Erythromycin and Lyme Disease Case File

Erythromycin and Lyme Disease Case File

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

❖ CASE 9

A 40-year-old male returned from a deer-hunting trip approximately 6 weeks ago, and presents to clinic with multiple complaints. He states that recently he has had worsening joint pain and “arthritis” in multiple joints that seems to move to different spots. Patient also complains of some numbness in his feet bilaterally. The patient denies any medical problems and he had a normal annual physical prior to hunting trip. On further questioning, he remembered having a rash on his body and the lesions were circular and appeared to be resolving in the center. He noted that he felt really bad once he got home with muscle ache (myalgias), joint ache (arthralgias), stiff neck, and severe headache. He also remembered that many of his hunting friends had experienced flea and tick bites and is quite sure he was bitten as well. The physical exam is essentially normal except some joint tenderness of left knee and right shoulder. After making your diagnosis you gave him a prescription for erythromycin.

◆ What is the most likely diagnosis?

◆ What is the biochemical mechanism of action of erythromycin?

ANSWERS TO CASE 9: ERYTHROMYCIN AND LYME DISEASE

Summary: A 40-year-old male who presents with migrating arthralgias and neurologic changes which were preceded by a centrally clearing circular rash, myalgias, and headache after a hunting trip where he was exposed to fleas and ticks.

◆ Diagnosis: Lyme disease

◆ Biochemical site of action of erythromycin: Inhibits bacterial protein biosynthesis at the translocation step of translation

CLINICAL CORRELATION

Lyme disease is a multisystem disease caused by the spirochete (spiral-shaped bacteria), Borrelia burgdorferi, nearly always transmitted by tick bite. Lyme disease is much more common in the New England area, usually during the late spring and early summer months. The deer tick is the main vector of transmission. The first stage is an acute infection with a red papule developing at the site of the bite, sometimes with lymphadenopathy and fever. The second stage includes disseminated infection and can lead to involvement of the heart, brain, joints, and skin. The typical target-shaped skin lesions may be seen. The third stage is a chronic infection and may last for years. Antibiotics are the treatment of choice, usually tetracyclines or penicillins.

APPROACH TO PROTEIN BIOSYNTHESIS

Objectives

1. Understand protein biosynthesis (initiation, elongation, translocation, termination).

2. Know key differences between prokaryotic and eukaryotic protein synthesis.

3. Know the role of ribosomal ribonucleic acid (rRNA).

4. Know the mechanism of action of different antibiotics with protein biosynthesis.

Definitions

A-site: The acceptor site on the ribosome into which an aminoacyl-charged transfer RNA (tRNA) is brought that has an anticodon that is complementary to the messenger RNA (mRNA) codon. The ribosome will then catalyze the formation of a peptide bond with the aminoacyl group on this tRNA and the growing peptide chain.

Anticodon: The three-base sequence on a tRNA that will base pair with the three-base sequence on the mRNA. The anticodon is specific for an amino acid; it translates the DNA sequence into an amino acid sequence in the protein produced.

Codon: The three-base sequence of an mRNA that code for a particular amino acid.

Elongation factors: Proteins required for bringing the aminoacyl-tRNA to the A-site, codon recognition, and translocation of the newly elongated peptidyl-tRNA from the A-site to the P-site.

Initiation factors: Proteins required for assembly of the ribosomal complex with mRNA and Met-tRNA so that protein synthesis can proceed.

Nucleolar organizing region (NOR): Area of the nucleolus where a great deal of rRNA transcription and synthesis occurs.

P-site: The peptidyl site on the ribosome to which Met-tRNA is brought to base pair with the mRNA sequence AUG. It is also the site to which the peptidyl RNA is moved in a process known as translocation following the formation of a new peptide bond.

Posttranscription modification of tRNA: The synthesis of tRNA involves modification of some uridine nucleotides to unusual nucleotides, such as pseudouridine, ribothymidine, and dihydrouridine.

Ribosomes: Complexes of proteins and rRNA on which protein synthesis occurs. There are two major subunits to all ribosomes, a larger subunit (50S for prokaryotes, 60S for eukaryotes) and a smaller subunit (30S for prokaryotes, 40S for eukaryotes).

DISCUSSION

The synthesis of proteins involves converting the nucleotide sequence of specific regions of DNA into mRNA (transcription) followed by the formation of peptide bonds in a complex set of reactions that occur on ribosomes (translation). The amino acids incorporated into the protein are first activated by being attached to a family of tRNA molecules, each of which recognizes, by complementary base-pairing interactions, particular sets of three nucleotides (codons) in the mRNA. Proteins are synthesized on ribosomes by linking amino acids together in the specific linear order stipulated by the sequence of codons in an mRNA. Ribosomes are compact ribonucleoprotein particles found in the cytosol of all cells. All ribosomes are composed of a small and a large subunit. The two subunits contain rRNAs of different lengths, as well as a different set of proteins. All ribosomes consist of two major rRNA molecules (23S and 16S rRNA in bacteria, 28S and 18S rRNA in eukaryotes) and one or two small RNAs. Small and large subunits are brought together by an mRNA molecule and protein synthesis starts immediately. After the protein is synthesized, the ribosomal subunits separate and are reused.

Protein synthesis is divided into three stages: initiation, elongation, and termination.

Chain initiation: Initiation requires the smaller ribosomal subunit, an initiation tRNA (with a 5'-CAU-3' anticodon), mRNA with its initiator codon (5'-AUG-3'), and several initiation factors, all of which form the ribosomal initiation complex. The initiation complex is near completion when a tRNA carrying the amino acid methionine hydrogen bonds to the AUG codon on the mRNA. When these components are in place, the larger ribosomal subunit joins the complex in such a way that the initiator met-tRNAmet is localized in the P- or peptidyl site (Figure 9-1). The chain initiation phase ends and a second amino acid can be inserted.

Chain elongation: Elongation occurs by bringing an aminoacyl-charged tRNA to the A- or acceptor site, followed by formation of the peptide bond and the translocation of a tRNA from the A- to the P-site. Proteins called elongation factors are the workhorses in the process of elongating the nascent polypeptide chain by one amino acid at a time. Translation elongation factors are involved in the following three-step cycle:

1. Codon recognition: A hydrogen bond forms between the mRNA codon and the anticodon of the next aminoacyl tRNA at the empty A-site of the ribosome.

2. Peptide bond formation: An enzyme called peptidyl transferase embedded in the large ribosomal subunit catalyzes the formation of a peptide bond between the polypeptide of the peptidyl-tRNA in the P-site and the newly arrived aminoacyl tRNA in the A-site.

3. Translocation: The tRNA in the P-site is ejected so that the newly formed peptidyl-tRNA located in the A-site can shift over to the P-site freeing up the A-site for the next aminoacyl-charged tRNA.

Chain termination: Elongation comes to an end when one (or more) of three stop codons (UAA, UAG, UGA) is encountered. A protein called a release factor binds directly to the termination codon in the A-site. The newly synthesized protein is hydrolyzed from the tRNA and both the tRNA and the protein are released from the ribosome.

Figure 9-1. Various sites of antibiotic action in protein synthesis.

A list of key differences between prokaryotes and eukaryotes with respect to protein synthesis is shown in Table 9-1. These include the existence of multiple eukaryotic initiation factors that facilitate the assembly of the ribosomal protein synthetic machinery, whereas there are only three for prokaryotes.

An initiation site on bacterial mRNA consists of the AUG initiation codon preceded with a gap of approximately 10 bases by the Shine-Dalgarno polypurine hexamer, whereas the 5′ Cap (a 7-methylguanylate residue in a 5′→5′ triphosphate linkage) acts as an initiation signal in eukaryotes. In prokaryotes, the first or N-terminal amino acid is a formyl-methionine (fMet), but in eukaryotes it is usually a simple methionine. Additionally, the size and nature of the prokaryotic ribosomes are quite different from the eukaryotic ribosomes.

Ribosomal RNA (rRNA) is a component of the ribosomes, the protein synthesis factories in the cell. rRNA molecules are extremely abundant, making up at least 80 percent of the RNA molecules found in a typical eukaryotic cell. Virtually all ribosomal proteins are in contact with rRNA. Most of the contacts between ribosomal subunits are made between the 16S and 23S rRNAs such that the interactions involving rRNA are a key part of ribosome function. The environment of the tRNA-binding sites is largely determined by rRNA. The rRNA molecules have several roles in protein synthesis. 16S rRNA plays an active role in the functions of the 30S subunit. It interacts directly with mRNA, with the 50S subunit, and with the anticodons of tRNAs in the P- and A-sites. Peptidyl transferase activity resides exclusively in the 23S rRNA. Finally, the rRNA molecules have a structural role. They fold into three-dimensional shapes that form the scaffold on which the ribosomal proteins assemble.

Many antibiotics act to inhibit protein synthesis in prokaryotes without affecting eukaryotic cells much. Several important targets in protein synthesis have been identified that are blocked by these agents to curb microorganism

Table 9-1

DIFFERENCES IN PROKARYOTIC AND

EUKARYOTIC PROTEIN SYNTHESIS

ORGANISM	PROKARYOTE	EUKARYOTE
Start	fMet-tRNAfMet	Met-tRNAiMet
Recognition sequence	Shine-Dalgarno sequence	5′ caps direct e-IFs
Initiation factors	IF-1, IF-2, IF-3	multiple e-Ifs (>10)
Elongation factors	EF-Tu, EF-G, EF-Ts	Multi-subunit eEF-1, eEF-2, eEF-3

growth (see Figure 9-1). Aminoglycosides interfere with binding of fMettRNA to the ribosome, thereby preventing correct initiation of protein synthesis and partially freezing the complex. Puromycin, on the other hand, causes premature termination of protein synthesis because it resembles tyrosyl-tRNA. It can bind to the A-site on the ribosome, and the peptidyl transferase will form a peptide bond between the growing peptide and puromycin. However, since there is no anticodon to bind to the mRNA, the peptidyl-puromycin is released from the ribosome following formation of the peptide bond, thus stopping the synthesis. Tetracyclines inhibit protein synthesis in bacteria by blocking the A-site on the ribosome and inhibiting binding of aminoacyl tRNAs. Macrolides (see below) bind to the 50S subunit near the P-site to cause conformational changes and inhibit translocation of the peptidyl tRNA from the A-site to the P-site. Lincosamides bind near the P-site and interfere with binding of the aminoacyl end of the AA-tRNA. They occupy the site or change the ribosomal conformation such that it destabilizes the ribosomes and the growing chains fall off the mRNA. Chloramphenicol inhibits protein synthesis by bacterial ribosomes by blocking peptidyl transfer. It inhibits peptide bond formation between AA-tRNA and the growing chain on the P-site by inhibiting peptidyl transferase. Neomycin, kanamycin, and gentamicin interfere with the decoding site in the vicinity of nucleotide 1400 in 16S rRNA of 30S subunit. This region interacts with the wobble base in the anticodon of tRNA and blocks self-splicing of group I introns. Streptomycin, a basic trisaccharide, causes misreading of the genetic code in bacteria at relatively low concentrations but can inhibit initiation at higher concentrations.

Macrolide antibiotics constitute a group of 12- to 16-membered lactone rings substituted with one or more sugar residues, some of which may be amino sugars. Macrolides such as erythromycin (Figure 9-2) are generally bacteriostatic, although some of these drugs are bactericidal only at very high concentrations. Gram-positive bacteria accumulate approximately 100 times more erythromycin than do gram-negative microorganisms. Cells are considerably more permeable to the nonionized form of the drug, which explains increased antimicrobial activity observed at alkaline pH. The newer macrolides have structural modifications, such as methylation of the nitrogen atom in the lactone ring, that improve acid stability and tissue penetration of these agents. Macrolides act by binding reversibly to the ribosomal subunits of sensitive microorganisms and thereby inhibiting protein synthesis. Resistance to macrolides in clinical isolates is most frequently a result of posttranscriptional methylation of an adenine residue of 23S ribosomal RNA, which leads to coresistance to macrolides. Other mechanisms of resistance involving cell impermeability or drug inactivation have also been detected. It is believed that erythromycin does not inhibit peptide bond formation directly but rather inhibits the translocation step wherein a newly synthesized peptidyl tRNA molecule moves from the acceptor site on the ribosome

Figure 9-2. Chemical structure of erythromycin.

to the peptidyl or donor site. Erythromycin, which does not reach the peptidyl transferase center, induces dissociation of peptidyl tRNAs containing six, seven, or eight amino acid residues.

COMPREHENSION QUESTIONS

For each of the following steps in prokaryotic protein synthesis (Questions [9.1] to [9.3]), indicate the most appropriate antibiotic (A–J) to inhibit the process.

A. Aminoglycosides

B. Chloramphenicol

C. Erythromycin

D. Gentamicin

E. Kanamycin

F. Lincosamides

G. Neomycin

H. Puromycin

I. Streptomycin

J. Tetracycline

[9.1] Transfer of the peptide from the peptidyl tRNA to the aminoacyl-tRNA and formation of a peptide bond.

[9.2] Binding of aminoacyl-tRNA in the A-site of the ribosomal complex.

[9.3] Translocation of the peptidyl tRNA from the A-site to the P-site.

[9.4] The 6-year-old son of a migrant worker is brought to a clinic with chills, headache, nausea, vomiting, and sore throat. The examining physician notes a persistent grayish colored membrane near the tonsils. History reveals that the patient has not been immunized against diphtheria. Diphtheria toxin is potentially lethal in this unimmunized patient because it causes which of the following?

A. Inactivates an elongation factor required for translocation in protein synthesis

B. Binds to the ribosome and prevents peptide bond formation

C. Prevents binding of mRNA to the 60S ribosomal subunit

D. Inactivates an initiation factor

E. Inhibits the synthesis of aminoacyl-charged tRNA

[9.5] Replication of a particular DNA sequence is noted to be under inhibitory control usually. However, when substance “A” is added, it binds to a repressor, rendering the repressor inactive and allowing transcription to occur. Which of the following terms describes agent “A”?

A. Histone

B. Operon

C. Polymerase

D. Transcriber

E. Inducer

Answers

[9.1] B. Chloramphenicol inhibits protein synthesis by inhibiting peptidyl transferase. This peptidyl group cannot be transferred to the aminoacyltRNA in the A-site.

[9.2] J. Tetracyclines bind to the A-site of the prokaryotic ribosome and prevent aminoacyl-tRNAs from binding. Thus protein synthesis is halted because new amino acids cannot be added to the growing protein.

[9.3] C. Erythromycin and other macrolide antibiotics bind the 50S subunit near the P-site and cause conformational changes that inhibit the translocation of peptidyl tRNA from the A-site to the P-site.

[9.4] A. Diphtheria toxin has two subunits. The B subunit binds to a cell surface receptor and facilitates the entry of the A subunit into the cell. The A subunit then catalyzes the ADP-ribosylation of elongation

factor 2 (EF2). EF2 is thus inhibited from participating in the translocation process of protein synthesis; hence, protein synthesis stops.

[9.5] E. An inducer is a small molecule that binds to and inactivates a repressor, which allows the sequence of DNA to be transcribed. An operon is a set of prokaryotic genes in close proximity that are coordinated as “all off” or “all on.” An inducer may act to “turn on” the operon. One classic example is the lac operon. When allolactose is present, it serves as an inducer, and the operon is turned on, allowing proteins to be formed that metabolize lactose.

BIOCHEMISTRY PEARLS

❖ The synthesis of proteins involves converting the nucleotide sequence of specific regions of DNA into mRNA (transcription), followed by the formation of peptide bonds in a complex set of reactions that occur on ribosomes (translation).

❖ Protein synthesis is divided into three stages: initiation, elongation, and termination.

❖ Ribosomal RNA (rRNA) is a component of the ribosomes, the protein synthetic factories in the cell.

❖ Many antibiotics take advantage of the differences of the rRNA between eukaryotic and prokaryotic cells.

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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