LESSON 1.3 WORKBOOK. Bacterial structures. Workbook Lesson 1.3

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1 Colonize the ability of bacteria to adapt to permanently inhabit our bodies. Capsule an external layer made of sugars that surrounds some bacteria. Cell wall an external layer surrounding the plasma membrane of most bacteria. Plasma (cell) membrane a membrane that separates the internal cell contents from the outside environment. LESSON 1.3 WORKBOOK Bacterial structures Before we can discuss processes used to identify infectious diseases, details about specific diseases, and immune responses, we need to become familiar with the structures of bacteria, viruses, and immune barriers. In this lesson we will focus on the structures of bacteria that directly relate to their ability to cause infection. As we will see, these structures in disease-causing microbes are often precisely adapted for survival in a host. The bacterial envelope Figure 1: The envelope of the bacteria helps to keep it camouflaged from the immune system. When bacteria attempt to grow in, or colonize, an organism, the immune system responds to the foreign invader immediately. The first bacterial component that the immune system recognizes as foreign, is its outer surface called the envelope. The bacterial envelope is composed of a capsule, cell wall, and plasma membrane (Figure 2). Since the immune system is so vigilant, pathogenic bacteria must try to stay one step ahead by continually modifying their surface components, so as to go undetected. The capsule is camouflage and protection from the environment The capsule is not essential to bacterial life and not all bacteria have one, but those that do, have an advantage in infecting their hosts. The capsule is composed of long chains of sugar molecules. It plays two roles: First, it is durable and 1. The bacterial envelope is composed of all of the following EXCEPT a. sputum b. capsule c. plasma membrane d. cell wall 27

2 Osmotic pressure the pressure on the plasma membrane as the result of water flowing from a dilute outside environment into more concentrated internal cell content. Pneumonia inflammation of the lung caused by microbes. Meningitis inflammation of the membranes covering the brain and the spinal cord. Transport channels protein complexes in the cell membrane used to transport ions and molecules across the membrane. so can protect the bacterium from physical stresses such as osmotic pressure. Second, it camouflages the bacteria from the immune system. A strategy that some bacteria use, is to make their capsule with the same sugars that are found in the host, and thus avoid being detected as foreign. The immune system therefore doesn t recognize the bacterium as a foreign interloper and doesn t target it for death. Examples of bacteria that use a capsule to evade immune system recognition are Streptococcus pyogenes which causes both strep throat and flesh eating disease, Streptococcus pneumoniae which causes pneumonia, and Neisseria meningitidis which causes meningitis. The cell wall protects the bacteria from environmental stresses In contrast to the capsule, most bacteria have some kind of cell wall. It is located internal to the capsule (if there is one) and external to the plasma membrane. The cell wall reinforces the cell membrane and protects it from environmental stress, and particularly osmotic pressure. If we think about the stresses bacteria face in their natural environment, the reasons they need a cell wall becomes clear. For example, intestinal bacteria, such as E. coli, are constantly exposed to bile salts that have detergent-like properties able to dissolve a lipid-containing cell membrane. The cell wall also plays a key role in causing infection, as we will see later in the course. As in eukaryotic cells, the plasma membrane in bacteria separates the cell from the environment Figure 2: The bacterial envelope is made up of the capsule (if present), the cell wall, and the plasma membrane. The bacterial plasma membrane, also called the cell membrane, lies internal to the cell wall. It is the definitive structure of a cell since it sequesters the molecules of life in a unit, separating it from the environment. Its main function is that of a selective permeability barrier that regulates the passage of substances into and out of the cell. The bacterial membrane allows passage of water and small uncharged molecules, but does not allow passage of larger nutrients or ions, except through special transport channels. One key way in which the bacterial plasma membrane is different from the eukaryotic plasma membrane, is that it carries out functions of energy generation, since bacteria don't have mitochondria. 2. Most bacteria have some kind of cell wall. The cell wall is an important structure for bacteria because a. it reinforces the cell membrane and protects it from environmental stress b. it exports toxins to the plasma membrane c. it provides camouflage d. it contains waxes that makes it impervious to many chemicals 28

3 Murein (peptidoglycan) a complex molecule (polymer) made of sugars and amino acids, that is the building block of the bacterial cell wall. The acid-fast solution to membrane protection: slow growing but very tough and well-camouflaged bacteria Acid-fast bacteria have cell walls that contain large amounts of waxes. This protective cover makes them impervious to many chemicals and able to avoid being killed by immune cells. The cost of this protection, is that they grow very slowly, probably because they cannot take up nutrients very rapidly. Only a few pathogenic bacteria are acid-fast. One notable example is the tuberculosis causing agent, Mycobacterium tuberculosis, which divides only once every 24 hours. This is very slow growth rate! For comparison, E. coli divides every 20 minutes. The acid-fast coating allows the Mycobacterium to wall itself off from the immune system when it infects the lungs. Its slow division rate, coupled with resistance to immune attack, results in infections that can persist for a long time. Figure 4: Gram-positive bacteria have a rigid, external cell wall made of murein layers (purple rectangles) and LTA (cells with thin green protrusions up through cell wall). The larger red bodies in the cell membrane are membrane proteins. Figure 3: Acid-fast bacteria have a waxy, durable coating, and they grow slowly. The Grampositive solution to membrane protection Most bacteria protect their cell membranes with a thick exterior cell wall that plays a critical role in maintaining rigidity. But the cell walls differ in structure and organization among different bacteria and there are two major types of cell wall. A staining technique, called Gram staining (named after the microbiologist who designed it), is used to determine the type of cell wall: Gram-positive or Gram-negative. In Gram-positive bacteria the wall is made of many layers of a polymer composed of sugars and amino acids, called murein. Bacteria are the only organisms to have murein, and so bacterial cell walls are easily recognized as foreign by the immune system. The murein on the surface of a Gram-positive bacterium can absorb and retain a purple dye during staining, hence the term Gram-positive. This technique is used to identify Gram-positive bacteria from an infected person. 3. Which statement represents the advantage and disadvantage of being acid-fast? a. it has a cell wall; it separates the cell from the environment b. it separates the cell from the environment; it has a cell wall c. it contains waxes; it reproduces slowly d. it reproduces slowly; it contains waxes 29

4 Peptides small proteins made of a few amino acids. Bacilli rod-like bacteria. Cocci sphere shaped bacteria Lipoteichoic acid (LTA) a polymer of modified lipids used to strengthen the Gram-positive cell wall. Lipopolysaccharides (LPS) large molecules composed of lipids and polysaccharides, critical in the making of the Gramnegative outer membrane. How does murein work? Murein is composed of sugar chains that are cross-linked to one another and to chains of peptides in an organization resembling a chain link fence. Layers of murein are wrapped around the length and width of the bacterium to form a sac. The murein sack is critical in maintaining bacterial shape such as bacilli or cocci. Figure 5: Murein The thick dense layer of murein allows bacteria to survive in is the target for many environments where the osmotic pressure (pressure on the membrane antibiotics. either from the inside or the outside) is high. This allows the bacteria to live in solutions that have a low or high salt concentration. However, if the murein is breached the cells may burst or shrink depending on the outside environment. Gram positive cell wall components can cause illness Gram-positive bacterial cell walls contain other unique polymers such as the lipid (fat) molecule lipoteichoic acid (LTA). Both murein and LTA are recognized as foreign by the immune system. The Gram-positive bacterium Staphylococcus aureus for instance, has a capsule that cannot always camouflage it from the immune system. The cell wall murein and LTA that lie beneath the capsule, are easily recognized by the immune system which then initiates a response to the invading bacteria. This gives rise to typical symptoms of bacterial infection such as fever. The Gram-negative solution to membrane protection Figure 6: Gram-stained cells: Gram-positive bacteria stain purple and Gram-negative bacteria stain pink. Gram-negative bacteria have adopted a radically different solution to the problem of how to protect their plasma membranes. Their cell wall also has a murein component for rigidity, but it is far less elaborate than in the Gram-positive cell wall. Instead, Gram-negative bacteria build a second membrane external to the murein wall (figure 7). This outer membrane is just like other typical biological membranes, except it contains molecules called lipopolysaccharides (LPS), that are unique to Gram-negative bacteria. The composition of this outer membrane doesn't allow Gram-negative bacteria to keep the purple Gram dye during the decolorization step. Instead they turn pink when the last pink dye is added after decolorization. 4. The following are true about the differences between Gram-positive and Gram-negative bacteria EXCEPT a. Gram-positive bacteria can keep a purple dye while Gramnegative bacteria cannot b. Gram-positive bacteria have an additional outer membrane external to the murein cell wall while Gram-negative bacteria have a rigid external cell wall made from murein c. Gram-positive have LTA while Gram-negative have LPS d. Gram-positives use their cell wall components to cause illness while Gram-negatives use their cell wall to inhibit antibiotics 30

5 Enzymes proteins produced by living organisms that are used to speed up chemical reactions. The unique LPS of Gram-negative bacteria can cause symptoms of disease Since LPS is the major component of the Gram-negative outer membrane, just like Gram-positive LTA, it is recognized as foreign and stimulates a strong immune response. LPS is very potent even small amounts of LPS in the bloodstream will cause the host to become severely ill. Each Gram-negative species has its own unique LPS. As a consequence, the immune system's response to the different kinds of LPS is specific as well. Figure 7: Gram-negative bacteria have an additional outer membrane, external to the murein cell wall. The outer membrane contains LPS. The red shapes in the inner and outer membranes are membrane proteins. The Gram-negative cell wall can inhibit antibiotics Between the inner and outer membrane of Gram-negative bacteria, in addition to the thin murein cell wall layer, there is a gel-like solution of enzymes. These enzymes can play important roles in infection. One enzyme in particular, beta-lactamase, can inactivate certain types of antibiotics like penicillins and cephalosporins, which interfere with the synthesis of the bacterial cell wall. This is one of the reasons why Gram-negative bacteria are somewhat more resistant to antibiotics than Gram-positives. Examples of the many disease-causing bacteria with Gram-negative cell walls are Escherichia coli O157:H7 that causes intestinal diarrhea (hamburger disease), and Haemophilus influenzae that causes flu-like symptoms. The complex architecture of the Gram-negative cell wall must work very well because in nature (but not necessarily in the human body) Gram-negatives outnumber Gram-positives! 31

6 Chemotaxis the targeted movement of an organism towards or away from a stimulus. Flagella How bacteria get around Why bacteria need to move Bacteria, just like other organisms, need to move in order to obtain food and to escape predators. Some bacteria are carried passively by fluid or air currents. Others have special long, whip-like appendages on their outer surface, called flagella, that allow them to be actively motile. Depending on the species, bacteria may have one or several flagella. Flagella allow bacteria to move toward substances that they need, such as nutrients, and away from those that they want to avoid such as toxins or immune cells that might kill them. This process of movement in response to the concentration of chemicals in their environment is called chemotaxis. How flagella help bacteria move When a bacterium has a single flagellum, its counter-clockwise rotation propels the bacterium forward, and a flicking motion causes reorientation. If the bacterium has several flagella, they form a bundle when all of them spin counterclockwise, and this propels the bacterium forward. This forward propulsion is called swimming. However, if any of the flagella begin to rotate clockwise, the bundle unwinds and movement becomes erratic causing the bacterium to tumble randomly and reorient its direction. Figure 8: Swimming allows bacteria to chemotax towards or away from a stimulus. 5. Flagella is to as pili is to. a. movement; attachment b. clockwise; counterclockwise c. counterclockwise; clockwise d. attachment; movement and immunity 32

7 Antigen a structure which can be recognized by the immune system as foreign. These two types of motion, swimming and tumbling, both occur during chemotaxis. When moving in a favorable direction such as towards nutrients, swimming predominates and tumbles are suppressed. However, when the bacterium's direction of motion is unfavorable (e.g., movement towards immune cells), tumbles are no longer suppressed and occur much more often, with the chance that the bacterium will thus be reoriented in a more favorable direction and then proceed to swim. How flagella impact infection Figure 9: Vibrio cell with flagella. Flagella can aid in the movement of pathogenic bacteria and their spread through the host. An example is Helicobacter pylori, which uses multiple flagella to propel itself through the mucus lining to reach the stomach epithelium. Flagella are also a target of the immune system, since their constituent protein flagellin, also called the bacterial H antigen, is a potent immune stimulator. Bacteria therefore have to continually modify their flagella in an attempt to camouflage themselves. For example, one of the Salmonella species that causes food poisoning (Salmonella enterica) has two different kinds of H protein, types 1 and 2. When S. enterica infects a host, the immune system will recognize and start building a response to the current H protein of the flagella, e.g., type 1. However, the bacteria have the ability to switch and start building their flagella with protein type 2 which the immune system has not recognized yet, giving the bacteria more time to replicate. Pili How bacteria stick around Some sites in the host are very inhospitable to infection. For instance the nasal cavity is continually being cleansed by sneezing, while swallowing washes contents of the mouth down into the harsh acid environment of the stomach. Bacteria that need to survive these challenges use special structures pili to attach to cells of the host or other bacteria to avoid being washed away. Figure 10: Pili are hair-like structures on the bacterial surface, used for attachment to surfaces. 33

8 Gonoccoci Gram-negative bacteria which cause gonorrhea. Gonorrhea sexually transmitted disease caused by gonoccoci, also called Neisseria gonorrhoeae. Figure 11: Gonorrhea causing bacteria use pili to adhere to the genital tract. How pili impact disease Pili are used to stick to surfaces Like flagella, pili also protrude from the cell wall, but unlike flagella they are usually used to attach to specific surfaces and rarely for movement. Pili are shorter than flagella and often distributed in large numbers over the entire surface of the bacteria, like little hairs. During attachment the tip of the pilus attaches to cells or other bacteria, in a process that resembles the use of grappling hooks or Velcro. In addition to these common pili some bacteria also use "sex pili" to form a tube between donor and recipient cells through which DNA is transferred between them. Like flagella, pili are often recognized by the immune system, and therefore many bacteria also continually vary the types of pili they produce. This enables them to keep one step ahead of the immune system. For example, gonococci that cause gonorrhea can make many different types of pili proteins. However, they only make a few at any given time. If the types of pili proteins that the bacteria make at a given time during infection, are easily recognized by the immune system, then the bacteria will be attacked and killed. In response to this immune pressure, the gonococci constantly switch pili, so that the immune system doesn't get a chance to recognize them. In this quick-change scenario the bacteria keep one step ahead of the immune system. This is why attempts to immunize against gonococci using a vaccine that recognizes only one kind of pilus protein have failed so far. Spores How bacteria survive hostile conditions When environmental stresses become too harsh, simply sticking to a surface is not enough. Some bacteria wait out harsh stresses, such as starvation, by forming spores. Spores are formed when bacteria lose their cellular content, form a protective coat around their DNA, and enter a dormant state. Each bacterium usually makes only one spore. When the spore finds that conditions have become favorable again, it gets reactivated and forms a live bacterium that can grow and divide. Figure 12: Spores are first formed inside the cell and are then released to the outside. 6. Spores help bacteria stay alive by a. allowing bacteria to be constantly active in harsh conditions. b. allowing bacteria to be dormant in harsh conditions and active in better ones. c. causing food poisoning. d. none of the above 34

9 Tetanus an infection caused by Clostridium tetani characterized by muscle spasms due to toxins produced by the bacteria. Botulism illness that results from toxins produced by the bacterium Clostridium botulinum. How spores impact disease Some particularly important pathogenic bacteria form spores. The Clostridium species that cause tetanus and botulism are key examples. Spores are highly resistant to desiccation, heat, and radiation. Tetanus spores can lie dormant in the soil for many years before becoming reactivated when they enter the body through a cut in the skin. Botulinum spores, on the other hand, are resistant to heat and can survive food canning processes. They can cause food poisoning when the food is eaten even years after preparation if the preservation method used was insufficient to kill them. When swallowed they can get reactivated in the intestines, and start growing and multiplying. Bacterial chromosome and plasmids bacterial genes Figure 13: Nucleoid, the dense, whitish area of DNA packing in the cytoplasm. Bacterial genes are in the nucleoid Bacteria do not have a nucleus like eukaryotic cells. Instead there is a DNA-rich area in the cytoplasm called the nucleoid, where the DNA is present in a compacted form. Also unlike eukaryotic cells, where the DNA is in the form of genes arranged on linear chromosomes; bacteria usually have their genes arranged on one or two circular chromosomes. Since the DNA is not enclosed in a membrane-bound nucleus, once transcription begins, the translational machinery in the cytoplasm has instant access to the transcript, and can start protein synthesis simultaneously. This is in contrast to eukaryotes where there is a delay, because the transcript has to be exported from the nucleus to the cytoplasm for translation to occur. Bacteria are thus better equipped to deal with changes in their environment, by making rapid changes in the proteins that are synthesized. Bacterial genes obviously play a key role in their ability to cause disease because they determine every single characteristic of the bacterium, such as whether they are Gram-positive or negative, have multiple types of flagella or pili, and whether they can form spores. 7. Plasmids a. are DNA molecules that are distinct and can replicate independently from chromosomal DNA. b. can transfer advantageous genes between bacteria through sex pili. c. are involved in the production of toxins. d. all of the above 35

10 Toxins molecules (usually proteins) produced by bacteria that are toxic to the host's cells, and a major reason for the symptoms of an infectious disease. Antibiotics molecules, produced usually by bacteria and fungi, that have the ability to suppress the growth of microbes or kill them. Bacterial genes are also found in plasmids Unlike eukaryotic cells, whose genes are solely confined to chromosomes, bacteria have additional genes located on small circular pieces of DNA called plasmids. Although these plasmids are not critical to the life of the bacterium itself, they may confer key advantages. In fact, the two most potent toxins known to man, tetanus and botulinum toxins, are produced from plasmids that are found in different Clostridium species. Figure 14: Plasmids (the pale yellow loops) are circular pieces of DNA found in the cytoplasm. Plasmids can also contain genes that make the bacteria resistant to specific antibiotics. The advantage of plasmid DNA, rather than chromosomal DNA, is that plasmids are relatively easy to transfer between bacteria, using their sex pili. This exchange of advantageous genes is of great benefit to the bacteria but causes a public health nightmare. Bacteria acquiring drug resistance genes from other bacteria can rapidly become problematic. Vibrio cholerae, which causes cholera, routinely acquires new genes via plasmids. If such species exchange genes they can give rise to a highly infectious species that is resistant to many more antibiotics. 36

11 STUDENT RESPONSES Bacterial structure Description How is it advantageous? Capsule Gram-positive cell wall Gram-negative cell wall Plasma membrane Murein LTA LPS Flagella Pili Spore Nucleoid Plasmid 37

12 STUDENT RESPONSES Some bacteria use pili "like Velcro". Sketch what Velcro looks like up close how could hair-like structures with a similiar shape help bacteria infect hosts? Would you expect the capsule to perform this same function? Why or why not? Remember to identify your sources Why do you think it is important to organize bacteria based on the different structures they have (e.g cell wall type, presence of capsule, etc.)? 38

13 TERMS TERM DEFINITION Antibiotics Molecules, produced usually by bacteria and fungi, that have the ability to suppress the growth of microbes or kill them. Antigen A structure which can be recognized by the immune system as foreign. Bacilli Rod-like bacteria. Botulism Illness that results from toxins produced by the bacterium Clostridium botulinum. Capsule An external layer made of sugars that surrounds some bacteria. Cell wall An external layer surrounding the plasma membrane of most bacteria. Chemotaxis The targeted movement of an organism towards or away from a stimulus. Cocci Sphere-shaped bacteria Colonize Ability of bacteria to adapt to permanently inhabit our bodies. Enzymes Proteins produced by living organisms that are used to speed up chemical reactions. Gonoccoci Gram-negative bacteria which cause gonorrhea. Gonorrhea Sexually transmitted disease caused by gonoccoci, also called Neisseria gonorrhoeae. Lipopolysaccharides (LPS) Large molecules composed of lipids and polysaccharides, critical in the making of the Gram-negative outer membrane. Lipoteichoic acid (LTA) A polymer of modified lipids used to strengthen the Gram-positive cell wall. Meningitis Inflammation of the membranes covering the brain and the spinal cord. Murein (peptidoglycan) A complex molecule (polymer) made of sugars and amino acids, that is the building block of the bacterial cell wall. 39

14 TERMS TERM DEFINITION Peptides Plasma (cell) membrane Pneumonia Tetanus Toxins Transport channels Turgor pressure Small proteins made of a few amino acids. A membrane that separates the internal cell contents from the outside environment. Inflammation of the lung caused by microbes. An infection caused by Clostridium tetani characterized by muscle spasms due to toxins produced by the bacteria. Molecules (usually proteins) produced by bacteria that are toxic to the host's cells, and a major reason for the symptoms of an infectious disease. Protein complexes in the cell membrane used to transport ions and molecules across the membrane. The pressure caused by the plasma membrane on the cell wall as the result of water flowing from a dilute outside environment into more concentrated internal cell content. 40