Advanced Immunology I (MI211) -1/23/04
Answer Key to Homework Problem Set #1
1. Inflammation is traditionally defined by the four Latin words calor, dolor, rubor, and tumor, meaning
heat, pain, redness, and swelling. Detail the molecular events that led to such characteristic features
of an infected site. (10 points)
Pathogens are detected by complement proteins and neutrophils in the blood and/or detected by
resident tissue phagocytes such as macrophages. Detection can result in phagocytosis, destruction of the
pathogen, and phagocyte activation. Activated macrophages secrete pro-inflammatory cytokines, such as
IL-1, IL-6, TNF-(tumor necrosis factor-), and chemokines, and lipid mediators, such as histamine,
prostaglandins and leukotrienes. Cytokines can activate other macrophages. Soluble mediators secreted by
macrophages also lead to 3 key changes in local blood vessels: (1) an increase in the vasculature diameter
which increases local blood flow (there is also a reduction in blood flow velocity), which can cause
contribute to heat and redness, (2) endothelial cells lining the blood vessels are also activated to express
TNF-and adhesion molecules that will trap circulating leukocytes, and (3) an increase in vascular
permeability of local blood vessels. This leads to the exit of fluid and proteins, such as IgG and
complement, from the blood vessels into the tissues. There is also infiltration of additional leukocytes, such
as neutrophils, recruited by chemokines expressed by endothelial cells and activated macrophages. This
influx of fluid and cells leads to edema or swelling. Heat and swelling puts pressure on the nerves and
contributes to pain and redness.
The complement cascade can also trigger inflammation. C3b coated bacteria can be phagocytosed
and activate phagocytes. C3a and C5a can act as chemoattractants to recruit additional leukocytes.
Complement proteins can also activate endothelial cells.
2. Antimicrobial peptides (AMP) are evolutionary ancient and effective ways hosts wield against their
invading pathogen. To-date, there are more than 800 eukaryotic antimicrobial peptides described.
Name two AMPs and provide experimental evidence on how each of these AMPs kills microbes.
Here are some examples:
A) By the inhibition of intracellular functions.
Example 1:
Short, proline-rich antibacterial peptides from insects, pyrrhocoricin, drosocin, and apidaecin interact with
the bacterial heat shock protein DnaK and inhibit two major functions of DnaK, ATPase activity and
refolding of misfolded proteins.
Kragol et al. (see Kragol et al, Biochemistry, 2001, 40: 3016-3026) showed that:
1) Biologically active pyrrhocoricin made of L-amino acids diminished the ATPase activity of
recombinant DnaK. The inactive D-pyrrhocoricin analogue and the membrane-active antibacterial
peptide cecropin A or magainin 2 failed to inhibit the DnaK-mediated phosphate release from
adenosine 5'-triphosphate (ATP). Pyrrhocoricin binding was not observed to the homologous DnaK
fragment of Staphylococcus aureus, a pyrrhocoricin nonresponsive strain.
2) pyrrhocoricin also inhibited the ability of Dnak to refold misfolded proteins. They showed that
incubation with L-pyrrhocoricin or drosocin reduced the enzymatic activity of alkaline phosphatase
and beta–galactosidase in live bacteria. In contrast, D-Pyrrhocoricin, magainin 2, or buforin II, an
antimicrobial peptide involved in binding to bacterial nucleic acids (see below), had only negligible
effect.
Example 2:
Binding to DNA and inhibit DNA synthesis.
1) In studies by Xiong and coworkers using thrombin-induced PMP-1 (tPMPs), S. aureus cells remained
viable long after rapid membrane permeabilization, indicating that disruption of membrane is not the major
contributor to tPMP antimicrobial activity. tPMP-mediated inhibition of DNA and/or RNA synthesis
corresponded temporally with cell death but was not observed until 30 or more minutes after membrane
permeabilization (see Xiong et al, J Infect Diseases 2002, 186: 668-677). Interestingly, staphylocidal
effects did not appear to result from global cellular dysfunctions, since protein synthesis was inhibited to an
equivalent extent in strains susceptible or resistant to tPMP-1. Moreover, pre-exposure to agents that
selectively inhibit protein synthesis (30 S or 50 S subunit inhibitors) or DNA metabolism (DNA gyrase)
mitigated subsequent tPMP-1 induced killing of an otherwise susceptible S. aureus strain in vitro. These
findings implicate a direct inhibition of nucleic acid synthesis by tPMPs. The relatively strong negative
charge of nucleic acids is consistent with the hypothesis that cationic peptides bind to and inhibit these
molecules, not unlike histone proteins.
2) Buforin II. Using FITC-labelled buforin II and a gel retardation experiment, Park et al (Biochemical and
Biophysical Research Communications 1998, 244: 253-257) showed that buforin II killed E. coli without
lysing the cell membrane. The gel-retardation experiment showed that buforin II bound to DNA and RNA
after penetrating the cell membranes, resulting in the rapid cell death.
B) Disruption of physicochemical properties of target membrane.
Examples, Magainin (Xenopus skin), tachyplesins (horseshoe crab hemolymph) and cecropin (Drosophila).
For a fine review on how the mode of interaction between AMP is strongly dependent on the
physicochemical properties of both the peptide and the target membrane, see Matsuzaki, Biochemica et
Biophysica Acta 1999, 1462:1-10. Basically, cationic antimicrobial peptides, such as magainin
electrostatically recognizes anionic lipids that are abundant in bacterial membranes, forming a peptide-lipid
supramolecular complex pore, whereas the peptide does not effectively bind to zwitterionic phospholipids
constituting the outer leaflets of mammalian cell membranes because of the low hydrophobicity of the
peptide.
C) Pore Formation:
Pores formed by magainin and protegrin in membranes were directly demonstrated by crystallization, see
Yang et al, Biophysical J 2000, 79: 2002-2009.