Zebrafish tsc1 and cxcl12a increase susceptibility to mycobacterial infection.

Regulation of host miRNA expression is a contested node that controls the host immune response to mycobacterial infection. The host must counter subversive efforts of pathogenic mycobacteria to launch a protective immune response. Here, we examine the role of miR-126 in the zebrafish-Mycobacterium marinum infection model and identify a protective role for infection-induced miR-126 through multiple effector pathways. We identified a putative link between miR-126 and the tsc1a and cxcl12a/ccl2/ccr2 signalling axes resulting in the suppression of non-tnfa expressing macrophage accumulation at early M. marinum granulomas. Mechanistically, we found a detrimental effect of tsc1a expression that renders zebrafish embryos susceptible to higher bacterial burden and increased cell death via mTOR inhibition. We found that macrophage recruitment driven by the cxcl12a/ccl2/ccr2 signalling axis was at the expense of the recruitment of classically activated tnfa-expressing macrophages and increased cell death around granulomas. Together, our results delineate putative pathways by which infection-induced miR-126 may shape an effective immune response to M. marinum infection in zebrafish embryos.

Vacuolar localisation of anthocyanin pigmentation in microgreen cotyledons of basil, cabbage and mustard greens does not impact on colonisation by Shiga-toxigenic Escherichia coli O157:H7.

Microgreens, the immature plants harvested after a few weeks of growth, are perceived as a heathy, nutritious food ingredient but may be susceptible to colonisation by human pathogens including Shiga-toxigenic Escherichia coli (STEC). Some microgreen cultivars accumulate anthocyanins or secrete essential oils which, when extracted or purified, have been reported to inhibit bacterial growth. Therefore, the impact of anthocyanins on bacterial colonisation by STEC (Sakai) was compared for three species that have pigmented cultivars: basil (Ocimum basilicum L.), cabbage (Brassica oleracea L.) and mustard greens (Brassica juncea L.). Inoculation with low concentrations of STEC (Sakai) (3 log10 colony forming units/ml (CFU/ml)) during seed germination resulted in extensive colonisation at the point of harvest, accumulating to âˆ¼ 8 log10 CFU/g FW in all cultivars. Bacterial colonies frequently aligned with anticlinal walls on the surface of epidermal cells of the cotyledons and, in basil, associated with peltate and capitate gland cells. Crude lysates of pigmented and non-pigmented basil cultivars had no impact on STEC (Sakai) growth rates, viability status or biofilm formation. Anthocyanins are located within plant vacuoles of these microgreen cultivars and did not affect colonisation by STEC (Sakai) and pigmentation therefore cannot be considered as a controlling factor in bacterial interactions.

Investigating the genetic components of tuber bruising in a breeding population of tetraploid potatoes.

BACKGROUND: Tuber bruising in tetraploid potatoes (Solanum tuberosum) is a trait of economic importance, as it affects tubers' fitness for sale. Understanding the genetic components affecting tuber bruising is a key step in developing potato lines with increased resistance to bruising. As the tetraploid setting renders genetic analyses more complex, there is still much to learn about this complex phenotype. Here, we used capture sequencing data on a panel of half-sibling populations from a breeding programme to perform a genome-wide association analysis (GWAS) for tuber bruising. In addition, we collected transcriptomic data to enrich the GWAS results. However, there is currently no satisfactory method to represent both GWAS and transcriptomics analysis results in a single visualisation and to compare them with existing knowledge about the biological system under study. RESULTS: When investigating population structure, we found that the STRUCTURE algorithm yielded greater insights than discriminant analysis of principal components (DAPC). Importantly, we found that markers with the highest (though non-significant) association scores were consistent with previous findings on tuber bruising. In addition, new genomic regions were found to be associated with tuber bruising. The GWAS results were backed by the transcriptomics differential expression analysis. The differential expression notably highlighted for the first time the role of two genes involved in cellular strength and mechanical force sensing in tuber resistance to bruising. We proposed a new visualisation, the HIDECAN plot, to integrate the results from the genomics and transcriptomics analyses, along with previous knowledge about genomic regions and candidate genes associated with the trait. CONCLUSION: This study offers a unique genome-wide exploration of the genetic components of tuber bruising. The role of genetic components affecting cellular strength and resistance to physical force, as well as mechanosensing mechanisms, was highlighted for the first time in the context of tuber bruising. We showcase the usefulness of genomic data from breeding programmes in identifying genomic regions whose association with the trait of interest merit further investigation. We demonstrate how confidence in these discoveries and their biological relevance can be increased by integrating results from transcriptomics analyses. The newly proposed visualisation provides a clear framework to summarise of both genomics and transcriptomics analyses, and places them in the context of previous knowledge on the trait of interest.

In situ control of root-bacteria interactions using optical trapping in transparent soil.

Bacterial attachment on root surfaces is an important step preceding the colonization or internalization and subsequent infection of plants by pathogens. Unfortunately, bacterial attachment is not well understood because the phenomenon is difficult to observe. Here we assessed whether this limitation could be overcome using optical trapping approaches. We have developed a system based on counter-propagating beams and studied its ability to guide Pectobacterium atrosepticum (Pba) cells to different root cell types within the interstices of transparent soils. Bacterial cells were successfully trapped and guided to root hair cells, epidermal cells, border cells, and tissues damaged by laser ablation. Finally, we used the system to quantify the bacterial cell detachment rate of Pba cells on root surfaces following reversible attachment. Optical trapping techniques could greatly enhance our ability to deterministically characterize mechanisms linked to attachment and formation of biofilms in the rhizosphere.

Plant species-dependent transmission of Escherichia coli O157:H7 from the spermosphere to cotyledons and first leaves.

The colonization of six edible plant species: alfalfa, broccoli, coriander, lettuce, parsley and rocket, by the human pathogen Shigatoxigenic Escherichia coli was investigated following two modes of artificial inoculation of seeds, by soaking or watering. The frequency and extent of colonization of cotyledons depended on the mode of inoculation, with three, rapidly germinating species being successfully colonized after overnight soaking, but slower germinating species requiring prolonged exposure to bacteria by watering of the surrounding growth media. Separate analysis of the cotyledons and leaves from individual plants highlighted that successful colonization of the true leaves was also species dependent. For three species, failure of transfer, or lack of nutrients or suitable microhabitat on the leaf surface resulted in infrequent bacterial colonization. Colonization of leaves was lower and generally in proportion to that in cotyledons, if present. The potential risks associated with consumption of leafy produce are discussed.

The innovation of the symbiosome has enhanced the evolutionary stability of nitrogen fixation in legumes.

Nitrogen-fixing symbiosis is globally important in ecosystem functioning and agriculture, yet the evolutionary history of nodulation remains the focus of considerable debate. Recent evidence suggesting a single origin of nodulation followed by massive parallel evolutionary losses raises questions about why a few lineages in the N2 -fixing clade retained nodulation and diversified as stable nodulators, while most did not. Within legumes, nodulation is restricted to the two most diverse subfamilies, Papilionoideae and Caesalpinioideae, which show stable retention of nodulation across their core clades. We characterize two nodule anatomy types across 128 species in 56 of the 152 genera of the legume subfamily Caesalpinioideae: fixation thread nodules (FTs), where nitrogen-fixing bacteroids are retained within the apoplast in modified infection threads, and symbiosomes, where rhizobia are symplastically internalized in the host cell cytoplasm within membrane-bound symbiosomes (SYMs). Using a robust phylogenomic tree based on 997 genes from 147 Caesalpinioideae genera, we show that losses of nodulation are more prevalent in lineages with FTs than those with SYMs. We propose that evolution of the symbiosome allows for a more intimate and enduring symbiosis through tighter compartmentalization of their rhizobial microsymbionts, resulting in greater evolutionary stability of nodulation across this species-rich pantropical legume clade.

Mycobacterium avium subsp. paratuberculosis exploits miRNA expression to modulate lipid metabolism and macrophage polarisation pathways during infection.

Pathogenic mycobacteria including Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease, manipulate host macrophages to persist and cause disease. In mycobacterial infection, highly plastic macrophages, shift between inflammatory M1 and permissive M2 phenotypes which alter the disease outcome and allow bacteria to survive intracellularly. Here we examine the impact of MAP infection on polarised macrophages and how increased lipid availability alters macrophage phenotype and bacterial persistence. Further, we assess if host microRNA (miRNA) are sensitive to macrophage polarisation state and how MAP can drive their expression to overcome innate responses. Using in vitro MAP infection, we find that increasing lipid availability through supplementing culture media with exogenous lipid increases cellular nitric oxide production. Lipid-associated miRs -19a, -129, -24, and -24-3p are differentially expressed following macrophage polarisation and lipid supplementation and are further regulated during MAP infection. Collectively, our results highlight the importance of host lipid metabolism in MAP infection and demonstrate control of miRNA expression by MAP to favour intracellular persistence.

Treatment of infection-induced vascular pathologies is protective against persistent rough morphotype Mycobacterium abscessus infection in zebrafish.

Mycobacterium abscessus infections are of increasing global prevalence and are often difficult to treat due to complex antibiotic resistance profiles. While there are similarities between the pathogenesis of M. abscessus and tuberculous mycobacteria, including granuloma formation and stromal remodelling, there are distinct molecular differences at the host-pathogen interface. Here we have used a zebrafish-M. abscessus model and host-directed therapies that were previously identified in the zebrafish-M. marinum model to identify potential host-directed therapies against M. abscessus infection. We find efficacy of anti-angiogenic and vascular normalizing therapies against rough M. abscessus infection, but no effect of anti-platelet drugs.

OXSR1 inhibits inflammasome activation by limiting potassium efflux during mycobacterial infection.

Pathogenic mycobacteria inhibit inflammasome activation to establish infection. Although it is known that potassium efflux is a trigger for inflammasome activation, the interaction between mycobacterial infection, potassium efflux, and inflammasome activation has not been investigated. Here, we use Mycobacterium marinum infection of zebrafish embryos and Mycobacterium tuberculosis infection of THP-1 cells to demonstrate that pathogenic mycobacteria up-regulate the host WNK signalling pathway kinases SPAK and OXSR1 which control intracellular potassium balance. We show that genetic depletion or inhibition of OXSR1 decreases bacterial burden and intracellular potassium levels. The protective effects of OXSR1 depletion are at least partially mediated by NLRP3 inflammasome activation, caspase-mediated release of IL-1ß, and downstream activation of protective TNF-α. The elucidation of this druggable pathway to potentiate inflammasome activation provides a new avenue for the development of host-directed therapies against intracellular infections.

Rough and smooth variants of Mycobacterium abscessus are differentially controlled by host immunity during chronic infection of adult zebrafish.

Prevalence of Mycobacterium abscessus infections is increasing in patients with respiratory comorbidities. After initial colonisation, M. abscessus smooth colony (S) variants can undergo an irreversible genetic switch into highly inflammatory, rough colony (R) variants, often associated with a decline in pulmonary function. Here, we use an adult zebrafish model of chronic infection with R and S variants to study M. abscessus pathogenesis in the context of fully functioning host immunity. We show that infection with an R variant causes an inflammatory immune response that drives necrotic granuloma formation through host TNF signalling, mediated by the tnfa, tnfr1 and tnfr2 gene products. T cell-dependent immunity is stronger against the R variant early in infection, and regulatory T cells associate with R variant granulomas and limit bacterial growth. In comparison, an S variant proliferates to high burdens but appears to be controlled by TNF-dependent innate immunity early during infection, resulting in delayed granuloma formation. Thus, our work demonstrates the applicability of adult zebrafish to model persistent M. abscessus infection, and illustrates differences in the immunopathogenesis induced by R and S variants during granulomatous infection.

Deciphering Molecular Host-Pathogen Interactions During Ramularia Collo-Cygni Infection on Barley.

Ramularia collo-cygni is the causal agent of Ramularia leaf spot disease (RLS) on barley and became, during the recent decades, an increasing threat for farmers across the world. Here, we analyze morphological, transcriptional, and metabolic responses of two barley cultivars having contrasting tolerance to RLS, when infected by an aggressive or mild R. collo-cygni isolate. We found that fungal biomass in leaves of the two cultivars does not correlate with their tolerance to RLS, and both cultivars displayed cell wall reinforcement at the point of contact with the fungal hyphae. Comparative transcriptome analysis identified that the largest transcriptional differences between cultivars are at the early stages of fungal colonization with differential expression of kinases, calmodulins, and defense proteins. Weighted gene co-expression network analysis identified modules of co-expressed genes, and hub genes important for cultivar responses to the two R. collo-cygni isolates. Metabolite analyses of the same leaves identified defense compounds such as p-CHDA and serotonin, correlating with responses observed at transcriptome and morphological level. Together these all-round responses of barley to R. collo-cygni provide molecular tools for further development of genetic and physiological markers that may be tested for improving tolerance of barley to this fungal pathogen.

The GpIA7 effector from the potato cyst nematode Globodera pallida targets potato EBP1 and interferes with the plant cell cycle.

The potato cyst nematode Globodera pallida acquires all of its nutrients from an elaborate feeding site that it establishes in a host plant root. Normal development of the root cells is re-programmed in a process coordinated by secreted nematode effector proteins. The biological function of the G. pallida GpIA7 effector was investigated in this study. GpIA7 is specifically expressed in the subventral pharyngeal glands of pre-parasitic stage nematodes. Ectopic expression of GpIA7 in potato plants affected plant growth and development, suggesting a potential role for this effector in feeding site establishment. Potato plants overexpressing GpIA7 were shorter, with reduced tuber weight and delayed flowering. We provide evidence that GpIA7 associates with the plant growth regulator StEBP1 (ErbB-3 epidermal growth factor receptor-binding protein 1). GpIA7 modulates the regulatory function of StEBP1, altering the expression level of downstream target genes, including ribonucleotide reductase 2, cyclin D3;1, and retinoblastoma related 1, which are down-regulated in plants overexpressing GpIA7. We provide an insight into the molecular mechanism used by the nematode to manipulate the host cell cycle and demonstrate that this may rely, at least in part, on hindering the function of host EBP1.

The GpIA7 effector from the potato cyst nematode Globodera pallida targets potato EBP1 and interferes with the plant cell cycle programme.

The potato cyst nematode Globodera pallida acquires all of its nutrients from an elaborate feeding site that it establishes in a host plant root. Normal development of the root cells is re-programmed in a process coordinated by secreted nematode effector proteins. The biological function of the G. pallida GpIA7 effector was investigated in this study. GpIA7 is specifically expressed in the subventral pharyngeal glands of pre-parasitic stage nematodes. Ectopic expression of GpIA7 in potato plants affected plant growth and development, suggesting a potential role for this effector in feeding site establishment. Potato plants overexpressing GpIA7 were shorter, with reduced tuber weight and delayed flowering. We provide evidence that GpIA7 associates with the plant growth regulator StEBP1 (ErbB-3 epidermal growth factor receptor-binding protein 1). GpIA7 modulates the regulatory function of StEBP1, altering the expression level of downstream target genes, including ribonucleotide reductase 2, cyclin D3;1 and retinoblastoma related 1, which are downregulated in plants overexpressing GpIA7. We provide an insight into the molecular mechanism used by the nematode to manipulate the host cell cycle and provide evidence that this may rely, at least in part, on hindering the function of host EBP1.

The role of l-arabinose metabolism for Escherichia coli O157:H7 in edible plants.

Arabinose is a major plant aldopentose in the form of arabinans complexed in cell wall polysaccharides or glycoproteins (AGP), but comparatively rare as a monosaccharide. l-arabinose is an important bacterial metabolite, accessed by pectolytic micro-organisms such as Pectobacterium atrosepticum via pectin and hemicellulose degrading enzymes. However, not all plant-associated microbes encode cell-wall-degrading enzymes, yet can metabolize l-arabinose, raising questions about their use of and access to the glycan in plants. Therefore, we examined l-arabinose metabolism in the food-borne pathogen Escherichia coli O157:H7 (isolate Sakai) during its colonization of plants. l-arabinose metabolism (araBA) and transport (araF) genes were activated at 18 °C in vitro by l-arabinose and expressed over prolonged periods in planta. Although deletion of araBAD did not impact the colonization ability of E. coli O157:H7 (Sakai) on spinach and lettuce plants (both associated with STEC outbreaks), araA was induced on exposure to spinach cell-wall polysaccharides. Furthermore, debranched and arabinan oligosaccharides induced ara metabolism gene expression in vitro, and stimulated modest proliferation, while immobilized pectin did not. Thus, E. coli O157:H7 (Sakai) can utilize pectin/AGP-derived l-arabinose as a metabolite. Furthermore, it differs fundamentally in ara gene organization, transport and regulation from the related pectinolytic species P. atrosepticum, reflective of distinct plant-associated lifestyles.

Mycobacterial infection-induced miR-206 inhibits protective neutrophil recruitment via the CXCL12/CXCR4 signalling axis.

Pathogenic mycobacteria actively dysregulate protective host immune signalling pathways during infection to drive the formation of permissive granuloma microenvironments. Dynamic regulation of host microRNA (miRNA) expression is a conserved feature of mycobacterial infections across host-pathogen pairings. Here we examine the role of miR-206 in the zebrafish model of Mycobacterium marinum infection, which allows investigation of the early stages of granuloma formation. We find miR-206 is upregulated following infection by pathogenic M. marinum and that antagomir-mediated knockdown of miR-206 is protective against infection. We observed striking upregulation of cxcl12a and cxcr4b in infected miR-206 knockdown zebrafish embryos and live imaging revealed enhanced recruitment of neutrophils to sites of infection. We used CRISPR/Cas9-mediated knockdown of cxcl12a and cxcr4b expression and AMD3100 inhibition of Cxcr4 to show that the enhanced neutrophil response and reduced bacterial burden caused by miR-206 knockdown was dependent on the Cxcl12/Cxcr4 signalling axis. Together, our data illustrate a pathway through which pathogenic mycobacteria induce host miR-206 expression to suppress Cxcl12/Cxcr4 signalling and prevent protective neutrophil recruitment to granulomas.

Functional Analysis of Shiga Toxin-Producing Escherichia coli Biofilm Components in Plant Leaves.

Plants represent alternative or secondary hosts for Shiga toxin-producing Escherichia coli (STEC), enabling transmission of the pathogens through the food chain on horticultural crops. This becomes a public health concern for plants that are eaten raw or minimally processed, such as leafy salad and fruits. STEC actively interact with plants as hosts, and so to determine the mechanistic basis to the interaction, it is necessary to assess STEC gene function in planta. Here, we describe analysis of an STEC biofilm component, curli, that plays a role in STEC colony formation in plant leaves. It also serves as a suitable example of the approaches required for qualitative and quantitative assessment of functional host colonization traits.

Exposure to the gut microbiota from cigarette smoke-exposed mice exacerbates cigarette smoke extract-induced inflammation in zebrafish larvae.

Cigarette smoke (CS)-induced inflammation leads to a range of diseases including chronic obstructive pulmonary disease and cancer. The gut microbiota is a major modifying environmental factor that determine the severity of cigarette smoke-induced pathology. Microbiomes and metabolites from CS-exposed mice exacerbate lung inflammation via the gut-lung axis of shared mucosal immunity in mice but these systems are expensive to establish and analyse. Zebrafish embryos and larvae have been used to model the effects of cigarette smoking on a range of physiological processes and offer an amenable platform for screening modifiers of cigarette smoke-induced pathologies with key features of low cost and rapid visual readouts. Here we exposed zebrafish larvae to cigarette smoke extract (CSE) and characterised a CSE-induced leukocytic inflammatory phenotype with increased neutrophilic and macrophage inflammation in the gut. The CSE-induced phenotype was exacerbated by co-exposure to microbiota from the faeces of CS-exposed mice, but not control mice. Microbiota could be recovered from the gut of zebrafish and studied in isolation in a screening setting. This demonstrates the utility of the zebrafish-CSE exposure platform for identifying environmental modifiers of cigarette smoking-associated pathology and demonstrates that the CS-exposed mouse gut microbiota potentiates the inflammatory effects of CSE across host species.

MacConkey broth purple provides an efficient MPN estimation method for Shigatoxigenic Escherichia coli.

MacConkey broth purple provides a more efficient method for Most Probable Number estimation for Shigatoxigenic Escherichia coli (E.coli) than the process of bacterial enrichment in buffered peptone water followed by detection on MacConkey agar, since it is a single-step process that gives comparable results in plant extracts.

Escherichia coli O157:H7 F9 Fimbriae Recognize Plant Xyloglucan and Elicit a Response in Arabidopsis thaliana.

Fresh produce is often a source of enterohaemorrhagic Escherichia coli (EHEC) outbreaks. Fimbriae are extracellular structures involved in cell-to-cell attachment and surface colonisation. F9 (Fml) fimbriae have been shown to be expressed at temperatures lower than 37 °C, implying a function beyond the mammalian host. We demonstrate that F9 fimbriae recognize plant cell wall hemicellulose, specifically galactosylated side chains of xyloglucan, using glycan arrays. E. coli expressing F9 fimbriae had a positive advantage for adherence to spinach hemicellulose extract and tissues, which have galactosylated oligosaccharides as recognized by LM24 and LM25 antibodies. As fimbriae are multimeric structures with a molecular pattern, we investigated whether F9 fimbriae could induce a transcriptional response in model plant Arabidopsis thaliana, compared with flagella and another fimbrial type, E. coli common pilus (ECP), using DNA microarrays. F9 induced the differential expression of 435 genes, including genes involved in the plant defence response. The expression of F9 at environmentally relevant temperatures and its recognition of plant xyloglucan adds to the suite of adhesins EHEC has available to exploit the plant niche.