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Lecture notes on general microbiology & immunology Prepared by By Lecturer of Microbiology & Immunology Faculty of Pharmacy South Valley University (2023/2024)  General bacteriology Bacterial cell...

Lecture notes on general microbiology & immunology Prepared by By Lecturer of Microbiology & Immunology Faculty of Pharmacy South Valley University (2023/2024)  General bacteriology Bacterial cell morphology Depending on their shape, bacteria are classified into: ▪ Cocci (singular coccus, from; kokkos, meaning berry)- are oval or spherical cells. ▪ Bacilli (singular bacillus, meaning rod shaped) are relatively straight, rod- shaped (cylindrical) cells. In some of the bacilli, the length of the cells may be equal to the width. Such bacillary forms are known as coccobacilli and must be carefully differentiated from cocci. Cocci are arranged in groups (clusters), pairs or chains. Similarly, bacilli can be arranged in chain, pair, and some bacilli are curved, comma shaped, flexuous/rigid spiral, or cuneiform shaped (fig. 1) Figure 1:Different morphology of bacteria and Gram staining property 1  Both cocci and bacilli are further classified based on Gram staining property into : Gram-positive cocci, Gram-negative cocci, Gram-positive bacilli, and Gram- negative bacilli. However, there are some bacteria that are weakly Gram stained and hence need special stains for their demonstration, such as: ▪ Spirochetes (Treponema and Leptospira)-thin spirally coiled bacilli. ▪ Mycoplasma (cell wall deficient free-living bacteria) do not possess a stable morphology. They occur as round or oval bodies and interlacing filaments. ▪ Rickettsiae and chlamydiae are obligate intracellular bacteria. Classification of bacteria depending on their morphology and Gram staining property. a) Gram-positive cocci arranged in Cluster - (Staphylococcus) Chain - (Streptococcus) Pairs, lanceolate shaped - (Pneumococcus) Pair or in short chain, spectacle shaped - (Enterococcus) Tetrads - (Micrococcus) Octate - Sarcina b) Gram-negative cocci arranged in Pairs, lens shaped - (Meningococcus) Pairs, kidney shaped - (Gonococcus) c) Gram-positive bacilli arranged in Chain (bamboo stick appearance) - (Bacillus anthracis) Chinese letter or cuneiform pattern - (Corynebacterium diphtheriae) Branched and filamentous form – (Actinomyces and Nocardia) d) Gram-negative bacilli arranged in Pleomorphic (various shapes) -(Haemophilus, Proteus) 2 Comma shaped - (Vibrio cholerae) Curved attached at their ends (s-shaped) Campylobacter and Helicobacter e) Others Chain Streptobacillus Spirally coiled, flexible Spirochetes Rigid spiral forms Spirillum Bacteria that lack cell wall Mycoplasma Bacterial cell anatomy Bacterial cell comprises of the following structures (Fig. 2) Figure 2: bacterial cell structure a) The outer layer or the envelope of a bacterial cell consists of a rigid cell wall and underlying plasma membrane (lipid bilayer). b) The cytoplasm contains cytoplasmic inclusions (mesosomes, ribosomes, inclusion granules, vacuoles). c) a diffuse nucleoid containing single circular chromosome. d) Some bacteria may possess additional cell wall appendages such as capsule, flagella and fimbriae. 3  1) Bacterial cell wall The internal osmotic pressure of most bacteria ranges from 5 to 20 atm as a result of solute concentration via active transport. In most environments, this pressure would be sufficient to burst the cell were it not for the presence of a high- tensile strength cell wall. The bacterial cell wall owes its strength to a layer composed of a substance variously referred to as murein, mucopeptide, or peptidoglycan. Most bacteria are classified as Gram-positive or Gram-negative according to their response to the Gram-staining procedure due to differences in their cell wall. This procedure was named for the histologist Hans Christian Gram, who developed this differential staining procedure to identify bacteria in infected tissues. In addition to providing osmotic protection, the cell wall plays an essential role in cell division as well as serving as a primer for its own biosynthesis. Peptidoglycan is a complex polymer consisting of three parts: a backbone, composed of alternating N- acetylglucosamine and N-acetylmuramic acid connected by β1→4 linkages; a set of identical tetrapeptide side chains attached to N-acetylmuramic acid; and a set of identical peptide (pentaglycine) cross-bridges. The tetrapeptide side chains have L-alanine at position 1 (attached to N- acetylmuramic acid), D-glutamate at position 2, and D-alanine at position 4. Position 3 is the most variable one: Most Gram-negative bacteria have diaminopimelic acid at this position, to which is linked the lipoprotein cell wall component. Gram-positive bacteria usually have L-lysine at position 3. Figure 3: Interconnected glycan chains form a very large three-dimensional molecule of peptidoglycan. 4  Figure 4: Chemical structure of N-acetylglucosamine (NAG) and N- acetylmuramic acid (NAM); the ring structures of the two molecules are glucose. Glycan chains are composed of alternating subunits of NAG and NAM joined by covalent bonds. Adjacent glycan chains are cross-linked via their tetrapeptide chains to create peptidoglycan. In Gram-positive bacteria, there are as many as 40 sheets of peptidoglycan, comprising up to 50% of the cell wall material; in Gram-negative bacteria, there appears to be only one or two sheets, comprising 5–10% of the wall material. Bacteria owe their shapes, which are characteristic of particular species, to their cell wall structure. ▪ Special Components of Gram-Positive Cell Walls: Most Gram-positive cell walls contain considerable amounts of teichoic and teichuronic acids, In addition, some Gram-positive walls may contain polysaccharide molecules. The term teichoic acids encompass all wall, membrane, 5 or capsular polymers containing glycerophosphate or ribitol phosphate residues. These polyalcohols are connected by phosphodiester linkages and usually have other sugars and d-alanine attached. Figure 5:(on the left) Teichoic acid structure. The segment of a teichoic acid made of phosphate, glycerol, and a side chain, R. R may represent D-alanine, glucose, or other molecules. (on the right) Teichoic and lipoteichoic acids of the Gram-positive envelope There are two types of teichoic acids: wall teichoic acid (WTA), covalently linked to peptidoglycan; and membrane teichoic acid (lipotechoic acid LTA), covalently linked to membrane glycolipid. Together with peptidoglycan, WTA and LTA make up a polyanionic network or matrix that provides functions relating to the elasticity, porosity, tensile strength, and electrostatic properties of the envelope. The teichoic acids constitute major surface antigens of those Gram-positive species that possess them, and their accessibility to antibodies has been taken as evidence that they lie on the outside surface of the peptidoglycan. Their activity is often increased, however, by partial digestion of the peptidoglycan; thus, much of the teichoic acid may lie between the cytoplasmic membrane and the peptidoglycan layer, possibly extending upward through pores in the latter. The teichuronic acids are similar polymers, but the repeat units include sugar acids (eg, N-acetylmannosuronic or d-glucosuronic acid) instead of phosphoric acids. 6 ▪ Special Components of Gram-Negative Cell Walls Gram-negative cell walls contain three components that lie outside of the peptidoglycan layer: outer membrane, lipopolysaccharide, and lipoprotein. Figure 6: Gram negative cell wall I. outer membrane: It is a bilayered structure; its inner leaflet resembles in composition that of the cytoplasmic membrane, and its outer leaflet contains a distinctive component, a lipopolysaccharide (LPS). the outer membrane has special channels, consisting of protein molecules called porins that permit the passive diffusion of low-molecular- weight hydrophilic compounds, such as sugars, amino acids, and certain ions. Large antibiotic molecules penetrate the outer membrane relatively slowly, which accounts for the relatively high resistance of Gram-negative bacteria to some antibiotics. 7  II. Lipopolysaccharide (LPS): The LPS of Gram-negative cell walls consists of a complex glycolipid, called lipid A, to which is attached a polysaccharide made up of a core and a terminal series of repeat units. The lipid A component is embedded in the outer leaflet of the membrane anchoring the LPS. LPS, which is extremely toxic to animals, has been called the endotoxin of Gram-negative bacteria because it is firmly bound to the cell surface and is released only when the cells are lysed. When LPS is split into lipid A and polysaccharide, all the toxicity is associated with the former. The O antigen is highly immunogenic in vertebrate animals. Antigenic specificity is conferred by the O antigen because this antigen is highly variable among species and even in strains within a species. III. Lipoprotein: Molecules of an unusual lipoprotein cross-link the outer membrane and peptidoglycan layers. The lipoprotein contains 57 amino acids, representing repeats of a 15-amino-acid sequence; it is peptide linked to DAP residues of the peptidoglycan tetrapeptide side chains. The lipid component, consisting of a diglyceride thioether linked to a terminal cysteine, is noncovalently inserted in the outer membrane. The function of the lipoprotein is to stabilize the outer membrane and anchor it to the peptidoglycan layer. IV. Periplasmic space: The space between the inner and outer membranes, called the periplasmic space, contains the peptidoglycan layer and a gel-like solution of proteins. The periplasmic proteins include binding proteins for specific substrates (eg, amino acids, sugars, vitamins, and ions), hydrolytic enzymes (eg, alkaline phosphatase and 5′-nucleotidase) that break down non-transportable substrates into transportable ones, and detoxifying enzymes (eg, β-lactamase and aminoglycoside- phosphorylase) that inactivate certain antibiotics. 8  The periplasm also contains high concentrations of highly branched polymers of d-glucose, 8 to 10 residues long, which are variously substituted with glycerol phosphate and phosphatidylethanolamine residues. These so-called membrane- derived oligosaccharides appear to play a role in osmoregulation because cells grown in media of low osmolarity increase their synthesis of these compounds 16- fold. Comparison of Features of Gram-Positive and Gram-Negative Bacterial cell wall Component Gram-Positive Gram-Negative Peptidoglycan Thick layer (40 sheets) Thin layer (2 sheets) Techoic acids Present Absent Outer membrane Absent Present Lipopolysaccharides Absent Present Porin protein Absent Present periplasm Absent Present sensitivity to lysozyme yes No 9