highlighting a unique mechanism by which bacterial xenogeneic regulators recognize and regulate foreign DNA.

FabR senses long-chain unsaturated fatty acids to control virulence in pathogen Edwardsiella piscicida.

Long-chain unsaturated fatty acids (UFAs) can serve as nutrient sources or building blocks for bacterial membranes. However, little is known about how UFAs may be incorporated into the virulence programs of pathogens. A previous investigation identified FabR as a positive regulator of virulence gene expression in Edwardsiella piscicida. Here, chromatin immunoprecipitation-sequencing coupled with RNA-seq analyses revealed that 10 genes were under the direct control of FabR, including fabA, fabB, and cfa, which modulate the composition of UFAs. The binding of FabR to its target DNA was facilitated by oleoyl-CoA and inhibited by stearoyl-CoA. In addition, analyses of enzyme mobility shift assay and DNase I footprinting with wild-type and a null mutant (F131A) of FabR demonstrated crucial roles of FabR in binding to the promoters of fabA, fabB, and cfa. Moreover, FabR also binds to the promoter region of the virulence regulator esrB for its activation, facilitating the expression of the type III secretion system (T3SS) in response to UFAs. Furthermore, FabR coordinated with RpoS to modulate the expression of T3SS. Collectively, our results elucidate the molecular machinery of FabR regulating bacterial fatty acid composition and virulence in enteric pathogens, further expanding our knowledge of its crucial role in host-pathogen interactions.

Identification of a novel transcriptional regulator, CorR, for copper stress response in Edwardsiella piscicida.

Copper plays a vital role in the host-pathogen interface, potentially making components of the bacterial copper response suitable targets for the development of innovative antimicrobial strategies. The anti-copper arsenal of intracellular pathogens has expanded as an adaptation to survive copper toxicity in order to escape intracellular killing by the host immune system. Herein, we employed transposon insertion sequencing to investigate the genetic mechanisms underlying the survival of Edwardsiella piscicida under copper stress. A novel transcriptional regulator, ETAE_2324 (named CorR), was identified to participate in the response to copper ions by controlling the expression of copA, the core component of cytoplasmic copper homeostasis. Furthermore, CorR regulated the expression of virulent determinant eseB, influencing the in vivo colonization of E. piscicida. Collectively, our results contribute to the comprehension of the underlying mechanism of the adaption of intracellular pathogens to copper stress during bacterial infections.IMPORTANCECopper ions play a pivotal role in the interaction between bacteria and the host during infection. The host's innate immune system employs copper ions for their bactericidal properties, thereby making bacterial copper tolerance a crucial determinant of virulence. Edwardsiella piscicida, a significant marine pathogen, has caused substantial losses in the global aquaculture industry. To comprehensively investigate how E. piscicida responds to copper stress, we utilized transposon insertion sequencing to explore genes associated with copper tolerance in culture media containing different concentrations of copper ions. A novel transcriptional regulator, CorR, was identified to respond to copper ions and regulates the expression of crucial components of copper homeostasis CopA, along with the essential virulence factor EseB. These findings offer valuable insights into the underlying mechanisms that govern bacterial copper tolerance and present novel perspectives for the development of vaccines and therapeutic strategies targeting E. piscicida.