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.

Enteric Nervous System-Derived IL-18 Orchestrates Mucosal Barrier Immunity.

Mucosal barrier immunity is essential for the maintenance of the commensal microflora and combating invasive bacterial infection. Although immune and epithelial cells are thought to be the canonical orchestrators of this complex equilibrium, here, we show that the enteric nervous system (ENS) plays an essential and non-redundant role in governing the antimicrobial protein (AMP) response. Using confocal microscopy and single-molecule fluorescence in situ mRNA hybridization (smFISH) studies, we observed that intestinal neurons produce the pleiotropic cytokine IL-18. Strikingly, deletion of IL-18 from the enteric neurons alone, but not immune or epithelial cells, rendered mice susceptible to invasive Salmonella typhimurium (S.t.) infection. Mechanistically, unbiased RNA sequencing and single-cell sequencing revealed that enteric neuronal IL-18 is specifically required for homeostatic goblet cell AMP production. Together, we show that neuron-derived IL-18 signaling controls tissue-wide intestinal immunity and has profound consequences on the mucosal barrier and invasive bacterial killing.

Ancestral ribonucleases back in motion for evolutionary-dynamics guided protein design.

The dynamics behavior of a protein is essential for its functionality. Here, Doucet et al. demonstrate how the evolutionary analysis of conformational pathways within a protein family serves to identify common core scaffolds that accommodate branch-specific functional regions controlled by flexibility switches, offering a model for evolutionary-dynamics based protein design.

The relevance of intestinal barrier dysfunction, antimicrobial proteins and bacterial endotoxin in metabolic dysfunction-associated steatotic liver disease.

BACKGROUND: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a leading cause of end-stage liver disease associated with increased mortality and cardiovascular disease. Obesity and diabetes are the most important risk factors of MASLD. It is well-established that obesity-associated insulin resistance leads to a situation of tissue lipotoxicity characterized by an accumulation of excess fat in non-fat tissues such as the liver, promoting the development of MASLD, and its progression into metabolic dysfunction-associated steatohepatitis. METHODS: Here, we aimed to review the impact of disrupted intestinal permeability, antimicrobial proteins and bacterial endotoxin in the development and progression of MASLD. RESULTS AND CONCLUSION: Recent studies demonstrated that obesity- and obesogenic diets-associated alterations of intestinal microbiota along with the disruption of intestinal barrier integrity, the alteration in antimicrobial proteins and, in consequence, an enhanced translocation of bacterial endotoxin into bloodstream might contribute to this pathological process through to impacting liver metabolism and inflammation.

Characterization of a novel Type-I Crustin (carcininPm2) from black tiger shrimp Penaeus monodon.

Carcinins are type-I crustins from crustaceans and play an important role in innate immune system. In this study, type-I crustins, carcininPm1 and carcininPm2, from the hemocytes of Penaeus monodon were identified. Comparison of their amino acid sequences and the phylogenetic tree revealed that they were closely related to the other crustacean carcinin proteins, but were clustered into different groups of the carcinin proteins. The full-length amino acids of carcininPm1 and carcininPm2 were 92 and 111 residues, respectively. CarcininPm1 and carcininPm2 were expressed mainly in hemocytes and intestine compared to the other tissues. The expression of carcininPm1 and carcininPm2 were dramatically increased in early time of bacterial challenged shrimp hemocytes. In contrast, the carcininPm1 and carcininPm2 were expressed in response to late state of YHV-infected shrimp hemocytes where the copy number of virus was high. The recombinant carcininPm2 (rcarcininPm2) but not its WAP domain (rcarcininPm2_WAP) exhibited antimicrobial activity against Vibrio harveyi and Vibrio parahaemolyticus AHPND but not other bacteria tested. The rcarcininPm2 was able to prolong the survival rate of VH-treated post larval shrimp from about 102 h to 156 h. These studies indicated that the carcininPm2 possessed the potential and challenges as antibacterial in innate immunity of shrimp.

Dental caries in children and vitamin D deficiency: a narrative review.

Dental caries represents one of the most prevalent health problems in childhood. Numerous studies have assessed that vitamin D deficiency is highly related to dental caries in primary and permanent teeth in children. The aim of this study is to elaborate a narrative review about proposed mechanisms by which vitamin D deficiency interacts with dental caries process in children. Vitamin D deficiency during pregnancy may cause intrauterine enamel defects, and through childhood is accompanied by insufficient activity of antibacterial peptides, decreased saliva secretion, and a low level of calcium in saliva.  Conclusion: In conclusion, vitamin D deficiency would increase the risk of caries in the primary and/or permanent dentition. Relationship between vitamin D deficiency and dental caries is evident enough for vitamin D deficiency to be considered as a risk factor for dental caries in children. Optimal levels of vitamin D throughout pregnancy and childhood may be considered an additional preventive measure for dental caries in the primary and permanent dentition.

Antibiotic Resistant Biofilms and the Quest for Novel Therapeutic Strategies.

Antimicrobial resistance (AMR) is one of the major leading causes of death around the globe. Present treatment pipelines are insufficient to overcome the critical situation. Prominent biofilm forming human pathogens which can thrive in infection sites using adaptive features results in biofilm persistence. Considering the present scenario, prudential investigations into the mechanisms of resistance target them to improve antibiotic efficacy is required. Regarding this, developing newer and effective treatment options using edge cutting technologies in medical research is the need of time. The reasons underlying the adaptive features in biofilm persistence have been centred on different metabolic and physiological aspects. The high tolerance levels against antibiotics direct researchers to search for novel bioactive molecules that can help combat the problem. In view of this, the present review outlines the focuses on an opportunity of different strategies which are in testing pipeline can thus be developed into products ready to use.

A conserved extracellular Ribo1 with broad-spectrum cytotoxic activity enables smut fungi to compete with host-associated bacteria.

Ribotoxins are secreted ribonucleases that specifically target and cleave the universally conserved sarcin-ricin loop sequence of rRNA, which leads to inhibition of protein biosynthesis and subsequently to cell death. We have identified and characterized a secreted Ribo1 protein of plant pathogenic smut fungi. Heterologous expression in different model systems showed that smut Ribo1 has cytotoxic activity against bacteria, yeast, host and nonhost plants. Recombinant expression of Ribo1 in Nicotiana benthamiana induced plant cell death; however, an active site mutant induced cell death only when expressed as a secreted protein. In the maize smut Ustilago maydis, transcription of Ribo1 is specifically induced in early infection stages. While a knockout mutant revealed that Ribo1 is dispensable for U. maydis virulence, the overexpression of Ribo1 in planta had a strong dominant negative effect on virulence and induced host defense responses including cell death. Our findings suggest a function of Ribo1 during the epiphytic development rather than for invasive colonization of the host. Accordingly, in the presence of the biocontrol bacteria Pantoea sp., which were isolated from maize leaves, the ribo1 knockout mutant was significantly impaired in virulence. Together, we conclude that Ribo1 enables smut fungi to compete with host-associated bacteria during epiphytic development.

Proteins released into the plant apoplast by the obligate parasitic protist Albugo selectively repress phyllosphere-associated bacteria.

Biotic and abiotic interactions shape natural microbial communities. The mechanisms behind microbe-microbe interactions, particularly those protein based, are not well understood. We hypothesize that released proteins with antimicrobial activity are a powerful and highly specific toolset to shape and defend plant niches. We have studied Albugo candida, an obligate plant parasite from the protist Oomycota phylum, for its potential to modulate the growth of bacteria through release of antimicrobial proteins into the apoplast. Amplicon sequencing and network analysis of Albugo-infected and uninfected wild Arabidopsis thaliana samples revealed an abundance of negative correlations between Albugo and other phyllosphere microbes. Analysis of the apoplastic proteome of Albugo-colonized leaves combined with machine learning predictors enabled the selection of antimicrobial candidates for heterologous expression and study of their inhibitory function. We found for three candidate proteins selective antimicrobial activity against Gram-positive bacteria isolated from A. thaliana and demonstrate that these inhibited bacteria are precisely important for the stability of the community structure. We could ascribe the antibacterial activity of the candidates to intrinsically disordered regions and positively correlate it with their net charge. This is the first report of protist proteins with antimicrobial activity under apoplastic conditions that therefore are potential biocontrol tools for targeted manipulation of the microbiome.

Salivary bactericidal/permeability-increasing protein: A novel inflammatory marker associated with periodontitis.

OBJECTIVES: The present case-control study aims to investigate the salivary levels of bactericidal/permeability-increasing protein (BPI) and interleukin-1beta (IL-1ß) in systemically healthy individuals with periodontitis and periodontally healthy for the evaluation of BPI's relation with periodontal inflammation and clinical diagnosis of periodontitis. MATERIALS AND METHODS: A total of 100 participants were enrolled in this study and divided into periodontitis (P group) (n = 50) and periodontally healthy (H group) (n = 50) groups based on their full-mouth periodontal examination results including plaque index, probing pocket depth, gingival index, bleeding on probing, and clinical attachment level. Unstimulated whole saliva was collected. Salivary BPI and IL-1ß levels were determined using an enzyme-linked immunosorbent assay. Receiver operating characteristic (ROC) curves were created to determine the diagnostic value of BPI. RESULTS: The levels of BPI and IL-1ß in saliva were significantly higher in the P group than in the H group (p<0.001). Moreover, salivary BPI and IL-1ß levels correlated significantly with all clinical periodontal parameters (all p<0.001). Interestingly, there was a strong positive correlation between salivary levels of BPI and IL-1ß (r=0.544, p<0.001). In addition, the results of the ROC curve analysis showed that BPI had a high diagnostic potential to distinguish periodontitis from healthy controls with an area under the curve value of 0.94% (p<0.000). CONCLUSION: The significantly higher salivary levels of BPI in periodontitis patients together with strong positive correlations between all periodontal parameters and salivary IL-1ß levels suggest that BPI may be involved in the inflammatory process of periodontal disease. CLINICAL RELEVANCE: The present study for the first time report that salivary BPI levels may serve as a potential biomarker of inflammation in periodontal disease. TRIAL REGISTRATION NUMBER: Thai Clinical Trials.gov (TCTR20211222008) (22 December 2021).

Antimicrobial Properties of a Peptide Derived from the Male Fertility Factor kl2 Protein of Drosophila melanogaster.

Antimicrobial peptides (AMPs) are important components of innate immunity. Here, we report the antimicrobial properties of a peptide derived from the Male fertility factor kl2 (MFF-kl2) protein of Drosophila melanogaster, which was identified as a functional analog of the mammalian antibacterial chemerin-p4 peptide. The antimicrobial activity of multifunctional chemerin is mainly associated with a domain localized in the middle of the chemerin sequence, Val66-Pro85 peptide (chemerin-p4). Using bioinformatic tools, we found homologs of the chemerin-p4 peptide in the proteome of D. melanogaster. One of them is MFF-p1, which is a part of the MFF kl2 protein, encoded by the gene male fertility factor kl2 (kl-2) located on the long arm of the Y chromosome. The second detected peptide (Z-p1) is a part of the Zizimin protein belonging to DOCK family, which is involved in cellular signaling processes. After testing the antimicrobial properties of both peptides, we found that only MFF-p1 possesses these properties. Here, we demonstrate its antimicrobial potential both in vitro and in vivo after infecting D. melanogaster with bacteria. MFF-p1 strongly inhibits the viable counts of E. coli and B. subtilis after 2 h of treatment and disrupts bacterial cells. The expression of kl-2 is regulated by exposure to bacteria and by the circadian clock.

A Lysozyme Murein Hydrolase with Broad-Spectrum Antibacterial Activity from Enterobacter Phage myPSH1140.

Bacteriophages and bacteriophage-derived peptidoglycan hydrolases (endolysins) present promising alternatives for the treatment of infections caused by multidrug resistant Gram-negative and Gram-positive pathogens. In this study, Gp105, a putative lysozyme murein hydrolase from Enterobacter phage myPSH1140 was characterized in silico, in vitro as well as in vivo using the purified protein. Gp105 contains a T4-type lysozyme-like domain (IPR001165) and belongs to Glycoside hydrolase family 24 (IPR002196). The putative endolysin indeed had strong antibacterial activity against Gram-negative pathogens, including E. cloacae, K. pneumoniae, P. aeruginosa, S. marcescens, Citrobacter sp., and A. baumannii. Also, an in vitro peptidoglycan hydrolysis assay showed strong activity against purified peptidoglycans. This study demonstrates the potential of Gp105 to be used as an antibacterial protein to combat Gram-negative pathogens.

Characterization of defense responses against bacterial pathogens in duckweeds lacking EDS1.

ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) mediates the induction of defense responses against pathogens in most angiosperms. However, it has recently been shown that a few species have lost EDS1. It is unknown how defense against disease unfolds and evolves in the absence of EDS1. We utilize duckweeds; a collection of aquatic species that lack EDS1, to investigate this question. We established duckweed-Pseudomonas pathosystems and used growth curves and microscopy to characterize pathogen-induced responses. Through comparative genomics and transcriptomics, we show that the copy number of infection-associated genes and the infection-induced transcriptional responses of duckweeds differ from other model species. Pathogen defense in duckweeds has evolved along different trajectories than in other plants, including genomic and transcriptional reprogramming. Specifically, the miAMP1 domain-containing proteins, which are absent in Arabidopsis, showed pathogen responsive upregulation in duckweeds. Despite such divergence between Arabidopsis and duckweed species, we found conservation of upregulation of certain genes and the role of hormones in response to disease. Our work highlights the importance of expanding the pool of model species to study defense responses that have evolved in the plant kingdom independent of EDS1.

The future of recombinant host defense peptides.

The antimicrobial resistance crisis calls for the discovery and production of new antimicrobials. Host defense peptides (HDPs) are small proteins with potent antibacterial and immunomodulatory activities that are attractive for translational applications, with several already under clinical trials. Traditionally, antimicrobial peptides have been produced by chemical synthesis, which is expensive and requires the use of toxic reagents, hindering the large-scale development of HDPs. Alternatively, HDPs can be produced recombinantly to overcome these limitations. Their antimicrobial nature, however, can make them toxic to the hosts of recombinant production. In this review we explore the different strategies that are used to fine-tune their activities, bioengineer them, and optimize the recombinant production of HDPs in various cell factories.

Antimicrobial defenses of table eggs: Importance of antibacterial proteins in egg white as a function of hen age in an extended production cycle.

The importance of egg natural defences to prevent bacterial contamination and their relation with hen age in extended production cycles were evaluated. Egg-white from eggs of different hen age groups (up 100-weeks-old) and lines (Hy-Line white and brown) were inoculated with Gram-positive Staphylococcus aureus or Gram-negative Salmonella Typhimurium, ranging from 103-106 CFU/mL. Our results show that concentrations of egg-white lysozyme and, particularly, ovotransferrin are important to modulate bacterial survival in a dose-dependent matter. Depending on protein concentration, their effect ranges from bactericidal to bacteriostatic, with a threshold for bacterial contamination that depends also on hen age and line. The concentrations of lysozyme and ovotransferrin increased with hen age (up to 2 and 22 w/w% of total protein, respectively), and eggs laid by older hens exhibited the greatest potential to prevent the growth of the highest Salmonella inoculum (106 CFU/mL). Salmonella-penetration experiments demonstrated that non-contaminated eggs display significantly higher concentrations of antimicrobial proteins. However, eggs from older hens needed a higher concentration of these proteins (>20% ovotransferrin) to prevent bacterial contamination, showing that antimicrobial protein concentrations in egg-whites was not the only factor influencing bacterial contamination. Finally, this study demonstrated that egg-white of eggs produced by old hens are less prone to contamination by Salmonella.

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.

Structure-Based Design of an RNase Chimera for Antimicrobial Therapy.

Bacterial resistance to antibiotics urges the development of alternative therapies. Based on the structure-function of antimicrobial members of the RNase A superfamily, we have developed a hybrid enzyme. Within this family, RNase 1 exhibits the highest catalytic activity and the lowest cytotoxicity; in contrast, RNase 3 shows the highest bactericidal action, alas with a reduced catalytic activity. Starting from both parental proteins, we designed a first RNase 3/1-v1 chimera. The construct had a catalytic activity much higher than RNase 3, unfortunately without reaching an equivalent antimicrobial activity. Thus, two new versions were created with improved antimicrobial properties. Both of these versions (RNase 3/1-v2 and -v3) incorporated an antimicrobial loop characteristic of RNase 3, while a flexible RNase 1-specific loop was removed in the latest construct. RNase 3/1-v3 acquired both higher antimicrobial and catalytic activities than previous versions, while retaining the structural determinants for interaction with the RNase inhibitor and displaying non-significant cytotoxicity. Following, we tested the constructs' ability to eradicate macrophage intracellular infection and observed an enhanced ability in both RNase 3/1-v2 and v3. Interestingly, the inhibition of intracellular infection correlates with the variants' capacity to induce autophagy. We propose RNase 3/1-v3 chimera as a promising lead for applied therapeutics.

Effects of Carbohydrate Mouth Rinsing on Salivary Lysozyme, Mood States and Running Performance Among Recreational Runners.

INTRODUCTION: Carbohydrate (CHO) mouth rinsing can enhance sports performance through a central action mediated by receptors in the mouth. This study examined the effect of a CHO mouth rinse on salivary lysozyme concentrations, mood states and running performances. METHODS: Ten males recreational runners were randomised to three running trials with a 1 week recovery period between the trials. Each trial involved running at 75% maximum heart rate (HRmax) for 1 h, followed by a 15 min time trial. The participants used a CHO mouth rinse, placebo (PLA) solution or control (CON, no solution) every 15 min during the exercise. Heart rate (HR), rating of perceived exertion (RPE) and mood states were recorded pre-, during and post-exercise. Saliva samples were collected pre-, post- and 1 h post-exercise. RESULTS: There was no significant interaction and time effect (P > 0.05) on the salivary lysozyme concentration and running performance, but it was significant (P < 0.05) for HR and RPE (increase in all trials). However, there was no significant difference (P > 0.05) in salivary lysozyme concentrations, running performances, HR values or RPE between the trials. Mood states were not significantly different (P > 0.05) between the trials, but one of the mood sub-scales showed a significant (P < 0.001) time effect (increase fatigue in all trials). CONCLUSION: CHO mouth rinsing did not affect physiological parameters, salivary lysozyme concentrations, mood states or running performance among recreational runners.

Structural and functional properties of antimicrobial protein L5 of Lysоbacter sp. XL1.

The Gram-negative bacterium Lysobacter sp. XL1 secretes into the extracellular space five bacteriolytic enzymes that lyse the cell walls of competing microorganisms. Of special interest are homologous lytic proteases L1 and L5. This work found protein L5 to possess Gly-Gly endopeptidase and N-acetylmuramoyl-L-Ala amidase activities with respect to staphylococcal peptidoglycan. Protein L5 was found to be capable of aggregating into amyloid-like fibril structures. The crystal structure of protein L5 was determined at a 1.60-Å resolution. Protein L5 was shown to have a rather high structural identity with bacteriolytic protease L1 of Lysobacter sp. XL1 and α-lytic protease of Lysobacter enzymogenes at a rather low identity of their amino acid sequences. Still, the structure of protein L5 was revealed to have regions that differed from their equivalents in the homologs. The revealed structural distinctions in L5 are suggested to be of importance in exhibiting its unique properties.

Purification and MIC analysis of antimicrobial proteins from Cucumis sativus L. seeds.

BACKGROUND: Cucumis sativus L. (cucumber), from the family Cucurbitaceae, is a therapeutic plant with various pharmacological benefits, broadly utilized as a part of complementary medicine (e.g., Unani, Ayurveda, Siddha, and Traditional Chinese). In light of past research discoveries, this plant had been chosen to consider its potential antibacterial action. METHODS: Extracts were purified by dialysis and ion exchange chromatography strategy and then assayed for antibacterial activity against four standard pathogenic bacterial strains known to cause foodborne infections and spoilage of food and herbal drugs. Antimicrobial peptides were extracted from seeds using a sodium phosphate citrate (pH 7.2) - CTAB cradle (pH 6.0). RESULTS: The highest protein concentration was seen with elute fractions 1 and 3 (370 mg/mL) compared with elute fractions 2 and 4 (340 mg/mL). Among the bacteria utilized, E. coli was clearly the most sensitive out of selected four strains. CONCLUSION: Our results suggest that Cucumis sativus L seeds extracts have significant potentials as new antimicrobial agents.