Comparative functional genomic analysis of two <i>Vibrio </i>phages reveals complex metabolic interactions with the host cell

Sequencing and annotation was performed for two giant double stranded DNA bacteriophages, φGrn1 and φSt2 of the Myoviridae family, considered to be of great interest for phage therapy against Vibrios in aquaculture live feeds. In addition, phage-host metabolic interactions and exploitation was studied by transcript profiling of selected viral and host genes. Comparative genomic analysis with other giant Vibrio phages was also performed to establish the presence and location of homing endonucleases highlighting distinct features for both phages. Phylogenetic analysis revealed that they belong to the schizoT4like clade. Although many reports of newly sequenced viruses have provided a large set of information, basic research related to the shift of the bacterial metabolism during infection remains stagnant. The function of many viral protein products in the process of infection is still unknown. Genome annotation identified the presence of several viral ORFs participating in metabolism, including a Sir2/cobB (sirtuin) protein and a number of genes involved in auxiliary NAD+ and nucleotide biosynthesis, necessary for phage DNA replication. Key genes were subsequently selected for detail study of their expression levels during infection. This work suggests a complex metabolic interaction and exploitation of the host metabolic pathways and biochemical processes, including a possible post-translational protein modification, by the virus during infection

Comparative genomic analysis of marine bacteriophages

Isolation and/or full characterization, including sequencing and annotations, was per-formed for five double stranded DNA bacteriophages of the Myoviridae and Si-phoviridae families, named φGrn1, φSt2, Aphrodite1, Athena1 and Ares1, considered to be of great interest for phage therapy against Vibrios. Phylogenetic analysis re-vealed that φGrn1 and φSt2 belong to the “schizoT4like” clade, Aphrodite1 to “phiKZlikevirus” clade, Athena1 to an unclassified clade of Myoviridae family and Ares1 to an unclassified clade of Siphoviridae family. In addition, phage–host meta-bolic interactions and exploitation was studied by transcript profiling of selected viral and host genes. Although many reports of newly sequenced viruses have provided a large set of information, basic research related to the shift of the bacterial metabolism during infection remains stagnant. The function of many viral protein products in the process of infection is still unknown. Regarding the two “schizoT4like” phages ge-nome annotation identified the presence of several viral open reading frames (ORFs) participating in metabolism, including a Sir2/cobB (sirtuin) protein and a number of genes involved in auxiliary NAD+ and nucleotide biosynthesis, necessary for phage DNA replication. Key genes were subsequently selected for detail study of their ex-pression levels during infection. Additionally comparative genomic analysis with other Vibrio phages was also performed to establish the presence and location of homing endonucleases, tRNAs and lysozymes, highlighting distinct features for them. This work suggests a complex metabolic interaction and exploitation of the host metabolic pathways and biochemical processes, including a possible post-translational protein modification, by “schizoT4like” viruses during infection. Distinctive features of the other 3 viruses are also studied and discussed. Phage therapy interest has been revived during the last decade in an attempt to tackle antibiotic resistant bacteria, but its appli-cation is hampered by the development of phage-resistant bacterial strains. Although bacterial intracellular molecular mechanisms of resistance development against phage infections have been well characterized over the years, the knowledge about defensive mechanisms that include alterations in membrane proteins remains inadequate. Bacte-ria can develop resistance against phages not only through costly constitutive muta-tions, but also by altering, shutting down or diminishing the expression of specific transmembrane channels. Transcriptional changes of membrane and transmembrane transporters of the Gram negative fish pathogen Vibrio alginolyticus were monitored on phage-resistant strains against 3 of the lytic Vibrio phages isolated and/or character-ized in the current thesis, belonging to “schizoT4like” and “phiKZlikevirus” clades of Myoviridae family and a lytic bacteriophage belonging to Siphoviridae family. Phage-resistant strains of V. alginolyticus revealed also phenotypic differences on growth rate and metabolic activity compared to the wild type strain. We correlated these differ-ences with changes in the transcription levels of sugar and amino acid ABC, PTS and other transporters. More specifically, the targeted transporters were downregulated, whereas some transcript levels were almost totally depleted. Changes of intracellular metabolites in the resistant strains were also monitored. Finally, we studied the tran-scriptional pattern of the biochemical pathways of TCA cycle and amino acid biosyn-thesis. Several defense mechanisms that bacteria may utilize in order to circumvent viral attack, have been reported in the literature, yet the mechanisms that regulate the metabolic reprogramming of phage-resistant strains remain rather unexplored. These results suggest that phage resistant bacteria are able to diminish the transcription levels of the membrane transporters and reprogram their metabolism. The present work pro-motes Vibrio sp. for studying the mechanisms that Gram negative bacteria may follow against their viral predators, emphasizing that phage resistant phenotype in Vibrio spe-cies can be a result of a metabolic adaptation and reproramming, while also strength-ens the concept for developing phage cocktails against resistant to antibiotics bacteria.Στην παρούσα διδακτορική διατριβή, πραγματοποιήθηκε απομόνωση και/ή πλήρης χαρακτηρισμός, συμπεριλαμβανομένου του γονιδιωμάτος και των γονιδίων τους, για πέντε λυτικούς βακτηριοφάγους των οικογενειών Myoviridae και Siphoviridae, όπου ονομάστηκαν φGrn1, φSt2, Aphrodite1, Athena1 and Ares1 και θεωρούνται καλοί υ-ποψήφιοι για εφαρμογή φαγοθεραπείας στις υδατοκαλλιέργειες απέναντι στα παθογό-να των ψαριών του γένους Vibrio. H φυλογενετική ανάλυση και η συγκριτική γονιδιωματική έδειξε ότι οι φGrn1 και φSt2 ανήκουν στον κλάδο “schizoT4like”, o Aphrodite1 στον κλάδο “phiKZlikevirus”, o Athena1 σε μη καταταγμένους σε κλά-δους φάγους της οικογένειας Myoviridae και ο Ares1 σε μη καταταγμένους σε κλά-δους φάγους της οικογένειας Siphoviridae. Μετά την αλληλούχιση των φGrn1 και φSt2, πραγματοποιήθηκε λειτουργική γονιδιωματική με ανάλυση των μεταγραφημά-των του ιού και του βακτηρίου, ώστε να μελετηθούν τα γονίδια που συμμετέχουν στην αλληλεπίδραση των δύο την ώρα της μόλυνσης. Αν και οι αναφορές με νεοαλληλου-χημένα γονιδιώματα έχουν προσφέρει μεγάλο και χρήσιμο όγκο πληροφορίας τα τε-λευταία χρόνια, η βασική έρευνα σχετιζόμενη με τον μεταβολικό χειρισμό που βιώνει ο ξενιστής από τον ιό την ώρα της μόλυνσης από τους “schizoT4like” φάγους απου-σιάζει. Η λειτουργία πολλών ανοιχτών αναγνωστικών πλαισίων των βακτηριοφαγικών γονιδιωμάτων παραμένει άγνωστη. Όμως, η in silico πρόβλεψη γονιδίων αποκάλυψε πολλά από αυτά που σχετίζονται με την βιοσύνθεση του NAD+, μία πρωτεΐνη με δρά-ση σιρτουίνης (Sir2/cobB πρωτεΐνη), αλλά και γονιδίων που συμμετέχουν στην βιο-σύνθεση νουκλεοτιδίων, απαραίτητα για τον πολλαπλασιασμό του φαγικού DNA. Αυτά τα γονίδια κλειδιά μελετήθηκαν περαιτέρω μέσω μεταγραφομικής ανάλυσης κα-τά τη διάρκεια της μόλυνσης. Ακόμα, η συγκριτική γονιδιωματική χρησιμοποιήθηκε για να εντοπιστούν και να χαρακτηριστούν μεταθετές ενδονουκλεάσες που φέρουν τα φαγικά γονιδιώματα, μαζί με τα tRNAs και τις φαγικές λυσοζύμες. Η παρούσα διδακτορική διατριβή προτείνει μία πολύπλοκη μεταβολική αλληλεπίδραση του δυαδικού συστήματος Vibrio-λυτικός φάγος, αλλά και έναν μεταβολικό χειρισμό που υπόκειται ο ξενιστής από τον ιό συμπεριλαμβανομένης μίας μεταμεταφραστικής πρωτεϊνικής αλληλεπίδρασης με στόχο την ενεργοποίηση πολλών ενζύμων. Μοναδικά και ενδια-φέροντα στοιχεία των άλλων τριών βακτηριοφάγων επίσης αναλύονται και συζητιού-νται. Ο βιολογικός έλεγχος παθογόνων βακτηρίων με βακτηριοφάγους, ή αλλιώς φαγοθεραπεία, έχει επανέλθει στο προσκήνιο τα τελευταία χρόνια σε μια προσπάθεια να αντιμετωπιστούν τα βακτήρια που είναι ανθεκτικά στα αντιβιοτικά. Παρόλα αυτά όμως η εφαρμογή καθυστερεί, με έναν από τους λόγους να είναι η ανθεκτικότητα που εμφανίζουν τα βακτήρια στους ιούς. Αν και πολλοί μοριακοί ενδοκυτταρικοί μηχανι-σμοί έχουνε περιγραφεί και μελετηθεί τα τελευταία χρόνια και ευθύνονται για την αν-θεκτικότητα, η έρευνα γύρω από μηχανισμούς άμυνας που συμπεριλαμβάνουν την αναδιάταξη των πρωτεϊνών-καναλιών της κυτταρικής μεμβράνης των ανθεκτικών στελεχών παραμένει ανεπαρκής. Τα βακτήρια μπορούν να αναπτύξουν ανθεκτικότητα σε λυτικούς φάγους, όχι μόνο μέσα από ενεργειακά δαπανηρές μεταλλάξεις, αλλά και μέσα από αλλαγές στη γονιδιακή έκφραση μεμβρανικών πρωτεϊνών που λειτουργούν ως υποδοχείς του ιού. Αλλαγές στη γονιδιακή έκφραση που παρατηρήθηκαν σε μεμ-βρανικές και διαμεμβρανικές πρωτεΐνες δείχνουν ότι μπορούν να προσφέρουν ανθε-κτικότητα έναντι λυτικών βακτηριοφάγων στο Vibrio alginolyticus. Τα ανθεκτικά στους ιούς στελέχη παρουσίασαν επίσης και φαινοτυπικές αλλαγές, όπως στο ρυθμό ανάπτυξης, αλλά και τους ενδοκυτταρικούς μεταβολίτες. Αυτές οι διαφορές συσχετί-στηκαν με αλλαγές στα σχετικά ποσοστά έκφρασης σακχάρων και αμινοξέων ABC, PTS και άλλων μεταφορέων. Ποιο συγκεκριμένα, οι μεταφορείς στόχοι έδειξαν πολύ μειωμένη έκφραση σε σχέση με τα μη ανθεκτικά στελέχη, ενώ κάποια επίπεδα έκφρα-σης ήταν κοντά στο μηδέν. Τέλος έγινε μελέτη στο μεταγραφικό πρότυπο βιοχημικών μονοπατιών που σχετίζονται με τον κύκλο του κιτρικού οξέος και τη βιοσύνθεση δια-φόρων απαραίτητων αμινοξέων. Πολλοί μηχανισμοί άμυνας των βακτηρίων έναντι των βακτηριοφάγων έχουν αναφερθεί ως τώρα, παρόλα αυτά οι μηχανισμοί που ενορ-χηστρώνουν τον μεταβολικό επαναπρογραμματισμό στα ανθεκτικά στελέχη παραμέ-νουν αρκετά ανεξερεύνητοι. Τα αποτελέσματα δείχνουν ότι τα ανθεκτικά στελέχη μπορούν να μειώσουν την μεταγραφή μεμβρανικών πρωτεϊνών, όπως οι μεταφορείς θρεπτικών, και να επαναπρογραμματίσουν τον μεταβολισμό τους. Η παρούσα διδα-κτορική διατριβή προωθεί το γένος Vibrio για μελέτη των μηχανισμών άμυνας των αρνητικών κατά Gram βακτηρίων, απέναντι στους φυσικούς τους εχθρούς, εστιάζο-ντας στην ανθεκτικότητα που προκύπτει ως αποτέλεσμα μεταβολικής προσαρμογής και επαναπρογραμματισμού, ενώ ταυτόχρονα ενισχύει την πεποίθηση ότι για την α-ντιμετώπιση της ανάπτυξης ανθεκτικότητας των βακτηρίων κατά τη φαγοθεραπεία, απαιτείται η χρήση φαγικών κοκτέιλ

Treatment of diabetes: Crossing to the other side

Type 2 diabetes mellitus affects nearly four hundred million people worldwide, and one of its major complications is cardiovascular disease. The evaluation of the effectiveness and safety of antidiabetic medication has been a challenging issue. Large clinical trials of new antidiabetic medications have used the non-inferiority approach to ensure primary safety of the drug before its incorporation into clinical practice. Currently, the trend is to prove superiority, that is, to prove that the new drug has additional beneficial effects to those of standard medications. In this review, we present the results of recent clinical trials on type 2 diabetes mellitus medications and outline what can be anticipated from ongoing clinical trials

Feeding level regulates the expression of some genes involved with programed cell death and remodeling in goat and sheep mammary tissue

Mammary tissue (MT) turnover is characterized by programed cell death and remodeling which might be affected by both feeding level and animal species. Thus, twenty-four dairy goats and the same number of sheep were assigned to three homogenous sub-groups per animal species and fed the same diet in quantities which met 70% (FL70), 100% (FL100) and 130% (FL130) of their daily energy and crude protein requirements. Individual MT samples were taken by biopsy from the animals on the 30th and 60th experimental day. The results showed, in the first sampling time, a significant reduction in the mRNA abundance for selected genes involved in programed cell death in both FL 70 fed goats (STAT3 and BECN1) and sheep (CASPASE8 and BECN1) compared with the respective FL100 groups. The FL130, in comparison with the FL100, caused a significant increase in transcripts accumulation of STAT3 gene in both sampling times and CASPASE8 gene in the second sampling time in goat MT, while the opposite happened for the mRNA expression of CASPASE8 and BECN1 genes in sheep MT, but only in the first sampling time. Moreover, a significant up regulation in the mRNA levels of MMP2 gene in MT of FL130 fed sheep was observed. The FL130, in comparison with the FL70, caused an enhancement in the mRNA expression levels of BECN1, CASPASE8, BAX and STAT3 genes in goat MT only. It was also shown that apoptosis and autophagy can be affected simultaneously by the feeding level. Overfeeding affects MT programed cell death and remodeling by a completely different way in goats than sheep. In conclusion, feeding level and animal species have strong effects on both MT programed cell death (apoptosis and autophagy) and remodeling but the molecular mechanisms need further investigation

The Effect of Forage-to-Concentrate Ratio on Schizochytrium spp.-Supplemented Goats: Modifying Rumen Microbiota

The inclusion of feed additives and the implementation of various nutritional strategies are studied to modify the rumen microbiome and consequently its function. Nevertheless, rumen enzymatic activity and its intermediate products are not always matched with the microbiome structure. To further elucidate such differences a two-phase trial using twenty-two dairy goats was carried out. During the first phase, both groups (20HF n = 11; high forage and 20HG n = 11; high grain) were supplemented with 20 g Schizochytrium spp./goat/day. The 20HF group consumed a diet with a forage:concentrate (F:C) ratio of 60:40 and the 20HG-diet consisted of a F:C = 40:60. In the second phase, the supplementation level of Schizochytrium spp. was increased to 40 g/day/goat while the F:C ratio between the two groups were remained identical (40HF n = 11; high forage and 40HG n = 11; high grain). By utilizing a next-generation sequencing technology, we monitored that the high microalgae inclusion level and foremost in combination with a high grains diet increased the unmapped bacteria within the rumen. Bacteroidetes and Prevotella brevis were increased in the 40HG -fed goats as observed by using a qPCR platform. Additionally, methanogens and Methanomassiliicoccales were increased in high microalgae-fed goats, while Methanobrevibacter and Methanobacteriales were decreased. Fibrolytic bacteria were decreased in high microalgae-fed goats, while cellulolytic activity was increased. Ammonia was decreased in high grains-fed goats, while docosapentaenoic and docosahexaenoic acids showed a lower degradation rate in the rumen of high forage-fed goats. The alteration of the F:C ratio in goats supplemented with Schizochytrium spp. levels modified both ruminal microbiota and enzymatic activity. However, there was no significant consistency in the relations between them

The Impact of Whole Sesame Seeds on the Expression of Key-Genes Involved in the Innate Immunity of Dairy Goats

Whole sesame seeds (WSS) are rich in both linoleic acid (LA) and lignans. However, their impact on the innate immunity of goats is not well studied. Twenty-four goats were divided into three homogeneous sub-groups; comprise one control (CON) and two treated (WWS5 and WWS10). In the treated groups, WSS were incorporated in the concentrates of the CON at 5 (WSS5) and 10% (WSS10) respectively, by partial substitution of both soybean meal and corn grain. The expression levels of MAPK1, IL6, TRIF, IFNG, TRAF3, and JUND genes in the neutrophils of WSS10 fed goats were reduced significantly compared with the CON. The same was found for the expression levels of IFNG and TRAF3 genes in the neutrophils of WSS5 fed goats. Both treated groups primarily affected the MYD88-independent pathway. The dietary supplementation of goats with WSS might be a good nutritional strategy to improve their innate immunity

Comprehensive Characterization of a Novel Bacteriophage, vB_VhaS_MAG7 against a Fish Pathogenic Strain of <i>Vibrio harveyi</i> 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

Alterations in the Rumen Particle-Associated Microbiota of Goats in Response to Dietary Supplementation Levels of Schizochytrium spp.

Fat rich microorganisms, such as microalgae Schizochytrium spp., are potential biotechnological tools in the modulation of rumen microbiome towards ecofriendly and high nutritional value end-products. However, limited in vivo trials have been reported on the topic. The aim of this study was to contribute to the knowledge on the effect of fat rich microalgae on the methanogenic and feed degrading particle-associated microbes in goats&rsquo; rumen content. For the trial, twenty-four goats were divided into four homogenous clusters (six goats/treatment) according to their fat corrected (4%) milk yield, body weight and age and individually were fed with alfalfa hay and concentrate feeds (F/C = 50/50). The concentrate of the control group (CON) contained no microalgae, while those of the treated groups were supplemented daily with 20 (ALG20), 40 (ALG40), and 60 (ALG60) g of Schizochytrium spp./goat. The relative abundances of total Archaea, methanogens, Methanomassiliicoccales, Methanobrevibacter spp., Methanosphaera stadmanae and Methanobacterium formicicum were significantly (p &lt; 0.05) decreased in microalgae-fed goats compared to the CON ones. Moreover, a significant decline in the relative abundances of Firmicutes, Ruminococcus flavefaciens, Butyrivibrio fibrosolvents, and Neocallimastigales in the rumen particle-associated microbiota of microalgae supplemented goats were observed. In conclusion, goats&rsquo; diets supplementation with Schizochytrium spp., could be considered a sustainable nutritional strategy for methanogens inhibition in their rumen particle-associated microbiota

Vibrio Phage Artemius, a Novel Phage Infecting Vibrio alginolyticus

Vibrio alginolyticus is an important pathogen of marine animals and has been the target of phage therapy applications in marine aquaculture for many years. Here, we report the isolation and partial characterization of a novel species of the Siphoviridae family, the Vibrio phage Artemius. The novel phage was species-specific and could only infect strains of V. alginolyticus. It could efficiently reduce the growth of the host bacterium at various multiplicities of infection as assessed by an in vitro lysis assay. It had a genome length of 43,349 base pairs. The complete genome has double-stranded DNA with a G + C content of 43.61%. In total, 57 ORFs were identified, of which 19 were assigned a predicted function. A genomic analysis indicated that Vibrio phage Artemius is lytic and does not harbor genes encoding toxins and antibiotic resistance determinants