Multi-kingdom microbiota analyses identify bacterial-fungal interactions and biomarkers of colorectal cancer across cohorts.

Despite recent progress in our understanding of the association between the gut microbiome and colorectal cancer (CRC), multi-kingdom gut microbiome dysbiosis in CRC across cohorts is unexplored. We investigated four-kingdom microbiota alterations using CRC metagenomic datasets of 1,368 samples from 8 distinct geographical cohorts. Integrated analysis identified 20 archaeal, 27 bacterial, 20 fungal and 21 viral species for each single-kingdom diagnostic model. However, our data revealed superior diagnostic accuracy for models constructed with multi-kingdom markers, in particular the addition of fungal species. Specifically, 16 multi-kingdom markers including 11 bacterial, 4 fungal and 1 archaeal feature, achieved good performance in diagnosing patients with CRC (area under the receiver operating characteristic curve (AUROC) = 0.83) and maintained accuracy across 3 independent cohorts. Coabundance analysis of the ecological network revealed associations between bacterial and fungal species, such as Talaromyces islandicus and Clostridium saccharobutylicum. Using metagenome shotgun sequencing data, the predictive power of the microbial functional potential was explored and elevated D-amino acid metabolism and butanoate metabolism were observed in CRC. Interestingly, the diagnostic model based on functional EggNOG genes achieved high accuracy (AUROC = 0.86). Collectively, our findings uncovered CRC-associated microbiota common across cohorts and demonstrate the applicability of multi-kingdom and functional markers as CRC diagnostic tools and, potentially, as therapeutic targets for the treatment of CRC.

Integrative microbiomics in bronchiectasis exacerbations.

Bronchiectasis, a progressive chronic airway disease, is characterized by microbial colonization and infection. We present an approach to the multi-biome that integrates bacterial, viral and fungal communities in bronchiectasis through weighted similarity network fusion ( https://integrative-microbiomics.ntu.edu.sg ). Patients at greatest risk of exacerbation have less complex microbial co-occurrence networks, reduced diversity and a higher degree of antagonistic interactions in their airway microbiome. Furthermore, longitudinal interactome dynamics reveals microbial antagonism during exacerbation, which resolves following treatment in an otherwise stable multi-biome. Assessment of the Pseudomonas interactome shows that interaction networks, rather than abundance alone, are associated with exacerbation risk, and that incorporation of microbial interaction data improves clinical prediction models. Shotgun metagenomic sequencing of an independent cohort validated the multi-biome interactions detected in targeted analysis and confirmed the association with exacerbation. Integrative microbiomics captures microbial interactions to determine exacerbation risk, which cannot be appreciated by the study of a single microbial group. Antibiotic strategies probably target the interaction networks rather than individual microbes, providing a fresh approach to the understanding of respiratory infection.

Genome sequencing of Rigidoporus microporus provides insights on genes important for wood decay, latex tolerance and interspecific fungal interactions.

Fungal plant pathogens remain a serious threat to the sustainable agriculture and forestry, despite the extensive efforts undertaken to control their spread. White root rot disease is threatening rubber tree (Hevea brasiliensis) plantations throughout South and Southeast Asia and Western Africa, causing tree mortality and severe yield losses. Here, we report the complete genome sequence of the basidiomycete fungus Rigidoporus microporus, a causative agent of the disease. Our phylogenetic analysis confirmed the position of R. microporus among the members of Hymenochaetales, an understudied group of basidiomycetes. Our analysis further identified pathogen's genes with a predicted role in the decay of plant cell wall polymers, in the utilization of latex components and in interspecific interactions between the pathogen and other fungi. We also detected putative horizontal gene transfer events in the genome of R. microporus. The reported first genome sequence of a tropical rubber tree pathogen R. microporus should contribute to the better understanding of how the fungus is able to facilitate wood decay and nutrient cycling as well as tolerate latex and utilize resinous extractives.

Interactome networks between the human respiratory syncytial virus (HRSV), the human metapneumovirus (ΗMPV), and their host: In silico investigation and comparative functional enrichment analysis.

BACKGROUND AND OBJECTIVES: Human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) are leading causes of upper and lower respiratory tract infections in non-immunocompetent subjects, yet the mechanisms by which they induce their pathogenicity differ significantly and remain elusive. In this study we aimed at identifying the gene interaction networks between the HRSV, HMPV respiratory pathogens and their host along with the different cell-signaling pathways associated with the above interactomes. MATERIALS AND METHODS: The Viruses STRING database (http://viruses.string-db.org/) was used for the identification of the host-viruses interaction networks. The two lists of the predicted functional partners were entered in the FunRich tool (http://www.funrich.org) for the construction of the Venn diagram and the comparative Funcional Enrichment Analysis (FEA) with respect to biological pathways. The sets of the common and unique human genes identified in the two networks were also analyzed. The computational predictions regarding the shared human genes in the host-HRSV and the host-HMPV interactomes were further evaluated via the analysis of the GSE111732 dataset. miRNA transcriptomics data were mapped to gene targets using the miRNomics pipeline of the GeneTrail2 database (https://genetrail2.bioinf.uni-sb.de/). RESULTS: Eleven out of twenty predicted human genes were common in the two interactomes (TLR4, SOCS3, SFXN1, AKT1, SFXN3, LY96, SFXN2, SOCS7, CISH, SOCS6, SOCS1). FEA of these common genes identified the kit receptor and the GH receptor signaling pathways as the most significantly enriched annotations. The remaining nine genes of the host-HRSV and the host-HMPV interaction networks were the IFIH1, DDX58, NCL, IRF3, STAT2, HSPA4, CD209, KLF6, CHKA and the MYD88, SOCS4, SOCS2, SOCS5 AKT2, AKT3, SFXN4, SFXN5 and TLR3 respectively. Distinct cell-signaling pathways were enriched per interactome. The comparative FEA highlighted the association of the host-HRSV functional partners with the negative regulation of RIG-I/MDA5 signaling. The analysis with respect to miRNAs mapping to gene targets of the GSE111732 dataset indicated that nine out of the eleven common host genes are either enriched or depleted in the sample sets (HRSV or HMPV infected) as compared with the reference set (non-infected), although with no significant scores. CONCLUSIONS: We have identified both shared and unique host genes as members of the HRSV and HMPV interaction networks. The disparate human genes likely contribute to distinct responses in airway epithelial cells.

N-Acyl Homoserine Lactones and Lux Solos Regulate Social Behaviour and Virulence of Pseudomonas syringae pv. actinidiae.

The phyllosphere is a complex environment where microbes communicate through signalling molecules in a system, generally known as quorum sensing (QS). One of the most common QS systems in Gram-negative proteobacteria is based on the production of N-acyl homoserine lactones (AHLs) by a LuxI synthase and their perception by a LuxR sensor. Pseudomonas syringae pv. actinidiae (Psa), the aetiological agent of the bacterial canker of kiwifruit, colonises plant phyllosphere before penetrating via wounds and natural openings. Since Psa genome encodes three LuxR solos without a cognate LuxI, this bacterium may perceive diffusible signals, but it cannot produce AHLs, displaying a non-canonical QS system. The elucidation of the mechanisms underlying the perception of environmental cues in the phyllosphere by this pathogen and their influence on the onset of pathogenesis are of crucial importance for a long-lasting and sustainable management of the bacterial canker of kiwifruit. Here, we report the ability of Psa to sense its own population density and the presence of surrounding bacteria. Moreover, we show that Psa can perceive AHLs, indicating that AHL-producing neighbouring bacteria may regulate Psa virulence in the host. Our results suggest that the ecological environment is important in determining Psa fitness and pathogenic potential. This opens new perspectives in the use of more advanced biochemical and microbiological tools for the control of bacterial canker of kiwifruit.

Burkholderia cepacia Complex Contact-Dependent Growth Inhibition Systems Mediate Interbacterial Competition.

Burkholderia species, including opportunistic pathogens in the Burkholderia cepacia complex (Bcc), have genes to produce contact-dependent growth inhibition (CDI) system proteins. CDI is a phenomenon in which Gram-negative bacteria use the toxic C terminus of a polymorphic surface-exposed exoprotein, BcpA, to inhibit the growth of susceptible bacteria upon direct cell-cell contact. Production of a small immunity protein, BcpI, prevents autoinhibition. Although CDI systems appear widespread in Gram-negative bacteria, their function has been primarily examined in several model species. Here we demonstrate that genes encoding predicted CDI systems in Bcc species exhibit considerable diversity. We also show that Burkholderia multivorans, which causes pulmonary infections in patients with cystic fibrosis, expresses genes that encode two CDI systems, both of which appear distinct from the typical Burkholderia-type CDI system. Each system can mediate intrastrain interbacterial competition and contributes to bacterial adherence. Surprisingly, the immunity-protein-encoding bcpI gene of CDI system 1 could be mutated without obvious deleterious effects. We also show that nonpathogenic Burkholderia thailandensis uses CDI to control B. multivorans growth during coculture, providing one of the first examples of interspecies CDI and suggesting that CDI systems could be manipulated to develop therapeutic strategies targeting Bcc pathogens.IMPORTANCE Competition among bacteria affects microbial colonization of environmental niches and host organisms, particularly during polymicrobial infections. The Bcc is a group of environmental bacteria that can cause life-threatening opportunistic infections in patients who have cystic fibrosis or are immunocompromised. Understanding the mechanisms used by these bacterial pathogens to compete with one another may lead to the development of more effective therapies. Findings presented here demonstrate that a Bcc species, Burkholderia multivorans, produces functional CDI system proteins and that growth of this pathogen can be controlled by CDI system proteins produced by neighboring Burkholderia cells.

The Yin and Yang of Streptococcus Lung Infections in Cystic Fibrosis: a Model for Studying Polymicrobial Interactions.

The streptococci are increasingly recognized as a core component of the cystic fibrosis (CF) lung microbiome, yet the role that they play in CF lung disease is unclear. The presence of the Streptococcus milleri group (SMG; also known as the anginosus group streptococci [AGS]) correlates with exacerbation when these microbes are the predominant species in the lung. In contrast, microbiome studies have indicated that an increased relative abundance of streptococci in the lung, including members of the oral microflora, correlates with impacts on lung disease less severe than those caused by other CF-associated microflora, indicating a complex role for this genus in the context of CF. Recent findings suggest that streptococci in the CF lung microenvironment may influence the growth and/or virulence of other CF pathogens, as evidenced by increased virulence factor production by Pseudomonas aeruginosa when grown in coculture with oral streptococci. Conversely, the presence of P. aeruginosa can enhance the growth of streptococci, including members of the SMG, a phenomenon that could be exacerbated by the fact that streptococci are not susceptible to some of the frontline antibiotics used to treat P. aeruginosa infections. Collectively, these studies indicate the necessity for further investigation into the role of streptococci in the CF airway to determine how these microbes, alone or via interactions with other CF-associated pathogens, might influence CF lung disease, for better or for worse. We also propose that the interactions of streptococci with other CF pathogens is an ideal model to study clinically relevant microbial interactions.

Transcriptional profiling of Pseudomonas aeruginosa and Staphylococcus aureus during in vitro co-culture.

BACKGROUND: Co-colonization by Pseudomonas aeruginosa and Staphylococcus aureus is frequent in cystic fibrosis patients. Polymicrobial infections involve both detrimental and beneficial interactions between different bacterial species. Such interactions potentially indirectly impact the human host through virulence, antibiosis and immunomodulation. RESULTS: Here we explored the responses triggered by the encounter of these two pathogens to identify early processes that are important for survival when facing a potential competitor. Transcriptional profiles of both bacteria were obtained after 3 h co-culture and compared to the respective mono-culture using RNAseq. Global responses in both bacteria included competition for nitrogen sources, amino acids and increased tRNA levels. Both organisms also induced lysogenic mechanisms related to prophage induction (S. aureus) and R- and F- pyocin synthesis (P. aeruginosa), possibly as a response to stress resulting from nutrient limitation or cell damage. Specific responses in S. aureus included increased expression of de novo and salvation pathways for purine and pyrimidine synthesis, a switch to glucose fermentation, and decreased expression of major virulence factors and global regulators. CONCLUSIONS: Taken together, transcriptomic data indicate that early responses between P. aeruginosa and S. aureus involve competition for resources and metabolic adaptations, rather than the expression of bacteria- or host-directed virulence factors.

Differential roles for pathogenicity islands SPI-13 and SPI-8 in the interaction of Salmonella Enteritidis and Salmonella Typhi with murine and human macrophages.

BACKGROUND: Salmonella pathogenicity island (SPI)-13 is conserved in many serovars of S. enterica, including S. Enteritidis, S. Typhimurium and S. Gallinarum. However, it is absent in typhoid serovars such as S. Typhi and Paratyphi A, which carry SPI-8 at the same genomic location. Because the interaction with macrophages is a critical step in Salmonella pathogenicity, in this study we investigated the role played by SPI-13 and SPI-8 in the interaction of S. Enteritidis and S. Typhi with cultured murine (RAW264.7) and human (THP-1) macrophages. RESULTS: Our results showed that SPI-13 was required for internalization of S. Enteritidis in murine but not human macrophages. On the other hand, SPI-8 was not required for the interaction of S. Typhi with human or murine macrophages. Of note, the presence of an intact copy of SPI-13 in a S. Typhi mutant carrying a deletion of SPI-8 did not improve its ability to be internalized by, or survive in human or murine macrophages. CONCLUSIONS: Altogether, our results point out to different roles for SPI-13 and SPI-8 during Salmonella infection. While SPI-13 contributes to the interaction of S. Enteritidis with murine macrophages, SPI-8 is not required in the interaction of S. Typhi with murine or human macrophages. We hypothesized that typhoid serovars have lost SPI-13 and maintained SPI-8 to improve their fitness during another phase of human infection.

Differential roles for pathogenicity islands SPI-13 and SPI-8 in the interaction of Salmonella Enteritidis and Salmonella Typhi with murine and human macrophages

BACKGROUND: Salmonella pathogenicity island (SPI)-13 is conserved in many serovars of S. enterica, including S. Enteritidis, S. Typhimurium and S. Gallinarum. However, it is absent in typhoid serovars such as S. Typhi and Paratyphi A, which carry SPI-8 at the same genomic location. Because the interaction with macrophages is a critical step in Salmonella pathogenicity, in this study we investigated the role played by SPI-13 and SPI-8 in the interaction of S. Enteritidis and S. Typhi with cultured murine (RAW264.7) and human (THP-1) macrophages. RESULTS: Our results showed that SPI-13 was required for internalization of S. Enteritidis in murine but not human macrophages. On the other hand, SPI-8 was not required for the interaction of S. Typhi with human or murine macrophages. Of note, the presence of an intact copy of SPI-13 in a S. Typhi mutant carrying a deletion of SPI-8 did not improve its ability to be internalized by, or survive in human or murine macrophages. CONCLUSIONS: Altogether, our results point out to different roles for SPI-13 and SPI-8 during Salmonella infection. While SPI-13 contributes to the interaction of S. Enteritidis with murine macrophages, SPI-8 is not required in the interaction of S. Typhi with murine or human macrophages. We hypothesized that typhoid serovars have lost SPI-13 and maintained SPI-8 to improve their fitness during another phase of human infection.

Metagenomic surveys of gut microbiota.

Gut microbiota of higher vertebrates is host-specific. The number and diversity of the organisms residing within the gut ecosystem are defined by physiological and environmental factors, such as host genotype, habitat, and diet. Recently, culture-independent sequencing techniques have added a new dimension to the study of gut microbiota and the challenge to analyze the large volume of sequencing data is increasingly addressed by the development of novel computational tools and methods. Interestingly, gut microbiota maintains a constant relative abundance at operational taxonomic unit (OTU) levels and altered bacterial abundance has been associated with complex diseases such as symptomatic atherosclerosis, type 2 diabetes, obesity, and colorectal cancer. Therefore, the study of gut microbial population has emerged as an important field of research in order to ultimately achieve better health. In addition, there is a spontaneous, non-linear, and dynamic interaction among different bacterial species residing in the gut. Thus, predicting the influence of perturbed microbe-microbe interaction network on health can aid in developing novel therapeutics. Here, we summarize the population abundance of gut microbiota and its variation in different clinical states, computational tools available to analyze the pyrosequencing data, and gut microbe-microbe interaction networks.

First principles of Hamiltonian medicine.

We introduce the field of Hamiltonian medicine, which centres on the roles of genetic relatedness in human health and disease. Hamiltonian medicine represents the application of basic social-evolution theory, for interactions involving kinship, to core issues in medicine such as pathogens, cancer, optimal growth and mental illness. It encompasses three domains, which involve conflict and cooperation between: (i) microbes or cancer cells, within humans, (ii) genes expressed in humans, (iii) human individuals. A set of six core principles, based on these domains and their interfaces, serves to conceptually organize the field, and contextualize illustrative examples. The primary usefulness of Hamiltonian medicine is that, like Darwinian medicine more generally, it provides novel insights into what data will be productive to collect, to address important clinical and public health problems. Our synthesis of this nascent field is intended predominantly for evolutionary and behavioural biologists who aspire to address questions directly relevant to human health and disease.

Bacterial lipopolysaccharide binding enhances virion stability and promotes environmental fitness of an enteric virus.

Enteric viruses, including poliovirus and reovirus, encounter a vast microbial community in the mammalian gastrointestinal tract, which has been shown to promote virus replication and pathogenesis. Investigating the underlying mechanisms, we find that poliovirus binds bacterial surface polysaccharides, which enhances virion stability and cell attachment by increasing binding to the viral receptor. Additionally, we identified a poliovirus mutant, VP1-T99K, with reduced lipopolysaccharide (LPS) binding. Although T99K and WT poliovirus cell attachment, replication, and pathogenesis in mice are equivalent, VP1-T99K poliovirus was unstable in feces following peroral inoculation of mice. Consequently, the ratio of mutant virus in feces is reduced following additional cycles of infection in mice. Thus, the mutant virus incurs a fitness cost when environmental stability is a factor. These data suggest that poliovirus binds bacterial surface polysaccharides, enhancing cell attachment and environmental stability, potentially promoting transmission to a new host.

Comparative transcriptomics reveals different strategies of Trichoderma mycoparasitism.

BACKGROUND: Trichoderma is a genus of mycotrophic filamentous fungi (teleomorph Hypocrea) which possess a bright variety of biotrophic and saprotrophic lifestyles. The ability to parasitize and/or kill other fungi (mycoparasitism) is used in plant protection against soil-borne fungal diseases (biological control, or biocontrol). To investigate mechanisms of mycoparasitism, we compared the transcriptional responses of cosmopolitan opportunistic species and powerful biocontrol agents Trichoderma atroviride and T. virens with tropical ecologically restricted species T. reesei during confrontations with a plant pathogenic fungus Rhizoctonia solani. RESULTS: The three Trichoderma spp. exhibited a strikingly different transcriptomic response already before physical contact with alien hyphae. T. atroviride expressed an array of genes involved in production of secondary metabolites, GH16 ß-glucanases, various proteases and small secreted cysteine rich proteins. T. virens, on the other hand, expressed mainly the genes for biosynthesis of gliotoxin, respective precursors and also glutathione, which is necessary for gliotoxin biosynthesis. In contrast, T. reesei increased the expression of genes encoding cellulases and hemicellulases, and of the genes involved in solute transport. The majority of differentially regulated genes were orthologues present in all three species or both in T. atroviride and T. virens, indicating that the regulation of expression of these genes is different in the three Trichoderma spp. The genes expressed in all three fungi exhibited a nonrandom genomic distribution, indicating a possibility for their regulation via chromatin modification. CONCLUSION: This genome-wide expression study demonstrates that the initial Trichoderma mycotrophy has differentiated into several alternative ecological strategies ranging from parasitism to predation and saprotrophy. It provides first insights into the mechanisms of interactions between Trichoderma and other fungi that may be exploited for further development of biofungicides.

Competition between two virulent Marek's disease virus strains in vivo.

Previous studies have demonstrated the presence of multiple strains of Marek's disease virus simultaneously circulating within poultry flocks, leading to the assumption that individual birds are repeatedly exposed to a variety of virus strains in their lifetime. Virus competition within individual birds may be an important factor that influences the outcome of co-infection under field conditions, including the potential outcome of emergence or evolution of more virulent strains. A series of experiments was designed to evaluate virus competition within chickens following simultaneous challenge with two virulent serotype 1 Marek's disease virus strains, using either pathogenically similar (rMd5 and rMd5/pp38CVI) or dissimilar (JM/102W and rMd5/pp38CVI) virus pairs. Bursa of Fabricius, feather follicle epithelium, spleen, and tumour samples were collected at multiple time points to determine the frequency and distribution of each virus present using pyrosequencing, immunohistochemistry and virus isolation. In the similar pair, rMd5 appeared to have a competitive advantage over rMd5/pp38CVI, which in turn had a competitive advantage over the less virulent JM/102W in the dissimilar virus pair. Dominance of one strain over the other was not absolute for either virus pair, as the subordinate virus was rarely eliminated. Interestingly, competition between two viruses with either pair rarely ended in a draw. Further work is needed to identify factors that influence virus-specific dominance to better understand what characteristics favour emergence of one strain in chicken populations at the expense of other strains.