Module 10 Lecture 1 2023 PDF

The Cell Biology of Bacterial Infection. -Antibiotic resistance is a huge global threat! -Must understand the bacterial life cycle to develop new therapeutic strategies. -I will first introduce bacteria -We will dissect the mechanism of cell entry/invasion -Look at host cell response to kill the bac...

The Cell Biology of Bacterial Infection. -Antibiotic resistance is a huge global threat! -Must understand the bacterial life cycle to develop new therapeutic strategies. -I will first introduce bacteria -We will dissect the mechanism of cell entry/invasion -Look at host cell response to kill the bacteria Antrax bacteria (green) being engulfed by a macrophage Camenzind G. Robinson, Sarah Guilman and Arthur Friedlander, United States Army Medical Research Institute of Infectious Diseases -Describe how the bacteria escapes to continue the infection. Antibiotic resistance is a major threat. https://antimicrobialsworkinggroup.org/antimicrobial-resistance/ Two flavours of bacteria -Gram negative bacteria have two membranes. Vibrio cholerae, Klebsiella pneumoniae -Gram positive have one, with a thick peptidoglycan wall. Staphylococcus aureus, Listeria monocytogenes -Transporters and secretion machinery exist in both. -Difficult to get compounds into bacteria that may kill them. Gram negative bacteria shed vesicles that help infect cells. 4 methods of OMV secretion: Clathrin, Caveolin, Lipid Raft, Membrane fusion -Cargo is selected within outer membrane vesicles. -Can protect the bacteria when they shed antibiotics, and act as a “decoy” when antigens are shed. Immune cell will try to kill OMV, and will be busy, so it won’t attack bacteria itself -Can facilitate infection when they contain virulence factors, DNA, RNA, immunomodulatory factors, and adhesins. -For bacteria infecting the gut, the lower pH of the stomach triggers periplasmic proteins to oligomerize and insert into the outer membrane as a pore. Fusion with target cell membrane acts to depolarize and permeabilize the cell, facilitating bacterial entry. (-So why was anyone surprised that mitochondria shed vesicles????) joint ancestors Bacterial Secretion Systems. Essential membrane embedded multi-protein machineries, enabling bacteria to obtain nutrients, communicate and facilitate disease through the delivery of virulence factors. originally developed for bacteria to receive nutrients, communicate etc. To date: 11 secretion systems discovered - they can transport virulence factors to the cytosol, or in some systems, can directly insert into the cell wall T9SS - promotes gum disease infection Trivadi et al. 2022 Gram + and – bacteria can inject proteins across PM and/or phagosomal membrane. Gram Negative Systems -Sec and Tat transport machinery is the oldest. Found in all kingdoms, archaea, bacteria and eukarya. Are mostly for biogenesis of bacterial proteins. -Other systems then evolved to cross the outer membrane, and into host cells. These are secretion systems to release virulence factors. -Type II system secretes toxins, small molecules (like cholera toxin), which disables host protein synthesis, leading to lethal infection. Common Tat and Sec machinery -Type III system secretes effector proteins, shares similarity to components with flagellar apparatus, evolutionary related. Effectors vary widely in function. -Type IV can transfer DNA and proteins. -Type V/VI translocate portions of themselves. Beta-barrel channels, or pores. Type III (T3SS) secretion system is like a piston can assist in conjugation (tranfer of DNA) can transfer plasmids, and if on plasmids you havve antibiotic resistance genes, you can gain resistance Type IV (T4SS) system to move DNA TYPE3 - Virulence factors in cytosol go through needle complex and can enter the host cell can also transcribe DNA, and create virulence factors INSIDE the host cells to help them take over. Unwinding of DNA for transfer. Secretion system in Gram-positive is not as well understood. -Again, Tat and Sec machinery function in these strains too. -Many proteins remain within the cell wall and facilitate bacterial binding to the host surface. they don’t cross the cell surface like Gram neg. -Getting contents across the cell wall is not so well understood, nor are the host receptors and machineries. Passive diffusion is often discussed. Professional phagocytic cells work to clear the bacteria and develop immunity. -Macrophages clear and rapidly degrade bacteria. They are professional degrading cells, highly efficient. Bacteria are neutralized and degraded before they can replicate or escape the endosome (later…). -Dendritic cells have slower degradation processes. -This allows the selection of antigens for presentation on class II MHC. Activates CD4+ T cells to generate antibodies. Nature Immunology 12, 1137–1138 (2011) Peptides from pathogens that enter the cell are presented to T-cells. Also critical to sample “self” to develop immune tolerance in the thymus. MHC Class 1 – all somatic cells: -Pathogens in the cytosol are degraded by proteasome -Peptides enter ER through TAP transporters. -Then loaded on MHC I proteins -Transport to cell surface -Bind and activate cytotoxic CD8+ T cells -MHC I are also found in endosome, this is called “cross-presentation”. MCH Class 2 – antigen-presenting cells (APC) including dendritic cells (DC), macrophages, and B cells: -Pathogens within the endosome/phagosome are degraded by proteases. -Peptides are loaded on MHC II proteins. -Transport to cell surface -Bind and activate CD4+ T cells for antibody production. Bacterial infection by phagocytosis must be specifically induced. Two processes: Zipper and Trigger -Bacteria infect non-phagocytic cells, so this process must be induced. -Zipper mechanism is where surface proteins on bacteria bind host cell proteins (receptors or adhesion proteins like integrins or cadherins) -Signaling cascades are induced to remodel lipids and actin. -Trigger mechanism initiates upon bacterial secretion of effectors that activate actin and lipid remodeling events. it injects its secretion factors across membranes, allowing the engulfment of the bacterial cells. Zheng et al. Critical Reviews in Microbiology, 42(5):1-19, 2015. Signaling by the bacteria induces PIP changes that drive engulfment. Lipid Remodelling - PIPs!! -None of these can happen without modulation of phosphorylated phosphatidylinositol (PIP)! -The phagosome is coated with a series of PIPs in a very localized and dynamic manner. -PI(4,5)P2 at the top, PI(3,4,5)P3 at the bottom of the phagocytic cup, ending in PI(3)P once internalized. -The presence of PIPs reinforces the recruitment and activation of actin remodeling machinery. Comparing the entry of Listeria and Shigella receptor actin bacteria overlapping receptor actin bacteria overlapping -Zipper mechanism shows structure of listeria internalin binding cadherin, scanning EM of bacteria on cell surface, and immunofluorescence images of Listeria entering into Vero cells (red: Met (signaling receptor within the cell); green, actin; and blue: bacteria). -Trigger mechanism shows the reconstitution of the Shigella type III secretion system; scanning electron micrograph of Shigella entering into cells, and immunofluorescence images of Shigella entering into Caco 2 cells (red: cortactin; green: actin; and blue: bacteria). Back to bacterial infection strategies……. Secreted effectors proteins can be modified by host and interfere with cell function. -Secreted proteins include phospholipid modifiers, kinases, proteases, etc. must bind to GTP Rab5 to open its confirmation -Often these must be “activated” by the host machinery. -3 examples are shown here - Rab5:GTP binding can alter conformation, activating the phospholipase. -Binding to an E2-ubiquitin complex stabilizes the active site of a protein kinase. -Binding to redox-related protein isomerases can activate protease activity. Bacterial effectors modulate actin in numerous ways. -Some are injected early to activate internalization. -Others are secreted later, to facilitate closure through depolymerization. -Some evolved to evade internalization into macrophages, but allow entry into non-phagocytic cells. -Other are secreted once the bacteria has escaped the endosome. These drive bacterial movement through the cytosol. Diverse bacterial lifestyle within cells. -Some bacteria replicate within their own specialized vacuolar compartment, like Salmonella or Legionella. -The early salmonella containing vacuole (SCV here) interact and/or fuse with other intracellular organelles. Hydrolytic content is removed through the recycling endosome, ensuring the bacteria doesn’t get degraded. -The replicating vacuolar structure for Salmonella has long extensions that are in contact with many organelles. -Sometimes it also escapes, thought to be induced by secretion of a pore forming toxin effector. Intersection with host cell biology machinery drives pathogen survival -Recent study shows a Salmonella effector SipA acts as an “R-SNARE” and recruits early endosomal SNAREs Syntaxins 8, 7 and 13, thereby keeping the bacteria out of the later endosomes. -Other Salmonella effectors alter retromer, and Mannose-6 phosphate transport, all aimed to stop this compartment from becoming hydrolytic. J Cell Biol. 2018 Oct 11. pii: jcb.201802155. Other bacteria must escape the phagosome to replicate -Shigella escapes within 10-15 minutes and has a cytosolic lifestyle. - The scape involves the recruitment of Rab11-postive recycling endosomes, but mechanisms are not clear. Bacteria can move within the cell by generating actin comet tails. Cell Host Microbe. 2013 September 11; 14(3): 242–255. Biophysical Journal Volume 89 September 2005 2146–2158 -Bacterial membrane proteins initiate recruitment of actin polymerization machinery. -This drives rapid movement through the cell that can even cut through mitochondria. -Actin comet tails can also drive the bacteria directly into the neighboring cell without risking detection by the immune system. The host response! Xenphagy to capture and kill cytosolic bacteria. - Phagosome rupture induced by the bacteria exposes glycans (beige blobs). -Glycans recruit proteins called Galectins (Gal), which sense damaged membrane and target them for degradation through an autophagic mechanism. -Galectins have a signature motif to recruit the autophagic adaptor NDP52. Other adaptors Optineurin and p62 play roles as well. -NDP52 recruits ubiquitin E3 ligases that ubiquitinate proteins on the broken membrane and on bacterial surface. P62 binds a ubiquitin ligase Traf6, and Optineurin binds DUBs – suggesting that the ubiquitin landscape is highly sculpted. -NDP52, optineurin and p62 have a LIR domain to interact with LC3, initiating cargo incorporation into an autophagosome to remove bacteria. Remember this slide from Module 5….. -The LC3 interacting motif is called a LIR domain. -The most commonly studied one is p62, but the others are growing in interest. -They link aggregated proteins into the phagophore lined with LC3-II. Liu et al. Front. Microbiol 2020 Infected cells die through different types of death. this is preffered form of cell death from POV of bacteria very inflammatory, membrane rapture -Apoptosis is immune body will silent form of death. recuit more immune cells Bacteria can escape within the apoptotic bodies and infect next cell this way. -Necrosis is death from damage, loss of ATP, membrane rupture, and is inflammatory. -Pyroptosis is a highly programmed form of death where the dying cell expresses and releases specific cytokines to recruit immune cells to help. Transcriptional programs involved, caspase cascades, etc. But bacteria evolved mechanisms to evade host death. Bacterial effectors block apoptotic machinery. -Effector colors match bacteria color scheme here -Different targets to promote host survival during infection, depending on the bacteria. -Note how it all revolves around the mitochondria, which holds the key to all forms of cell death. Bacteria can also activate pro-survival pathways help cell proloferate and grow so that bacteria can develop and spread Summary -Covered the basics of bacterial infection -Dissected the mechanism of cell entry/invasion -Looked (briefly) at host cell response to kill the bacteria -Described how the bacteria escapes and delays cell death/inflammation to continue the infection.