A compendium of multi-omics data illuminating host responses to lethal human virus infections.

Human infections caused by viral pathogens trigger a complex gamut of host responses that limit disease, resolve infection, generate immunity, and contribute to severe disease or death. Here, we present experimental methods and multi-omics data capture approaches representing the global host response to infection generated from 45 individual experiments involving human viruses from the Orthomyxoviridae, Filoviridae, Flaviviridae, and Coronaviridae families. Analogous experimental designs were implemented across human or mouse host model systems, longitudinal samples were collected over defined time courses, and global multi-omics data (transcriptomics, proteomics, metabolomics, and lipidomics) were acquired by microarray, RNA sequencing, or mass spectrometry analyses. For comparison, we have included transcriptomics datasets from cells treated with type I and type II human interferon. Raw multi-omics data and metadata were deposited in public repositories, and we provide a central location linking the raw data with experimental metadata and ready-to-use, quality-controlled, statistically processed multi-omics datasets not previously available in any public repository. This compendium of infection-induced host response data for reuse will be useful for those endeavouring to understand viral disease pathophysiology and network biology.

The germ theory revisited: A noncentric view on infection outcome.

The germ theory states that pathogenic microorganisms are responsible for causing infectious diseases. The theory is inherently microbe-centric and does not account for variability in disease severity among individuals and asymptomatic carriership-two phenomena indicating an important role for host variability in infection outcome. The basic tenet of the germ theory was recently challenged, and a radically host-centric paradigm referred to as the "full-blown host theory" was proposed. According to this view, the pathogen is reduced to a passive environmental trigger, and the development of disease is instead due to pre-existing immunodeficiencies of the host. Here, we consider the factors that determine disease severity using established knowledge concerning evolutionary biology, microbial pathogenesis, and host-pathogen interactions. We note that the available data support a noncentric view that recognizes key roles for both the causative microbe and the host in dictating infection outcome.

An ELISA-based method for Galleria mellonella apolipophorin-III quantification.

Mammalian models, such as murine, are used widely in pathophysiological studies because they have a high degree of similarity in body temperature, metabolism, and immune response with humans. However, non-vertebrate animal models have emerged as alternative models to study the host-pathogen interaction with minimal ethical concerns. Galleria mellonella is an alternative model that has proved useful in studying the interaction of the host with either bacteria or fungi, performing drug testing, and assessing the immunological response to different microorganisms. The G. mellonella immune response includes cellular and humoral components with structural and functional similarities to the immune effectors found in higher vertebrates, such as humans. An important humoral effector stimulated during infections is apolipophorin III (apoLp-III), an opsonin characterized by its lipid and carbohydrate-binding properties that participate in lipid transport, as well as immunomodulatory activity. Despite some parameters, such as the measurement of phenoloxidase activity, melanin production, hemocytes counting, and expression of antimicrobial peptides genes are already used to assess the G. mellonella immune response to pathogens with different virulence degrees, the apoLp-III quantification remains to be a parameter to assess the immune response in this invertebrate. Here, we propose an immunological tool based on an enzyme-linked immunosorbent assay that allows apoLp-III quantification in the hemolymph of larvae challenged with pathogenic agents. We tested the system with hemolymph coming from larvae infected with Escherichia coli, Candida albicans, Sporothrix schenckii, Sporothrix globosa, and Sporothrix brasiliensis. The results revealed significantly higher concentrations of apoLp-III when each microbial species was inoculated, in comparison with untouched larvae, or inoculated with phosphate-buffered saline. We also demonstrated that the apoLp-III levels correlated with the strains' virulence, which was already reported. To our knowledge, this is one of the first attempts to quantify apoLp-III, using a quick and easy-to-use serological technique.

Proteomic and Lipidomic Profiling of Calves Experimentally Co-Infected with Influenza D Virus and Mycoplasma bovis: Insights into the Host-Pathogen Interactions.

The role of Influenza D virus (IDV) in bovine respiratory disease remains unclear. An in vivo experiment resulted in increased clinical signs, lesions, and pathogen replication in calves co-infected with IDV and Mycoplasma bovis (M. bovis), compared to single-infected calves. The present study aimed to elucidate the host-pathogen interactions and profile the kinetics of lipid mediators in the airways of these calves. Bronchoalveolar lavage (BAL) samples collected at 2 days post-infection (dpi) were used for proteomic analyses by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, lipidomic analyses were performed by LC-MS/MS on BAL samples collected at 2, 7 and 14 dpi. Whereas M. bovis induced the expression of proteins involved in fibrin formation, IDV co-infection counteracted this coagulation mechanism and downregulated other acute-phase response proteins, such as complement component 4 (C4) and plasminogen (PLG). The reduced inflammatory response against M. bovis likely resulted in increased M. bovis replication and delayed M. bovis clearance, which led to a significantly increased abundance of oxylipids in co-infected calves. The identified induced oxylipids mainly derived from arachidonic acid; were likely oxidized by COX-1, COX-2, and LOX-5; and peaked at 7 dpi. This paper presents the first characterization of BAL proteome and lipid mediator kinetics in response to IDV and M. bovis infection in cattle and raises hypotheses regarding how IDV acts as a co-pathogen in bovine respiratory disease.

Dual RNA sequencing of Helicobacter pylori and host cell transcriptomes reveals ontologically distinct host-pathogen interaction.

Helicobacter pylori is a highly successful pathogen that poses a substantial threat to human health. However, the dynamic interaction between H. pylori and the human gastric epithelium has not been fully investigated. In this study, using dual RNA sequencing technology, we characterized a cytotoxin-associated gene A (cagA)-modulated bacterial adaption strategy by enhancing the expression of ATP-binding cassette transporter-related genes, metQ and HP_0888, upon coculturing with human gastric epithelial cells. We observed a general repression of electron transport-associated genes by cagA, leading to the activation of oxidative phosphorylation. Temporal profiling of host mRNA signatures revealed the downregulation of multiple splicing regulators due to bacterial infection, resulting in aberrant pre-mRNA splicing of functional genes involved in the cell cycle process in response to H. pylori infection. Moreover, we demonstrated a protective effect of gastric H. pylori colonization against chronic dextran sulfate sodium (DSS)-induced colitis. Mechanistically, we identified a cluster of propionic and butyric acid-producing bacteria, Muribaculaceae, selectively enriched in the colons of H. pylori-pre-colonized mice, which may contribute to the restoration of intestinal barrier function damaged by DSS treatment. Collectively, this study presents the first dual-transcriptome analysis of H. pylori during its dynamic interaction with gastric epithelial cells and provides new insights into strategies through which H. pylori promotes infection and pathogenesis in the human gastric epithelium. IMPORTANCE: Simultaneous profiling of the dynamic interaction between Helicobacter pylori and the human gastric epithelium represents a novel strategy for identifying regulatory responses that drive pathogenesis. This study presents the first dual-transcriptome analysis of H. pylori when cocultured with gastric epithelial cells, revealing a bacterial adaptation strategy and a general repression of electron transportation-associated genes, both of which were modulated by cytotoxin-associated gene A (cagA). Temporal profiling of host mRNA signatures dissected the aberrant pre-mRNA splicing of functional genes involved in the cell cycle process in response to H. pylori infection. We demonstrated a protective effect of gastric H. pylori colonization against chronic DSS-induced colitis through both in vitro and in vivo experiments. These findings significantly enhance our understanding of how H. pylori promotes infection and pathogenesis in the human gastric epithelium and provide evidence to identify targets for antimicrobial therapies.

Pathogen evolution: Protective microbes act as a double-edged sword.

Vaccines and infection can sometimes cause incomplete immunity, which allows for pathogen re-infection with decreased disease severity but also contributes to the evolution of pathogen virulence. A new study demonstrates that incomplete immunity from resident protective microbes results in similar evolutionary trajectories.

Cell-to-cell heterogeneity drives host-virus coexistence in a bloom-forming alga.

Algal blooms drive global biogeochemical cycles of key nutrients and serve as hotspots for biological interactions in the ocean. The massive blooms of the cosmopolitan coccolithophore Emiliania huxleyi are often infected by the lytic E. huxleyi virus, which is a major mortality agent triggering bloom demise. This multi-annual "boom and bust" pattern of E. huxleyi blooms suggests that coexistence is essential for these host-virus dynamics. To investigate host-virus coexistence, we developed a new model system from an E. huxleyi culture that recovered from viral infection. The recovered population coexists with the virus, as host cells continue to divide in parallel to viral production. By applying single-molecule fluorescence in situ hybridization (smFISH) to quantify the fraction of infected cells, and assessing infection-specific lipid biomarkers, we identified a small subpopulation of cells that were infected and produced new virions, whereas most of the host population could resist infection. To further assess population heterogeneity, we generated clonal strain collections using single-cell sorting and subsequently phenotyped their susceptibility to E. huxleyi virus infection. This unraveled substantial cell-to-cell heterogeneity across a continuum of susceptibility to resistance, highlighting that infection outcome may vary depending on the individual cell. These results add a new dimension to our understanding of the complexity of host-virus interactions that are commonly assessed in bulk and described by binary definitions of resistance or susceptibility. We propose that phenotypic heterogeneity drives the host-virus coexistence and demonstrate how the coexistence with a lytic virus provides an ecological advantage for the host by killing competing strains.

Pathogen evolution following spillover from a resident to a migrant host population depends on interactions between host pace of life and tolerance to infection.

Changes to migration routes and phenology create novel contact patterns among hosts and pathogens. These novel contact patterns can lead to pathogens spilling over between resident and migrant populations. Predicting the consequences of such pathogen spillover events requires understanding how pathogen evolution depends on host movement behaviour. Following spillover, pathogens may evolve changes in their transmission rate and virulence phenotypes because different strategies are favoured by resident and migrant host populations. There is conflict in current theoretical predictions about what those differences might be. Some theory predicts lower pathogen virulence and transmission rates in migrant populations because migrants have lower tolerance to infection. Other theoretical work predicts higher pathogen virulence and transmission rates in migrants because migrants have more contacts with susceptible hosts. We aim to understand how differences in tolerance to infection and host pace of life act together to determine the direction of pathogen evolution following pathogen spillover from a resident to a migrant population. We constructed a spatially implicit model in which we investigate how pathogen strategy changes following the addition of a migrant population. We investigate how differences in tolerance to infection and pace of life between residents and migrants determine the effect of spillover on pathogen evolution and host population size. When the paces of life of the migrant and resident hosts are equal, larger costs of infection in the migrants lead to lower pathogen transmission rate and virulence following spillover. When the tolerance to infection in migrant and resident populations is equal, faster migrant paces of life lead to increased transmission rate and virulence following spillover. However, the opposite can also occur: when the migrant population has lower tolerance to infection, faster migrant paces of life can lead to decreases in transmission rate and virulence. Predicting the outcomes of pathogen spillover requires accounting for both differences in tolerance to infection and pace of life between populations. It is also important to consider how movement patterns of populations affect host contact opportunities for pathogens. These results have implications for wildlife conservation, agriculture and human health.

Alpha1-antitrypsin impacts innate host-pathogen interactions with Candida albicans by stimulating fungal filamentation.

Our immune system possesses sophisticated mechanisms to cope with invading microorganisms, while pathogens evolve strategies to deal with threats imposed by host immunity. Human plasma protein α1-antitrypsin (AAT) exhibits pleiotropic immune-modulating properties by both preventing immunopathology and improving antimicrobial host defence. Genetic associations suggested a role for AAT in candidemia, the most frequent fungal blood stream infection in intensive care units, yet little is known about how AAT influences interactions between Candida albicans and the immune system. Here, we show that AAT differentially impacts fungal killing by innate phagocytes. We observed that AAT induces fungal transcriptional reprogramming, associated with cell wall remodelling and downregulation of filamentation repressors. At low concentrations, the cell-wall remodelling induced by AAT increased immunogenic ß-glucan exposure and consequently improved fungal clearance by monocytes. Contrastingly, higher AAT concentrations led to excessive C. albicans filamentation and thus promoted fungal immune escape from monocytes and macrophages. This underscores that fungal adaptations to the host protein AAT can differentially define the outcome of encounters with innate immune cells, either contributing to improved immune recognition or fungal immune escape.

A standardized wound infection model for antimicrobial testing of wound dressings in vitro.

To investigate the effectiveness of antimicrobial agents against wound infections, experiments using either 2D cultures with planktonic microorganisms or animal infection models are frequently carried out. However, the transferability of the results to human skin is limited by the lack of complexity of the 2D models or by the poor translation of the results from animal models. Hence, there is a need for wound infection models capable of assessing antimicrobial agents. In this study, an easily standardized wound infection model was established. This model consists of a mechanically wounded human skin model on a collagen matrix infected with various clinically relevant bacteria. Infection of the model led to recognition of the pathogens and induction of an inflammatory response. The untreated infection spread over time, causing significant tissue damage. By applying an antimicrobial-releasing wound dressing, the bacterial load could be reduced and the success of the treatment could be further measured by a decrease in the inflammatory reaction. In conclusion, this wound infection model can be used to evaluate new antimicrobial therapeutics as well as to study host-pathogen interactions.

Changes in the Expression of Proteins Associated with Neurodegeneration in the Brains of Mice after Infection with Influenza A Virus with Wild Type and Truncated NS1.

Influenza type A virus (IAV) infection is a major cause of morbidity and mortality during influenza epidemics. Recently, a specific link between IAV infection and neurodegenerative disease progression has been established. The non-structural NS1 protein of IAV regulates viral replication during infection and antagonizes host antiviral responses, contributing to influenza virulence. In the present study, we have prepared a mouse lung-to-lung adapted to the NS1-truncated virus (NS80ad). Transcriptome analysis of the gene expression in the lungs revealed that infection with wild-type A/WSN/33 (WSN), NS80, and NS80ad viruses resulted in different regulation of genes involved in signaling pathways associated with the cell proliferation, inflammatory response, and development of neurodegenerative diseases. NS1 protein did not influence the genes involved in the RIG-I-like receptor signaling pathway in the brains. Lethal infection with IAVs dysregulated expression of proteins associated with the development of neurodegenerative diseases (CX3CL1/Fractalkine, Coagulation factor III, and CD105/Endoglin, CD54/ICAM-1, insulin-like growth factor-binding protein (IGFBP)-2, IGFBP-5, IGFBP-6, chitinase 3-like 1 (CHI3L1), Myeloperoxidase (MPO), Osteopontin (OPN), cystatin C, and LDL R). Transcription of GATA3 mRNA was decreased, and expression of MPO was inhibited in the brain infected with NS80 and NS80ad viruses. In addition, the truncation of NS1 protein led to reduced expression of IGFBP-2, CHI3L1, MPO, and LDL-R proteins in the brains. Our results indicate that the influenza virus influences the expression of proteins involved in brain function, and this might occur mostly through the NS1 protein. These findings suggest that the abovementioned proteins represent a promising target for the development of potentially effective immunotherapy against neurodegeneration.

Molecular Mechanisms of the Stripe Rust Interaction with Resistant and Susceptible Wheat Genotypes.

Rust fungi cause significant damage to wheat production worldwide. In order to mitigate disease impact and improve food security via durable resistance, it is important to understand the molecular basis of host-pathogen interactions. Despite a long history of research and high agricultural importance, still little is known about the interactions between the stripe rust fungus and wheat host on the gene expression level. Here, we present analysis of the molecular interactions between a major wheat pathogen-Puccinia striiformis f. sp. tritici (Pst)-in resistant and susceptible host backgrounds. Using plants with durable nonrace-specific resistance along with fully susceptible ones allowed us to show how gene expression patterns shift in compatible versus incompatible interactions. The pathogen showed significantly greater number and fold changes of overexpressed genes on the resistant host than the susceptible host. Stress-related pathways including MAPK, oxidation-reduction, osmotic stress, and stress granule formation were, almost exclusively, upregulated in the resistant host background, suggesting the requirement of the resistance-countermeasure mechanism facilitated by Pst. In contrast, the susceptible host background allowed for broad overrepresentation of the nutrient uptake pathways. This is the first study focused on the stripe rust pathogen-wheat interactions, on the whole transcriptome level, from the pathogen side. It lays a foundation for the better understanding of the resistant/susceptible hosts versus pathogenic fungus interaction in a broader sense.

When the microbiome shapes the host: immune evolution implications for infectious disease.

The microbiome includes both 'mutualist' and 'pathogen' microbes, regulated by the same innate immune architecture. A major question has therefore been: how do hosts prevent pathogenic infections while maintaining beneficial microbes? One idea suggests hosts can selectively activate innate immunity upon pathogenic infection, but not mutualist colonization. Another idea posits that hosts can selectively attack pathogens, but not mutualists. Here I review evolutionary principles of microbe recognition and immune activation, and reflect on newly observed immune effector-microbe specificity perhaps supporting the latter idea. Recent work in Drosophila has found a surprising importance for single antimicrobial peptides in combatting specific ecologically relevant microbes. The developing picture suggests these effectors have evolved for this purpose. Other defence responses like reactive oxygen species bursts can also be uniquely effective against specific microbes. Signals in other model systems including nematodes, Hydra, oysters, and mammals, suggest that effector-microbe specificity may be a fundamental principle of host-pathogen interactions. I propose this effector-microbe specificity stems from weaknesses of the microbes themselves: if microbes have intrinsic weaknesses, hosts can evolve effectors that exploit those weaknesses. I define this host-microbe relationship as 'the Achilles principle of immune evolution'. Incorporating this view helps interpret why some host-microbe interactions develop in a coevolutionary framework (e.g. Red Queen dynamics), or as a one-sided evolutionary response. This clarification should be valuable to better understand the principles behind host susceptibilities to infectious diseases. This article is part of the theme issue 'Sculpting the microbiome: how host factors determine and respond to microbial colonization'.

How infection-triggered pathobionts influence virulence evolution.

Host-pathogen interactions can be influenced by the host microbiota, as the microbiota can facilitate or prevent pathogen infections. In addition, members of the microbiota can become virulent. Such pathobionts can cause co-infections when a pathogen infection alters the host immune system and triggers dysbiosis. Here we performed a theoretical investigation of how pathobiont co-infections affect the evolution of pathogen virulence. We explored the possibility that the likelihood of pathobiont co-infection depends on the evolving virulence of the pathogen. We found that, in contrast to the expectation from classical theory, increased virulence is not always selected for. For an increasing likelihood of co-infection with increasing pathogen virulence, we found scenario-specific selection for either increased or decreased virulence. Evolutionary changes, however, in pathogen virulence do not always translate into similar changes in combined virulence of the pathogen and the pathobiont. Only in one of the scenarios where pathobiont co-infection is triggered above a specific virulence level we found a reduction in combined virulence. This was not the case when the probability of pathobiont co-infection linearly increased with pathogen virulence. Taken together, our study draws attention to the possibility that host-microbiota interactions can be both the driver and the target of pathogen evolution. This article is part of the theme issue 'Sculpting the microbiome: how host factors determine and respond to microbial colonization'.

In silico agent-based modeling approach to characterize multiple in vitro tuberculosis infection models.

In vitro models of Mycobacterium tuberculosis (Mtb) infection are a valuable tool for examining host-pathogen interactions and screening drugs. With the development of more complex in vitro models, there is a need for tools to help analyze and integrate data from these models. To this end, we introduce an agent-based model (ABM) representation of the interactions between immune cells and bacteria in an in vitro setting. This in silico model was used to simulate both traditional and spheroid cell culture models by changing the movement rules and initial spatial layout of the cells in accordance with the respective in vitro models. The traditional and spheroid simulations were calibrated to published experimental data in a paired manner, by using the same parameters in both simulations. Within the calibrated simulations, heterogeneous outputs are seen for bacterial count and T cell infiltration into the macrophage core of the spheroid. The simulations also predict that equivalent numbers of activated macrophages do not necessarily result in similar bacterial reductions; that host immune responses can control bacterial growth in both spheroid structure dependent and independent manners; that STAT1 activation is the limiting step in macrophage activation in spheroids; and that drug screening and macrophage activation studies could have different outcomes depending on the in vitro culture used. Future model iterations will be guided by the limitations of the current model, specifically which parts of the output space were harder to reach. This ABM can be used to represent more in vitro Mtb infection models due to its flexible structure, thereby accelerating in vitro discoveries.

A fungal core effector exploits the OsPUX8B.2-OsCDC48-6 module to suppress plant immunity.

Proteins containing a ubiquitin regulatory X (UBX) domain are cofactors of Cell Division Cycle 48 (CDC48) and function in protein quality control. However, whether and how UBX-containing proteins participate in host-microbe interactions remain unclear. Here we show that MoNLE1, an effector from the fungal pathogen Magnaporthe oryzae, is a core virulence factor that suppresses rice immunity by specifically interfering with OsPUX8B.2. The UBX domain of OsPUX8B.2 is required for its binding to OsATG8 and OsCDC48-6 and controls its 26 S proteasome-dependent stability. OsPUX8B.2 and OsCDC48-6 positively regulate plant immunity against blast fungus, while the high-temperature tolerance heat-shock protein OsBHT, a putative cytoplasmic substrate of OsPUX8B.2-OsCDC48-6, negatively regulates defense against blast infection. MoNLE1 promotes the nuclear migration and degradation of OsPUX8B.2 and disturbs its association with OsBHT. Given the high conservation of MoNLE1 among fungal isolates, plants with broad and durable blast resistance might be generated by engineering intracellular proteins resistant to MoNLE1.

Quantitative pathogenicity and host adaptation in a fungal plant pathogen revealed by whole-genome sequencing.

Knowledge of genetic determinism and evolutionary dynamics mediating host-pathogen interactions is essential to manage fungal plant diseases. Studies on the genetic architecture of fungal pathogenicity often focus on large-effect effector genes triggering strong, qualitative resistance. It is not clear how this translates to predominately quantitative interactions. Here, we use the Zymoseptoria tritici-wheat model to elucidate the genetic architecture of quantitative pathogenicity and mechanisms mediating host adaptation. With a multi-host genome-wide association study, we identify 19 high-confidence candidate genes associated with quantitative pathogenicity. Analysis of genetic diversity reveals that sequence polymorphism is the main evolutionary process mediating differences in quantitative pathogenicity, a process that is likely facilitated by genetic recombination and transposable element dynamics. Finally, we use functional approaches to confirm the role of an effector-like gene and a methyltransferase in phenotypic variation. This study highlights the complex genetic architecture of quantitative pathogenicity, extensive diversifying selection and plausible mechanisms facilitating pathogen adaptation.