C1&2 Key Concepts and Learning Objectives F2024 PDF

Classes 1&2: The bacterial cell envelope A cell wall is composed primarily of a molecule called peptidoglycan. This protects bacteria cells from turgor pressure. Turgor pressure is a force pressing outwards from the inside of a bacteria cell. Turgor pressure results from a bacterial cell being plac...

Classes 1&2: The bacterial cell envelope A cell wall is composed primarily of a molecule called peptidoglycan. This protects bacteria cells from turgor pressure. Turgor pressure is a force pressing outwards from the inside of a bacteria cell. Turgor pressure results from a bacterial cell being placed in a hypotonic solution and the resultant water that moves inside of the bacterial cell. If there is no cell wall or if the cell wall is damaged, turgor pressure can kill the bacterial cell by damaging its membrane. Peptidoglycan is composed of both sugars (-glycan; NAM & NAG) and amino acids (peptido-; five in a monomer). A single monomer of peptidoglycan is composed of (in this order): NAM, NAG, L-alanine (L-A), D-glutamic acid (D-E), m-diaminopimelic acid (m-Da), D-alanine (D-A), D-alanine (D-A). NAM stands for N-acetylmuramic acid and NAG stands for N-acetylglucosamine. In order for peptidoglycan to protect the bacterial cell, it must be covalently linked together into a huge polymer. This covalent bond occurs between the m-Da of one peptidoglycan monomer and the first D-A (the 4th amino acid) of the other peptidoglycan monomer. Because carbon can only have four bonds, the last/terminal D-A must leave (this is the 5th amino acid). The enzyme that forms the bond between m-Da and D-A is called a transpeptidase, because it is an enzyme (-ase) that forms the bond (-peptid-) between amino acids across (trans-) from one another. When you discussed amino acids in BIOL 133 or other classes, you talked about L-amino acids. They also have a mirror image called D-amino acids. Bacteria use many D-amino acids to make peptidoglycan. This difference enables us to clinically target bacterial peptidoglycan (D-amino acids) without affecting human proteins that contain L-amino acids. For the purposes of this class, there are two major mechanisms by which antibiotics can disrupt the cell wall. (There are other mechanisms, but this requires a fuller understanding of bacterial cell wall biogenesis, which is beyond the scope of this class.) The first mechanism is by direct inhibition of the enzyme transpeptidase. This is how penicillin (and related antibiotics) work. If the transpeptidase is inhibited, peptidoglycan cannot be crosslinked (the bond between m-Da and D-A). Thus, the cell wall is compromised. The second mechanism is by preventing the terminal D-alanine from leaving (the fifth amino acid) during cross-bridge formation. This is how vancomycin works. This antibiotic binds to both D-alanines present in the peptidoglycan and prevents the fifth from leaving. A key idea to remember is that cell walls are continually being remodeled and repaired, as bacteria grow in size and divide (e.g., grow in number). Thus, this is not a static polymer. This idea highlights why antibiotics that target the cell wall can be used against many bacterial species. Despite the commonality between peptidoglycan, bacteria can have many different cell envelope structures, including differences in the amount of peptidoglycan. A cell envelope consists of the inner membrane and any structures external to it (e.g., peptidoglycan, outer membrane, capsule, S-layer, etc.). Gram-positive, Gram-negative, and mycobacteria are the three types of cell envelopes we will discuss. (This language is slightly imprecise, as we’ll soon see.) The positively-charged (cationic) dyes called crystal violet (purple) and safranin (pink) can be used to determine whether a bacterium is Gram-positive (stays purple) or Gram-negative (becomes pink). We’ll talk about this more in the lab (and you’ll perform it several times); this is also discussed below. The Gram stain does not tell you anything about the genetics of the bacterium. It only tells you whether there are a lot of sugars (e.g., peptidoglycan, arabinogalactan, arabinomannan, etc.) in the cell envelope (Gram-positive, purple) or whether there are few sugars (e.g., peptidoglycan) in the cell envelope (Gram-negative, pink). Gram-negative (pink) bacteria have a small peptidoglycan layer (usually only a few layers) and two cell membranes. Gram-positive (purple) bacteria have a large peptidoglycan layer (imagine this as between 80-120 layers of peptidoglycan) and a single membrane. In the case of mycobacteria (Gram-positive, purple), they have several layers of peptidoglycan and sugars called arabian, galactan (also, together, called arabinogalactan), and arabinomannan. They also have two cell membranes, and these polysaccharides are in the middle (much like with the Gram-negative structure). Mycobacteria also have a waxy outer layer composed primarily of a type of lipid called mycolic acid. Also, they have a capsule (which we’ll discuss later for other bacteria). A capsule is another layer (external to the outer membrane or outer layer, in mycobacterium) that is primarily composed (in most species) of polysaccharides. Thus, Gram staining usually tells you whether bacteria have one or two cell membranes - EXCEPT for mycobacteria. This is a key exception. Why do these dyes retain differently? Iodine and crystal violet (the purple dye) can form a very large complex when added to water. Once formed, this complex is unable to diffuse back through several layers of peptidoglycan and/or arabinogalactan, but it can easily diffuse out of the Gram-negative cell envelope, because it is only a few layers thick. If you think of multiple layers of peptidoglycan as layer cloth/material, you can see how it would be really difficult for molecules to be washed out of all of the layers if they are near/in an inner layer. Thus, cell envelopes don’t retain dye just as a function of electrostatic interaction between cell envelope components and the cationic dye, but size-limited diffusion plays a huge role here. That is why (in the lab), you’ll see that it is possible to over-wash the dye and have an inconclusive Gram stain. Polysaccharides aren’t the only component of bacterial cell envelopes. Gram-positive bacteria have long molecules called teichoic acids. These can crosslink peptidoglycan layers to each other (wall teichoic acids, WTA) or they can crosslink peptidoglycan layers to each other and attach them to the cell membrane (lipoteichoic acids, LTA). A functionally similar molecule (through structurally distinct) in Gram-negative bacteria is called lipoprotein, and this protein connects both the inner and outer membranes to the limited peptidoglycan within the periplasmic space (the space between the inner and outer membranes). Another component that is present in many Gram-positive and Gram-negative bacteria (except lab-adapted stains) is called an S-layer. A S-layer is composed of repeating units of one or two proteins (usually glycoproteins) that form a repeating lattice structure. These proteins are among the most abundant in a bacterial cell, and they are non-covalently attached to the rest of the bacterial envelope. In the case of Gram-positive non-mycobacteria, S-layer proteins are associated with the peptidoglycan. While in Gram-negative bacteria, they are likely associated with the lipid lipopolysaccharide (LPS), which is a potent endotoxin and can contribute to septic shock. Much like LPS is a major defining lipid present in Gram-negative bacteria, mycolic acids in the outer membrane [or outer layer (OL)] of mycobacteria are a defining characteristic of this group of bacteria. The presence of mycolic acids give mycobacteria a waxy texture. The S-layer for mycobacteria is thought to be attached to the outer layer (OL) or outer membrane (OM), which contains the mycolic acids. Finally, another (external-most layer) called a capsule can be found in both Gram-positive (including mycobacteria) and Gram-negative species. If present, the capsule will be the external-most layer; the capsule is generally composed of polysaccharides, but some species might include other components (e.g., proteins, smaller peptides, lipids, etc.). The presence of LPS in the OM of Gram-negative bacteria acts as a permeability barrier, due (mainly) to its amphipathic nature. Molecules that are amphipathic have both hydrophobic and hydrophilic characteristics. More specifically, the hydrophobic lipid portion of LPS, like all lipids, can restrict the diffusion of hydrophilic molecules across the membrane. However, because LPS also has an external hydrophilic sugar molecule, this can also be a barrier to the diffusion of hydrophobic molecules, which can usually pass through membranes more freely. This is one of the many reasons why Gram-negative bacteria are much harder to treat therapeutically. All life has to have some way of getting things outside the cell into the cell. The converse is also true (inside to out). As such, no cell envelope [cell wall + cell membrane(s)] can be completely closed off! Thus, the cell envelope must balance these protective functions while still enabling size/charge-limited diffusion of molecules near the inner membrane or into the periplasmic space. To achieve this, Gram-negative bacteria and Gram-positive mycobacteria have porins. Porins are trimeric, transmembrane protein channels that enable passive diffusion of molecules based on size and charge. Porins enable the passive transport of small, hydrophilic molecules from the outside of the cell into the periplasmic space (the space between cell membranes). Porins limit the efficacy of large antibiotics and the internal diameter, expression levels, and internal charge can all be associated with resistance to one or more antibiotics. The internal diameter of porins is approximately 1.5 nm (on average), and they can enable diffusion of molecules around 600 Da or less. S-layers must also still enable molecules to get close to the cell. S-layers in general have openings of 2-8 nm in diameter. Thus, S-layers are predicted to toggle resistance to the host immune response and environmental perturbations more so than providing direct resistance to larger antibiotics. While capsules frequently protect bacteria from environmental and immunological stressors, these too must be somewhat permeable, as we’ll see in later classes. In addition to changes to the terminal amino acids of peptidoglycan (the fifth D-alanine is often D-lactate in vancomycin-resistant bacteria), there are other mechanisms by which bacteria can resist antibiotics. One is through modulation of porin expression levels, the internal diameter of the porin, and/or the internal charge of the porin channel (as mentioned above). The only Gram-positive bacteria that have porins are mycobacteria. Remember, despite being Gram-positive, they have two cell membranes (the IM and OL). Another example is efflux pumps. These can be found in both Gram-negative and Gram-positive bacteria. These pumps perform active transport of molecules from the inside of the cell (or periplasmic space) to the outside of the cell. Clearly, the structure of the efflux pumps would differ depending on whether the bacteria have one or two cell membranes. LOs: Classes 1&2 Explain the function(s) of the bacterial cell wall. Describe how structural differences between Gram-positive, Gram-negative, and mycobacteria envelopes could affect antibiotic efficacy and transport into and out of the cytoplasm. (This means you also need to know what is common/different between all three.) Hypothesize how bacteria could evolve resistance to antibiotics, based on the structure of the cell envelope. Hypothesize how loss of specific cell-wall or cell envelope components would affect bacterial structure and viability. Draw a labeled schematic of Gram-positive, Gram-negative, and mycobacterial envelopes and peptidoglycan. Explain the mechanisms behind vancomycin, penicillin, and lysozyme. Hypothesize, based on bacterial characteristics and phenotype, the mechanism of a novel antibiotic.