Pelvic Inflammatory Disease Case File

Pelvic Inflammatory Disease Case File

Eugene C.Toy, MD, William E. Seifert, Jr., PHD, Henry W. Strobel, PHD, Konrad P. Harms, MD

❖ CASE 10

A 20-year-old female presents to the ER with complaints of fever, pelvic pain, and some nausea and vomiting increasing over the last 2 days. She denies diarrhea or sick contacts. She is currently sexually active with a new partner. On examination she has a temperature of 38.9°C (102°F) and appears ill. She has moderate bilateral lower abdominal tenderness and minimal guarding without rebound or distention. Bowel sounds are present and normal. Pelvic exam revealed a foul-smelling discharge through cervix with severe cervical motion tenderness and bilateral adnexal tenderness. Cervical cultures were obtained. Patient was begun on a quinolone antibiotic.

◆ What is the most likely diagnosis?

◆ What is the biochemical mechanism of action of the quinolone?

◆ What is the role of deoxyribonucleic acid (DNA) topoisomerases?

ANSWERS TO CASE 10: PELVIC INFLAMMATORY

DISEASE

Summary: A 20-year-old female with history of new sexual partner, fever, abdominal and pelvic pain, foul-smelling discharge through cervical os, and severe cervical motion tenderness.

◆ Most likely diagnosis: Pelvic inflammatory disease

◆ Biochemical mechanism of action of quinolone: Inhibits DNA gyrase

◆ Function of topoisomerases: Enzymes that assist in formation of superhelices and regulate the breaking and rejoining of DNA chains

CLINICAL CORRELATION

Pelvic inflammatory disease (PID) is usually an acute infection affecting the fallopian tubes and possibly the uterus and ovaries. It is generally sexually transmitted, caused by organisms such as Chlamydia or Neisseria gonorrhoeae (gonorrhea). The diagnosis is made clinically based on the typical history and physical examination. A purulent cervical discharge is highly suggestive. Nearly all patients have cervical motion tenderness, that is, pain with motion and palpation of the cervix. The treatment is with antibiotics. Quinolone antibiotics have been popular in the past; however, increasing bacterial resistance, particularly in Southeast Asia and California, has rendered these agents less desirable. Complications of PID include infertility or ectopic pregnancy (pregnancy in the tube) as consequence of tubal damage.

APPROACH TO DNA TOPOLOGY

Objectives

1. Know about DNA superhelices.

2. Understand the role of topoisomerases and DNA gyrase.

3. Understand the importance of histones and nonhistone proteins.

4. Be familiar with nucleosomes and polynucleosomes.

5. Know about the chromosomal structure.

Definitions

DNA gyrase: A type II topoisomerase enzyme present in bacteria that introduces negative supercoils into the DNA double helix in advance of the replication fork.

Histones: Proteins containing a large number of positively charged amino acids (lysine, arginine) that associate with DNA to form nucleosomes.

Nucleosome: Disk-shaped particles that consist of a core of histone protein around which DNA is wrapped. They are a structural unit of chromatin.

Supercoiling: The act of DNA winding on itself as a result of unwinding caused by replication forks.

Topoisomerase: Enzymes that control the amount of supercoiling in DNA. Type I topoisomerases will cleave one strand of DNA to relieve supercoiling, whereas type II 

topoisomerases will cleave both strands of the DNA double helix.

DISCUSSION

The quinolones are broad-spectrum synthetic antibiotic drugs that contain the 4-quinolone ring (Figure 10-1). The first quinolone, nalidixic acid, was synthesized in 1962. More recently, a family of fluoroquinolones has been produced that contain a fluorine substituent at position 6 and a carboxylic acid moiety in the 3 position of the basic ring structure. R1, R7, and X are substituted with different side chains for the purpose of increasing bioavailability of the compound. A typical example of a fluoroquinolone is ciprofloxacin. The quinolone antibiotics target bacterial DNA gyrase in many gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and the like.

The normal biological functioning of DNA-like ribonucleic acid (RNA) transcription and DNA replication occurs only if it is in the proper topological state. In duplex DNA, the two strands are wound about each other once every 10 bp, that is, once every turn of the helix. Double-stranded circular DNA can form either negative supercoils when the strands are underwound or positive supercoils when they are overwound (Figure 10-2). Negative supercoiling introduces a torsional stress that promotes unwinding or separation of the right-handed B-DNA double helix, while positive supercoiling overwinds such a helix.

Supercoiling is controlled by a remarkable group of enzymes known as topoisomerases, which alter the topology of the circular DNA but not its

Figure 10-1. Structural formula for a fluoroquinolone. R1, R7, and X are side chains.

Figure 10-2. Positive and negative supercoiling in circular DNA.

covalent structure. There are two classes of topoisomerases. Type I topoisomerases relax DNA from negative supercoils formed by the action of type II topoisomerase by creating transient single-strand breaks in DNA without any expense of ATP. Type II topoisomerases (also called DNA gyrases) change DNA topology by making transient double-strand breaks in DNA and require ATP consumption (Figure 10-3).

During DNA replication, type II topoisomerase, or TOPO II, plays an important role in the fork progression by continuous removal of the excessive positive supercoils that stem from the unwinding of the DNA strands. TOPO II has the ability to cut both strands of a double-stranded DNA molecule, pass another portion of the duplex through the cut, and reseal the cut in a process that uses ATP. Hydrolysis of ATP by TOPO IIs inherent ATPase activity powers the conformational changes that are critical for the enzyme’s operation. Based on the DNA substrate, TOPO II can change a positive supercoil into a negative supercoil or increase the number of negative supercoils by two.

The DNA gyrase of E. coli is composed of two 105,000-dalton A subunits and two 95,000-dalton B subunits encoded by gyrA and gyrB genes, respectively. The A subunits, which carry out the strand-cutting function of the gyrase, are the site of action of the quinolones. DNA gyrase inhibition disrupts DNA replication and repair, transcription, bacterial chromosome separation during division, and other cell processes involving DNA. The drugs inhibit gyrase-mediated DNA supercoiling at similar concentrations that are required to inhibit bacterial growth (0.1 to 10 μg/mL). Mutations in gyrA gene that encodes the A subunit of the polypeptide can confer resistance to these drugs. Eukaryotic cells lack DNA gyrase but have a similar type of

Figure 10-3. DNA gyrase action and quinolone inhibition.

topoisomerase that can remove positive supercoils from eukaryotic DNA to prevent its tangling during replication. Quinolones inhibit eukaryotic topoisomerase at much higher concentrations (100 to 1000 μg/mL).

Although bacterial DNA is compacted as large circular chromosomes with a single replication origin, most eukaryotic DNA is much more highly organized and is associated with many proteins to form chromatin that contains multiple replication origins. The general structure of chromatin has been

found to be remarkably similar in the cells of all eukaryotes. The most abundant proteins associated with eukaryotic DNA (somewhat more than half its mass) are histones, a family of basic proteins rich in the positively charged amino acids lysine and/or arginine, which interact with the negatively charged phosphate groups in DNA. There are five types of histones that are evolutionarily conserved: H1, H2A, H2B, H3, and H4. Eight histone molecules (two each of H2A, H2B, H3, and H4) form an ellipsoid approximately 11 nm long and 6.5 nm in diameter. DNA coiled around the surface of this ellipsoid is termed the nucleosome core particle and has approximately 1¾ turns or 166 bp before it proceeds on to the next (Figure 10-4). The complex of histones plus DNA resembles a bead like structure and is called a nucleosome.

Figure 10-4. Schematic representation of nucleosomes showing DNA wrapped around a core of histones H2A, H2B, H3, and H4. Histone H1 associates with the linker region of DNA.

Composed of DNA and histones, nucleosomes are approximately 10 nm in diameter and are primary structural units of chromatin. Between every two nucleosomes there is a stretch of DNA, the linker region, which varies in length from 15 to over 55 bp. Histone H1 can associate with the linker region to aid in folding of DNA into more complex chromatin structures. The question of how the highly ordered nucleosome is formed can be explained by the fact that nucleosome assembly is facilitated by molecular chaperones. In the presence of nucleoplasmin (an acidic protein) and DNA topoisomerase I (nicking-closing enzyme) the nucleosome assembly proceeds rapidly without histone precipitation. Nucleoplasmin binds to histones but not to DNA or nucleosomes. It functions as a molecular chaperone to bring histones and DNA together in a controlled fashion and prevents their nonspecific aggregation through their otherwise strong electrostatic interactions. The nickingclosing enzyme acts to provide the nucleosome with its preferred level of supercoiling. Six nucleosomes are packed per turn further into a spiral or solenoid to form a regular array of polynucleosomes of 30 nm length.

As a 30-nm fiber, the typical human chromosome would be 0.1 cm in length and could span the nucleus more than 100 times. Clearly, there must be a still higher level of folding. This higher order packaging is one of the most fascinating but also most poorly understood aspects of chromatin. Chromosomes are generally decondensed during interphase. Several studies of interphase chromosomes

have suggested that each long DNA molecule in a chromosome is divided into a large number of discrete domains that are folded differently. The regions that are least condensed correlate well with the regions that are actively synthesizing RNA. All chromosomes adopt a highly condensed conformation during mitosis and reflect a coarse heterogeneity of chromosome structure. Other complex chromosomal structures found in eukaryotes are in histone-depleted metaphase chromosomes. These structures form radial loops around a central fibrous protein called scaffold. Most of these loops are organized into condensed 300 Å filaments but the exact nature of these formations is not known. Nonhistone proteins, whose hundreds of varieties constitute approximately 10 percent of chromosomal proteins, are thought to be involved in these processes.

COMPREHENSION QUESTIONS

[10.1] A 38-year-old woman, who works as an administrative assistant for a large company, opened a package and found a suspicious white powder. Analysis of the powder indicates that it contained traces of the bacterium Bacillus anthracis. The woman was treated with ciprofloxacin, an effective antibiotic. Ciprofloxacin’s mechanism of action is best described as an inhibition of which of the following?

A. Bacterial dihydrofolate reductase

B. Bacterial peptidyl transferase activity

C. Bacterial RNA polymerase

D. DNA gyrase

E. DNA polymerase III

[10.2] The Rubenstein-Taybi syndrome (RTS) is a genetic disease that is characterized by distinctive facial features, broad thumbs, broad big toes, and mental retardation. The affected gene is CBP (CREB-binding protein gene), which codes for a transcriptional activator. The RTS phenotype is best expressed by a haploinsufficiency model, in which two functional copies of the gene are required to produce sufficient CBP for proper development. CBP has a histone acetyltransferase activity, which does which of the following ?

A. Inhibits RNA polymerase II

B. Helps expose the promoters of genes

C. Inhibits the splicing of heterogeneous nuclear RNA (hnRNA) to messenger RNA (mRNA)

D. Prevents the addition of a poly-A tail to mRNA

E. Activates the formation of nucleosomes

[10.3] Acetylation and deacetylation of lysine residues on histone proteins provide one mechanism by which transcription can be activated or repressed. Which one of the histone proteins is least likely to participate in this process?

A. H1

B. H2A

C. H2B

D. H3

E. H4

Answers

[10.1] D. Ciprofloxacin is a fluoroquinolone antibiotic that will inhibit the strand-cutting function of A subunit of DNA gyrase, a bacterial topoisomerase II that introduces negative supercoils ahead of the replication fork. This disrupts DNA replication and repair, transcription, bacterial chromosome separation, and other bacterial processes involving DNA. At much higher concentrations, the type II topoisomerases of eukaryotic cells can be inhibited.

[10.2] B. When lysine residues in the N-terminal portion of histones are acetylated, it decreases the positive charge of the histone proteins and weakens the interaction between the histones and the DNA. As a result, the nucleosomes are “opened up” and lead to gene activation.

[10.3] A. Nucleosomes are disk shaped particles that consist of a core of histone protein around which DNA is wrapped. A short linker region of DNA joins nucleosomes. The core of the nucleosomes is made up of two copies each of histones H2A, H2B, H3, and H4. These histone proteins have N-terminal “tails” that contain lysine residues that can be reversibly acetylated, affecting the electrostatic interaction of the DNA with the histones. Histone H1 is not part of the nucleosomes core, therefore acetylation would likely not affect the protein–DNA interaction of the nucleosomes.

BIOCHEMISTRY PEARLS

❖ The quinolone antibiotics target bacterial DNA gyrase in many gram-negative bacteria.

❖ Supercoiling is controlled by topoisomerases which alter the topology of the circular DNA but not its covalent structure.

❖ Type I topoisomerases relax DNA from negative supercoils formed by the action of type II topoisomerase by creating transient single-strand breaks in DNA.

❖ Type II topoisomerases (also called DNA gyrases) change DNA topology by making transient double-strand breaks in DNA.

❖ Histones are abundant proteins associated with eukaryotic DNA and are a family of basic proteins rich in the positively charged amino acids lysine and/or arginine, which interact with the negative charges of DNA.

References

Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell, 4th ed. New York and London: Garland, 2002. 

Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology, 4th ed. New York: Freeman, 2000. 

Petri WA. Anti-microbial agents. In: Goodman AG, Gilman LS, eds. The Pharmacological Basis of Therapeutics, 10th ed. New York: McGraw-Hill, 2001. 

Prescott LM, Harley JP, Klein DA. Microbiology, 3rd ed. Boston, MA: W.C. Brown, 1996. 

Voet D, Voet JG, Pratt, CW. Fundamentals of Biochemistry, upgrade ed. New York: John Wiley, 2002.

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