Antimicrobial Peptides Originating from Expression Libraries of Aurelia aurita and Mnemiopsis leidyi Prevent Biofilm Formation of Opportunistic Pathogens.

The demand for novel antimicrobial compounds is rapidly growing due to the rising appearance of antibiotic resistance in bacteria; accordingly, alternative approaches are urgently needed. Antimicrobial peptides (AMPs) are promising, since they are a naturally occurring part of the innate immune system and display remarkable broad-spectrum activity and high selectivity against various microbes. Marine invertebrates are a primary resource of natural AMPs. Consequently, cDNA expression (EST) libraries from the Cnidarian moon jellyfish Aurelia aurita and the Ctenophore comb jelly Mnemiopsis leidyi were constructed in Escherichia coli. Cell-free size-fractionated cell extracts (<3 kDa) of the two libraries (each with 29,952 clones) were consecutively screened for peptides preventing the biofilm formation of opportunistic pathogens using the crystal violet assay. The 3 kDa fraction of ten individual clones demonstrated promising biofilm-preventing activities against Klebsiella oxytoca and Staphylococcus epidermidis. Sequencing the respective activity-conferring inserts allowed for the identification of small ORFs encoding peptides (10-22 aa), which were subsequently chemically synthesized to validate their inhibitory potential. Although the peptides are likely artificial products from a random translation of EST inserts, the biofilm-preventing effects against K. oxytoca, Pseudomonas aeruginosa, S. epidermidis, and S. aureus were verified for five synthetic peptides in a concentration-dependent manner, with peptide BiP_Aa_5 showing the strongest effects. The impact of BiP_Aa_2, BiP_Aa_5, and BiP_Aa_6 on the dynamic biofilm formation of K. oxytoca was further validated in microfluidic flow cells, demonstrating a significant reduction in biofilm thickness and volume by BiP_Aa_2 and BiP_Aa_5. Overall, the structural characteristics of the marine invertebrate-derived AMPs, their physicochemical properties, and their promising antibiofilm effects highlight them as attractive candidates for discovering new antimicrobials.

Microbial community changes correlate with impaired host fitness of Aurelia aurita after environmental challenge.

Climate change globally endangers certain marine species, but at the same time, such changes may promote species that can tolerate and adapt to varying environmental conditions. Such acclimatization can be accompanied or possibly even be enabled by a host's microbiome; however, few studies have so far directly addressed this process. Here we show that acute, individual rises in seawater temperature and salinity to sub-lethal levels diminished host fitness of the benthic Aurelia aurita polyp, demonstrated by up to 34% reduced survival rate, shrinking of the animals, and almost halted asexual reproduction. Changes in the fitness of the polyps to environmental stressors coincided with microbiome changes, mainly within the phyla Proteobacteria and Bacteroidota. The absence of bacteria amplified these effects, pointing to the benefit of a balanced microbiota to cope with a changing environment. In a future ocean scenario, mimicked by a combined but milder rise of temperature and salinity, the fitness of polyps was severely less impaired, together with condition-specific changes in the microbiome composition. Our results show that the effects on host fitness correlate with the strength of environmental stress, while salt-conveyed thermotolerance might be involved. Further, a specific, balanced microbiome of A. aurita polyps supports the host's acclimatization. Microbiomes may provide a means for acclimatization, and microbiome flexibility can be a fundamental strategy for marine animals to adapt to future ocean scenarios and maintain biodiversity and ecosystem functioning.

The Native Microbiome is Crucial for Offspring Generation and Fitness of Aurelia aurita.

All multicellular organisms are associated with microbial communities, ultimately forming a metaorganism. Several studies conducted on well-established model organisms point to immunological, metabolic, and behavioral benefits of the associated microbiota for the host. Consequently, a microbiome can influence the physiology of a host; moreover, microbial community shifts can affect host health and fitness. The present study aimed to evaluate the significance and functional role of the native microbiota for life cycle transitions and fitness of the cnidarian moon jellyfish Aurelia aurita A comprehensive host fitness experiment was conducted studying the polyp life stage and integrating 12 combinations of treatments with microbiota modification (sterile conditions, foreign food bacteria, and potential pathogens). Asexual reproduction, e.g., generation of daughter polyps, and the formation and release of ephyrae were highly affected in the absence of the native microbiota, ultimately resulting in a halt of strobilation and ephyra release. Assessment of further fitness traits showed that health, growth, and feeding rate were decreased in the absence and upon community changes of the native microbiota, e.g., when challenged with selected bacteria. Moreover, changes in microbial community patterns were detected by 16S rRNA amplicon sequencing during the course of the experiment. This demonstrated that six operational taxonomic units (OTUs) significantly correlated and explained up to 97% of fitness data variability, strongly supporting the association of impaired fitness with the absence/presence of specific bacteria. Conclusively, our study provides new insights into the importance and function of the microbiome for asexual reproduction, health, and fitness of the basal metazoan A. auritaIMPORTANCE All multicellular organisms are associated with a diverse and specific community of microorganisms; consequently, the microbiome is of fundamental importance for health and fitness of the multicellular host. However, studies on microbiome contribution to host fitness are in their infancy, in particular, for less well-established hosts such as the moon jellyfish Aurelia aurita Here, we studied the impact of the native microbiome on the asexual reproduction and on further fitness traits (health, growth, and feeding) of the basal metazoan due to induced changes in its microbiome. We observed significant impact on all fitness traits analyzed, in particular, in the absence of the protective microbial shield and when challenged with marine potentially pathogenic bacterial isolates. Notable is the identified crucial importance of the native microbiome for the generation of offspring, consequently affecting life cycle decisions. Thus, we conclude that the microbiome is essential for the maintenance of a healthy metaorganism.

Potential role of host-derived quorum quenching in modulating bacterial colonization in the moon jellyfish Aurelia aurita.

Multicellular organisms can be regarded as metaorganisms, comprising of a macroscopic host interacting with associated microorganisms. Within this alliance, the host has to ensure attracting beneficial bacteria and defending against pathogens to establish and maintain a healthy homeostasis. Here, we obtained several lines of evidence arguing that Aurelia aurita uses interference with bacterial quorum sensing (QS) - quorum quenching (QQ) - as one host defense mechanism. Three A. aurita-derived proteins interfering with bacterial QS were identified by functionally screening a metagenomic library constructed from medusa-derived mucus. Native expression patterns of these host open reading frames (ORFs) differed in the diverse life stages (associated with different microbiota) pointing to a specific role in establishing the developmental stage-specific microbiota. Highly increased expression of all QQ-ORFs in germ-free animals further indicates their impact on the microbiota. Moreover, incubation of native animals with pathogenic bacteria induced expression of the identified QQ-ORFs arguing for a host defense strategy against confronting bacteria by interference with bacterial QS. In agreement, immobilized recombinant QQ proteins induced restructuring of polyp-associated microbiota through changing abundance and operational taxonomic unit composition. Thus, we hypothesize that additional to the immune system host-derived QQ-activities potentially control bacterial colonization.

Highly Effective Inhibition of Biofilm Formation by the First Metagenome-Derived AI-2 Quenching Enzyme.

Bacterial cell-cell communication (quorum sensing, QS) represents a fundamental process crucial for biofilm formation, pathogenicity, and virulence allowing coordinated, concerted actions of bacteria depending on their cell density. With the widespread appearance of antibiotic-resistance of biofilms, there is an increasing need for novel strategies to control harmful biofilms. One attractive and most likely effective approach is to target bacterial communication systems for novel drug design in biotechnological and medical applications. In this study, metagenomic large-insert libraries were constructed and screened for QS interfering activities (quorum quenching, QQ) using recently established reporter strains. Overall, 142 out of 46,400 metagenomic clones were identified to interfere with acyl-homoserine lactones (AHLs), 13 with autoinducer-2 (AI-2). Five cosmid clones with highest simultaneous interfering activities were further analyzed and the respective open reading frames conferring QQ activities identified. Those showed homologies to bacterial oxidoreductases, proteases, amidases and aminotransferases. Evaluating the ability of the respective purified QQ-proteins to prevent biofilm formation of several model systems demonstrated highest inhibitory effects of QQ-2 using the crystal violet biofilm assay. This was confirmed by heterologous expression of the respective QQ proteins in Klebsiella oxytoca M5a1 and monitoring biofilm formation in a continuous flow cell system. Moreover, QQ-2 chemically immobilized to the glass surface of the flow cell effectively inhibited biofilm formation of K. oxytoca as well as clinical K. pneumoniae isolates derived from patients with urinary tract infections. Indications were obtained by molecular and biochemical characterizations that QQ-2 represents an oxidoreductase most likely reducing the signaling molecules AHL and AI-2 to QS-inactive hydroxy-derivatives. Overall, we propose that the identified novel QQ-2 protein efficiently inhibits AI-2 modulated biofilm formation by modifying the signal molecule; and thus appears particularly attractive for medical and biotechnological applications.

Composition of Bacterial Communities Associated with Aurelia aurita Changes with Compartment, Life Stage, and Population.

The scyphozoan Aurelia aurita is recognized as a key player in marine ecosystems and a driver of ecosystem change. It is thus intensely studied to address ecological questions, although its associations with microorganisms remain so far undescribed. In the present study, the microbiota associated with A. aurita was visualized with fluorescence in situ hybridization (FISH) analysis, and community structure was analyzed with respect to different life stages, compartments, and populations of A. aurita by 16S rRNA gene amplicon sequencing. We demonstrate that the composition of the A. aurita microbiota is generally highly distinct from the composition of communities present in ambient water. Comparison of microbial communities from different developmental stages reveals evidence for life stage-specific community patterns. Significant restructuring of the microbiota during strobilation from benthic polyp to planktonic life stages is present, arguing for a restructuring during the course of metamorphosis. Furthermore, the microbiota present in different compartments of the adult medusa (exumbrella mucus and gastric cavity) display significant differences, indicating body part-specific colonization. A novel Mycoplasma strain was identified in both compartment-specific microbiota and is most likely present inside the epithelium as indicated by FISH analysis of polyps, indicating potential endosymbiosis. Finally, comparison of polyps of different populations kept under the same controlled laboratory conditions in the same ambient water showed population-specific community patterns, most likely due the genetic background of the host. In conclusion, the presented data indicate that the associated microbiota of A. aurita may play important functional roles, e.g., during the life cycle.

Novel reporter for identification of interference with acyl homoserine lactone and autoinducer-2 quorum sensing.

Two reporter strains were established to identify novel biomolecules interfering with bacterial communication (quorum sensing [QS]). The basic design of these Escherichia coli-based systems comprises a gene encoding a lethal protein fused to promoters induced in the presence of QS signal molecules. Consequently, these E. coli strains are unable to grow in the presence of the respective QS signal molecules unless a nontoxic QS-interfering compound is present. The first reporter strain designed to detect autoinducer-2 (AI-2)-interfering activities (AI2-QQ.1) contained the E. coli ccdB lethal gene under the control of the E. coli lsrA promoter. The second reporter strain (AI1-QQ.1) contained the Vibrio fischeri luxI promoter fused to the ccdB gene to detect interference with acyl-homoserine lactones. Bacteria isolated from the surfaces of several marine eukarya were screened for quorum- quenching (QQ) activities using the established reporter systems AI1-QQ.1 and AI2-QQ.1. Out of 34 isolates, two interfered with acylated homoserine lactone (AHL) signaling, five interfered with AI-2 QS signaling, and 10 were demonstrated to interfere with both signal molecules. Open reading frames (ORFs) conferring QQ activity were identified for three selected isolates (Photobacterium sp., Pseudoalteromonas sp., and Vibrio parahaemolyticus). Evaluation of the respective heterologously expressed and purified QQ proteins confirmed their ability to interfere with the AHL and AI-2 signaling processes.

Facets of diazotrophy in the oxygen minimum zone waters off Peru.

Nitrogen fixation, the biological reduction of dinitrogen gas (N2) to ammonium (NH4(+)), is quantitatively the most important external source of new nitrogen (N) to the open ocean. Classically, the ecological niche of oceanic N2 fixers (diazotrophs) is ascribed to tropical oligotrophic surface waters, often depleted in fixed N, with a diazotrophic community dominated by cyanobacteria. Although this applies for large areas of the ocean, biogeochemical models and phylogenetic studies suggest that the oceanic diazotrophic niche may be much broader than previously considered, resulting in major implications for the global N-budget. Here, we report on the composition, distribution and abundance of nifH, the functional gene marker for N2 fixation. Our results show the presence of eight clades of diazotrophs in the oxygen minimum zone (OMZ) off Peru. Although proteobacterial clades dominated overall, two clusters affiliated to spirochaeta and archaea were identified. N2 fixation was detected within OMZ waters and was stimulated by the addition of organic carbon sources supporting the view that non-phototrophic diazotrophs were actively fixing dinitrogen. The observed co-occurrence of key functional genes for N2 fixation, nitrification, anammox and denitrification suggests that a close spatial coupling of N-input and N-loss processes exists in the OMZ off Peru. The wide distribution of diazotrophs throughout the water column adds to the emerging view that the habitat of marine diazotrophs can be extended to low oxygen/high nitrate areas. Furthermore, our statistical analysis suggests that NO2(-) and PO4(3-) are the major factors affecting diazotrophic distribution throughout the OMZ. In view of the predicted increase in ocean deoxygenation resulting from global warming, our findings indicate that the importance of OMZs as niches for N2 fixation may increase in the future.

Towards a molecular code for individuality in the absence of MHC: screening for individually variable genes in the urochordate Ciona intestinalis.

Urochordates possess several well described allorecognitions systems, the molecular nature of those is not yet understood. A prerequisite for any self-/nonself discimination system is the presence of a group of highly variable molecules, which should vary between individuals. Using suppression subtractive hybridisation (SSH) we surveyed Ciona intestinalis for individually variable genes. Our search so far identified two genes, ciS7 and ciMETA2, which both display an unexpected high degree of intra- and interindividual variability, code for secreted proteins, and contain multiple domains suitable for protein-protein interactions. The possible role of these molecules in allorecognition is discussed.