Invasion and Intracellular Growth PDF

Mondays @14:00-16:00 Introduction to the biology of human pathogenic bacteria 1. Microbes & Humans 1. 2. 3. 4. 5. 2. Koch & Defining Pathogens 1. 2. 3. 3. The microbiota – background Skin microbiota Oral and respiratory tract microbiota Genitourinary tract and gut microbiota Early life microbiota de...

Mondays @14:00-16:00 Introduction to the biology of human pathogenic bacteria 1. Microbes & Humans 1. 2. 3. 4. 5. 2. Koch & Defining Pathogens 1. 2. 3. 3. The microbiota – background Skin microbiota Oral and respiratory tract microbiota Genitourinary tract and gut microbiota Early life microbiota development Koch & some history (+ Anthrax) Koch’s postulates Molecular Koch’s postulates Pathogenesis & Virulence 1. 2. 3. 4. Virulence & ‘measuring’ virulence Opportunistic vs. primary pathogens Stages of pathogenesis Acquisition of virulence/pathogenic factors 1 Mondays @14:00-16:00 Introduction to the biology of human pathogenic bacteria 4. Host Defence Systems 1. 2. 3. 4. 5. 5. Natural/physical barriers Innate immune responses Phagocytosis & complement Adaptive immune responses Microbiota-associated anti-infection responses Defence Systems of Pathogens 1. 2. 3. 4. 5. Avoidance strategies – background Evasion of AMPs and IgA Prevention of phagocytosis and complement Molecular mimicry Antigenic/phase variation 2 Mondays @14:00-16:00 Introduction to the biology of human pathogenic bacteria 6. Adhesion 6. 7. 8. 9. 7. Invasion & Intracellular Growth 1. 2. 3. 4. 8. Significance of bacterial adhesion Pili Adhesins (MSCRAMMS) Other adhesins The intracellular lifestyle Invasion Intracellular survival & cell-to-cell spread Obligate intracellular pathogens Bacterial Toxins 1. 2. 3. 4. Types of toxins (endo vs. exotoxins) Endotoxins (LPS) Exotoxin and modes of action Examples of important bacterial toxins 3 Information on upcoming lectures Module: Molecular biology of infection (WZ2179) Virology lectures: Seminarraum @ Trogerstrasse 30, EG, Munich, MRI campus. Only for the lecture on December 5th: (Zoom only) https://tum-conf.zoomx.de/j/67965756639?pwd=QWpoV3JLUHh4RVNZc0YyRGlFY2M1UT09 Last lecture of 2023 ‘Bacterial toxins’: online only (usual Zoom link) 4 From an estimated 13·7 million infection-related deaths in 2019, there were 7·7 million deaths associated with the 33 bacterial pathogens (both resistant and susceptible to antimicrobials) across 11 infectious syndromes 5 Introduction to the biology of human pathogenic bacteria 4th December 2023 Invasion and intracellular growth Prof. Dr. Romana Gerner Chair of Clinical Microbiome 6 Extracellular vs. Intracellular lifecycle *Extracellular (most) bacteria do not have the capacity to survive the intracellular environment or to induce their own uptake by most host cells* Attachment Extracellular pathogen Resist phagocytosis Resist action of complement Acquire nutrients ETEC (Enterotoxigenic E. coli) Pseudomonas aeruginosa Bacillus anthracis Staphylococcus aureus Haemophilus influenzae Streptococcus pyogenes Mycoplasma sp. Colonisation, Entry Attachment Invasion Intracellular pathogen Resist intracellular destruction Acquire nutrients Mycobacterium spp. Listeria monocytogenes Salmonella spp. Brucella spp. Chlamydia spp. Rickettsia spp. General Invasion Once adhesion is successful, invasion can proceed Invasion involves dissemination of a pathogen throughout local tissues or the body Pathogens may produce exoenzymes or toxins, which serve as virulence factors that allow them to colonize and damage host tissues as they spread deeper into the body Pathogens may also produce virulence factors that protect them against immune system defenses A pathogen’s specific virulence factors determine the degree of tissue damage that occurs More details covered in other lectures! 8 The intracellular ‘lifestyle’ Intracellular pathogens achieve invasion by entering the host’s cells and reproducing In general, these bacteria are mostly shielded from humoral antibodies and can be eliminated primarily by a cellular immune response Also exploit nutrients in the host cell Rare competition from other microbes However, these bacteria must possess specialized mechanisms to protect them from the harsh effects of the lysosomal enzymes encountered within the cell Obligate intracellular pathogens = can only reproduce inside of host cells Facultative intracellular pathogens = can reproduce either inside or outside of host cells 9 Examples of intracellular pathogens Obligate Intracellular Pathogens Chlamydia spp. Mycobacterium leprae Coxiella burnettii Anaplasma phagocytophilum Ehrlichia chaffeensis Facultative Intracellular Pathogens Legionella pneumophila Mycobacterium tuberculosis Listeria monocyotogenes Salmonella spp. Entero-invasive/ adherent-invasive E. coli (EIEC/ AIEC) Neisseria spp. Brucella spp. Shigella spp. 10 Endocytosis (Invasion) Entry (invasion) into a host cell can occur by endocytosis For most kinds of host cells, pathogens use one of two different mechanisms for endocytosis and entry One mechanism relies on effector proteins secreted by the pathogen → effector proteins trigger entry into host cell Second mechanism relies on surface proteins expressed on the pathogen that bind to receptors on the host cell → resulting in entry Many intracellular pathogens thrive inside one of the most efficient cell types of antimicrobial defense: macrophages and DCs 11 Invasion strategies: ‘Trigger’ & ‘Zipper’ Trigger mechanism (a) bacteria attaching to host cell surface deliver effector proteins into host cell via specialized secretion systems (b) effectors interact with cellular targets that regulate host cell actin dynamics, causing membrane ruffling beneath the invading bacteria (c) eventually leads to bacterial engulfment and internalization into a membrane-bound vacuole through a macropinocytosis-like process Zipper mechanism (a) bacterial surface proteins bind receptors on host cell membrane, activating signaling cascades that control actin cytoskeleton (b) actin-driven pseudopods which fuse in a zippering membrane process progressively entrap bacteria (c) bacteria are progressively engulfed until a phagosome is formed 12 Invasion via membrane ruffling Salmonella and Shigella use effector proteins when invading intestinal epithelial cells When these pathogens come into contact with epithelial cells in the intestine → secrete effector molecules that cause protrusions of membrane ruffles that bring the bacterial cell in Process called membrane ruffling 13 Effectors that induce membrane ruffles Salmonella Pathogenicity Island 1 (SPI1)-encoded T3SS translocates effectors that drive “trigger”-mediated invasion of host cells subset of T3SS1 effectors (SipA, SipC, SopB, SopE, SopE2) act in concert to induce massive localised rearrangements of actin and the plasma membrane (and activate signaling pathways) 14 Pathogen-host receptor mediated entry After digestion of contaminated food, Yersinia pseudotuberculosis traverses through the gastrointestinal tract until the terminal ileum Present on their surface is the outer membrane (OM) protein invasion plays a crucial role during first phases of infection by facilitating efficient translocation across the intestinal epithelial barrier by binding beta-1 integrins expressed on surface of host cells - ZIPPER 15 Intracellular survival (1) Avoidance of the microbicidal lysosome is central to all intracellular survival strategies Achieved by either subverting endosomal trafficking and remodeling of the phagosome into a hospitable vacuole Promoting phagosomal membrane disruption and escaping to the cytosol 16 Intracellular survival: life in the vacuole Most intracellular pathogens use the vacuole formed after host cell uptake as a survival and replication niche Reflects that proliferation in a membrane-bound compartment confers an important selective advantage to the microbe enhanced protection against immune recognition probably plays a key role However, inherent challenges to a vacuolar lifestyle need for reprogramming endosomal traffic to avoid destruction by lysosomes vacuole remodeling to allow acquisition of nutrients to support pathogen replication 17 Intracellular survival/transit Once in the small intestine, Salmonella translocate across specialized cells called M (microfold) cells of Peyer’s patches or actively invade epithelial cells by the secretion of effector proteins through the SPI-1 encoded T3SS-1 What’s next..? 18 Intracellular survival: subverting phagocytosis Some host cells, such as immune cells and phagocytes, actively endocytose pathogens in a process called phagocytosis remember host defense lecture! Although phagocytosis allows the pathogen to gain entry to the host cell, in most cases, the host cell kills and degrades the pathogen by using digestive enzymes Normally, when a pathogen is ingested by a phagocyte, it is enclosed within a phagosome in the cytoplasm phagosome fuses with a lysosome to form a phagolysosome, where digestive enzymes kill the pathogen However, some intracellular pathogens have the ability to survive and multiply within phagocytes 19 Intracellular survival: within the phagosome Mycobacterium tuberculosis, Legionella pneumophila, and Salmonella species use a slightly different mechanism to evade being digested by the phagocyte → prevent fusion of phagosome with lysosome remain alive and dividing within the phagosome 20 Salmonella survival in macrophages After crossing the epithelial barrier, Salmonella are engulfed by macrophages → (i) secrete effector proteins into cytosol via the SPI-2 encoded T3SS-2 and prevent fusion of the phagosome with the lysosome (ii) within the SCV (Salmonella containing vacuole), Salmonella proliferate resulting in cytokine secretion (iii) macrophage undergoes apoptosis, and Salmonella escapes innate immune system 21 Intracellular survival: phagosome lysis L. monocytogenes and Shigella produce proteins that lyse the phagosome before it fuses with the lysosome Allows bacteria to escape into cytoplasm to multiply e.g. Shigella escape from phagosome/endosome by secretion of IpaB protein → Once inside phagosome/endosome, IpaB/C complex on tip of T3SS is inserted into membrane → Virulence factors are secreted into cytoplasm via T3SS, triggering host cell apoptosis, cytoskeleton rearrangement etc. → Virulence factors are also secreted into phagosome/endosome via assembled T3SS → Secreted IpaB protein forms a tetramer complex with inserted membrane and ion channels form → IpaB ion channels allows inflow/outflow of small molecules → Phagosome/endosome disrupted 22 An activity that is lethal for microorganisms. a molecular syringe through which bacteria can inject proteins directly into eukaryotic cells. IpaB assembles with IpaC into a pore complex that binds cholesterol and inserts into cell membranes during invasion, whereas another effector, IpaD, enhances the efficiency of insertion of the complex 29,30. Therefore, the IpaB–IpaC pore complex may be involved in disruption of the vacuolar membrane and subsequent bacterial escape. However, efficiently from vacuoles in epithelial cells. There is little information about how Rickettsia spp. and B. pseudomallei escape from the vacuole. BPSS1539, a protein of unknown function, does not have a role during invasion of epithelial cells by B. pseudomallei, but seems to facilitate bacterial escape35. Rickettsia spp. produce haemolysin C and phospholipases, which, at least for Rickettsia prowazekii, seem to play a part in escape from Intracellular lifestyle of cytosolic pathogens Bacteria free in the cytosol Entry Plasma membrane Cell–cell spread Bacteria free in the cytosol Cell cytosol Replication Plasma membrane Cell cytosol Replication Bacteria Internalization in primary vacuole Secretion of bacterial escape proteins Disruption of vacuole membrane Actin Cell polymerization protrusion Internalization in secondary vacuole Secretion of bacterial escape proteins Disruption of double-membrane vacuole Figure 1 | The intracellular lifestyle of cytosolic pathogens. During entry into the host cell, bacteria are engulfed in a Reviewsby| Microbiology primary vacuole. Once inside the vacuole, bacteria secrete proteins that facilitate escape fromNature the vacuole disrupting the vacuolar membrane. Bacteria replicate once free in the cytosol. With the exception of Francisella tularensis, all Nat. Rev. Microbiol. 2009 cytosolic bacteria polymerize actin at the bacterial pole and are therefore capable of intracellular and intercellular motility. During cell-to-cell spread, bacteria are enclosed in a secondary double-membrane vacuole. Bacteria secrete proteins that disrupt both membranes, allowing the bacteria to escape into the cytosol of an adjacent cell. The bacteria then replicate and continue their intercellular spread, disseminating the infection. 23 Cell-to-cell spread A subset of intracellular pathogens disseminate within non-phagocytic cells, such as epithelial and endothelial cells via cell-to-cell spread Listeria monocytogenes, Shigella flexneri, Rickettsia spp., and Burkholderia spp. Pathogens utilize host cell actin cytoskeleton to move in the cytosol of infected cells and project into adjacent cells through formation of membrane protrusions Formed protrusions resolve into vacuoles from which pathogen escapes, thereby gaining access to the cytosol of adjacent cells Weddle & Agaisse. PLOS Pathog. 2018. doi: 10.1371/journal.ppat.1007380 24 Cell-to-cell spread: Listeria Eating food that has been contaminated (raw milk products) with L. monocytogenes can result in life-threatening infections (pregnant women!) Bacteria first invade small intestinal epithelial cells → L. monocytogenes can move from one host cell to another allows infection to reach other organs 25 Cell-to-cell spread: Listeria (a) Listeria invades host cells via a zipper mechanism interaction of surface internalins InlA and InlB with host cell surface receptors E-cadherin and Met (b) Escapes from phagosome before fusion with lysosome occurs action of secreted proteins, pore-forming toxin LLO, and phosphatidylinositide phospholipase C (PI-PLC) (c) Replicates in cell cytosol, and (d) Spreads by actin polymerization propels the bacteria unidirectionally (e) Promotes cell-to-cell spreading of Listeria (f) Rupture of two-membrane vacuole is mediated by action of LLO and PC-PLC 26 Obligate intracellular bacteria Obligate intracellular bacteria cannot live outside host cells Most are unable to carry out energy metabolism and lack many biosynthetic pathways → entirely dependent on host cell to supply them with ATP (adenosine triphosphate) and other intermediate molecules Obligate intracellular bacteria cannot be grown in artificial media (agar plates/broths), but require viable eukaryotic host cells e.g., cell culture, embryonated eggs, and susceptible animals But, there are some exceptions! 27 Obligate pathogen: Chlamydia Chlamydial growth cycle involves transformation between distinct forms elementary body (EB) and reticulate body (RB) Highly infectious EB attaches to epithelial cells and induces ingestion by host cell EB are metabolically inactive and represent the extracellular Chlamydia growth form Once ingested into a phagosome, fusion of the phagosome with the host lysosome is prevented ensures EB survival EB reorganises within the phagosome into a metabolically active RB RBs are noninfectious but can replicate and do so by binary fission Several stimuli, including antibiotics and cytokines, can drive chlamydia into a persistent state lasts until removal of exogenous stressor If persistence is avoided, or if infection is reactivated from persistence, the RB will ultimately reorganise back into EB → released from host cell to infect surrounding epithelial cells 28 Obligate pathogen: Coxiella burnetii Coxiella burnetii is an obligate intracellular bacterium that causes acute and chronic infections Agent of Q fever After inhalation by a host, C. burnetii invades and replicates within alveolar macrophages without alerting the innate immune system ‘stealth pathogen’ Inside macrophages, bacterium replicates within a compartment that is very similar to a phagolysosome, termed the Coxiellacontaining vacuole (CCV) Although an intracellular parasite under natural conditions, C. burnetii is culturable using specially designed axenic media 29 Obligate pathogen: Rickettsiales Group of obligate intracellular vector-borne Gram-negative bacteria that include many organisms of clinical and agricultural importance Anaplasma spp. Ehrlichia chaffeensis Rickettsia spp. Orientia tsutsugamushi Each have a different method of intracellular survival 30 Host defence against intracellular pathogens Recognition of pathogens through PAMPs (e.g. TLR, RIG/MDA5, NOD...) R E V organelles I E W S and pathogens are delivered to Autophagy: A degradative pathway by which cytosolic content, lysosomes as part of cellular homeostasis and innate immunity. a L. monocytogenes High LLO Cell cytosol LLO ATG5 Vacuole lysis ActA Phospholipase C Autophagy Low LLO SLAPs Autophagosome Induction ATG5 IcsA IcsB IcsA BopA Autolysosome — degradation of contents FCV d F. tularensis Nat. Rev. Microbiol. 2007 & 2009 b S. flexneri c B. pseudomallei Perspectives Only a limited num niche for replicatio due to the failure of to use cytosolic sub bicidal factors, or t also clear that the c in different cell ty explain the tropism human host. We th of the constituents o the constituents are infectious agents. Although this R cytosolic pathoge show that a wider ra niche during steps Mycobacterium ma undergoes actin-ba terial species pers 31 partment. Howeve M. leprae that wer Learning Objectives (1) Describe what is meant by the term invasion Understand the benefits for bacterial pathogens living inside host cells Describe the difference between facultative vs. obligate intracellular pathogens and provide some examples Describe the 2 method employed by pathogens to invade host cells Briefly describe the processes of ‘trigger’ and ‘zipper’ mediated entry Explain the process of membrane ruffling using Salmonella as an example Understand how Y. pseudotuberculosis invades host cells 32 Learning Objectives (2) Describe the general principles employed by bacteria to avoid intracellular (lysosome) killing Describe how bacterial pathogens (and give examples of specific species) subvert phagocytosis via living within the phagosome and via phagosome lysis Describe the process of cell-to-cell spread used by Listeria Understand why obligate intracellular pathogens need host cells to survive and the difficulties in working with them Describe the obligate pathogen Chlamydia’s life cycle Understand the processes by which Coxiella and Rickettsiales survive inside host cells Host defense mechanismsvagainst intracellular pathogens 33