the TCT fragments, producing dose-response curves ... only natural origin in bacteriai peptidoglycan. ..... used in our previous study (Luker etal.. 1993a) ..... phorus pentachloride. .... phosphate (BOP) and 12eq of diisopropylethylamine (DIPEA).
Molecular Microbiology (1995) 16(4), 733-743
Tracheal cytotoxin structural requirements for respiratory epithelial damage in pertussis Kathryn E. Luker,' ^ Andrew N. Garland R. Marshall^ and William E. Goldman^* 'Department of fs/toiecutar Microbiotogy, and ^Department of Motecutar Bioiogy and Pharmacotogy, Washington University Schoot of Medicine. 660 South Euctid Avenue, St. Louis, f\/tissouri 63110-1093. USA. ^Department of Chemistry, t-iarvard University. Cambridge, Massachusetts 02138, USA.
Summary The respiratory epithelial pathology of pertussis (whooping cough) can be reproduced by tracheal cytotoxin (TCT), a disaccharide-tetrapeptide released by Bordetella pertussis. TCT is a muramyl peptide, a class of peptidoglycan-derived compounds which have many biological activities including adjuvanticity, somnogenicity, pyrogenicity, and cytotoxicity. The structural requirements for muramyl peptides to produce some of these biological effects have been partially characterized. Using in vitro assays with respiratory epithelial cells and tissue, we have previously determined that the disaccharide moiety of TCT is not involved in toxicity and that the side-chain functional groups of diaminopimelic acid (A2pni) are crucial for toxicity. In this study, we determine the importance of every amino acid, functional group and chiral centre in the peptide portion of TCT. Although lactyl tetrapeptides are the most toxic of the TCT fragments, producing dose-response curves identical to TCT, the smallest analogues of TCT which are active in our assay are of the form X-Y-(D)Glu-meso-Ajpm, where X may be an amino acid or a blocking group. Within this active substructure, main-chain chirality and all functional groups are essential for toxicity. This definition of the core region of TCT indicates that the TCT interaction site is unlike almost all other muramyl peptide interaction sites for which structure-activity data are available.
Introduction Tracheal cytotoxin (TCT) is one of several exotoxins Recerved 19 December, 1994. revised 5 January, 1995; accepted 17 January, 1995, 'For correspondence. E-mail: goldman@borcim, wustl.edu: Tel. (314) 362 2742; Fax (314) 362 4879, 1995 Blackwell Science Ltd
produced by the respiratory pathogen Bordetetta pertussis, a Gram-negative bacterium. 6, pertussis is the causative agent of the pertussis syndrome, or whooping cough, and specifically colonizes and destroys the ciliated cells of the respiratory epithelium (Mallory and Hornor, 1912). In the absence of ciliary activity, coughing becomes the oniy means of clearing mucus, bacteria, and inflammatory debris from the respiratory tract, TCT, purified to homogeneity from B. pertussis culture supernatant, is capable of reproducing the specific damage to ciliated cells observed during pertussis (Cookson ef aL. 1989a). The characteristic changes in the ciliated cell population include rounding, decrease in the number of cilia per cell, bloating of mitochondria, and eventual extrusion of the ciliated cells (Goldman and Henvaldt. 1985). This cytopathology is produced tn vitro when TCT is tested on explanted hamster (Goldman and Henwaldt, 1985) and human (Wilson etat.. 1991) respiratory epithelium. TCT, with a molecular mass of 921 Da, is a disaccharide-tetrapeptide monomer of peptidoglycan, the polymeric cell wall of bacteria. The structure of TCT is A/-acetylglucosaminyl-1,6-anhydro-/\/-acetylmuramyl-(L)alanyl-v-(D)-glutamyl-meso~diaminopimelyl-(D)-alanine (Cookson et at.. 1989b) (Fig. 1). Although this peptidoglycan monomer structure is common to all Gramnegative bacteria, only Bordeteita spp. (Cookson and Goldman. 1987) and Neisseria gonorrhoeae (Sinha and Rosenthal. 1980) have been shown to release TCT into the supernatant during growth in broth cuiture. When compared with typical gene-encoded peptides, the peptidic portion of TCT is unusual in the alternating chirality of the :i-carbons, the side-chain linkage of glutamic acid, and the presence of diaminopimelic acid (Ajpm). These structural features mark TCT as a member of the class of compounds called muramyl peptides, which have their only natural origin in bacteriai peptidoglycan. Muramyl peptides have a variety of biological activities in addition to cytotoxicity, including adjuvanticity, enhancement of cell-mediated immunity, pyrogenicity. arthritogenicity, and induction of slow-wave sleep (for a review see Adam and Lederer, 1984). A number of studies have examined the structure-activity relationships for these biological activities, although most of that work has been limited by the availability of appropriate muramy! peptide analogues, TCT toxicity is most evident in respiratory tract tissue, but it can also be quantitatively studied with cultured hamster tracheal epithelial (HTE) cells, a proliferating,
734
K. E. Lui• 0.46 cm (Applied Biosystems), with isocratic eiution with 0,1% TFA in water, flow 1,0 ml min V b. Calculated based on M + H determined by mass spectrometry.
none of the non-toxic analogues could compete with TCT for binding to the putative TCT-receptor site. Because of small variations in the cellular responses to TCT. which causes half-maximal toxicity to range from 0.15 nM to 0,45 |,iM, results are reported as analogue toxicity relative to TCT within a given experiment. Relative toxicity is calculated as the ratio of the concentrations of TCT and analogue which correspond to a half-maximal
effect in that experiment. As described in the Experimental procedures, TCT and two analogues, 1 and 10, were tested for stability during exposure to HTE cells as in toxicity assays. This was carried out to determine whether the toxicity of these analogues could be ascribed to chemically modified forms which might have been produced during incubation with HTE cells. However, none of the three compounds were observed to be altered by this treatment.
Table 2. Relative toxicities of TCT fragments.
Analogue
Stnjcture iactyl-(L)-Ala-Y-(D)-Glu-meso-A2pm-(o)-Ala (derived from peptidoglycan} lactyl-(L)-Aia-7-(D)-Glu-meso-A2pm-(D)-Ala lactyl-(L)-Ata-7-(D)-Glu-meso-A2pm (i-)-Ala-y-(D)-Glu-fneso-A2pm-(D)-Ala (L)-Ala-y-(o}-Glu-meso-A2pm
Mean relative toxicity" 1,7* 1,7* 3* 3* 0,04 0,03 0,3 0,4 0,05 0,05 < 0,003
