Lecture 1-4
Define Sterilization and Disinfection.
Sterilization: the total destruction of all microbes including more resilient forms like spores, non-enveloped viruses and fungi.
Disinfection: the physical procedures or chemical agents used to destroy microbes or render them inert
Compare and contrast different methods of disinfection.
Heat (high larval disinfectant): dry heat (incineration \ dry oven), moist heat (boiling/autoclaving), moist heat is more effeicent. At temps above the growth range heat energy destroys proteins and nucleic acids, dry hear also dessicates cells
Radiation (high level disinfectant): UV radiation (crosslinks nucleic acids rendering organisms unable to replicate genetic material), ionizing radiation (X-ray or microwave radiation create strand breaks in nucleic acids rendering organisms unable to replicate genetic information)
Filtration (low level disinfectant): filtration traps microorganisms, as fluid passes through a filter, organisms above a certain size threshold are trapped in the pores, air can be filtered using a high-efficiency particulate air (HEPA) filter, particularly useful for sterilizing liquids
Antiseptics: chemical agent that is used to kill microbes on living tissue
Disinfectants: chemical agent that is used to kill microbes on a non-living surface
• Gram positive is made up of
○ Capsule (not all species): made up of polysaccharides
○ S-layer (not all species): made of proteins
§ Some cells have both, others have one or other and still others have none
○ Thick cell wall: 9 amino acid cross-links in peptidoglycan, Lipoteichoic acids (polysaccharide of glycerol phosphate or ribitol phosphate linked via phosphodiester bonds) increase cell wall
○ Periplasm
○ Plasma membrane
• Gram negative is made up of
○ Capsule (not all species): made of polysaccharides
○ Outer membrane: outer leaflet is lipopolysaccharide (LPS), inner leaflet normal phospholipid
○ This cell wall: 4 amino acid crosslinks in peptidoglycan, no techoic acid
○ Periplasm: in gram (-) cells the periplasm is defined as the space between the outer and inner membranes
○ Plasma membrane
• Fimbriae
○ Short, helical, filamentous protein structures
○ Plentiful, can be up to 1000 per cell
○ Enable organisms to stick to surfaces or each other
○ Not all bacteria have fimbriae
• Pili
• Attach cells to surfaces
• Thin filaments of proteins made of a protein monomer called pilin
• Longer than fimbriae
• Sometimes allow movement via twitching motility
• Often involved in helping bacteria evade the components of the innate immune system
• One specific type, the conjugal pilus, allows for exchange of DNA from one bacteria to another
Flagella
§ Long, helical protein filaments attached to a complex integral membrane protein "machine"
§ Protein filament is made from a protein monomer called flagellin
§ Cells can have one flagellum or many flagellum
§ Can be arranged differently
§ Rotate to propel cell
• Proton passage drives rotation
• Clockwise or counterclockwise
• Movement is directed
§ Different arrangements
• Monotrichuous: single polar flagellum
• Amphitricuous: a flagellum at each pole
• Lophotricuous: multiple flagella localized at the cell pole
• Peritrichous: multiple flagella inserted at many locations in the cell membrane
• Capsules and slime layers
§ Polysaccharide layers
§ May be thick or thin, rigid or flexible
§ Capsules are closely associated with the bacteria and difficult to wash off
§ Slime layers are loosely associated with bacteria and easily removed
§ Both function to :
□ Assist in attachment to surfaces
□ Protect against phagocytosis
□ Resist desiccation
• Secretion systems allow secretion of proteins from bacteria to the extracellular environment or into host cell
• Seven types (I-VII)
§ Sex pilus is similar to type IV secretion system
• Essential for bacterial pathogenicity
• Plasmids
• Extra-chromosomal molecule of DNA, not required for cell survival in all conditions
§ Smaller than chromosome
• Replicated by the cells DNA polymerase
• They often confer important phenotypes to the cell
• Can confer the ability to metabolize certain sugars, kill other bacteria and much more
• Nature of genomes
• DNA sequence is not static
§ Mutations of single bases
§ Large deletion
§ Large insertions of sequence
□ Transferred from other species
□ New functions useful on particular situations
§ Maintained via interactions with environment
□ Survival determined by having appropriate genes for specific environment
• Horizontal gene transfer
• DNA transfer between organisms that is due bacterial reproduction
• Horizontal DNA transfer can change the genotype and phenotype of a cell
• Three mechanisms
§ Transformation: transfer of naked DNA
§ Conjugation: cell to cell, require cell contact
□ Transfer of plasmids
□ Transfer of portions of the chromosome (hfr).
§ Transduction: transfer of chromosomal DNA via bacteriophage
□ Generalized: virulent phage transfers any piece of DNA randomly
□ Specialized: temperate phage transfers specific DNA sequences close to insertion site
• Nucleic acid information
• Information is encoded by the order of nucleic acids
§ DNA
□ Adenine, guanine, cytosine, thymine
• Is transcribed into:
§ RNA
□ Adenine, guanine, cytosine, uracil
• Is translated into
§ Protein
□ Amino acids
• Defining a gene
• Alignment of a bacterial structure gene with its mRNA transcript
• Genes are organized into operons
§ Operon: a unit of genetic material that functions in a coordinated manner by means of an operator, a promoter, and one or more structual genes
• Operons are organized into regulons
§ Regulon: a system of genes, formed by one or more operons, that have a common regulatory element
• Transcription: bacterial RNA polymerase
• RNA polymerase is a large molecular machine
§ 5 proteins in the core complex (holoenzyme)
□ (2) a : interacts with regulatory proteins
□ B : catalytic activity, hold s on to the RNA polymerase
□ B^′: involved binding and staying bound to DNA
□ w : stablizes the complex
• Sigma factors
• Recognizes promoter region of gene
• Released soon after the start of transcription
• Multiple sigma factor
• Sigma 70 - rpoD principal sigma factor
• Sigma 54 - rpoN nitrogen-regulated gene transcription
• Sigma 32 - rpoH heat-shock gene transcription
• Sigma S - rpoS stationary phase gene transcription
• Sigma F - rpoF flagellar operon transcription
• Sigma E - rpoE oxidative stress gene transcription
• Sigma Fecl - fecl transcription of genes for iron dicitrate transport
• Translation
• The process of translating mRNA sequence into amino sequences
• 3 types of RNA are involveed
§ mRNA - messenger RNA, codes the sequence of amino acids
§ rRNA - ribosomal RNA, in combination with protein makes up the ribosome "machine"
§ tRNA - shuffles amino acids to the ribosome
• Translation in bacteria can begin before completion of the full mRNA transcript
• Ribosome = protein polymerase
• Very large molecular machine
§ 2 subunits, 30s & 50s
§ 52 proteins, 3 rRNAs
□ Mostly rRNA
§ Can bind 1 mRNA + 3 tRNAs
• Differences from eukaryotic ribosome make this a good target for antibiotics
• Regulating gene expression
• Microbes respond to changing environment
§ To survive adverse conditions'
§ To thrive in good conditions
§ To compete for a niche
§ To move to a better niche
• Response to the environment requires
§ Sensing the environment
§ Changing gene expression
• Metabolic regulation
• Many proteins are enzymes that carry out biochemical reactions
§ Constitutive proteins are needed at the same level all the time
§ Microbial genomes encode many proteins that are NOT needed all the time
• Regulation of protein production and function helps conserve the cells energy and resources
• Two major levels of regulation
§ Post-translation regulation: controls the activity of preexisting enzymes
□ Typically a protein is modified to change its activity
□ Very rapid process (seconds)
§ Regulates of gene expression: controls the amount of an enzyme
□ Gene expression: transcription of gene into mRNA followed by translation of mRNA into protein
□ Generally regulation occurs at the level of transcription by controlling the amount of mRNA
□ Sometimes regulation can occur at the translation level by controlling whether an mRNA is translated
□ Slower process (minutes)
• Sensing the environment
• Two-component signal
• Sensor kinase in the cell membrane
§ Binds to environmental signal
§ Activates itself via phosphorlyation
• Response regulator
§ Activated via phosphorylation by sensor kinase
§ Mediates "the response" by altering gene transcription
• Regulating gene expression
• Repressor/negative regulator: bind to regulatory sequences in the DNA and prevent transcription of target genes
§ Most often repressors block sigma factor from binding promoter
§ Repressors can require co-factors to repress or be released from binding site
•
• Activator/positive regulator: bind to regulatory sequences in the DNA and transcription of target genes
§ Often acts to recruit or stabilize sigma factor interaction with a promoter
□ Can also alter DNA structure to allow binding of sigma factors
§ Activators can require co-factors to bind DNA
•
• Sigma factors
• Regulate transcription of all genes
§ Sigma 70 initiates transcription at most genes
§ Sporulation in B. subtilis - Bs sigma 28
§ Stationary phase - sigma 38
§ Heat-shock and stress response - sigma 32 (rpoH)
§ Flagellar synthesis - sigma 28
• Control of ratio of sigma factors determines global control of protein synthesis
§ These can be controlled by altered transcription, translation, proteolysis and anti-sigma factors
Explain the difference between the adaptive and PPPPPPPP immunity
Know the cells of the immune system, understand how they develop from their progenitor cells and the role each plays in an immune response
Be aware of the primary and secondary Lymph organs.
Identify the innate barriers to infection.
Define PAMPs and Understand the role of PRRs in the immune system.
Excluding non-self from self
Preventing infection by microorganisms means recognizing and excluding the non-self (invader) from self (human cell).
Physical barrier systems: prevent microorganisms from having access
Innate immune system: recognizes conserved microorganism elements and responds rapidly to remove the microorganisms
Adaptive immune system: recognize traits particular to specific micro and acts to remove them
Barriers to infection
Skin
Keratin, oil, antimicrobial secretions of skin flora
Dead skin cells, washing, remove attached cells
Respiratory tract
Mucocilliary clearence
Mucous
Ciliated cells
Eyes
Washing by tears
Lysozyme - enzyme that breaks down cell walls
Digestive tract
Acidity of stomach
Bile - breaks down lipid bilayers
Normal flora - prevent colonization
Genitourinary tract
Washing by urine
Acidity of urine
Lysozyme
Vaginal lactic acid - low pH inhibits bacterial growth
Antimicrobial peptides found in mucous secretions
Chemical barriers
Antimicrobial molecules
All act on non-self particles
Inhibit growth/metabolism
Lyse microorganism
Promote immune response
Many are found in either secretions or bodily fluids on the surfaces that have contact with invaders
Innate immune response
Initial response to an invader
General response triggered by components common to many pathogens
Includes
Physical barriers to infection
Antimicrobial molecules
Complement protein cascade
Cells of the innate immune response
Innate protections are activated by PAMPs
Pathogen Associated Molecular Patterns
Repetive structures of the microbial surface or genome
Ex. LPS, LTA, flagellin, peptidoglycan
Adaptive immune response
Secondary response
Specific to a specific pathogen
Immune system reacts to specific immunogens
Immunogen: a protein or carbohydrate that is recognized and sufficient to illict an immune response. An immunogen may contain one or more antigens
Antigens: a molecule that is recognized by a single specific antibody or T-cell receptor (TCR) or t-cells
Retains "memory" of those antigens
Humoral immunity
Production of antibody proteins
Each binds a different antigen
Cellular immunity
T cells that bind different antigens
Control antibody production
Kill intracellularly infected cells
Cells of the immune system
All blood cells derive from a pluripotent stem cell
Pluripotent stem differentiates into different stem cells for different lineages of blood cells
Development begins during fetal development and continues throughout life
Stem cells reside primarily within bone marrow
Hematopoietic and mesenchymal stromal stem cell
Differentiation results from interaction of the stem cell with the stromal cells of the bone marrow
Stroma and other cells release colony stimulating factors that stimulate stem cell differentiation
Innate immune cells
Develop from myeloid progenitor cell
Neutrophils: phagocytose and kill bacteria
Antigen presenting cells (APCs):
Antigen: foreign material that promote production of antibody
Phagocytose and kill bacteria
Present antigens to T-cells via class II MHC proteins
Derive from monocytes
Macrophages: found in tissue, spleen, lymph nodes and other organs. Initiate inflammatory response
Dendritic cells: lymph nodes and tissue. Most potent APC. Initiates and modulates T- cell response
Adaptive immune cells - lymphocytes
B-cells
Produce antibody
Internalize antigen, process antigen and present antigen to T-cells to expand immune response
Activated B-cells develop into
Memory cells: which circulate until activated by a specific antigen
Plasma cells: which produce large amounts of antibody
T-cells
CD-4: control, suppress, and activate immune and inflammatory response by cell-cell interaction or release of cytokins
Helper T-cells
CD-8: also release cytokins, but known for their role in recognizing virally infected cellsm tissue transplant and tumor cells
Cytotoxic killer T-cells
Surface markers of lymphocytes
Lymphocytes are often classified by surface markers
Subsets of both B-cells and T-cells present with different surface proteins
Subsets may have a more defined role in T-cells or B-cell response
Ex. Plasma cells present some surface markers that memory cells do not
Other cells of the immune system
Natural killer cells
Innate immune cells but derive from lymphoid precursor
Large granular lymphocytes that kill antibody decorated cells, virus-infected cells, or umor cells
Eosinophils and basophils
Granular cells involved in parasite defense, and allergic response
Basophils release histamine
Eosinophils are phagocytic cells, but do not play a role in response to bacterial pathogens
Cell-cell communication in immune system
Mediated by chemical signals:
Cytokins
Chemokines: small proteins that activate and attract immune cells in a site
Colony stimulating factors (CSF): glycoproteins that signal differentiations and proliferation of stem cell to immune cell
Growth factors (GF): proteins that stimulate proliferation of fibroblasts (cells involved in wound healing)
Cells recognize chemical signals by surface receptors and respond appropriately
Certain signals are recognized by certain cells and cause specific responses
Lymphatic/lymph system
A network of organs, lymph nodes, lymph ducts, and lymph vessels that make and move lymph from tissue to the bloodstream
Function
Maintains fluid balance of the tissues
Absorbs fats and fat soluble vitamins from the small intestine, delivering them to tissues
Production and transport of immune cells
Organs of the lymph system
Primary lymphoid organs
Bone marrow & thymus
Sites of initial lymphocyte differentiation
Thymus most important in t cell differentiation
Secondary lymphoid organs
Lymph nodes, spleen
Mucosa associated lymphoid tissue (MALT)
Peyers patches and mesenteric lymph nodes in the gut
Bronchus assoc lymp tissue, tonsils etc.
Where B-cells, T-cells reside and respond immune challenge
Lymph circulation
Open circulatory system: moves toward the heart
Lymph is a fluid composed of water and immune cells
Lymph collects bacteria, fats, proteins and cell waste
Enters the lymph system via lymphatic capillaries
Can be found near blood capillaries
Pumped by smooth muscle contraction up vessels to lymph organs and trunks
Lymph organs generally remove pathogenic organisms
Lymph drains into the circulatory system at lymph ducts
The lymph system at work
M cell samples intestinal track
M cell presents antigen to B cell, T cell and/or macrophage
Stimulated immune cells migrate to the peyer's patch stimulating resident B and T cells
These cells travel through lymph system to the blood stream
Thus, a systemic response can result from a local challenge
Innate immune response
Initial response to an invader
Includes
Physical barriers to infection
Antimicrobial molecules
Complement protein cascade
Cells of the innate immune response
Innate protections are activated by PAMPs
Pathogenic Associated Molecular Patterns
Repetive structures of the microbial surface or genome
Ex. LPS, LTA, flagellin, peptidoglycan, etc.
Pathogen Associate Molecular Patterns
Note the items in this incomplete list are either
Unique to microorganisms
Very different microorganisms
Pathogen Recognition Receptors
Cells of the innate immune system can be activateed by:
Direct interactions with PAMPs mediated by pathogen recognition receptors (PRRs)
Indirect activation via interaction with cytokines released by other cells (immune cells or other cell types)
There are multiple PRRs and more are being discovered as research on the immune system progresses
PRRs can be found on the cell surface, in a cell endosome, or in the cytoplasm of the cell
The distribution of sensors is important as the immune system needs to be able to detect both extracellular (i.e. bacteria, fungi) and intracellular (i.e. bacteria, viruses) invaders
The majority of PRRs are part of a protein family called Toll-Like Receptors or TLRs
Note that not all TRLs are present in all immune cells and that some non-immune cells express TLRs
Interaction of a PAMP with a PRR activates cell signaling cascades that result in the construction of the inflammasome
Stimulates localized inflammation by promoting the activation of IL-1B and IL-18
Transcription factors promote the expression of antimicrobial peptides, interferon a and B, and cytokines that activate both the innate and adaptive immune response