Expression Of Antimicrobial Proteins Research Articles

The Shigella flexneri effector IpaH1.4 facilitates RNF213 degradation and protects cytosolic bacteria against interferon-induced ubiquitylation.

A central signal that marshals host defense against many infections is the lymphocyte-derived cytokine interferon-gamma (IFNγ). The IFNγ receptor is expressed on most human cells and its activation leads to the expression of antimicrobial proteins that execute diverse cell-autonomous immune programs. One such immune program consists of the sequential detection, ubiquitylation, and destruction of intracellular pathogens. Recently, the IFNγ-inducible ubiquitin E3 ligase RNF213 was identified as a pivotal mediator of such a defense axis. RNF213 provides host protection against viral, bacterial, and protozoan pathogens. To establish infections, potentially susceptible intracellular pathogens must have evolved mechanisms that subdue RNF213-controlled cell-autonomous immunity. In support of this hypothesis, we demonstrate here that a causative agent of bacillary dysentery, Shigella flexneri, uses the type III secretion system (T3SS) effector IpaH1.4 to induce the degradation of RNF213. S. flexneri mutants lacking IpaH1.4 expression are bound and ubiquitylated by RNF213 in the cytosol of IFNγ-primed host cells. Linear (M1-) and lysine-linked ubiquitin is conjugated to bacteria by RNF213 independent of the linear ubiquitin chain assembly complex (LUBAC). We find that ubiquitylation of S. flexneri is insufficient to kill intracellular bacteria, suggesting that S. flexneri employs additional virulence factors to escape from host defenses that operate downstream from RNF213-driven ubiquitylation. In brief, this study identified the bacterial IpaH1.4 protein as a direct inhibitor of mammalian RNF213 and highlights evasion of RNF213-driven immunity as a characteristic of the human-tropic pathogen Shigella .

C/EBPε and its acetylation in PMN enhance the tolerance to trauma.

Severe trauma can lead to numerous serious complications, threating the well-being and vitality of the afflicted. The quantity and functionality of PMNs undergo rapid transformations in response to severe trauma, playing a pivotal role in the trauma response. The absence of CCAAT/enhancer-binding protein ε (C/EBPε) profoundly impairs the functionality of polymorphonuclear neutrophils (PMNs), a function of paramount importance in trauma. In this study, by generating mice with C/EBPε knocked out or overexpressed, we substantiate that C/EBPε ensures the restoration of PMN function, enhancing the expression of antimicrobial proteins and thereby promoting trauma recovery. Furthermore, diminished expression of C/EBPε is observed in trauma patients, with levels displaying a negative correlation with ISS and APACHE II scores, suggesting its potential as a prognostic indicator for clinical treatment. Mechanistically, we uncover the upregulation of SIRT1 and the inhibition of P300 participating in the suppression of C/EBPε acetylation, consequently reducing the resilience of mice to trauma. As therapeutic interventions, whether through the sole administration of PMN, NAM treatment, or their combination, all result in an increased survival rate in traumatic mice. In conclusion, our study elucidates the role of C/EBPε in enhancing the resilience to trauma and identifies C/EBPε acetylation as a critical regulatory mechanism, offering potential therapeutic approaches involving PMN transfusion and NAM treatment.

Supplemental Psyllium Fiber Increases Antimicrobial Proteins via the Tuft Cell-ILC2 Circuit and Type II Immune Response in the Mouse Small Intestine

Dietary fibers regulate intestinal barrier function; however, the precise mechanisms remain unclear. This study investigated the effects of psyllium fibers on antimicrobial protein expression, focusing on the type II immunity and tuft cell-group 2 innate lymphoid cell (ILC2) circuit in the small intestine of the mouse. Supplemental psyllium fiber upregulated antimicrobial proteins, such as small proline-rich protein 2A (SPRR2A) and resistin-like beta (RELMβ), in mouse small intestine, evidently affecting cecal microbiota composition. The psyllium fibers also increased the RNA and protein expression of molecules related to ILC2 and tuft cells, such as IL-13, IL-25, DCLK1, Gfi-1b, SH2 domain-containing protein 3C, and Spi-B. In addition, ILC2 inhibitor (disulfiram) and bitter taste receptor blocker administration reduced psyllium-induced SPRR2A and RELMβ expression. Collectively, psyllium supplementation upregulates antimicrobial proteins such as SPRR2A and RELMß via the type II immune response and tuft cell-ILC2 circuit in the mouse small intestine.

Anti-psoriasis activities of hydroxytyrosol on HaCaT cells under psoriatic inflammation in vitro

Background Psoriasis is a prevalent chronic inflammatory dermatosis, which can significantly impact life quality of patients. The treatment of psoriasis is no cure and novel therapeutic options are urgently needed. Hydroxytyrosol (HT) possesses multiple biological activities, such as anti-inflammatory and anti-proliferation properties, suggesting its potential to counteract hallmarks of psoriasis. However, its role in the regulation of psoriasis remains unknown. Objective In the current study, we explored the anti-proliferative activity and anti-inflammatory responses of HT in psoriatic keratinocytes in vitro. Methods We used M5 cytokines cocktail, which includes tumor necrosis factor (TNF)-α, oncostatin-M, interleukin (IL)-17A, IL-1α, and IL-22, to simulate HaCaT cells to establish the cell model of psoriasis and explore the effects of HT on psoriasis in vitro. Results This study showed that HT exerted potent anti-inflammatory effect via influencing the expression of IL-6, IL-8, and TNF-α in M5-induced cell model of psoriasis. Moreover, it suppressed the expression of antimicrobial proteins in psoriatic keratinocytes. Additionally, it inhibited cell proliferation in psoriasis cell model. Conclusions Altogether, our results suggested that HT has anti-psoriasis effects in vitro and HT may be a promising therapeutic agent in psoriasis treatment.

FABP4 in Paneth cells regulates antimicrobial protein expression to reprogram gut microbiota

ABSTRACT Antimicrobial proteins possess a broad spectrum of bactericidal activity and play an important role in shaping the composition of gut microbiota, which is related to multiple diseases such as metabolic syndrome. However, it is incompletely known for the regulation of defensin expression in the gut Paneth cells. Here, we found that FABP4 in the Paneth cells of gut epithelial cells and organoids can downregulate the expression of defensins. FABP4fl/flpvillinCreT mice were highly resistance to Salmonella Typhimurium (S.T) infection and had increased bactericidal ability to pathogens. The FABP4-mediated downregulation of defensins is through degrading PPARγ after K48 ubiquitination. We also demonstrate that high-fat diet (HFD)-mediated downregulation of defensins is through inducing a robust FABP4 in Paneth cells. Firmicutes/Bacteroidetes (F/B) ratio in FABP4fl/flpvillinCreT mice is lower than control mice, which is opposite to that in mice fed HFD, indicating that FABP4 in the Paneth cells could reprogram gut microbiota. Interestingly, FABP4-mediated downregulation of defensins in Paneth cells not only happens in mice but also in human. A better understanding of the regulation of defensins, especially HFD-mediated downregulation of defensin in Paneth cells will provide insights into factor(s) underlying modern diseases. Abbreviations: FABP4: Fatty acid binding protein 4; S. T: SalmonellaTyphimurium; HFD: High-fat diet; Defa: α-defensin; 930HD5: Human α-defensin 5; HD6: Human α-defensin 6;F/B: Firmicutes/Bacteroidetes; SFB: Segmental filamentousbacteria; AMPs: Antimicrobial peptides; PPARγ:Peroxisome proliferator-activated receptor γ; P-PPAR:Phosphorylated PPAR; Dhx15: DEAD-box helicase 15; 935EGF: Epidermal growth factor; ENR: Noggin andR-spondin 1; CFU: Colony forming unit; Lyz1: Lysozyme1; Saa1: Serum amyoid A 1; Pla2g2a: Phospholipase A2,group IIA; MMP-7: Matrix metalloproteinase; AU-PAGE:Acid-urea polyacrylamide gel electrophoresis; PA: Palmitic 940acid; GPR40: G-protein-coupled receptor; GF: Germ-free;EGF: Epidermal growth factor; LP: Lamina propria; KO:Knock out; WT: Wild-type.

C/N-Dependent Element Bioconversion Efficiency and Antimicrobial Protein Expression in Food Waste Treatment by Black Soldier Fly Larvae.

The black soldier fly (BSF), Hermetia illucens, has emerged as a promising species for waste bioconversion and source of antimicrobial proteins (AMPs). However, there is a scarcity of research on the element transformation efficiency and molecular characterization of AMPs derived from waste management. Here, food waste treatment was performed using BSF larvae (BSFL) in a C/N ratio of 21:1–10:1, with a focus on the C/N-dependent element bioconversion, AMP antimicrobial activity, and transcriptome profiling. The C-larvae transformation rates were found to be similar among C/Ns (27.0–35.5%, p = 0.109), while the N-larvae rates were different (p = 0.001), with C/N 21:1–16:1 (63.5–75.0%) being higher than C/N 14:1–10:1 (35.0–45.7%). The C/N ratio did not alter the antimicrobial spectrum of AMPs, but did affect the activities, with C/N 21:1 being significantly lower than C/N 18:1–10:1. The lysozyme genes were found to be significantly more highly expressed than the cecropin, defensin, and attacin genes in the AMP gene family. Out of 51 lysozyme genes, C/N 18:1 and C/N 16:1 up-regulated (p < 0.05) 14 and 12 genes compared with C/N 21:1, respectively, corresponding to the higher activity of AMPs. Overall, the element bioconversion efficiency and AMP expression can be enhanced through C/N ratio manipulation, and the C/N-dependent transcriptome regulation is the driving force of the AMP difference.

Transformation of food waste to source of antimicrobial proteins by black soldier fly larvae for defense against marine Vibrio parahaemolyticus

Insect biorefinery by black soldier fly larvae (BSFL), Hermetia illucens, has emerged as an innovative technique for the valorization of food waste. However, despite BSFL being an attractive natural source of antimicrobial proteins (AMPs), there is a scarcity of research on the antimicrobial activity and transcriptome expression of AMPs derived from BSFL following waste treatment. In the present study, food waste treatment was performed by BSFL with a substrate C/N ratio ranging from 21:1 to 10:1, marine Vibrio parahaemolyticus (VP) was selected as the model aquaculture pathogen, the antimicrobial activities of AMPs in vitro and zebrafish in vivo were examined, and the molecular mechanism of the C/N-dependent AMP difference was expounded. Findings were made that the AMP extract of C/N16:1 resulted in relatively higher antimicrobial activity in vitro than that of other C/Ns. Further, the AMPs of C/N16:1 exhibited a promising in vivo defense effect for elevating the 96-h survival rate of zebrafish from 0% to 39% after VP infection, comparable to the animal antibiotic sulfamethoxidine. The results of transcriptome analysis reveal that lysozymes were the highest expressed components in the AMP gene family. The C/N16:1 BSFL significantly up-regulated 12 out of 51 lysozyme genes compared with C/N21:1, which likely contributed to the improvement of AMP antimicrobial activity. Further, C/N16:1 significantly up-regulated the expression of lysozyme, glycosyl hydrolase and muscle protein genes compared with C/N21:1, which likely enhanced the defense ability of the immune system, the utilization of the starch-like substrate, and the mobility of the larvae, thereby facilitating the larval transformation and AMP production. Overall, such results indicate that waste C/N ratio interacted with the activity and expression of BSFL AMPs through transcriptome regulation, and the BSFL AMPs derived from food waste could be used for the defense against marine pathogens to support the sustainable development of aquaculture.

Yorkie Negatively Regulates the Expression of Antimicrobial Proteins by Inducing Cactus Transcription in Prawns Macrobrachium nipponense.

The Hippo signaling pathway controls organ size and immune system in Drosophila and mammals. Yorkie acts as a transcriptional co-activator in the Hippo pathway and cross-talks with other essential pathways. In this study, a Yorkie gene and two Cactus isoforms (designated as MnYorkie, MnCactus-a, and MnCactus-b, respectively) were isolated and characterized from oriental river prawns (Macrobrachium nipponense). Results showed that MnYorkie includes 1620 bp open reading frame and encodes a protein of 539 amino acids (aa). MnCactus-a (377 aa) and MnCactus-b (471 aa) were produced by alternative splicing. MnYorkie and MnCactus were continuously expressed in all selected tissues. Upon Gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Vibrio parahaemolyticus stimulation, the mRNA levels of MnYorkie and MnCactus in hemocytes and intestines underwent time-dependent enhancement. RNA interference studies showed that MnYorkie silencing remarkably downregulated the transcription of MnCactus but upregulated the expression of seven immune-related genes. In addition, MnYorkie silencing in vivo decreased the susceptibility of prawns to bacterial challenge. After S. aureus and V. parahaemolyticus infection, the survival rate of prawns increased significantly from 2 to 6 days, which corresponded to the period of MnYorkie knockdown. All these findings suggested that MnYorkie in the Hippo pathway might exhibit remarkable biological roles in the immune defense of M. nipponense by negatively regulating the expression of immune-related genes and promoting the transcription of MnCactus.

Transcriptional Landscaping Identifies a Beige Adipocyte Depot in the Newborn Mouse.

The present study sought to identify gene networks that are hallmarks of the developing inguinal subcutaneous adipose tissue (iWAT) and the interscapular brown adipose tissue (BAT) in the mouse. RNA profiling revealed that the iWAT of postnatal (P) day 6 mice expressed thermogenic and lipid catabolism transcripts, along with the abundance of transcripts associated with the beige adipogenesis program. This was an unexpected finding, as thermogenic BAT was believed to be the only site of nonshivering thermogenesis in the young mouse. However, the transcriptional landscape of BAT in P6 mice suggests that it is still undergoing differentiation and maturation, and that the iWAT temporally adopts thermogenic and lipolytic potential. Moreover, P6 iWAT and adult (P56) BAT were similar in their expression of immune gene networks, but P6 iWAT was unique in the abundant expression of antimicrobial proteins and virus entry factors, including a possible receptor for SARS-CoV-2. In summary, postnatal iWAT development is associated with a metabolic shift from thermogenesis and lipolysis towards fat storage. However, transcripts of beige-inducing signal pathways including β-adrenergic receptors and interleukin-4 signaling were underrepresented in young iWAT, suggesting that the signals for thermogenic fat differentiation may be different in early postnatal life and in adulthood.

Proteomics Mapping of the ISGylation Landscape in Innate Immunity.

During infection, pathogen sensing and cytokine signaling by the host induce expression of antimicrobial proteins and specialized post-translational modifications. One such protein is ISG15, a ubiquitin-like protein (UBL) conserved among vertebrates. Similar to ubiquitin, ISG15 covalently conjugates to lysine residues in substrate proteins in a process called ISGylation. Mice deficient for ISGylation or lacking ISG15 are strongly susceptible to many viral pathogens and several intracellular bacterial pathogens. Although ISG15 was the first UBL discovered after ubiquitin, the mechanisms behind its protective activity are poorly understood. Largely, this stems from a lack of knowledge on the ISG15 substrate repertoire. To unravel the antiviral activity of ISG15, early studies used mass spectrometry-based proteomics in combination with ISG15 pulldown. Despite reporting hundreds of ISG15 substrates, these studies were unable to identify the exact sites of modification, impeding a clear understanding of the molecular consequences of protein ISGylation. More recently, a peptide-based enrichment approach revolutionized the study of ubiquitin allowing untargeted discovery of ubiquitin substrates, including knowledge of their exact modification sites. Shared molecular determinants between ISG15 and ubiquitin allowed to take advantage of this technology for proteome-wide mapping of ISG15 substrates and modification sites. In this review, we provide a comprehensive overview of mass spectrometry-based proteomics studies on protein ISGylation. We critically discuss the relevant literature, compare reported substrates and sites and make suggestions for future research.

The microbiota coordinates diurnal rhythms in innate immunity with the circadian clock

Environmental light cycles entrain circadian feeding behaviors in animals that produce rhythms in exposure to foodborne bacteria. Here, we show that the intestinal microbiota generates diurnal rhythms in innate immunity that synchronize with feeding rhythms to anticipate microbial exposure. Rhythmic expression of antimicrobial proteins was driven by daily rhythms in epithelial attachment by segmented filamentous bacteria (SFB), members of the mouse intestinal microbiota. Rhythmic SFB attachment was driven by the circadian clock through control of feeding rhythms. Mechanistically, rhythmic SFB attachment activated an immunological circuit involving group 3 innate lymphoid cells. This circuit triggered oscillations in epithelial STAT3 expression and activation that produced rhythmic antimicrobial protein expression and caused resistance to Salmonella Typhimurium infection to vary across the day-night cycle. Thus, host feeding rhythms synchronize with the microbiota to promote rhythms in intestinal innate immunity that anticipate exogenous microbial exposure.

Hepatopancreas-Specific Lectin Participates in the Antibacterial Immune Response by Regulating the Expression of Antibacterial Proteins.

The hepatopancreas is an important digestive and immune organ in crustacean. There were low but stable numbers of microbes living in the hemolymph of crustacean, whereas the organs (including hepatopancreas) of crustacean were immersed in the hemolymph. It is very important to study the immune mechanism of the hepatopancreas against bacteria. In this study, a novel CTL (HepCL) with two CRDs, which was mainly expressed in the hepatopancreas, was identified in red swamp crayfish (Procambarus clarkii). HepCL binds to bacteria in vitro and could enhance bacterial clearance in vivo. Compared with the C-terminal CRD of HepCL (HepCL-C), the N-terminal CRD (HepCL-N) showed weaker bacterial binding ability in vitro and stronger bacterial clearance activity in vivo. The expression of some antimicrobial proteins, such as FLP, ALF1 and ALF5, was downregulated under knockdown of HepCL or blocked with Anti-HepCL after challenge with Vibrio in crayfish. These results demonstrated that HepCL might be involved in the antibacterial immune response by regulating the expression of antimicrobial proteins.

Lessons Learned from Hereditary 1,25-Dihydroxyvitamin D–Resistant Rickets Patients on Vitamin D Functions

We summarize here lessons learned from studies on skeletal and extra-skeletal functions of vitamin D in hereditary 1,25-dihydroxyvitamin D–resistant rickets (HVDRR) patients with a mutant, nonfunctioning vitamin D receptor (VDR). During childhood, HVDRR patients are dependent on intestinal VDR, demonstrate low intestinal fraction calcium absorption, and have a bone calcium accretion rate that leads to hypocalcemia and rickets. After puberty, there is recovery in intestinal calcium absorption and in bone calcium accretion and structure. HVDRR monocytes and lymphocytes show impairment in the expression of antimicrobial proteins and demonstrate a proinflammatory cytokine profile. However, HVDRR patients do not exhibit increased rates of infections or inflammatory diseases. Vitamin D deficiency is associated with asthmatic exacerbations. Surprisingly, HVDRR patients do not usually develop asthma. They have normal allergic tests and lung functions and are protected against provoked bronchial hyperactivity. HVDRR patients have decreased IL-5 levels in their exhaled breath condensate. Given that IL-5 is a key cytokine in the development of airway inflammation and hyperactivity and that VDR is important for IL-5 generation, it is plausible that low lung IL-5 protects HVDRR patients from asthma. Vitamin D metabolites have suppressive effects on the renin angiotensin system. However, no HVDRR patient showed hypertension or echocardiographic pathology, and their renin angiotensin metabolites were normal. The VDR is expressed throughout the reproductive system, suggesting a role in reproduction. However, the reproductive history of HVDRR patients is normal despite the lack of a normal VDR. HVDRR patients provide a unique opportunity to study the role of the VDR and the role of vitamin D in various human systems.

Inclusion of Oat and Yeast Culture in Sow Gestational and Lactational Diets Alters Immune and Antimicrobial Associated Proteins in Milk.

Simple SummaryThis study investigated the impact that supplementing sow’s gestation and lactation feed with oat alone or together with brewer’s yeast has on milk proteins and piglet growth and health. Oat and yeast supplements increased abundance of several milk proteins involved in immune protection. Piglets born from either the oat- or yeast-supplemented sows had decreased incidence of diarrhea after weaning. The average birth weights for piglets born of dams that consumed Oat were significantly greater than those that did not. However, piglets born to sows that consumed yeast in combination with oat weighed less at weaning and gained the least amount of weight post-weaning. These data suggest that oat, and to a lesser extent, yeast, added to maternal diets during gestation and lactation can positively impact milk, growth, and health of offspring but given in combination can potentially negatively affect piglet weight gain.Maternal diet supplementation with pro- and prebiotics is associated with decreased incidence of diarrhea and greater piglet performance. This study investigated the impact adding whole ground oat as a prebiotic, alone or in combination with a probiotic, yeast culture (YC) (Saccharomyces cerevisiae), to sow gestation and lactation rations had on milk protein composition, piglet growth, and incidence of post-weaning diarrhea (PWD). Diets: control (CON), CON + yeast culture (YC) [5 g/kg], CON + oat (15% inclusion rate) (Oat) or CON+ YC [5 g/kg] + Oat (15%) were fed the last 30 days of gestation and throughout lactation (18–21 days). Shotgun proteome analysis of day 4 and 7 postpartum milk found 36 differentially abundant proteins (P-adj < 0.1) in both Oat and YC supplemented sows relative to CON. Notable was the increased expression of antimicrobial proteins, lactoferrin and chitinase in milk of Oat and YC sows compared to CON. The levels of IgA, IgM (within colostrum and milk) and IgG (within milk) were similar across treatments. However, colostral IgG levels in Oat-supplemented sows were significantly lower (p < 0.05) than that of the control sows, IgG from Oat-supplemented sows displayed greater reactivity to E. coli-antigens compared with CON and YC. Piglets from sows that consumed Oat alone or in combination weighed significantly more (p < 0.05) at birth compared to CON and YC. However, piglets in the Oat + YC group weighed less at weaning and had the lowest weight gain (p < 0.05) postweaning, compared with CON. Taken together with the observation that piglets of either YC- or Oat-fed sows had less PWD compared to CON and YC+ Oat suggests that Oat or YC supplementation positively impacts piglets through expression of certain milk-associated immune and antimicrobial proteins.

The RNA helicase Dhx15 mediates Wnt-induced antimicrobial protein expression in Paneth cells

RNA helicases play roles in various essential biological processes such as RNA splicing and editing. Recent in vitro studies show that RNA helicases are involved in immune responses toward viruses, serving as viral RNA sensors or immune signaling adaptors. However, there is still a lack of in vivo data to support the tissue- or cell-specific function of RNA helicases owing to the lethality of mice with complete knockout of RNA helicases; further, there is a lack of evidence about the antibacterial role of helicases. Here, we investigated the in vivo role of Dhx15 in intestinal antibacterial responses by generating mice that were intestinal epithelial cell (IEC)-specific deficient for Dhx15 (Dhx15 f/f Villin1-cre, Dhx15ΔIEC). These mice are susceptible to infection with enteric bacteria Citrobacter rodentium (C. rod), owing to impaired α-defensin production by Paneth cells. Moreover, mice with Paneth cell-specific depletion of Dhx15 (Dhx15 f/f Defensinα6-cre, Dhx15ΔPaneth) are more susceptible to DSS (dextran sodium sulfate)-induced colitis, which phenocopy Dhx15ΔIEC mice, due to the dysbiosis of the intestinal microbiota. In humans, reduced protein levels of Dhx15 are found in ulcerative colitis (UC) patients. Taken together, our findings identify a key regulator of Wnt-induced α-defensins in Paneth cells and offer insights into its role in the antimicrobial response as well as intestinal inflammation.

Tryptophan-derived metabolites and BAK1 separately contribute to Arabidopsis postinvasive immunity against Alternaria brassicicola

Nonhost resistance of Arabidopsis thaliana against the hemibiotrophic fungus Colletotrichum tropicale requires PEN2-dependent preinvasive resistance and CYP71A12 and CYP71A13-dependent postinvasive resistance, which both rely on tryptophan (Trp) metabolism. We here revealed that CYP71A12, CYP71A13 and PAD3 are critical for Arabidopsis’ postinvasive basal resistance toward the necrotrophic Alternaria brassicicola. Consistent with this, gene expression and metabolite analyses suggested that the invasion by A. brassicicola triggered the CYP71A12-dependent production of indole-3-carboxylic acid derivatives and the PAD3 and CYP71A13-dependent production of camalexin. We next addressed the activation of the CYP71A12 and PAD3-dependent postinvasive resistance. We found that bak1-5 mutation significantly reduced postinvasive resistance against A. brassicicola, indicating that pattern recognition contributes to activation of this second defense-layer. However, the bak1-5 mutation had no detectable effects on the Trp-metabolism triggered by the fungal penetration. Together with this, further comparative gene expression analyses suggested that pathogen invasion in Arabidopsis activates (1) CYP71A12 and PAD3-related antifungal metabolism that is not hampered by bak1-5, and (2) a bak1-5 sensitive immune pathway that activates the expression of antimicrobial proteins.

Functional analysis of Medicago-derived pathogen-induced gene promoters for usage in transgenic alfalfa

Three gene promoters from the model legume species Medicago truncatula and Medicago sativa (alfalfa) pathogenesis-related (PR) proteins (MtPR5, MtPR10, and MsPR10) were isolated and investigated using in silico and in situ approaches. For the functional analysis of these promoters, plant transformation vectors linking promoter sequences with the β-glucuronidase (GUS) reporter gene were constructed and utilized to generate transgenic alfalfa. Histochemical GUS staining established that the PR5 and both PR10 promoters were functional in alfalfa and induce GUS expression primarily in the root tissue. When transgenic alfalfa leaves were inoculated with a diverse set of alfalfa pathogens, either Phoma medicaginis, Colletotrichum trifolii, or Pseudomonas syringae pv. syringae, the promoters were responsive to induction by both bacterial and fungal pathogens. Gene expression analysis also indicated that all three PR promoters were pathogen-induced and upregulate transgene expression. Additionally, several putative transcription regulator elements (REs) responsible for the observed pathogen-induced promoter activity were predicted in each of the promoter sequences. These characterized Medicago PR promoters may be a unique and valuable tool for pathogen-induced transgenic expression of antimicrobial proteins or other genes enhancing disease resistance in alfalfa.

Characterization and genome sequencing of three Aeromonas hydrophila-specific phages, CF8, PS1, and PS2.

Aeromonas hydrophila is an important finfish pathogen, besides being an opportunistic human pathogen. In the present study, the genomes of three A. hydrophila-specific phages, CF8, PS1, and PS2, were isolated, characterized and sequenced. Transmission electron microscopy showed that all three phages had typical Myoviridae morphology. The linear dsDNA genomes of CF8, PS1, and PS2 were 238,150 bp, 237,367 bp, and 240,447 bp in length, with a GC content of 42.2%, 38.8%, and 38.8%, respectively. The low sequence similarity (67.6% - 69.8% identity with 27.0% - 29.0% query coverage) to other phage genomes in the NCBI database indicated the novel nature of the CF8, PS1, and PS2 genomes. A total of 244, 247, and 250 open reading frames (ORFs) were predicted in the CF8, PS1, and PS2 genome, respectively. During the annotation process, functional predictions were made for 28-31 ORFs, while the rest were classified as "hypothetical proteins" with yet unknown functions. Genes for tRNAs were also detected in all phage genomes. As all three phages in the present study had a very narrow host range with lytic activity against only one strain of A. hydrophila, these phages could be good candidates for phage typing applications. Moreover, the endolysin- and lytic-transglycosylase-encoding genes could be used for recombinant cloning and expression of anti-microbial proteins.

Nutrient Excess Triggers the Expression of the Penicillium chrysogenum Antifungal Protein PAFB.

Nutrient limitation and nonfavorable growth conditions have been suggested to be major triggers for the expression of small, cysteine-rich antimicrobial proteins (AMPs) of fungal origin, e.g., the Penicillium chrysogenum antifungal protein (PAF), the Aspergillus giganteus antifungal protein (AFP), the Aspergillus niger antifungal protein (AnAFP). Therefore, these AMPs have been considered to be fungal secondary metabolite products. In contrast, the present study revealed that the expression of the PAF-related AMP P. chrysogenum antifungal protein B (PAFB) is strongly induced under nutrient excess during the logarithmic growth phase, whereas PAFB remained under the detection level in the supernatant of cultures grown under nutrient limitation. The efficiency of the pafB-promoter to induce PAFB expression was compared with that of two P. chrysogenum promoters that are well established for recombinant protein production: the paf-promoter and the xylose-inducible promoter of the xylanase gene, xylP. The inducibility of the pafB-promoter was superior to that of the xylP-promoter yielding comparable PAFB amounts as under the regulation of the paf-promoter. We conclude that (i) differences in the expression regulation of AMPs suggest distinct functional roles in the producer beyond their antifungal activity; and (ii) the pafB-promoter is a promising tool for recombinant protein production in P. chrysogenum, as it guarantees strong gene expression with the advantage of inducibility.

Localized expression of antimicrobial proteins mitigates huanglongbing symptoms in Mexican lime

Citrus huanglongbing (HLB) is a devastating disease associated with Candidatus Liberibacter asiaticus spp. (CLas), a bacterium restricted to the sieve tube system of the phloem that is transmitted by the psyllid vector, Diaphorina citri. In this study, the human antimicrobial peptides, lysozyme and β-defensin 2, were targeted to the vascular tissue of Mexican lime (Citrus x aurantifolia [Christm.] Swingle) by fusion to a phloem-restricted protein. Localized expression was achieved, via Agrobacterium tumefaciens-mediated transformation of the stem, which led to protein expression and mobilization within the vascular tissue of heterotrophic tissues. HLB-infected plants were monitored for 360 days. Lower bacteria titers were observed in plants expressing either β-defensin 2, lysozyme, or the combination thereof, and these plants had increased photosynthesis, compared to untreated control trees. Thus, targeting of antimicrobial proteins to the vascular tissue was effective in decreasing CLas titer, and alleviating citrus greening symptoms. Based on these findings, this strategy could be used to effectively treat plants that are already infected with bacterial pathogens that reside in the phloem translocation stream.