Structure and function of infectious agents Lecture 2 Introductory AVBS 3001 Gary Muscatello BVSc PhD The University of Sydney Page 1 COPYRIGHT COMMONWEALTH OF AUSTRALIA Copyright Regulation WARNING This material has been reproduced and communicated to you by or on behalf of the University of Sydney pursuant to Part VB of the Copyright Act 1968 (the Act). The material in this communication may be subject to copyright under the Act. Any further reproduction or communication of this material by you may be the subject of copyright protection under the Act. Do not remove this notice The University of Sydney Page 2 Lecture Goals – Structure and function of infectious agents – Taxonomy and nomenclature – Basic morphology and structure • Bacteria • Virus • Fungi • Metazoan parasites • Protozoa – Relationship between structure, function and classification – Significance of various structural and functional properties The University of Sydney Page 3 Taxonomy – Science of biological classification – Consists of three separate but interrelated parts – Classification – arrangement of organisms into groups (plural = taxa; singular = taxon) – Nomenclature – assignment of names to taxa – Identification – determination of taxon to which an isolate belongs – Taxonomy allows us to identify organisms based on shared characteristics The University of Sydney Page 4 Classification – Arranges organisms into groups whose members share many characteristics • First such classification in 18th century developed by Linnaeus – Phenotypic characteristics – Still used for cellular organisms – This approach to classification does not necessarily provide information on evolutionary relatedness. – Phylogenetic classification – Allows us to examine the line of evolutionary descent • Genetic analysis and sequencing • DNA-DNA hybridisation • ‘Housekeeping’ gene – Hierarchical family trees – Modern taxonomy combines phenotypic and genotypic analysis The University of Sydney Page 5 DNA-DNA hybridisation Known bacterium Heat Unknown bacterium + 0% Homology The University of Sydney 100% Homology Page 6 Phylogenetic trees The University of Sydney Page 7 Classification hierarchy Kingdom Phylum Related classes within a kingdom Class Related orders within a phylum Order Related families within a class Family Related tribes within an order Tribe Related genera within a family Genus Related species = ≥ 97% sequence homology Species The University of Sydney Related strains = ≥ 99% sequence homology Page 8 Five Kingdom System multicellular, walled eukaryotic cells, photoautotrophs multicellular and unicellular, walled eukaryotic cells, absorptive nutrition multi-cellular, wall-less eukaryotic cells, ingestive nutrition unicellular eukaryotes, varied types of nutrition all prokaryotes The University of Sydney Page 9 Hierarchical nomenclature Standard suffixes used to name given taxa Organisms Metazoan parasites Protozoa Phylum Fungi Algae -mycota -phyta Bacteria Class -ea -mycetes -phyceae -al Order -ida -ales -ales -ales -idea -aceae -aceae -aceae -ieae -ieae Family -idae Tribe -ini Further subdivision or superdivison – refine or include related organisms Example: subspecies = subdivision at the species level based on small but consistent difference existing between strains within a species. Abbreviated subsp. or ssp. (i.e. Streptococcus equi subsp. equi) The University of Sydney Page 10 Binomial nomenclature – Naming living cellular organisms at the species level – The genus followed by the species, capital initial letter for the genus and uncapitalised species name/designation – Italics or underline – Can abbreviate the genus when there is no risk of confusion • Escherichia coli = E. coli – If an organism is only classified to the genus level we use the non italic species abbreviation sp. – Escherichia sp. – Can describe multiple related species in the one genus by using the plural species abbreviation spp. – Salmonella spp. – Higher taxa, no set rules – Prokaryotes commonly italic or underline these higher taxa with the initial letter capital • Enterobacteriaceae The University of Sydney Page 11 Viral nomenclature – Non cellular so can’t use the binomial system – Grouped into families – Virion morphology – Replication strategy – Nucleic acid type – Further subdivision related to host species affected and clinical disease – – – – Order (suffix – virales) Family (suffix- viridae) Subfamily ( suffix – virinae) Genus (suffix – virus) – Use vernacular (common) term to name species so NO ITALICS (or underlines) required. – Italics or underline needed for higher taxa, with an initial capital letter – i.e. Herpesviridae The University of Sydney Page 12 Strain typing – Strain: an organism within a species which is a clonal descendent from a single isolate – Bacteria – We can use the term strain to define organism within a species with particular phenotypic or genotypic trait – Phenotyping methods – Genotyping methods – All very useful in epidemiological studies – Trace the source of an infectious agent – Explain disease manifestation The University of Sydney Takai et al. 1999 Page 13 Morphology of Bacterial Cells Bacillus Coccus Coccobacillus Diplococci Streptococci Staphylococci Spirillum Chain of bacilli The University of Sydney Filamentous bacillus Curved rod Spore-forming rod Page 14 Features of the Bacterial Cell Genome (Nucleoid) Ribosome Capsule Cell membrane Cell wall Mesosome Inclusion granules Plasmid Periplasmic space Cytoplasm Pili Flagella The University of Sydney Page 15 Cytoplasmic Membrane – Phospholipid bilayer – Osmotic barrier – Responsible for many functions performed by organelles in eukaryotic cells – Antibiotics specific for cytoplasmic membrane – Polymyxin B • Membrane permeable The University of Sydney Page 16 Cell Wall – Rigid carbohydrate-based structure 20-50 nm thick – Maintains shape and protects cytoplasmic membrane – 2 forms Firmicutes The University of Sydney Gracilicutes Page 17 Gram Stain – First step in identification of bacterial species – Demonstrates a basic difference in cell wall structure Basic Dye (Crystal violet) Decolourise (Acetone) Mordant/Trapping dye (Iodine) Counter Stain (Safranin) = Gram positive bacterium = Gram negative bacterium The University of Sydney Page 18 Reason for Differential Stain – Gram positive organisms retain the crystal violet-I2 – Gram-negative organisms show the counterstain – Gram-positive staining is dependent on cell wall integrity - older cultures can appear Gram-negative The University of Sydney Page 19 Gram Staining Gram negative (rods) The University of Sydney Gram positive (cocci in chains) Page 20 Gram Positive Cell Wall Lipoteichoic acid Peptidoglycan Cell membrane – Lipoteichoic acid – Long chain polymers – Bound to cell membrane and/or peptidoglycan – Antigenic specificity The University of Sydney Page 21 Gram Negative Cell Wall Lipopolysaccharide (LPS) Lipoprotein Peptidoglycan Cell Membrane – Lipopolysaccharide (LPS) – Protect cell against enzymatic attack – Endotoxins – Aids survival in the small intestine – O antigen used in serotype identification The University of Sydney Page 22 Growth of Bacteria in vitro The University of Sydney Page 23 Nutrient Requirements of Bacteria – Water – Carbon and Energy Source – Nitrogen and Sulphur – Phosphorus – Other Elements The University of Sydney Page 24 Temperature Requirements – Psychrophiles <20°C – Mesophiles 20 - 55°C – Thermophiles 55 - 80°C The University of Sydney Page 25 Aerobic and Anaerobic Metabolism – – – – – Aerobic Metabolism = Respiration Anaerobic Metabolism = Fermentation Obligate or strict Aerobes Obligate or strict Anaerobes Microaerophilic – Reduced O2 tension QuickTime™ and a Photo - JPEG decompressor are needed to see this picture. – Facultative Anaerobes – Fermentation in the absence of O2 – Aerotolerant – Fermentation in the absence and presence of O2 The University of Sydney Page 26 Bacterial Metabolism – Utilise a very broad range of environmental conditions and substrates – Varying metabolic activity used to identify species – Products can contribute to pathogenesis – Exotoxins – Selective toxicity used in chemotherapy of bacterial diseases – Antimicrobials effect synthesis of macromolecules The University of Sydney Page 27 Diagnostic applications Rods Enterobacteriaeceae Cocci or coccobacilli Acinetobacter Oxidase NEGATIVE Growth on MACA OF Test Oxidative Oxidase POSITIVE Aerobic growth OF Test Fermentative OF Test No reaction or Non fermentative Oxidase POSITIVE Blood agar No growth on MACA Oxidase NEGATIVE Microaerophilic Growth under special conditions Requires NAD and/or haemin Obligate anaerobe No growth Pseudomonas Actinobacillus Aeromonas Pasteurella Mannheimia Avibacterium Plesiomonas Vibrio (curved) Bordetella Alcaligenes Moraxella OF Test Oxidative Flavobacterium Neisseria (cocci) Brahnamella (cocci) OF Test Fermentative Pasteurella Mannheimia OF Test No reaction or Non fermentative Moraxella Brucella Brucella Campylobacter (curved rods) Brucella Taylorella Mycoplasma Actinobacillus Haemophilus Bacteroides Fusobacterium Dichelobacter Mycoplasma Leptospira Borrelia Chlamydia Rickettsiales Coxiella Determinative keys/tables use metabolic differences to given sources/atmospheres to help differentiate and identify bacterial pathogens The University of Sydney Page 28 Recognition of fungi in the clinical diagnostic laboratory – Morphological features of importance – – – – Yeasts Moulds Dimorphic Asexual spores The University of Sydney Page 29 KINGDOM Myceteae Class Chytridiomycetes Asexual Spores Motile Asexual Spores Nonmotile Div. Mastigomycota (water moulds) Div. Amastigomycota Sexual Spores Present Ascospores within asci, from Ascomycetes Sexual Spores Absent Class Oomycetes Zygospore Class Deuteromycetes (fungi imperfecti) Class Zygomycetes Ascospore Class Ascomycetes Basidiospore Class Basidiomycetes Pathogenesis related to invasion and mycotoxins The University of Sydney Page 30 Viruses – Non-cellular – Obligate intracellular parasites – Need to be in a cell to replicate – Use host metabolism – Possess one type of nucleic acid – DNA or RNA – Single or double strained – Host cell identification (tropisms) – Lock and key – Host cell protein and virus surface proteins The University of Sydney Page 31 Virion Infectious virus particle Cubical/icosahedral Helical The University of Sydney Page 32 Enveloped and non-enveloped – Some viral families are characterised by an envelope which surrounds the capsid Capsid – Envelope contains: – Host phospholipids Herpesvirus – Glycoproteins of viral origin – Lipid rich – Sensitive to lipid solvents – More fragile – Non enveloped virus Envelope – More resilient Rotavirus – Capsid cell surface proteins • Similar functions to the glycoproteins – Variation in protective immunity – Envelope viruses needs humoral and cell mediated – Non-enveloped needs only humoral The University of Sydney Page 33 DNA viruses – Most DNA viruses are double-stranded – Most replicate in the nucleus (except poxvirus) – Stable and less prone to mutation – Persistent infection – May integrate into the cell genome Poxvirus – Temporal regulation of gene transcription – Early genes → DNA replication – Late gene → Structural proteins The University of Sydney Page 34 RNA viruses – Most RNA viruses are single-stranded – Linear and can be segmented – Positive or negative sense – Positive sense can go straight into protein synthesis • Identical to viral mRNA – Most replicate in the cytoplasm – Labile and prone to mutation – Influenza antigenic variation • Antigenic drift The University of Sydney Influenza virus Page 35 Describing viruses Orthomyxoviridae Enveloped -ssRNA helical virus Influenza virus Poxviridae Enveloped dsDNA complex virus Poxvirus The University of Sydney Page 36 Metazoan parasites – Arthropods – Insects • Flies, mosquitoes and lice – Acarines • Ticks and mites – Helminths – Nematodes • Roundworms – Cestodes • Tapeworms – Trematodes • Flukes The University of Sydney Psoroptes Wool mite Ascaris suum (pig roundworm) Page 37 – Ectoparasites Arthropods – Live on the hosts surface – Some stages can cause disease in the host – Vectors !!! – Classification based on structure – Insect is more complex and has 6 legs – Acarines are simple and have 8 legs – Functional and disease significance • Feeding, transmission of agents and damage • Mouthparts – Life-cycles – Complete (pupation) or incomplete metamorphism – Larva and pupa (complete) • Can sometime be parasitic – Nymph stages (incomplete) The University of Sydney Page 38 Helminths – Nematodes – Endoparasites – Cylindrical multi-cellular eukaryotes – Classification • Reproductive structures and external cuticle features – Mouth pieces • Adaptation to feeding and damaging the host – Direct or in-direct life cycles – Direct = a single host animal species (definitive host) – In-direct = non definitive host (intermediate host) – Life-cycles can influence disease spread and control The University of Sydney Page 39 – Cestodes Helminths – Endoparasitic segmented flatworms • Tapeworm – 3 basic parts • Scolex – Anchors to mucosa of the host • Neck • Strobila – Segments – Release eggs or detach – Nutrition absorbed through the cuticle – Disease associated with attachment and occupation • Usually the gut – Can have in-direct life cycles • Some involve arthropods (i.e. fleas) The University of Sydney Page 40 Helminths – Trematodes – Endoparasitic non-segmented flatworms • Flukes – Monogenean and digenean – Liver fluke most significant • In-direct life cycle • Lymnaeid snail – Young migrate through liver – Adult develops in bile duct – Lays eggs → Faeces The University of Sydney Page 41 Protozoa – Single cell eukaryotes – Cell envelope – Attachment • Adhesions, flagella or cilia • Contribute to survival and pathogenesis • Classification – Tropism – – – – • Blood cells – Maleria (Plasmodium spp.), Babesia spp. Giardia Inter or intracellular in the host Various modes of motility Reproduce asexually and sexually Direct or in-direct life cycles – Tissue cysts in intermediate hosts – Cysts can also be used in classification Babesia – Cysts can usually survive outside the host for some time The University of Sydney Page 42 The University of Sydney Page 43
