Microbial Pathogenesis and Host Immune Response
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This document provides an overview of the human microbiome, detailing the interactions between microbial communities and host immune responses. It emphasizes the importance of the microbiome in maintaining health, its role in host defense mechanisms, and the potential implications for diseases when this relationship is disrupted. Additionally, it discusses various technologies for exploring host-pathogen interactions and methodologies for analyzing microbial communities.
Microbial Pathogenesis and Host Immune Response
- 1. Sample to Insight Microbial Pathogenesis and Host Immune Responses Krishnan Allampallam, Ph.D. Senior Global Market Manager, QIAGEN 1
- 2. Sample to Insight Legal disclaimer 2 • QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of a disease. • For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.QIAGEN.com or can be requested from QIAGEN Technical Services or your local distributor.
- 3. Sample to Insight Agenda Introduction to the microbiome Host-pathogen interactions Host defense mechanisms Technologies for exploring host-pathogen interactions 3 1 2 3 4
- 4. Sample to Insight Agenda Introduction to the microbiome The host-pathogen interaction The host defense mechanisms Technologies for exploring host-pathogen interactions 4 1 2 3 4
- 5. Sample to Insight Microbiome: Definition and background 5 What does “microbiome” mean? “The microbiome is defined as the collective genomes of the microbes (composed of bacteria, bacteriophage, fungi, protozoa, and viruses) that live inside and on the human body.” -NIH, 2012 Microbiota refers to the collection of microbial organisms that inhabits a certain environment Metagenomics is the study of the collective genomes of microorganisms from a sample without cultivation (Lederberg and McCray 2001, The NIH HMP Working Group) Kuczynski et al. Nature Reviews Genetics 13,47-58 (January 2012)
- 6. Sample to Insight NIH Human Microbiome Project (HMP) 6 Microorganisms cluster by body site Cataloguing efforts by the NIH Human Microbiome Project suggest: • ~10,000 organisms live with us • ~ 8 ×106 genes in this “second genome” Identifying microbiota in healthy individuals revealed: • Different body sites have unique communities • Race, age, gender, weight or ethnicity have an effect 1 Hoffmann A.R., et al. “The Microbiome: The Trillionsof MicroorganismsThat Maintain Healthand Cause Disease in Humansand Companion Animals.” Vet Pathol.2015 2 http://commonfund.nih.gov/hmp/index 3 Structure, functionand diversity of the health human microbiome. The HumanMicrobiome Project Consortium. Nature,486, 207 -214 (14 June 2012). doi: 10.1038/nature11234
- 7. Sample to Insight Human microbiome 7 Trillions of microorganisms that maintain health and cause disease in humans The gut microbiota is profoundly altered during pregnancy • Microbial diversity is changed during pregnancy • Shift in bacterial diversity is unrelated to health state • Newborn babies are born with a distinct gut microbiome Hoffmann A.R., et al. “The Microbiome: TheTrillionsof MicroorganismsThat Maintain Health and Cause Disease in Humansand Companion Animals.” Vet Pathol. 2015 Koren O. et al. “Host Remodeling of the Gut Microbiome and Metabolic Changesduring Pregnancy”, Cell, 2012, 150,470 Different body sites have unique communities • Each area of the body has its own microbiome
- 8. Sample to Insight Agenda Introduction to the microbiome Host-pathogen interactions Host defense mechanisms Technologies for exploring host-pathogen interactions 8 1 2 3 4
- 9. Sample to Insight Host-microbiota interaction • The host provides the microbiota a niche with a stable nutrient supply • The microbiota performs essential functions for host physiology, including metabolic, digestive, and immune mechanisms • Regulate the host’s metabolic function and energy balance • Provide the host with the capacity to hydrolyze complex plant sugars, synthesize vitamins, and detoxify xenobiotics in a mutualistic context • Affect the most fundamental of host physiological phenotypes, the rate of aging itself 9 Dual necessity: Peacefully coexisting to achieve a mutually beneficial relationship. Commensal gut microbiota protects the host from infection via both direct and indirect mechanisms. The symbiosis between microbes and humans provides a stable and common metabolic pattern and well-balanced physiological homeostasis. Caroline Heintz andWilliam Mair, “You Are What You Host: MicrobiomeModulationof the AgingProcess”, 2014, Cell, 156:408 Kamada N, et al. “Role of the gut microbiota inimmunity and inflammatory disease”. 2013, Nat Rev Immunol. 13(5):321-35. Review. 9
- 10. Sample to Insight Host-microbiota interaction 10 Direct and indirect protection mechanisms of microbiota Kamada N, et al. “Role of the gut microbiota inimmunity and inflammatory disease”. 2013, Nat Rev Immunol. 13(5):321-35. Review.
- 11. Sample to Insight Host-microbiota interaction 11 Direct and indirect protection mechanisms of microbiota Kamada N, et al. “Role of the gut microbiota inimmunity and inflammatory disease”. 2013, Nat Rev Immunol. 13(5):321-35. Review.
- 12. Sample to Insight Host-microbiota interaction 12 Direct and indirect protection mechanisms of microbiota Kamada N, et al. “Role of the gut microbiota inimmunity and inflammatory disease”. 2013, Nat Rev Immunol. 13(5):321-35. Review.
- 13. Sample to Insight Host-microbiota interaction 13 The big question? How, what, who in the microbiota does what, when and how to the immune system? Kamada N, et al. “Role of the gut microbiota inimmunity and inflammatory disease”. 2013, Nat Rev Immunol. 13(5):321-35. Review. • Identify microbiota • Virulence factors • Antibiotic resistance genes
- 14. Sample to Insight Crosstalk between the microbiome and the immune system • Impact lymphoid structure development and epithelial function • Enhance innate immunity to pathogens • Shape T cell subsets • Provide protective roles against systemic infection • Influence invariant T cells and innate lymphoid cells • Trigger inflammation in immunocompromised hosts • Protect against autoimmune diseases 14 How does microbiota shape immunity: The cellular and molecular mediators Lora V. Hooper et al., InteractionsBetween the Microbiota andthe Immune System. 2012,Science 336:1268
- 15. Sample to Insight Crosstalk between the microbiome and the immune system “The mammalian immune system plays an essential role in maintaining homeostasis with resident microbial communities, thus ensuring that the mutualistic nature of the host-microbial relationship is maintained.” The critical controls of the immune system 1. Exerts control over stratification and compartmentalization of the microbiota 2. Exerts control over microbiota composition The immune system exerts critical controls over microbiota composition, diversity, and location. 15 The control of immune system over microbiota Lora V. Hooper et al., InteractionsBetween the Microbiota andthe Immune System. 2012,Science 336:1268
- 16. Sample to Insight The importance of microbiota in human health and diseases 16 When the mutualistic relationship between the host and microbiota is disrupted, the gut microbiota can cause or contribute to diseases. Gut • Intestinal infections • Obesity • Inflammatory Bowel Disease Airway • Pneumonia and other respiratory infections • Chronic Obstructive Pulmonary Disease • Cystic Fibrosis Urogenital • Bacterial vaginosis • Urinary Tract Infections • Sexually Transmitted Disease Blood • Sepsis/bloodstreaminfections Oral • Periodontitis • Gingivitis http://commonfund.nih.gov/hmp/index
- 17. Sample to Insight The host responses It is important to note that commensal bacteria do not always protect against pathogenic infection and in certain contexts they can facilitate it. Under certain conditions, particular bacterial populations can acquire pathogenic properties. 17 The challenge that the host faces How does the host discriminate between symbiotic and pathogenic bacteria to adjust its level of immune response?
- 18. Sample to Insight Agenda Introduction to the microbiome The host-pathogen interaction The host defense mechanisms Technologies for exploring host-pathogen interactions 18 1 2 3 4
- 19. Sample to Insight The immune responses: Innate and adaptive 19 The innate immune system is the first line of defense against pathogens and is initiated by genome-encoded pattern recognition receptors (PRRs) that recognize PAMP • Non-specific and does not confer long-lasting immunity (memory) • Immune cells: dendritic cells (DCs) and macrophages, intestinal epithelial cells and myofibroblasts The adaptive immunity is highly specific, confers long lasting immunity, and is adaptable • Cooperation between the molecules and cells of the innate immune system mounts an effective immune response • Key players: T cells – Th1, Th2 or Th17 cell
- 20. Sample to Insight Innate immune responses: Toll-like receptors 20 Pattern recognition receptors(PRRs) include Toll-like receptors (TLRs), NOD-likereceptors (NLRs) and RIG1- like receptors (RLRs), etc. • PRRs signaling cascades result in nuclear factor (NF)-kB activation of gene transcription and production of pro- inflammatory mediators • PRRs also play a crucial role in the crosstalk between innate and adaptive immune responses by promoting antigen presenting cell maturation and T cell activation • TLRs induce the expression of genes required for the inflammatory response, including inflammatory cytokines, chemokines, antimicrobial molecules, and major histocompatibility (MHC) and costimulatory molecules important for adaptive immune activation
- 21. Sample to Insight Adaptive immune responses 21 Adaptive responses in the gut • Th1 and Th2 cells • Th17 cells • Regulatory T cells During active inflammation, Th0 cells differentiate into T helper cells Th1, Th17 and Th2 • Th1 cells produce interferon (IFN)-γ and tumor necrosis factor (TNF)-α • Th2 cells are a major source of IL-13 • Th17 cells release IL-17 and IL-21 Important adaptive immune cells pDC detect viral antigen and release type I IFN • Activation of mDC, mV, CD4 , and CD8 T cells • FRCs secrete chemokines and T cell survival factors • FDCs coordinate B cell migration and B cell and CD4 T cell interactions
- 22. Sample to Insight Host defense mechanisms: Summary 22 Lance W. Peterson and David Artis, “Intestinal epithelial cells: regulatorsof barrier functionand immune homeostasis”, Nat Rev Immunol. 2014, 14(3):141-53.
- 23. Sample to Insight Agenda Introduction to the microbiome The host-pathogen interaction The host defense mechanisms Technologies for exploring host-pathogen interactions 23 1 2 3 4
- 24. Sample to Insight Technologies for exploring host-pathogen interactions 24 Characterize the composition and function of the gut microbiome and the contribution of microbes to human health. • Genomics – characterize DNA • Transcriptomics – characterize RNA • Metabolomics – characterize small molecules • Proteomics – characterize proteins and peptides • Are changes in the composition of the microbiome are associated with or cause a disease? • How do microbial functionschange in a disease at the DNA, RNA, protein, and metabolite levels? • How do metabolic processes change in a disease? • How does an intervention or a treatment affect the composition and function of the microbial community? Key questions:
- 25. Sample to Insight Technologies for exploring host-pathogen interactions 25 16S rRNA gene sequencing 18S rRNA gene sequencing Total DNA sequencing (shotgun) Bacteria and Archaea Fungus /Yeast Viruses Gene content RNA expression profiling (transcriptomics) Gene expression Metabolite characterization Mass spectroscopy (metabolomics) Identify relative frequencies and pathways Human microbiome sample Extract DNA Extract RNA Extract small molecules What organisms are present and what is their relative abundance? What are the functions of the community?
- 26. Sample to Insight Sample prep requirements for successful microbiome studies There are several areas where sample prep inefficiencies can bias a microbiome/metagenomics study: • Insufficient homogenization of sample matrix (to dislodge/disrupt cell:substrate interactions) • Insufficient cell lysis Can bias downstream analysis toward ‘the easily disrupted’ population(s) • Poor nucleic acid quality, extensive shearing • Insufficient inactivation of nucleases/proteases Unintended destruction of template molecules of interest • Insufficient solubilization of analyte(s) of interest/separation from interacting cellular components • Unintended precipitation of nucleic acids via complexation with matrix-derived metals, bioactive amines • Low-yielding binding interactions with purification matrices • Co-purification of small molecule inhibitors of amplification reactions (e.g., PCR) Decreases efficiency of amplification or can completely inhibit library prep reactions
- 27. Sample to Insight High Inhibitors Difficult Lysis MO BIO’s sample prep solutions Easy Difficult Lysis Inhibitors Low High Blood, animal tissue & cells Pure microbial cultures Soil microbesLeaf tissue Stool & gut microbes Biofilm FFPE Tissue Food cultures
- 28. Sample to Insight MO BIO’s sample prep solutions Cell lysis with homogenous gene/community representation Mechanical homogenization with tailored lysis buffer formulations (DNA and RNA, DNA or RNA only, protein) Increased purity Inhibitor Removal Technology® – IRT (“Power” line of kits) Customizable throughput From single silica spin filters to unique non-fouling ClearMag™ beads in high-throughput, automated applications
- 29. Sample to Insight Key research question How can we identify the microbiome and monitor innate and adaptive immune responses? 29
- 30. Sample to Insight Technologies to analyze microbial communities 30 • Culture • Gene cloning (Pan 16S rRNA) and Sanger sequencing • Microarray • MALDI • Next-generation sequencing • 16S rRNAsequencing • Whole genome sequencing • qPCR - target dependent • 16S rRNAgene • Other relevant gene (antibiotic resistance gene, virulence factor gene)
- 31. Sample to Insight Limitations of current pathogen detection methods • Time consuming (Involve multiple steps, 5-7 days) • Cannot identify all pathogens Majority are non-culturable • Culture conditions are different • Require extensive microbiological training and expertise • Varying protocols for identification • Waste generation 31
- 32. Sample to Insight 32 Rapid • Detection in less than 3 hours • Amenable to routine testing Sensitive • Can detect low copy numbers Standardized • Automated protocols • Stable chemical design Specific • Only detects target sequence Benefits of real-time PCR for detection of microorganisms
- 33. Sample to Insight Technologies for exploring host-pathogen interactions 33 Microbial DNA qPCR Assay/Assay Detect microbial species, virulence genes, or antibiotic resistance genes • MO BIO Isolation Kits • QIAamp DNA Microbiome Kit • QIAamp Fast DNA Stool Mini Kit • QIAamp UCP Pathogen Mini Kit • QIAamp UCP PurePathogen Blood Kit • QIAamp MinElute Media Kit RNA expression
- 34. Sample to Insight Allprotect Tissue Reagents • QIAamp DNA Microbiome Kit • QIAamp UCP Pathogen Mini Kit • QIAamp DNA Stool Mini Kit • MO BIO DNA Kits REPLI-g Single Cell Kit • GeneRead Library Prep Kits • GeneRead Size Selection Kit • GeneRead Library Quant System • Microbial DNA qPCR Arrays • Microbial DNA qPCR Assays • Custom Microbial DNA qPCR Arrays Sample to Insight workflow Detecting microbial metagenomes: a complete solution 34 QIAGEN provides next-generation sequencing technologies for metagenomics, as well as qPCR assays and arrays for verification of sequencing results and screening for specific bacterial species, virulence factor genes, and antibiotic resistance genes. Sample Collection DNA purification DNA amplification Library preparation Verification by qPCR
- 35. Sample to Insight Microbial DNA qPCR assay pipeline >600 bacteria identification assay 8 Fungi identification assay 1 Protist identification assay 87 Antibiotic resistance genes 87 Virulence factor genes 18 Arrays 35 Reveal the mysteries of the microbiome with over 600 assays that target species- specific or gene-specific microbial DNA • Antibiotic Resistance Genes • Bacterial Vaginosis • Biodefense • Food testing: Dairy • Food testing: Meat • Food testing: Poultry • Food testing: Seafood • Food testing: Vegetable • Intestinal Infections • Intestinal Infections 2 • Metabolic Disorders • Oral Disease • Respiratory Infections • Respiratory viral • Sepsis • Urinary Tract Infections • Vaginal Flora • Water Analysis DNA Purification Detection by qPCR Data analysis
- 36. Sample to Insight 36 Microbial qPCR assays and arrays for identification and profiling use probes and primers against 16s rRNA variable region. Success with the 3Cs: Content, custom and control Content: Largest microbiome portfolio; experimentally verified 580 assays Custom: Select 8 to 384 microbial species for simultaneous detection / profiling Control: Integrated controls ensure reliability of results Sensitivity: Can detect as low as 10 copy numbers; data available
- 37. Sample to Insight Technologies for exploring host-pathogen interactions 37 RT2 Profiler Arrays • Most relevant genes in biological and disease pathways • Gene lists identified through state-of-the-art bioinformatics and text-mining tools • Integrated controls for genomic DNA contamination, normalization and PCR processes • Web-based data analysis software at no additional cost • Compatible with most real-time PCR instruments Profiling gene expression from immune cells (especially cytokines, chemokines and other immune molecules) can be interpreted into immune system “status” • Inflammation? • Early • Chronic • Resolution • Type of response (bacterial, viral, other)? Sample Collection Sample Preparation DNA Purification Gene expression analysis Data analysis
- 38. Sample to Insight Host response profiling: gene expression 38 Profile 84 or 370 genes simultaneously with RT2 Profiler PCR Arrays • Antifungal Response • Antiviral Response • Antibacterial Response • Innate & Adaptive Immune Responses • Inflammatory Cytokines & Receptors • Dendritic & Antigen-Presenting Cells • Inflammasomes • Th1 & Th2 Responses • Toll-Like Receptor SignalingPathway • IFN-a/b Response • NFkB Signaling • NFkB Signaling Targets • MAPK Signaling • PI3K/AKT Signaling • 140+ pathways, including customarrays • Wet-bench validated assays for all genes of 13 species
- 39. Sample to Insight Date analysis 39 • Free complete and easy analysis with web/excel-based software • Multiple analysis formats to interpret gene expression results Volcano Plot Scatter Plot Clustergram
- 40. Sample to Insight Summary and questions Introduction to the microbiome • Human microbiome • The complex host-microbe relationship • The impact of microbiome on human health and disease Host-pathogen interactions • The protection mechanisms of microbiota • The control of immune system over microbiota Host defense mechanisms • Innate immune response • Adaptive immune response Technologies for exploring host-pathogen interactions • Characterize the composition and function of the gut microbiome • Profile innate and adaptive immune response 40 1 2 3 4