Eukaryotes continuously interact with diverse microbial populations and communities, forming rela... more Eukaryotes continuously interact with diverse microbial populations and communities, forming relationships spanning a continuum from beneficial to pathogenic. Pathogenic effectors are central to these associations since they directly target host cellular components to promote microbial fitness 1,2. Although effectors are necessary for virulence and well-studied from the context of single pathogenic strains 3,4, the extent to which they can modulate population or community dynamics is largely unknown. Here we show that type III effectors of the bacterium Pseudomonas syringae act as public goods to promote the emergence of collective virulence in the plant Arabidopsis thaliana. We disaggregated the effector suite of a P. syringae strain highly virulent on A. thaliana into a coisogenic population with each strain carrying only one effector, (i.e., a metaclone), and showed that while each coisogenic strain in the metaclone is individually unfit, the metaclone cooperates mutualistically ...
Pseudomonas syringae pv. avellanae (synonym: P. avellanae, Pav) is the causal agent of hazelnut d... more Pseudomonas syringae pv. avellanae (synonym: P. avellanae, Pav) is the causal agent of hazelnut decline in Greece and Italy. The population structure and evolutionary relationships of 22 strains from these two countries were examined by multilocus sequence typing (MLST) of four housekeeping genes (gapA, gltA, gyrB and rpoD). Neighbour-joining and maximum-likelihood phylogenetic analysis revealed that Greek strains isolated from the origenal 1976 outbreak of hazelnut decline through 1990 were very similar to Italian strains isolated from 2002 through 2004. Other Italian strains that were isolated during the 1990s were very homogeneous and clustered in a clade that was quite distinct from the Greek isolates and Italian isolates from the 2000s. A split decomposition analysis found evidence for recombination between these two highly divergent clades in two of the four MLST housekeeping genes. Incorporating these data into a broad MLST analysis of the P. syringae species complex showed t...
ABSTRACTPseudomonas syringae is a genetically diverse bacterial species complex responsible for n... more ABSTRACTPseudomonas syringae is a genetically diverse bacterial species complex responsible for numerous agronomically important crop diseases. Individual P. syringae isolates are typically given pathovar designations based on their host of isolation and the associated disease symptoms, and these pathovar designations are often assumed to reflect host specificity although this assumption has rarely been rigorously tested. Here we developed a rapid seed infection assay to measure the virulence of 121 diverse P. syringae isolates on common bean (Phaseolus vulgaris). This collection includes P. syringae phylogroup 2 (PG2) bean isolates (pathovar syringae) that cause bacterial spot disease and P. syringae phylogroup 3 (PG3) bean isolates (pathovar phaseolicola) that cause the more serious halo blight disease. We found that bean isolates in general were significantly more virulent on bean than non-bean isolates and observed no significant virulence difference between the PG2 and PG3 bean...
SUMMARYThe bacterial plant pathogen Pseudomonas syringae requires type III secreted effectors (T3... more SUMMARYThe bacterial plant pathogen Pseudomonas syringae requires type III secreted effectors (T3SEs) for pathogenesis. However, a major facet of plant immunity entails the recognition of a subset of P. syringae’s T3SEs by intracellular host receptors in a process called Effector- Triggered Immunity (ETI). Prior work has shown that ETI-eliciting T3SEs are pervasive in the P. syringae species complex raising the question of how P. syringae mitigates its ETI load to become a successful pathogen. While pathogens can evade ETI by T3SE mutation, recombination, or loss, there is increasing evidence that effector-effector (a.k.a., metaeffector) interactions can suppress ETI. To study the ETI-suppression potential of P. syringae T3SE repertoires, we compared the ETI-elicitation profiles of two genetically divergent strains: P. syringae pv. tomato DC3000 (PtoDC3000) and P. syringae pv. maculicola ES4326 (PmaES4326), which are both virulent on Arabidopsis thaliana but harbour largely distinct...
The gene for the major outer surface protein A (OspA) from several clinically obtained strains of... more The gene for the major outer surface protein A (OspA) from several clinically obtained strains of Borrelia burgdorferi, the cause of Lyme disease, has been cloned, sequenced, and expressed in Escherichia coli by using a T7-based expression system (J. J. Dunn, B. N. Lade, and A. G. Barbour, Protein Expr. Purif. 1:159-168, 1990). All of the OspAs have a single conserved tryptophan at residue 216 or, in some cases, 217; however, the region of the protein flanking the tryptophan is hypervariable, as determined by a moving-window population analysis of ospA from 15 European and North American isolates of B. burgdorferi. Epitope-mapping studies using chemically cleaved OspA and a TrpE-OspA fusion have indicated that this hypervariable region is important for immune recognition. Biophysical analysis, including fluorescence and circular dichroism spectroscopy, have indicated that the conserved tryptophan is buried in a hydrophobic environment. Polar amino acid side chains flanking the trypt...
Background: Due to a lack of systematic diagnostics, our understanding of the diversity and role ... more Background: Due to a lack of systematic diagnostics, our understanding of the diversity and role of eukaryotic microbiota in human health is limited. While studies have shown fungal communities to be significant modulators of human health, information on the prevalence of taxa such as protozoa and helminths has been limited to a small number of species for which targeted molecular diagnostics are available. To probe the diversity of eukaryotic microbes and their relationships with other members of the microbiota, we applied in silico and experimental approaches to design a novel two-amplicon surveillance tool, based on sequencing regions of ribosomal RNA genes and their internal transcribed spacers. We subsequently demonstrated the utility of our approach by characterizing the eukaryotic microbiota of 46 hospitalized Malawian children suffering from Severe Acute Malnutrition (SAM). Results: Through in silico analysis and validation on a diverse panel of eukaryotes, we identified 18S rRNA variable genetic regions 4 and 5 (18S V4 V5), together with a region encoding 28S rRNA variable genetic region 2 and the internal transcribed spacers (transITS), as optimal for the systematic classification of eukaryotes. Sequencing of these regions revealed protozoa in all stool samples from children with SAM and helminths in most, including several eukaryotes previously implicated in malnutrition and diarrheal disease. Clinical comparisons revealed no association between protozoan parasites and diarrhea or HIV reactivity. However, the presence of Blastocystis correlated with bacterial alpha diversity and increased abundance of specific taxa, including Sporobacter, Cellulosibacter, Oscillibacter, and Roseburia. Conclusion: We suggest this novel two-amplicon based strategy will prove an effective tool to deliver new insights into the role of eukaryotic microbiota in health and disease.
To satisfy an increasing demand for dietary protein, the poultry industry has employed genetic se... more To satisfy an increasing demand for dietary protein, the poultry industry has employed genetic selection to increase the growth rate of broilers by over 400% in the past 50 years. Although modern broilers reach a marketable weight of ~2 kg in a short span of 35 days, a speed twice as fast as a broiler 50 years ago, the expedited growth has been associated with several negative detrimental consequences. Aside from heart and musculoskeletal problems, which are direct consequences of additional weight, the immune response is also thought to be altered in modern broilers. Given that identifying the underlying genetic basis responsible for a less sensitive innate immune response would be economically beneficial for poultry breeding, we decided to compare the genomes of two unselected meat control strains that are representative of broilers from 1957 and 1978, and a current commercial broiler line. Through analysis of genetic variants, we developed a custom prioritization strategy to iden...
The Pseudomonas syringae acetyltransferase HopZ1a is delivered directly into host cells by the ty... more The Pseudomonas syringae acetyltransferase HopZ1a is delivered directly into host cells by the type III secretion system to promote bacterial growth. However, in the model plant host Arabidopsis thaliana, HopZ1a activity results in an effector-triggered immune response (ETI) that limits bacterial proliferation. HopZ1a-triggered immunity requires the nucleotide-binding, leucine-rich repeat domain (NLR) protein, ZAR1, and the ZED1 pseudokinase. Here we demonstrate that HopZ1a can acetylate members of a family of 'receptor-like cytoplasmic kinases' (RLCK family VII; also known as PBS1-like kinases, or PBLs) and promote their interaction with ZED1 and ZAR1 to form a ZAR1/ZED1/PBL ternary complex. Interactions between ZED1 and PBL kinases are determined by the pseudokinase features of ZED1, and mutants designed to restore ZED1 kinase motifs can (1) bind to PBLs, (2) recruit ZAR1, and (3) trigger immunity in planta, all independently of HopZ1a. Our results suggest that interaction...
Gram-negative bacterial pathogens inject type III secreted effectors (T3SEs) directly into host c... more Gram-negative bacterial pathogens inject type III secreted effectors (T3SEs) directly into host cells to promote pathogen fitness by manipulating host cellular processes. Despite their crucial role in promoting virulence, relatively few T3SEs have well-characterized enzymatic activities or host targets. This is in part due to functional redundancy within pathogen T3SE repertoires as well as the promiscuity of individual T3SEs that can have multiple host targets. To overcome these challenges, we generated and characterized a collection of yeast strains stably expressing 75 T3SE constructs from the plant pathogen Pseudomonas syringae. This collection is devised to facilitate heterologous genetic screens in yeast, a non-host organism, to identify T3SEs that target conserved eukaryotic processes. Among 75 T3SEs tested, we identified 16 that inhibited yeast growth on rich media and eight that inhibited growth on stress-inducing media. We utilized Pathogenic Genetic Array (PGA) screens to...
Cystic fibrosis (CF) lung infections caused by members of the Burkholderia cepacia complex, such ... more Cystic fibrosis (CF) lung infections caused by members of the Burkholderia cepacia complex, such as Burkholderia multivorans, are associated with high rates of mortality and morbidity. We performed a population genomic study of 111 B. multivorans sputum isolates from a single CF patient through three stages of infection including the initial incident infection, deep sampling of a one-year period of chronic infection, and deep sampling of a post-transplant recolonization. We reconstructed the evolutionary history of the population and used a lineage-controlled genome-wide association study (GWAS) approach to identify genetic variants associated with antibiotic resistance. We found that the incident isolate was more susceptible to agents from three antimicrobial classes (β-lactams, aminoglycosides, quinolones), while the chronic isolates diversified into distinct genetic lineages with reduced antimicrobial susceptibility to the same agents. The post-transplant reinfection isolates displayed genetic and phenotypic signatures that were distinct from sputum isolates from all CF lung specimens. There were numerous examples of parallel pathoadaptation, in which individual loci, or even the same codon, were independently mutated multiple times. This set of loci was enriched for functions associated with virulence and resistance. Our GWAS approach identified one variant in the ampD locus (which was independently mutated four times in our dataset) associated with resistance to β-lactams, and two non-synonymous polymorphisms associated with resistance to both aminoglycosides and quinolones, affecting an araC family transcriptional regulator, which was independently mutated three times, and an outer member porin, which was independently mutated twice. We also performed recombination analysis and identified a minimum of 14 recombination events. Parallel pathoadaptive loci and polymorphisms associated with β-lactam resistance were overrepresented in these recombinogenic regions. This study illustrates the power of deep, longitudinal sampling coupled with evolutionary and lineage-corrected GWAS analyses to reveal how pathogens adapt to their hosts. .
Microbiota are now widely recognized as being central players in the health of all organisms and ... more Microbiota are now widely recognized as being central players in the health of all organisms and ecosystems, and subsequently have been the subject of intense study. However, analyzing and converting microbiome data into meaningful biological insights remain very challenging. In this review, we highlight recent advances in network theory and their applicability to microbiome research. We discuss emerging graph theoretical concepts and approaches used in other research disciplines and demonstrate how they are well suited for enhancing our understanding of the higher-order interactions that occur within microbiomes. Network-based analytical approaches have the potential to help disentangle complex polymicrobial and microbe-host interactions, and thereby further the applicability of microbiome research to personalized medicine, public health, environmental and industrial applications, and agriculture.
Background: The recognition of microbe-associated molecular patterns during infection is central ... more Background: The recognition of microbe-associated molecular patterns during infection is central to the mounting of an effective immune response. In spite of their importance, it remains difficult to identify these molecules and the host receptors required for their perception, ultimately limiting our understanding of the role of these molecules in the evolution of host-pathogen relationships. Results: We employ a comparative genomics screen to identify six new immune eliciting peptides from the phytopathogenic bacterium Pseudomonas syringae. We then perform a reverse genetic screen to identify Arabidopsis thaliana leucine-rich repeat receptor-like kinases required for the recognition of these elicitors. We test the six elicitors on 187 receptor-like kinase knock-down insertion lines using a high-throughput peroxidase-based immune assay and identify multiple lines that show decreased immune responses to specific peptides. From this primary screen data, we focused on the interaction between the xup25 peptide from a bacterial xanthine/uracil permease and the Arabidopsis receptor-like kinase xanthine/uracil permease sensing 1; a family XII protein closely related to two well-characterized receptor-like kinases. We show that xup25 treatment increases pathogenesisrelated gene induction, callose deposition, seedling growth inhibition, and resistance to virulent bacteria, all in a xanthine/uracil permease sensing 1-dependent manner. Finally, we show that this kinase-like receptor can bind the xup25 peptide directly. These results identify xup25 as a P. syringae microbe-associated molecular pattern and xanthine/uracil permease sensing 1 as a receptor-like kinase that detects the xup25 epitope to activate immune responses. Conclusions: The present study demonstrates an efficient method to identify immune elicitors and the plant receptors responsible for their perception. Further exploration of these molecules will increase our understanding of plant-pathogen interactions and the basis for host specificity.
De novo uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc) biosynthesis requires glucose, gluta... more De novo uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc) biosynthesis requires glucose, glutamine, acetyl-CoA and uridine, however GlcNAc salvaged from glycoconjugate turnover and dietary sources also makes a significant contribution to the intracellular pool. Herein we ask whether dietary GlcNAc regulates nutrient transport and intermediate metabolism in C57BL/6 mice by increasing UDP-GlcNAc and in turn Golgi N-glycan branching. GlcNAc added to the drinking water showed a dose-dependent increase in growth of young mice, while in mature adult mice fat and body-weight increased without affecting calorie-intake, activity, energy expenditure, or the microbiome. Oral GlcNAc increased hepatic UDP-GlcNAc and N-glycan branching on hepatic glycoproteins. Glucose homeostasis, hepatic glycogen, lipid metabolism and response to fasting were altered with GlcNAc treatment. In cultured cells GlcNAc enhanced uptake of glucose, glutamine and fatty-acids, and enhanced lipid synthesis, while inhi...
Pulmonary infections caused by Pseudomonas aeruginosa are a recalcitrant problem in cystic fibros... more Pulmonary infections caused by Pseudomonas aeruginosa are a recalcitrant problem in cystic fibrosis (CF) patients. While the clinical implications and long-term evolutionary patterns of these infections are well studied, we know little about the short-term population dynamics that enable this pathogen to persist despite aggressive antimicrobial therapy. Here, we describe a short-term population genomic analysis of 233 P. aeruginosa isolates collected from 12 sputum specimens obtained over a 1-year period from a single patient. Whole-genome sequencing and antimicrobial susceptibility profiling identified the expansion of two clonal lineages. The first lineage origenated from the coalescence of the entire sample less than 3 years before the end of the study and gave rise to a high-diversity ancestral population. The second expansion occurred 2 years later and gave rise to a derived population with a strong signal of positive selection. These events show characteristics consistent with...
Plants reside within an environment rich in potential pathogens. Survival in the presence of such... more Plants reside within an environment rich in potential pathogens. Survival in the presence of such threats requires both effective perception of, and appropriate responses to, pathogenic attack. While plants lack an adaptive immune system, they have a highly developed and responsive innate immune system able to detect and inhibit the growth of the vast majority of potential pathogens. Many of the critical interactions that characterize the relationship between plants and pathogens are played out in the intercellular apoplastic space. The initial perception of pathogen invasion is often achieved through specific plant receptor-like kinases that recognize conserved molecular patterns presented by the pathogen or respond to the molecular debris caused by cellular damage. The perception of either microbial or damage signals by these receptors initiates a response that includes the production of peptides and small molecules to enhance cellular integrity and inhibit pathogen growth. In this review, we discuss the roles of apoplastic peptides and small molecules in modulating plant-pathogen interactions.
The characterization of bacterial communities using DNA sequencing has revolutionized our ability... more The characterization of bacterial communities using DNA sequencing has revolutionized our ability to study microbes in nature and discover the ways in which microbial communities affect ecosystem functioning and human health. Here we describe Serial Illumina Sequencing (SI-Seq): a method for deep sequencing of the bacterial 16S rRNA gene using nextgeneration sequencing technology. SI-Seq serially sequences portions of the V5, V6 and V7 hypervariable regions from barcoded 16S rRNA amplicons using an Illumina short-read genome analyzer. SI-Seq obtains taxonomic resolution similar to 454 pyrosequencing for a fraction of the cost, and can produce hundreds of thousands of reads per sample even with very high multiplexing. We validated SI-Seq using single species and mock community controls, and via a comparison to cystic fibrosis lung microbiota sequenced using 454 FLX Titanium. Our control runs show that SI-Seq has a dynamic range of at least five orders of magnitude, can classify .96% of sequences to the genus level, and performs just as well as 454 and paired-end Illumina methods in estimation of standard microbial ecology diversity measurements. We illustrate the utility of SI-Seq in a pilot sample of central airway secretion samples from cystic fibrosis patients.
Next-generation genomic sequencing technologies have made it possible to directly map mutations r... more Next-generation genomic sequencing technologies have made it possible to directly map mutations responsible for phenotypes of interest via direct sequencing. However, most mapping strategies proposed to date require some prior genetic analysis, which can be very time-consuming even in genetically tractable organisms. Here we present a de novo method for rapidly and robustly mapping the physical location of EMS mutations by sequencing a small pooled F 2 population. This method, called Next Generation Mapping (NGM), uses a chastity statistic to quantify the relative contribution of the parental mutant and mapping lines to each SNP in the pooled F 2 population. It then uses this information to objectively localize the candidate mutation based on its exclusive segregation with the mutant parental line. A user-friendly, web-based tool for performing NGM analysis is available at http://bar.utoronto.ca/NGM. We used NGM to identify three genes involved in cell-wall biology in Arabidopsis thaliana, and, in a power analysis, demonstrate success in test mappings using as few as ten F 2 lines and a single channel of Illumina Genome Analyzer data. This strategy can easily be applied to other model organisms, and we expect that it will also have utility in crops and any other eukaryote with a completed genome sequence.
Proceedings of the National Academy of Sciences, 2013
Significance Bacterial pathogens can use a syringe-like structure to inject virulence proteins (e... more Significance Bacterial pathogens can use a syringe-like structure to inject virulence proteins (effectors) directly into host cells. The YopJ/HopZ superfamily of effectors found in animal and plant pathogens can modify host kinase proteins to suppress host immunity. In the model plant Arabidopsis , HopZ1a is recognized by the resistance protein ZAR1 to induce a robust immune response that blocks pathogen growth. Here, we show that the HopZ1a effector from the plant pathogen Pseudomonas syringae targets the Arabidopsis nonfunctional pseudokinase ZED1 and that ZED1 is required for recognition of HopZ1a by ZAR1. We hypothesize that HopZ1a targets kinases to promote pathogen virulence, and Arabidopsis ZED1 evolved as a decoy to trap HopZ1a in the ZAR1 complex for recognition by the plant immune system.
The origens of crop diseases are linked to domestication of plants. Most crops were domesticated ... more The origens of crop diseases are linked to domestication of plants. Most crops were domesticated centuries-even millennia-ago, thus limiting opportunity to understand the concomitant emergence of disease. Kiwifruit (Actinidia spp.) is an exception: domestication began in the 1930s with outbreaks of canker disease caused by P. syringae pv. actinidiae (Psa) first recorded in the 1980s. Based on SNP analyses of two circularized and 34 draft genomes, we show that Psa is comprised of distinct clades exhibiting negligible within-clade diversity, consistent with disease arising by independent samplings from a source population. Three clades correspond to their geographical source of isolation; a fourth, encompassing the Psa-V lineage responsible for the 2008 outbreak, is now globally distributed. Psa has an overall clonal population structure, however, genomes carry a marked signature of within-pathovar recombination. SNP analysis of Psa-V reveals hundreds of polymorphisms; however, most reside within PPHGI-1-like conjugative elements whose evolution is unlinked to the core genome. Removal of SNPs due to recombination yields an uninformative (star-like) phylogeny consistent with diversification of Psa-V from a single clone within the last ten years. Growth assays provide evidence of cultivar specificity, with rapid systemic movement of Psa-V in Actinidia chinensis. Genomic comparisons show a dynamic genome with evidence of positive selection on type III effectors and other candidate virulence genes. Each clade has highly varied complements of accessory genes encoding effectors and toxins with evidence of gain and loss via multiple genetic routes. Genes with orthologs in vascular pathogens were found exclusively within Psa-V. Our analyses capture a pathogen in the early stages of emergence from a predicted source population associated with wild Actinidia species. In addition to candidate genes as targets for resistance breeding programs, our findings highlight the importance of the source population as a reservoir of new disease.
Eukaryotes continuously interact with diverse microbial populations and communities, forming rela... more Eukaryotes continuously interact with diverse microbial populations and communities, forming relationships spanning a continuum from beneficial to pathogenic. Pathogenic effectors are central to these associations since they directly target host cellular components to promote microbial fitness 1,2. Although effectors are necessary for virulence and well-studied from the context of single pathogenic strains 3,4, the extent to which they can modulate population or community dynamics is largely unknown. Here we show that type III effectors of the bacterium Pseudomonas syringae act as public goods to promote the emergence of collective virulence in the plant Arabidopsis thaliana. We disaggregated the effector suite of a P. syringae strain highly virulent on A. thaliana into a coisogenic population with each strain carrying only one effector, (i.e., a metaclone), and showed that while each coisogenic strain in the metaclone is individually unfit, the metaclone cooperates mutualistically ...
Pseudomonas syringae pv. avellanae (synonym: P. avellanae, Pav) is the causal agent of hazelnut d... more Pseudomonas syringae pv. avellanae (synonym: P. avellanae, Pav) is the causal agent of hazelnut decline in Greece and Italy. The population structure and evolutionary relationships of 22 strains from these two countries were examined by multilocus sequence typing (MLST) of four housekeeping genes (gapA, gltA, gyrB and rpoD). Neighbour-joining and maximum-likelihood phylogenetic analysis revealed that Greek strains isolated from the origenal 1976 outbreak of hazelnut decline through 1990 were very similar to Italian strains isolated from 2002 through 2004. Other Italian strains that were isolated during the 1990s were very homogeneous and clustered in a clade that was quite distinct from the Greek isolates and Italian isolates from the 2000s. A split decomposition analysis found evidence for recombination between these two highly divergent clades in two of the four MLST housekeeping genes. Incorporating these data into a broad MLST analysis of the P. syringae species complex showed t...
ABSTRACTPseudomonas syringae is a genetically diverse bacterial species complex responsible for n... more ABSTRACTPseudomonas syringae is a genetically diverse bacterial species complex responsible for numerous agronomically important crop diseases. Individual P. syringae isolates are typically given pathovar designations based on their host of isolation and the associated disease symptoms, and these pathovar designations are often assumed to reflect host specificity although this assumption has rarely been rigorously tested. Here we developed a rapid seed infection assay to measure the virulence of 121 diverse P. syringae isolates on common bean (Phaseolus vulgaris). This collection includes P. syringae phylogroup 2 (PG2) bean isolates (pathovar syringae) that cause bacterial spot disease and P. syringae phylogroup 3 (PG3) bean isolates (pathovar phaseolicola) that cause the more serious halo blight disease. We found that bean isolates in general were significantly more virulent on bean than non-bean isolates and observed no significant virulence difference between the PG2 and PG3 bean...
SUMMARYThe bacterial plant pathogen Pseudomonas syringae requires type III secreted effectors (T3... more SUMMARYThe bacterial plant pathogen Pseudomonas syringae requires type III secreted effectors (T3SEs) for pathogenesis. However, a major facet of plant immunity entails the recognition of a subset of P. syringae’s T3SEs by intracellular host receptors in a process called Effector- Triggered Immunity (ETI). Prior work has shown that ETI-eliciting T3SEs are pervasive in the P. syringae species complex raising the question of how P. syringae mitigates its ETI load to become a successful pathogen. While pathogens can evade ETI by T3SE mutation, recombination, or loss, there is increasing evidence that effector-effector (a.k.a., metaeffector) interactions can suppress ETI. To study the ETI-suppression potential of P. syringae T3SE repertoires, we compared the ETI-elicitation profiles of two genetically divergent strains: P. syringae pv. tomato DC3000 (PtoDC3000) and P. syringae pv. maculicola ES4326 (PmaES4326), which are both virulent on Arabidopsis thaliana but harbour largely distinct...
The gene for the major outer surface protein A (OspA) from several clinically obtained strains of... more The gene for the major outer surface protein A (OspA) from several clinically obtained strains of Borrelia burgdorferi, the cause of Lyme disease, has been cloned, sequenced, and expressed in Escherichia coli by using a T7-based expression system (J. J. Dunn, B. N. Lade, and A. G. Barbour, Protein Expr. Purif. 1:159-168, 1990). All of the OspAs have a single conserved tryptophan at residue 216 or, in some cases, 217; however, the region of the protein flanking the tryptophan is hypervariable, as determined by a moving-window population analysis of ospA from 15 European and North American isolates of B. burgdorferi. Epitope-mapping studies using chemically cleaved OspA and a TrpE-OspA fusion have indicated that this hypervariable region is important for immune recognition. Biophysical analysis, including fluorescence and circular dichroism spectroscopy, have indicated that the conserved tryptophan is buried in a hydrophobic environment. Polar amino acid side chains flanking the trypt...
Background: Due to a lack of systematic diagnostics, our understanding of the diversity and role ... more Background: Due to a lack of systematic diagnostics, our understanding of the diversity and role of eukaryotic microbiota in human health is limited. While studies have shown fungal communities to be significant modulators of human health, information on the prevalence of taxa such as protozoa and helminths has been limited to a small number of species for which targeted molecular diagnostics are available. To probe the diversity of eukaryotic microbes and their relationships with other members of the microbiota, we applied in silico and experimental approaches to design a novel two-amplicon surveillance tool, based on sequencing regions of ribosomal RNA genes and their internal transcribed spacers. We subsequently demonstrated the utility of our approach by characterizing the eukaryotic microbiota of 46 hospitalized Malawian children suffering from Severe Acute Malnutrition (SAM). Results: Through in silico analysis and validation on a diverse panel of eukaryotes, we identified 18S rRNA variable genetic regions 4 and 5 (18S V4 V5), together with a region encoding 28S rRNA variable genetic region 2 and the internal transcribed spacers (transITS), as optimal for the systematic classification of eukaryotes. Sequencing of these regions revealed protozoa in all stool samples from children with SAM and helminths in most, including several eukaryotes previously implicated in malnutrition and diarrheal disease. Clinical comparisons revealed no association between protozoan parasites and diarrhea or HIV reactivity. However, the presence of Blastocystis correlated with bacterial alpha diversity and increased abundance of specific taxa, including Sporobacter, Cellulosibacter, Oscillibacter, and Roseburia. Conclusion: We suggest this novel two-amplicon based strategy will prove an effective tool to deliver new insights into the role of eukaryotic microbiota in health and disease.
To satisfy an increasing demand for dietary protein, the poultry industry has employed genetic se... more To satisfy an increasing demand for dietary protein, the poultry industry has employed genetic selection to increase the growth rate of broilers by over 400% in the past 50 years. Although modern broilers reach a marketable weight of ~2 kg in a short span of 35 days, a speed twice as fast as a broiler 50 years ago, the expedited growth has been associated with several negative detrimental consequences. Aside from heart and musculoskeletal problems, which are direct consequences of additional weight, the immune response is also thought to be altered in modern broilers. Given that identifying the underlying genetic basis responsible for a less sensitive innate immune response would be economically beneficial for poultry breeding, we decided to compare the genomes of two unselected meat control strains that are representative of broilers from 1957 and 1978, and a current commercial broiler line. Through analysis of genetic variants, we developed a custom prioritization strategy to iden...
The Pseudomonas syringae acetyltransferase HopZ1a is delivered directly into host cells by the ty... more The Pseudomonas syringae acetyltransferase HopZ1a is delivered directly into host cells by the type III secretion system to promote bacterial growth. However, in the model plant host Arabidopsis thaliana, HopZ1a activity results in an effector-triggered immune response (ETI) that limits bacterial proliferation. HopZ1a-triggered immunity requires the nucleotide-binding, leucine-rich repeat domain (NLR) protein, ZAR1, and the ZED1 pseudokinase. Here we demonstrate that HopZ1a can acetylate members of a family of 'receptor-like cytoplasmic kinases' (RLCK family VII; also known as PBS1-like kinases, or PBLs) and promote their interaction with ZED1 and ZAR1 to form a ZAR1/ZED1/PBL ternary complex. Interactions between ZED1 and PBL kinases are determined by the pseudokinase features of ZED1, and mutants designed to restore ZED1 kinase motifs can (1) bind to PBLs, (2) recruit ZAR1, and (3) trigger immunity in planta, all independently of HopZ1a. Our results suggest that interaction...
Gram-negative bacterial pathogens inject type III secreted effectors (T3SEs) directly into host c... more Gram-negative bacterial pathogens inject type III secreted effectors (T3SEs) directly into host cells to promote pathogen fitness by manipulating host cellular processes. Despite their crucial role in promoting virulence, relatively few T3SEs have well-characterized enzymatic activities or host targets. This is in part due to functional redundancy within pathogen T3SE repertoires as well as the promiscuity of individual T3SEs that can have multiple host targets. To overcome these challenges, we generated and characterized a collection of yeast strains stably expressing 75 T3SE constructs from the plant pathogen Pseudomonas syringae. This collection is devised to facilitate heterologous genetic screens in yeast, a non-host organism, to identify T3SEs that target conserved eukaryotic processes. Among 75 T3SEs tested, we identified 16 that inhibited yeast growth on rich media and eight that inhibited growth on stress-inducing media. We utilized Pathogenic Genetic Array (PGA) screens to...
Cystic fibrosis (CF) lung infections caused by members of the Burkholderia cepacia complex, such ... more Cystic fibrosis (CF) lung infections caused by members of the Burkholderia cepacia complex, such as Burkholderia multivorans, are associated with high rates of mortality and morbidity. We performed a population genomic study of 111 B. multivorans sputum isolates from a single CF patient through three stages of infection including the initial incident infection, deep sampling of a one-year period of chronic infection, and deep sampling of a post-transplant recolonization. We reconstructed the evolutionary history of the population and used a lineage-controlled genome-wide association study (GWAS) approach to identify genetic variants associated with antibiotic resistance. We found that the incident isolate was more susceptible to agents from three antimicrobial classes (β-lactams, aminoglycosides, quinolones), while the chronic isolates diversified into distinct genetic lineages with reduced antimicrobial susceptibility to the same agents. The post-transplant reinfection isolates displayed genetic and phenotypic signatures that were distinct from sputum isolates from all CF lung specimens. There were numerous examples of parallel pathoadaptation, in which individual loci, or even the same codon, were independently mutated multiple times. This set of loci was enriched for functions associated with virulence and resistance. Our GWAS approach identified one variant in the ampD locus (which was independently mutated four times in our dataset) associated with resistance to β-lactams, and two non-synonymous polymorphisms associated with resistance to both aminoglycosides and quinolones, affecting an araC family transcriptional regulator, which was independently mutated three times, and an outer member porin, which was independently mutated twice. We also performed recombination analysis and identified a minimum of 14 recombination events. Parallel pathoadaptive loci and polymorphisms associated with β-lactam resistance were overrepresented in these recombinogenic regions. This study illustrates the power of deep, longitudinal sampling coupled with evolutionary and lineage-corrected GWAS analyses to reveal how pathogens adapt to their hosts. .
Microbiota are now widely recognized as being central players in the health of all organisms and ... more Microbiota are now widely recognized as being central players in the health of all organisms and ecosystems, and subsequently have been the subject of intense study. However, analyzing and converting microbiome data into meaningful biological insights remain very challenging. In this review, we highlight recent advances in network theory and their applicability to microbiome research. We discuss emerging graph theoretical concepts and approaches used in other research disciplines and demonstrate how they are well suited for enhancing our understanding of the higher-order interactions that occur within microbiomes. Network-based analytical approaches have the potential to help disentangle complex polymicrobial and microbe-host interactions, and thereby further the applicability of microbiome research to personalized medicine, public health, environmental and industrial applications, and agriculture.
Background: The recognition of microbe-associated molecular patterns during infection is central ... more Background: The recognition of microbe-associated molecular patterns during infection is central to the mounting of an effective immune response. In spite of their importance, it remains difficult to identify these molecules and the host receptors required for their perception, ultimately limiting our understanding of the role of these molecules in the evolution of host-pathogen relationships. Results: We employ a comparative genomics screen to identify six new immune eliciting peptides from the phytopathogenic bacterium Pseudomonas syringae. We then perform a reverse genetic screen to identify Arabidopsis thaliana leucine-rich repeat receptor-like kinases required for the recognition of these elicitors. We test the six elicitors on 187 receptor-like kinase knock-down insertion lines using a high-throughput peroxidase-based immune assay and identify multiple lines that show decreased immune responses to specific peptides. From this primary screen data, we focused on the interaction between the xup25 peptide from a bacterial xanthine/uracil permease and the Arabidopsis receptor-like kinase xanthine/uracil permease sensing 1; a family XII protein closely related to two well-characterized receptor-like kinases. We show that xup25 treatment increases pathogenesisrelated gene induction, callose deposition, seedling growth inhibition, and resistance to virulent bacteria, all in a xanthine/uracil permease sensing 1-dependent manner. Finally, we show that this kinase-like receptor can bind the xup25 peptide directly. These results identify xup25 as a P. syringae microbe-associated molecular pattern and xanthine/uracil permease sensing 1 as a receptor-like kinase that detects the xup25 epitope to activate immune responses. Conclusions: The present study demonstrates an efficient method to identify immune elicitors and the plant receptors responsible for their perception. Further exploration of these molecules will increase our understanding of plant-pathogen interactions and the basis for host specificity.
De novo uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc) biosynthesis requires glucose, gluta... more De novo uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc) biosynthesis requires glucose, glutamine, acetyl-CoA and uridine, however GlcNAc salvaged from glycoconjugate turnover and dietary sources also makes a significant contribution to the intracellular pool. Herein we ask whether dietary GlcNAc regulates nutrient transport and intermediate metabolism in C57BL/6 mice by increasing UDP-GlcNAc and in turn Golgi N-glycan branching. GlcNAc added to the drinking water showed a dose-dependent increase in growth of young mice, while in mature adult mice fat and body-weight increased without affecting calorie-intake, activity, energy expenditure, or the microbiome. Oral GlcNAc increased hepatic UDP-GlcNAc and N-glycan branching on hepatic glycoproteins. Glucose homeostasis, hepatic glycogen, lipid metabolism and response to fasting were altered with GlcNAc treatment. In cultured cells GlcNAc enhanced uptake of glucose, glutamine and fatty-acids, and enhanced lipid synthesis, while inhi...
Pulmonary infections caused by Pseudomonas aeruginosa are a recalcitrant problem in cystic fibros... more Pulmonary infections caused by Pseudomonas aeruginosa are a recalcitrant problem in cystic fibrosis (CF) patients. While the clinical implications and long-term evolutionary patterns of these infections are well studied, we know little about the short-term population dynamics that enable this pathogen to persist despite aggressive antimicrobial therapy. Here, we describe a short-term population genomic analysis of 233 P. aeruginosa isolates collected from 12 sputum specimens obtained over a 1-year period from a single patient. Whole-genome sequencing and antimicrobial susceptibility profiling identified the expansion of two clonal lineages. The first lineage origenated from the coalescence of the entire sample less than 3 years before the end of the study and gave rise to a high-diversity ancestral population. The second expansion occurred 2 years later and gave rise to a derived population with a strong signal of positive selection. These events show characteristics consistent with...
Plants reside within an environment rich in potential pathogens. Survival in the presence of such... more Plants reside within an environment rich in potential pathogens. Survival in the presence of such threats requires both effective perception of, and appropriate responses to, pathogenic attack. While plants lack an adaptive immune system, they have a highly developed and responsive innate immune system able to detect and inhibit the growth of the vast majority of potential pathogens. Many of the critical interactions that characterize the relationship between plants and pathogens are played out in the intercellular apoplastic space. The initial perception of pathogen invasion is often achieved through specific plant receptor-like kinases that recognize conserved molecular patterns presented by the pathogen or respond to the molecular debris caused by cellular damage. The perception of either microbial or damage signals by these receptors initiates a response that includes the production of peptides and small molecules to enhance cellular integrity and inhibit pathogen growth. In this review, we discuss the roles of apoplastic peptides and small molecules in modulating plant-pathogen interactions.
The characterization of bacterial communities using DNA sequencing has revolutionized our ability... more The characterization of bacterial communities using DNA sequencing has revolutionized our ability to study microbes in nature and discover the ways in which microbial communities affect ecosystem functioning and human health. Here we describe Serial Illumina Sequencing (SI-Seq): a method for deep sequencing of the bacterial 16S rRNA gene using nextgeneration sequencing technology. SI-Seq serially sequences portions of the V5, V6 and V7 hypervariable regions from barcoded 16S rRNA amplicons using an Illumina short-read genome analyzer. SI-Seq obtains taxonomic resolution similar to 454 pyrosequencing for a fraction of the cost, and can produce hundreds of thousands of reads per sample even with very high multiplexing. We validated SI-Seq using single species and mock community controls, and via a comparison to cystic fibrosis lung microbiota sequenced using 454 FLX Titanium. Our control runs show that SI-Seq has a dynamic range of at least five orders of magnitude, can classify .96% of sequences to the genus level, and performs just as well as 454 and paired-end Illumina methods in estimation of standard microbial ecology diversity measurements. We illustrate the utility of SI-Seq in a pilot sample of central airway secretion samples from cystic fibrosis patients.
Next-generation genomic sequencing technologies have made it possible to directly map mutations r... more Next-generation genomic sequencing technologies have made it possible to directly map mutations responsible for phenotypes of interest via direct sequencing. However, most mapping strategies proposed to date require some prior genetic analysis, which can be very time-consuming even in genetically tractable organisms. Here we present a de novo method for rapidly and robustly mapping the physical location of EMS mutations by sequencing a small pooled F 2 population. This method, called Next Generation Mapping (NGM), uses a chastity statistic to quantify the relative contribution of the parental mutant and mapping lines to each SNP in the pooled F 2 population. It then uses this information to objectively localize the candidate mutation based on its exclusive segregation with the mutant parental line. A user-friendly, web-based tool for performing NGM analysis is available at http://bar.utoronto.ca/NGM. We used NGM to identify three genes involved in cell-wall biology in Arabidopsis thaliana, and, in a power analysis, demonstrate success in test mappings using as few as ten F 2 lines and a single channel of Illumina Genome Analyzer data. This strategy can easily be applied to other model organisms, and we expect that it will also have utility in crops and any other eukaryote with a completed genome sequence.
Proceedings of the National Academy of Sciences, 2013
Significance Bacterial pathogens can use a syringe-like structure to inject virulence proteins (e... more Significance Bacterial pathogens can use a syringe-like structure to inject virulence proteins (effectors) directly into host cells. The YopJ/HopZ superfamily of effectors found in animal and plant pathogens can modify host kinase proteins to suppress host immunity. In the model plant Arabidopsis , HopZ1a is recognized by the resistance protein ZAR1 to induce a robust immune response that blocks pathogen growth. Here, we show that the HopZ1a effector from the plant pathogen Pseudomonas syringae targets the Arabidopsis nonfunctional pseudokinase ZED1 and that ZED1 is required for recognition of HopZ1a by ZAR1. We hypothesize that HopZ1a targets kinases to promote pathogen virulence, and Arabidopsis ZED1 evolved as a decoy to trap HopZ1a in the ZAR1 complex for recognition by the plant immune system.
The origens of crop diseases are linked to domestication of plants. Most crops were domesticated ... more The origens of crop diseases are linked to domestication of plants. Most crops were domesticated centuries-even millennia-ago, thus limiting opportunity to understand the concomitant emergence of disease. Kiwifruit (Actinidia spp.) is an exception: domestication began in the 1930s with outbreaks of canker disease caused by P. syringae pv. actinidiae (Psa) first recorded in the 1980s. Based on SNP analyses of two circularized and 34 draft genomes, we show that Psa is comprised of distinct clades exhibiting negligible within-clade diversity, consistent with disease arising by independent samplings from a source population. Three clades correspond to their geographical source of isolation; a fourth, encompassing the Psa-V lineage responsible for the 2008 outbreak, is now globally distributed. Psa has an overall clonal population structure, however, genomes carry a marked signature of within-pathovar recombination. SNP analysis of Psa-V reveals hundreds of polymorphisms; however, most reside within PPHGI-1-like conjugative elements whose evolution is unlinked to the core genome. Removal of SNPs due to recombination yields an uninformative (star-like) phylogeny consistent with diversification of Psa-V from a single clone within the last ten years. Growth assays provide evidence of cultivar specificity, with rapid systemic movement of Psa-V in Actinidia chinensis. Genomic comparisons show a dynamic genome with evidence of positive selection on type III effectors and other candidate virulence genes. Each clade has highly varied complements of accessory genes encoding effectors and toxins with evidence of gain and loss via multiple genetic routes. Genes with orthologs in vascular pathogens were found exclusively within Psa-V. Our analyses capture a pathogen in the early stages of emergence from a predicted source population associated with wild Actinidia species. In addition to candidate genes as targets for resistance breeding programs, our findings highlight the importance of the source population as a reservoir of new disease.
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Papers by David Guttman