Host Species Research Articles

Diversity of Host Species and Optimized Cultivation Practices for Enhanced Bioactive Compound Production in Cordyceps militaris

Cordyceps militaris, recognized for its diverse and potent medicinal properties, plays a critical role in herbal medicine. This study investigates the genus Cordyceps, particularly focusing on C. militaris, a species widely utilized in East Asian traditional medicine for its therapeutic properties. C. militaris is rich in bioactive compounds, including cordycepin, adenosine, polysaccharides, ergosterol, and mannitol, which contribute to its broad physiological activities. The research aims to explore the diversity of host species in the cultivation of C. militaris and assess their impact on the biological activity and chemical composition of the fungus. The study highlights the variability in the efficacy of bioactive compounds due to different cultivation conditions and host species, emphasizing the need for standardized cultivation practices. Advances in genetic engineering and fermentation technology have significantly enhanced the production of these metabolites, supporting the sustainable scale-up of C. militaris cultivation. Future research should continue to explore the molecular mechanisms of its bioactive compounds, identify new clinical applications, and improve production efficiency and environmental sustainability. This comprehensive review provides insights for researchers, healthcare professionals, and cultivators on optimizing C. militaris for medicinal and commercial applications.

Virulence and Immune Evasion Strategies of FMDV: Implications for Vaccine Design.

Foot-and-mouth disease (FMD) is globally recognized as a highly economically devastating and prioritized viral disease affecting livestock. Vaccination remains a crucial preventive measure against FMD. The improvement of current vaccine platforms could help control outbreaks, leading to the potential eradication of the disease. In this review, we describe the variances in virulence and immune responses among FMD-susceptible host species, specifically bovines and pigs, highlighting the details of host-pathogen interactions and their impact on the severity of the disease. This knowledge serves as an important foundation for translating our insights into the rational design of vaccines and countermeasure strategies, including the use of interferon as a biotherapeutic agent. Ultimately, in this review, we aim to bridge the gap between our understanding of FMDV biology and the practical approaches to control and potentially eradicate FMD.

Mosquito Feeding Habits in Coastal French Guiana: Mammals in the Crosshairs?

Pathogens transmitted by mosquitoes (Diptera, Culicidae) in sylvatic or urban cycles involve wild or domestic animals and humans, driven by various mosquito species with distinct host preferences. Understanding mosquito-host associations is crucial for ecological insights and pathogen surveillance. In this study, we analyzed mosquito blood meals from coastal French Guiana by amplifying and sequencing host DNA from blood-fed females. Using the 12S ribosomal RNA gene and Sanger sequencing, we identified blood meals from 26 mosquito species across six genera, with 59% belonging to the Culex genus. Nanopore sequencing of selected samples showed 12 mosquito species with one to three mixed blood-meal sources. Mammals were the primary hosts (88%), followed by birds (7%), squamates (3%), and amphibians (2%), indicating a strong preference for mammalian hosts. A total of 46 vertebrate host species were identified, demonstrating high host diversity. This research provides insights into mosquito host usage and highlights the complexities of monitoring arboviruses of public health concern.

Simple and accurate genomic classification model for distinguishing between human and pig Staphylococcus aureus

Staphylococcus aureus (S. aureus) can cause various infections in humans and animals, contributing to high morbidity and mortality. To prevent and control cross-species transmission of S. aureus, it is necessary to understand the host-associated genetic variants. We performed a two-stage genome-wide association study (GWAS) including initial screening and further validation to compare genomic differences between human and pig S. aureus, aiming to identify host-associated determinants. Our multiple GWAS analyses found six consensus significant k-mers associated with host species, providing novel genetic evidence for distinguishing human from pig S. aureus. The best k-mer predictor achieved a high classification accuracy of 98.12% on its own and had extremely high resolution similar to the SNPs-based phylogeny, offering a very simple target for predicting the cross-species transmission risk of S. aureus. The final k-mer model revealed that 90% of S. aureus isolates from farm workers were predicted as livestock origin, suggesting a high risk of cross-species transmission. Bayesian inference revealed different cross-species transmission directions, with the human-to-pig transmission for ST5 and the pig-to-human transmission for ST398. Our findings provide a simple and accurate k-mer model for identifying and predicting the cross-species transmission risk of S. aureus.

Fungal community structure in bees: influence of biome and host species

Fungal community structure in bees: influence of biome and host species

Parasites Diversity, Abundance, Prevalence, and Richness Infecting Didelphis aurita (Didelphimorphia: Didelphidae) in the Atlantic Rainforest, Brazil.

Parasites are key players in ecosystems, influencing population sizes and food webs, yet the impact of environmental factors on their diversity is not well understood. The Atlantic rainforest in Brazil, particularly the Parque Estadual das Fontes do Ipiranga (PEFI), exemplifies a biodiversity hotspot facing significant deforestation, housing diverse animal species such as the synanthropic Brazilian common opossum (Didelphis aurita), which serves as a reservoir for multiple zoonotic pathogens. In this study, we investigated parasite diversity, abundance, prevalence, and richness in free-living D. aurita in the PEFI, São Paulo, Brazil. From January 2015 to January 2017, 101 fecal samples of D. aurita were collected in two areas of PEFI, at the Instituto de Pesquisas Ambientais (IPA) and the Parque de Ciência e Tecnologia (Cientec), and analyzed using three different parasitological methods. In total, 99% of the samples were positive for at least one parasite. The most prevalent parasite belonged to the order Strongylida (82%), followed by Cruzia sp. (77%), the latter having a significantly higher prevalence at IPA. In contrast, Acanthocephala showed greater prevalence at Cientec. Co-infections were common, with some individuals harboring up to seven different parasites. Our findings reveal significant parasite diversity in the D. aurita population at PEFI, including both helminths and protozoan trophozoites, some of which are reported for the first time in this host species. Further research is essential for accurate species identification of the observed parasites.

Arbuscular mycorrhizal fungal spore communities and co-occurrence networks demonstrate host-specific variation throughout the growing season.

Microbial community assembly involves a series of ecological filtering mechanisms that determine the composition of microbial communities. While the importance of both broad and local level factors on microbial communities has been reasonably well studied, this work often is limited to single observations and neglects to consider how communities change over time (i.e., seasonal variation). Because seasonal variation is an important determinant of community assembly and determines the relative importance of community assembly filters, this represents a key knowledge gap. Due to their close associations with seasonal variation in plant growth and fitness, arbuscular mycorrhizal (AM) fungi are useful groups for assessing the importance of seasonal dynamics in microbial community assembly. We tested how seasonal variation (spring vs. summer), plant life history stage (vegetative vs. flowering), and host plant species (Baptisia bracteata var. leucophaea & Andropogon gerardii) influenced AM fungal spore community assembly. AM fungal spore community temporal dynamics were closely linked to plant host species and life history stage. While AM fungal spore communities demonstrated strong turnover between the spring (e.g., higher sporulation) and late summer (e.g., higher diversity), the strength and direction of these changes was modified by host plant species. Here we demonstrate the importance of considering temporal variation in microbial community assembly, and also show how plant-microbe interactions can modify seasonal trends in microbial community dynamics.

Characterization of four novel actinospore types of fish parasitic myxozoans and the occurrence of Branchiodrilus hortensis and Ophidonais serpentina from fish farms of Hungary

Six distinct actinospore types were identified in the intestinal epithelium of oligochaetes collected from the Szigetbecse and Makád fish farms of Ráckeve Danube Arm Fishing Association, in Hungary. Four new types: triactinomyxon type, raabeia type, aurantiactinomyxon type 1, and aurantiactinomyxon type 2, were described morphologically and molecularly from three invertebrate host species: Limnodrilus hoffmeisteri, Ophidonais serpentina, and Tubifex tubifex. The 18S ribosomal DNA (rDNA) analysis revealed that these new types of actinospores did not match any myxospore sequences available in GenBank. Phylogenetic analysis placed the triactinomyxon type within the Myxobolus clade that parasitizes cyprinid fish, while the raabeia type and aurantiactinomyxon type 2 were both placed within the Myxobolus clade associated with Perciformes fish. Aurantiactinomyxon type 1 was clustered in a clade containing gill-infecting Henneguya spp. from Esociformes fish. However, no myxospores have been found yet to link to the newly sequenced actinospores reported in this survey. This study also reports the first occurrence of two oligochaetes species, Branchiodrilus hortensis and Ophidonais serpentina in Hungary, specifically in fish farms of Ráckeve Danube Arm Fishing Association. Moreover, this is the first report on the involvement of Ophidonais serpentina in the life cycle of myxozoans.

Uncovering new Firmicutes species in vertebrate hosts through metagenome-assembled genomes with potential for sporulation.

Metagenome-assembled genomes (MAGs) have contributed to identifying non-culturable microorganisms and understanding their ecological functions. MAGs offer an advantage in investigating sporulation-associated genes, especially given the difficulty of isolating many species residing in the gut microbiota of multiple hosts. Bacterial sporulation is a key survival mechanism with implications for pathogenicity and biotechnology. Here, we investigate MAGs from vertebrate hosts, emphasizing taxonomic identification and identifying sporulation-associated genes in potential novel species within the Firmicutes phylum. We identified potential new species in the classes Clostridia (Borkfalkiaceae, Lachnospiraceae, Monoglobaceae, and Oscillospiraceae families) and Bacilli (Bacillaceae and Erysipelotrichaceae families) through phylogenetic and functional pathway analyses, highlighting their sporulation potential. Our study covers 146 MAGs, 124 of them without refined taxonomic assignments at the family level. We found that Clostridia and Bacilli have unique sporulation gene profiles in the refined family MAGs for cattle, swine, poultry, and human hosts. The presence of genes related to Spo0A regulon, engulfment, and spore cortex in MAGs underscores fundamental mechanisms in sporulation processes in currently uncharacterized species with sporulation potential from metagenomic dark matter. Furthermore, genomic analyses predict sporulation potential based on gene presence, genome size, and metabolic pathways involved in spore formation. We emphasize MAGs covering families not yet characterized through the phylogenetic analysis, and with extensive potential for spore-forming bacteria within Clostridia, Bacilli, UBA4882, and UBA994 classes. These findings contribute to exploring spore-forming bacteria, which provides evidence for novel species diversity in multiple hosts, their adaptive strategies, and potential applications in biotechnology and host health.IMPORTANCESpores are essential for bacterial survival in harsh environments, facilitating their persistence and adaptation. Exploring sporulation-associated genes in metagenome-assembled genomes (MAGs) from different hosts contributes to clinical and biotechnological domains. Our study investigated the extent of genes associated with bacterial sporulation in MAGs from poultry, swine, cattle, and humans, revealing these genes in uncultivated bacteria. We identified potential novel Firmicutes species with sporulation capabilities through phylogenetic and functional analyses. Notably, MAGs belonging to Clostridia, Bacilli, and unknown classes, namely UBA4882 and UBA994, remained uncharacterized at the family level, which raises the hypothesis that sporulation would also be present in these genomes. These findings contribute to our understanding of microbial adaptation and have implications for microbial ecology, underlining the importance of sporulation in Firmicutes across different hosts. Further studies into novel species and their sporulation capability can contribute to bacterial maintenance mechanisms in various organisms and their applications in biotechnology studies.

Reinboldiella koreana sp. nov. (Ceramiales, Rhodophyta) is the correct name for the specimens previously known as Reinboldiella schmitziana in Korea

Epiphytic algae are important components of marine ecosystems. Despite the importance of epiphytic algae in marine ecosystems, our knowledge of these taxa is still limited. The marine red algal genus <i>Reinboldiella</i>, which is distributed mainly in the northwestern Pacific region, is characterized by tiny thalli that are epiphytic on various other red algae. Six species of the genus have been reported in Japan, Taiwan, and Korea. In this study, 50 Korean Reinboldiella specimens, previously known as <i>Reinboldiella schmitziana</i>, were collected from 11 localities and reexamined to verify their taxonomic status. We describe a new species, <i>Reinboldiella koreana</i> sp. nov., based on <i>rbc</i>L phylogeny and morphological observations. A total of four grateloupiacean species (<i>Pachymeniopsis lanceolata</i>, <i>P. elliptica</i>, <i>Grateloupia turuturu</i>, and <i>G. asiatica</i>) were identified as host species of <i>R. koreana</i> sp. nov. Due to the restricted host range of <i>R. koreana</i> sp. nov., a phylogenetic tree was reconstructed using the <i>cox</i>1 marker to confirm the co-phylogenetic relationships between the epiphytic algae and their grateloupiacean hosts. Two of the four cox1 haplotypes of <i>R. koreana</i> were found in different host species, regardless of which haplotype they were. It indicates that there are no co-phylogenetic relationships between <i>R. koreana</i> and host species.

Persistent biological invasions alter ecological network topology, impacting disease transmission during community assembly

Ecological networks experiencing persistent biological invasions may exhibit distinct topological properties, complicating the understanding of how network topology affects disease transmission during invasion-driven community assembly. We developed a trait-based network model to assess the impact of network topology on disease transmission, measured as community- and species-level disease prevalence. We found that trait-based feeding interactions between host species determine the frequency distribution of the niche of co-occurring species in steady-state communities, being either bimodal or multimodal. The width of the growth kernel influences the degree-biomass relationship of species, being either weakly positive or strongly negative. When this relationship is weakly positive, species-level disease prevalence is primarily correlated with biomass. However, when the degree-biomass relationship is strongly negative, species-level disease prevalence is determined by the difference between a host species’ in-degree and out-degree closeness centrality. At the community level, disease prevalence is generally amplified by increasing host richness, community biomass, and the standard deviation of interaction generality, while it is diluted by higher network connectance. Our framework verifies the amplification effects of host richness during invasion-driven community assembly and offers valuable insights for estimating disease prevalence based on host network topology.

Evolutionary characterization of the establishment of H6 influenza viruses in domestic geese in China: implications for the position of the host in the ecosystem

Abstract Geese, both wild and domestic, are generally considered part of the natural reservoir for influenza A viruses. The highly pathogenic H5 Goose/Guangdong avian influenza virus lineage that is still causing outbreaks worldwide was first detected in domestic geese in 1996. However, while wild geese might have a somewhat restricted role in the influenza ecosystem, the role of domestic geese is little studied. Here, 109 H6 viruses isolated from domestic geese during 2001–2018 in southern China had their phylogeny, evolutionary dynamics, and molecular signatures characterized to examine the role of domestic geese. Our findings demonstrated that all geese H6 viruses were derived from H6 viruses established in ducks and that they subsequently formed three distinct hemagglutinin lineages. Rapid evolution of the hemagglutinin genes was not detected after the duck-to-goose transmissions of H6 viruses that then circulated in geese. Despite long-term persistence in geese, H6 viruses were rarely observed to transmit back to ducks or terrestrial poultry and never exchanged genes with viruses from other subtypes. Most geese H6 viruses maintained the primary molecular signatures of their duck precursors. This study raises the possibility that, rather than being part of the natural reservoir, domestic geese might be more like an aberrant host species for influenza A viruses, and perhaps a “dead-end” host.

Phlebotomine sand flies (Diptera: Psychodidae) of Bosnia and Herzegovina: distribution, ecology and environmental preferences

Sand flies (Diptera: Phlebotominae) are the principal vectors for the protozoan parasites Leishmania spp. and for phleboviruses. The sand fly fauna on the Balkan Peninsula, including Bosnia and Herzegovina (BIH), is diverse and the circulation of Leishmania infantum as well as phleboviruses has been proven. However, recent data on the sand fly fauna in BIH are scarce. In this study, we surveyed understudied regions in central and northeastern BIH to update the sand fly distribution and gain insights into the ecological and environmental factors shaping their appearance. CDC light trapping was conducted in 2022 and 2023 and a combination of morphological and molecular methods (cytochrome oxidase I barcoding) was performed for species identifications. We mapped the currently known distribution, modelled climatic suitability patterns and performed environmental analyses by applying machine learning methods. In addition, we analyzed blood meals by host gene sequencing and MALDI-TOF peptide mass mapping and screened for Leishmania spp. DNA and Phlebovirus RNA.Altogether, 591 sand flies of four species were trapped, predominantly Phlebotomus neglectus (97 %), but also Ph. balcanicus, Ph. mascittii, and Ph. papatasi. Records of seven sand fly species known to be endemic were plotted onto distribution maps based on 101 datapoints, identifying Ph. neglectus as the overall predominant species. The environmental analyses of sand fly species indicated variation in altitudinal, thermal, and precipitation conditions across the sand fly-positive sites. Phlebotomus simici, Phlebotomus tobbi, and Sergentomyia minuta are typically found exclusively in Mediterranean and subtropical climate zones, whereas other species typically inhabit continental regions. The Inverse Distance Weighted (IDW) interpolation of sand fly species numbers and Shannon entropy values suggested the southeastern coastal region of BIH as a primary focus for sand fly occurrence. This finding was corroborated by modeled average climatic suitability patterns for sand flies, depicting four distinct meso-regions for sand fly occurrence. The results of the ensemble method highlight the importance of annual precipitation to distinguish between positive and negative sand fly trapping sites in BIH. In total, 55 blood meals of two sand fly species, Ph. neglectus and Ph. balcanicus, were analyzed and five host species identified.Our comprehensive assessment of ecological and environmental preferences of sand flies in BIH may support further entomological surveys and help to better understand and evaluate potential hot spots of disease transmission in the country.

Development of a novel triplex-PCR assay for the identification of feline hemoplasma species and survey of hemoplasma species in cats in Türkiye

Three hemoplasma species, Mycoplasma haemofelis, Candidatus Mycoplasma haemominutum, and Candidatus Mycoplasma turicensis, have been identified in domestic and wild felids. M. haemofelis may cause severe clinical manifestations in domestic cats, whereas others can lead to mild infections. Identification of these pathogens is done using molecular diagnostic tools like conventional-PCR or real-time PCR. However, these have disadvantages, such as the failure to differentiate species or high cost. This study aimed to develop a triplex-PCR method for the diagnosis and discrimination of feline hemoplasma species. Furthermore, it is aimed at providing molecular data on the epidemiology of feline hemoplasma species in Türkiye, where there is a paucity of information on these pathogens. Triplex-PCR primers amplifying the 16S rRNA gene regions of M. haemofelis (1022 bp), Ca. Mycoplasma haemominutum (607 bp), and Ca. Mycoplasma turicensis (456 bp) species were designed and optimized. Moreover, the detection limits of the method were also determined and it was found that the primers could detect 0.001 ng/μL amount of DNA for M. haemofelis, 0.0001 ng/μL for Ca. Mycoplasma haemominutum, and 0.0002 ng/μL for Ca. Mycoplasma turicensis in the sample. 286 cat blood samples obtained from Sivas province were researched for feline hemoplasma species. Feline hemoplasma species were detected in samples of 29 out of 286 cats (10.23 %). Five samples (1.74 %) were infected with only M. haemofelis, whereas 22 (7.69 %) were only infected with Ca. Mycoplasma haemominutum. Two samples (0.69 %) were found to be infected with both M. haemofelis and Ca. Mycoplasma haemominutum. Ca. Mycoplasma turicensis was not detected in this study. A triplex-PCR method that can be used for the identification and species differentiation of feline hemoplasma species in hosts was developed. Moreover, hemoplasma species were found to be circulating in cats in the study area, and it is recommended that veterinarians and animal owners take the necessary precautions to protect themselves and their cats from these pathogens.

Lernaea cf. cyprinacea infection in agile frog (Rana dalmatina) tadpoles in Hungary.

The ectoparasitic copepod Lernaea cyprinacea (anchor worm) has more than 100 host species among teleost fishes and affects cyprinids both in fish farms and natural waters. In addition, while L. cyprinacea infection in amphibians has been recorded in Asia, North and South America, there is no data available in the literature on their presence in Europe. In this study, we first reported L. cf. cyprinacea parasitising an anuran tadpole in Europe. Specimens of L. cf. cyprinacea were observed attached to a tadpole of the agile frog (Rana dalmatina), which was caught during fishing of crucian carp fingerlings (Carassius carassius) from a small fish pond in Hungary during the summer of 2012. The infected tadpole was collected from a rearing pond, where juvenile crucian carps were kept. The tadpole was inspected in the laboratory, and digital photos were taken. The parasites were found attached to the body-tail junction and to the leg of the tadpole (at Gosner stage 41). The parasite species was identified as L. cf. cyprinacea based on morphological traits.

A cross-sectional study on phlebotomine sand flies in relation to disease transmission in the Republic of Kosovo.

Sand flies (Diptera: Psychodidae: Phlebotominae) are blood-feeding insects that transmit the protozoan parasites Leishmania spp. and various arboviruses. The Balkan region, including the Republic of Kosovo, harbours a diverse sand fly fauna. Vector species of Leishmania infantum as well as phleboviruses are endemic; however, recent data are scarce. We performed a cross-sectional study to update the current sand fly distribution in Kosovo and assess biological as well as environmental factors associated with sand fly presence. CDC light trapping was conducted at 46 locations in 2022 and 2023, specifically targeting understudied regions in Kosovo. Individual morphological species identification was supported by molecular barcoding. The occurrence data of sand flies was used to create distribution maps and perform environmental analyses, taking elevation, wind speed and climate-related factors into account. In addition, PCR-based blood meal analysis and pathogen screening were conducted. Overall, 303 specimens of six sand fly species were trapped, predominated by Phlebotomus neglectus (97%). Barcodes from eight of nine known endemic sand fly species were obtained. Combining our data with previous surveys, we mapped the currently known sand fly distribution based on more than 4000 specimens at 177 data points, identifying Ph. neglectus and Ph. perfiliewi as the predominant species. Environmental analyses depicted two geographical groups of sand flies in Kosovo, with notable differences between the species. In total, 223 blood meals of five sand fly species were analysed. Of seven identified host species, the predominant blood meal source was observed to be cattle, but the DNA of dogs and humans, among others, was also detected. This study assessed biological as well as ecological factors of sand fly occurrence, which should help better understand and evaluate potential hot spots of disease transmission in Kosovo.

Pathogenic and genomic characterization of Ostreid herpesvirus 1 associated with mass mortalities of blood cockle (Anadara kagoshimensis)

Ostreid herpesvirus 1 (OsHV-1) is an infectious viral disease of bivalve mollusks with a wide geographic distribution across five continents. Since its initial identification in 1991, the disease has been identified as the cause of mortality in more than ten species of bivalve mollusks. The transmission of OsHV-1 is thought to be facilitated by water. In 2019 and 2021, mass mortalities of blood cockle (Anadara kagoshimensis) hatchery broodstock associated with OsHV-1 infection were reported. The presence of OsHV-1 DNA was confirmed in all hatchery samples with levels exceeding 106 copies per milligram of DNA. OsHV-1 was not detected in the wild populations from which the broodstock was collected, indicating that the virus was introduced into the hatchery with seawater from the open ocean. The results of the histopathological examination indicated that tissue lesions and associated hemocyte infiltration were most prevalent in the connective tissues of the digestive tract, mantle, gills and foot of the diseased A. kagoshimensis. In situ hybridization (ISH) signals of OsHV-1 DNA were frequently observed in infiltrated hemocytes, putative fibroblasts, and on occasion, in muscle cells with weak staining. Transmission electron microscopy analysis demonstrated the presence of herpes-like viral particles within the nucleus of infected cells. The genomes of the OsHV-1 variants from cases 2019 and 2021 were successfully resolved through direct high-throughput sequencing of infected tissues. A phylogenetic analysis of the complete genome sequences of 32 OsHV-1 variants revealed that the segregation of the virus was primarily structured by host species and geographic origin, with some exceptions. We propose that the sympatric distribution of different host species and the potential occurrence of cross-species transmission of OsHV-1 are responsible for the inconsistency between the phylogenetic segregation and their host species. This is the first report on the pathogenic and genomic characterization of OsHV-1 infecting A. kagoshimensis. The epidemiological and phylogenetic analyses offer new insights into the evolution of the virus and provide valuable information for the prevention and control of the disease.

New data on the morphology of Ascarophis parupenei (Nematoda: Cystidicolidae), an intestinal parasite of the marine fish Parupeneus indicus in the Indian Ocean, revealed by SEM.

Scanning electron microscopy studies of three paratype specimens (two males and one female) of Ascarophis parupenei Moravec, Orecchia & Paggi, 1988, an intestinal parasite of the marine fish Parupeneus indicus (Shaw) in the Indian Ocean, made it possible to redescribe in detail the cephalic structures of this nematode as well as the morphology of the male caudal end. The presence of non-bilobed sublabia, phasmids in both males and females, bifurcated deirids, well-developed precloacal cuticular ridges (area rugosa) and a ventral median caudal protuberance in the male are reported in this species for the first time. The general morphology and measurements of A. parupenei somewhat resemble those of Rasheedia novaecaledoniensis Moravec & Justine, 2018 described from the same host species (P. indicus) from off New Caledonia, but both species clearly differ from each other by the shape and structure of pseudolabia and the anterior portion of the digestive tract, belonging thus to different nematode families (Cystidicolidae and Physalopteridae, respectively).

Discovery and Heterologous Expression of Functional 4-O-Dimethylallyl-l-tyrosine Synthases from Lichen-Forming Fungi.

Fungal aromatic prenyltransferases are a family of biosynthetic enzymes that catalyze the prenylation of a range of aromatic substrates during the biosynthesis of bioactive indole alkaloids, diketopiperazines, and meroterpenoids. Their broad substrate scope and soluble nature make these enzymes particularly adept for applications in biocatalysis; for example, the enzymatic derivatization of aromatic drugs improves their bioactivity. Here, we investigated four putative aromatic prenyltransferases from lichen-forming fungi, an underexplored group of organisms that produce more than 1,000 unique metabolites. We successfully expressed two enzymes, annotated as dimethylallyltryptophan synthases, from two lichen species in the heterologous host A. oryzae. Based on their in vivo activity, we hypothesize that these enzymes are in fact 4-O-dimethylallyl-l-tyrosine synthases. Our extensive bioinformatic analysis further confirmed that these and related lichen aromatic prenyltransferases are likely not active on indoles but rather on aromatic polyketides and phenylpropanoids, major metabolites in lichens. Overall, our work provides new insights into fungal aromatic prenyltransferases at the family level and enables future efforts aimed at identifying new candidates for biocatalytic transformations of aromatic compounds.

Limb Perfusion Delivery of a rAAV1 Alpha-1 Antitrypsin Vector in Non-Human Primates Is Safe but Insufficient for Therapy.

α-1 antitrypsin (AAT) deficiency is an inherited, genetic condition characterized by reduced serum levels of AAT and increased risk of developing emphysema and liver disease. AAT is normally synthesized primarily in the liver, but muscle-targeting with a recombinant adeno-associated virus (rAAV) vector for α-1 antitrypsin (AAT) gene therapy has been used to minimize liver exposure to the virus and hepatotoxicity. Clinical trials of direct intramuscular (IM) administration of rAAV1-hAAT have demonstrated its overall safety and transgene expression for 5 years. However, the failure to reach the therapeutic target level after 100 large-volume (1.5 mL) IM injections of maximally concentrated vector led us to pursue a muscle-targeting approach using isolated limb perfusion. This targets the rAAV to a greater muscle mass and allows for a higher total volume (and thereby a higher dose) than is tolerable by multiple direct IM injections. Limb perfusion has been shown to be feasible in non-human primates using the rAAV1 serotype and a ubiquitous promoter expressing an epitope-tagged AAT matched to the host species. In this study, we performed a biodistribution and preclinical safety study in non-human primates with a clinical candidate rAAV1-human AAT (hAAT) vector at doses ranging from 3.0 × 1012 to 1.3 × 1013 vg/kg, bracketing those used in our clinical trials. We found that limb perfusion delivery of rAAV1-hAAT was safe and showed a biodistribution pattern similar to previous studies. However, serum levels of AAT obtained with high-dose limb perfusion still reached only ~50% of the target serum levels. Our results suggest that clinically effective AAT gene therapy may ultimately require delivery at doses between 3.5 × 1013-1 × 1014 vg/kg, which is within the dose range used for approved rAAV gene therapies. Muscle-targeting strategies could be incorporated when delivering systemic administration of high-dose rAAV gene therapies to increase transduction of muscle tissues and reduce the burden on the liver, especially in diseases that can present with hepatotoxicity such as AAT deficiency.