Publications

4606

Background The chicken is the most abundant food animal in the world. However, despite its importance, the chicken gut microbiome remains largely undefined. Here, we exploit culture-independent and culture-dependent approaches to reveal extensive taxonomic diversity within this complex microbial community. Results We performed metagenomic sequencing of fifty chicken faecal samples from two breeds and analysed these, alongside all (n = 582) relevant publicly available chicken metagenomes, to cluster over 20 million non-redundant genes and to construct over 5,500 metagenome-assembled bacterial genomes. In addition, we recovered nearly 600 bacteriophage genomes. This represents the most comprehensive view of taxonomic diversity within the chicken gut microbiome to date, encompassing hundreds of novel candidate bacterial genera and species. To provide a stable, clear and memorable nomenclature for novel species, we devised a scalable combinatorial system for the creation of hundreds of well-formed Latin binomials. We cultured and genome-sequenced bacterial isolates from chicken faeces, documenting over forty novel species, together with three species from the genus Escherichia, including the newly named species Escherichia whittamii. Conclusions Our metagenomic and culture-based analyses provide new insights into the bacterial, archaeal and bacteriophage components of the chicken gut microbiome. The resulting datasets expand the known diversity of the chicken gut microbiome and provide a key resource for future high-resolution taxonomic and functional studies on the chicken gut microbiome.

AbstractCandidatusLiberibacter asiaticus (CLas) is one the causative agents of greening disease in citrus, an unccurable, devastating disease of citrus worldwide. CLas is vectored byDiaphorina citri, and the understanding of the molecular interplay between vector and pathogen will provide additional basis for the development and implementation of successful management strategies. We focused in the molecular interplay occurring in the gut of the vector, a major barrier for CLas invasion and colonization. We investigated the differential expression of vector and CLas genes by analyzing ade novoreference metatranscriptome of the gut of adult psyllids fed of CLas-infected and healthy citrus plants for 1-2, 3-4 and 5-6 days. CLas regulates the immune response of the vector affecting the production of reactive species of oxygen and nitrogen, and the production of antimicrobial peptides. Moreover, CLas overexpressedperoxiredoxinin a protective manner. The major transcript involved in immune expression was related to melanization, aCLIP-domain serine proteasewe believe participates in the wounding of epithelial cells damaged during infection, which is supported by the down-regulation ofpangolin. We also detected that CLas modulates the gut peristalsis of psyllids through the down-regulation oftitin, reducing the elimination of CLas with faeces. The up-regulation of the neuromodulatorarylalkylamine N-acetyltransferaseimplies CLas also interferes with the double brain-gut communication circuitry of the vector. CLas colonizes the gut by expressing twoType IVb pilin flpgenes and several chaperones that can also function as adhesins. We hypothesized biofil formation occurs by the expression of the cold shock protein of CLas. We also describe the interplay during cell invasion and modification, and propose mechanisms CLas uses to invade the host hemocel. We identified several specific targets for the development of strategies directed to interfere with the successful utilization of the psyllid vector by this pathogen.Author SummaryHuanglongbing (HLB) or greening is an incurable disease causing severe damage to citrus production, making citrus industrial activity unsustainable in several countries around the world. HLB is caused by three species ofCandidatusLiberibacter.Ca.L. asiaticus (CLas), vectored by the psyllidDiaphorina citri, is the prevalent species. Attempts to apply new technologies in the development of strategies for disease and pest management are been made. However, we still miss basic information on this system to efficiently apply the current technologies and envisage the implementation of new approaches for pest control, despite the relevant scientific contribution available. One major gap is regarded to the molecular interplay between CLas and its vector. We focused our attention in the molecular interplay occurring at the first relevant interaction of CLas andD. citri, represented by the gut barrier. We report the transcriptional activity of CLas during the invasion and establishment of the infection in the gut of the vector, as well as the transcriptional activity of the vector in response to the infection. We identified several host genes that are targeted and regulated by CLas as well as several CLas genes that are promising targets for the application of new management strategies.

AbstractAnaplasmosis, caused by infection with bacteria of the genusAnaplasmais an important veterinary and zoonotic disease. The characterization of transmission has concentrated on ticks and little is known about non-tick vectors of livestock anaplasmosis. This study investigated the presence ofAnaplasmaspp. in camels in northern Kenya and whether the hematophagous camel ked,Hippobosca camelina, acts as a vector. Camels (n =976) and > 10,000 keds were sampled over a three-year study period and the presence ofAnaplasmaspecies was determined by PCR-based assays targeting theAnaplasmataceae16S rRNA gene. Camels were infected by‘CandidatusAnaplasma camelii’occurring from 63 - 78% during the dry (September 2017), wet (June-July 2018), and late wet seasons (July-August 2019). 10 - 29% of camel keds harbored ‘Ca. Anaplasma camelii’ acquired from infected camels during blood feeding. We determined whetherAnaplasmapositive camel keds could transmit ‘Ca. Anaplasma camelii’ to small laboratory animals via blood-feeding. We show competence in pathogen transmission and subsequent infection in mice and rabbits by both direct detection in blood smears and subsequent molecular identification by PCR. Transmission of ‘Ca. Anaplasma camelii’ to mice (8 - 47%) and rabbits (25%) occurred readily after ked bites. Hence, we demonstrate, for the first time, the potential ofH. camelinaas a vector of anaplasmosis. This key finding provides the basis for establishing ked control programmes for improvement of livestock and human health.Author summaryHematophagous flies such as Tabanids andStomoxys, among other biting flies, are mechanical transmitters of various pathogens such as African trypanosomes andAnaplasmaspecies. However, little is known about the role of common camel-specific biting keds (also known as camel flies or louse flies, genusHippobosca) in pathogen transmission. Keds inflict painful bites to access host blood, and in the process may transmit bacterial hemopathogens frequently detected in both camels and their keds. We confirmed by experimental blood-feeding, gene amplification, and amplicon sequencing that camel keds can transmit “CandidatusAnaplasma camelii” from naturally-infected camels to healthy mice and rabbits. The high prevalence of camel anaplasmosis throughout the year in northern Kenya could be explained by the infestation camel-specificH. camelina, whose capacity as efficient fliers, unlike ticks, promotes disease transmission and maintenance within and among camel herds. Although this study focused on the transmission ofAnaplasmasp. by camel keds, it is possible that other hemopathogens could also be transmitted by these flies through a similar mechanism. Notably, in the absence of their preferred hosts, keds occasionally bite humans and other vertebrates they come across in order to acquire bloodmeals, and in the process could transmit zoonotic pathogens.

Comparison of the metabolic changes prior to symptom development upon infection with Candidatus Liberibacter asiaticus (CLas), the bacterium associated with citrus greening disease, between citrus hosts with different tolerances is lacking. The objective of this study was to compare the early response of Lisbon lemon (Citrus limon) and Washington navel orange (Citrus sinensis [L.] Osbeck), two citrus species commercially important to California, to CLas through graft inoculation. Here, we compare the transcriptome, proteome, and metabolome response, using RNA sequencing, liquid chromatography tandem mass spectrometry, and 1H nuclear magnetic resonance, respectively, from our two recently published studies examining the response of the lemon and navel oranges separately, and introduce new micronutrient data from inductively coupled plasma mass spectrometry analysis, focusing on lemons at 10 and 14 weeks postgrafting (wpg), and navels at 8 and 18 wpg, prior to symptom development. Several micronutrients accumulated in presymptomatic infected lemons compared with controls, whereas little change was observed in the navels. Photosynthesis proteins were substantially altered by CLas infection in navels, with fewer changes observed in lemons. The metabolome differed between control and infected navels throughout infection, although differences between control and infected lemons were identified only after symptom expression. Taken together, these findings highlight differences in response to CLas between two varieties with differing tolerances. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

AbstractNonulosonic acids (NulOs) are a family of acidic carbohydrates with a nine-carbon backbone, which include different related structures, such as sialic acids. They have mainly been studied for their relevance in animal cells and pathogenic bacteria. Recently, sialic acids have been discovered as an important compound in the extracellular matrix of virtually all microbial life and in “CandidatusAccumulibacter phosphatis”, a well-studied polyphosphate-accumulating organism, in particular. Here, bioaggregates highly enriched with these bacteria (approx. 95% based on proteomic data) were used to study the production of NulOs in an enrichment of this microorganism. Fluorescence lectin-binding analysis, enzymatic quantification, and mass spectrometry were used to analyze the different NulOs present, showing a wide distribution and variety of these carbohydrates, such as sialic acids and bacterial NulOs, in the bioaggregates. Phylogenetic analysis confirmed the potential of “Ca. Accumulibacter” to produce different types of NulOs. Proteomic analysis showed the ability of “Ca. Accumulibacter” to reutilize and reincorporate these carbohydrates. This investigation points out the importance of diverse NulOs in non-pathogenic bacteria, which are normally overlooked. Sialic acids and other NulOs should be further investigated for their role in the ecology of “Ca. Accumulibacter” in particular, and biofilms in general.Key Points•“Ca. Accumulibacter” has the potential to produce a range of nonulosonic acids.•Mass spectrometry and lectin binding can reveal the presence and location of nonulosonic acids.•The role of nonulosonic acid in non-pathogenic bacteria needs to be studied in detail.