E H Beachey

General Concepts and Principles of Bacterial Adherence in Animals and Man

Adherence of bacteria to tissue surfaces has recently gained increasing attention as an important initial event in the pathogenesis of bacterial infections (Ofek and Beachey, 1980). The infectious process in animals and man can be envisioned as a stepwise process in which bacteria must first adhere to a tissue surface. The failure to adhere would result simply in their being swept away in the fluids which constantly bathe the tissue surfaces. Adherence of pathogenic organisms is then followed by colonization and eventual invasion of the surface either by a toxin produced by the colonizing organisms or by the bacteria themselves. In the deeper tissues, the attachment of the bacteria to phagocytic cells results in their ingestion and destruction. Organisms whose surfaces are not recognized by antibody and complement or phagocytic cells can multiply unimpeded to produce systemic infections. These steps in the infectious process are depicted diagramatically in Fig. 1.1.

Surface sugars of animal cells as determinants of recognition in bacterial adherence

Adherence of Escherichia coli to D -mannose-like residues on epithelial cells may be an important step in bacterial infection. This raises the possibility of using simple sugars, such as methyl α - D -mannoside in the prevention of infection by certain gram-negative bacteria.

Lipoteichoic acid and M protein: dual adhesins of group A streptococci

The roles of lipoteichoic acid (LTA) and M protein in the adherence of group A streptococci to human cells were investigated. Both M+ and M- streptococci bound to pharyngeal and buccal epithelial cells in similar numbers. Streptococcal attachment was inhibited by LTA, but not by the pepsin-extracted, amino-terminal half of M protein (pep M), suggesting that M protein does not mediate attachment to these cells. However, a purified, recombinant, intact M protein did block attachment of streptococci to buccal cells. Using synthetic peptides, the inhibitory domain was localized to a region of intact M protein that is within or near the bacterial cell wall. Evidence is presented to suggest that on the surface of streptococci this region of the M protein is probably not accessible for interactions with host cell receptors and that M protein does not mediate attachment to buccal or pharyngeal cells. In contrast, approximately 10-times more M+ streptococci bound to Hep-2 cells than did M- streptococci and pep M protein blocked binding of streptococci to Hep-2 cells. The data suggest that at least two streptococcal adhesins, LTA and M protein, are involved in the adherence of streptococci to certain cells and that the relative contributions of these adhesins to the attachment process depends on the type of host cells used to study adherence.

Protective and autoimmune epitopes of streptococcal M proteins

Several rheumatogenic serotypes of streptococcal M protein have been shown to contain both protective and cardiac tissue crossreactive epitopes. By synthesizing peptides copying different regions of M protein polypeptides, we were able to localize the protective and heart crossreactive epitopes. Some epitopes are only opsonic, some are only crossreactive, whereas others are both opsonic and tissue crossreactive. Multivalency of vaccines can be obtained by synthesizing protective peptides of one M serotype in tandem with protective peptides of other M serotypes. Such hybrid peptides evoke protective immune responses against the related streptococci without evoking tissue crossreactive immunity.

Bacterial adhesion: modulation by antibiotics with primary targets other than protein synthesis.

By inhibiting dihydropteroate synthetase and dihydrofolate reductase, sulfonamides and trimethoprim inhibit the synthesis of folate coenzymes and thereby affect the central pathway of the metabolism of C1 compounds (25). The sub-MIC of trimethoprim was shown to reduce the fimbriation, hemagglutination, and epithelial cell adhesion of several Escherichia coli strains (49, 55, 61) through the inhibition of fimbrial subunit synthesis (49). Trimethoprim, which is known to regulate RNA synthesis in E. coli (53), may act similarly on bacterial regulatory mechanisms at low doses, giving priority to the synthesis of proteins that are indispensable for growth and division. It is therefore plausible that other components of the bacterial surface are affected by trimethoprim. The altered membrane structure of E. coli probably reflects the pleiotropic effects of sub-MICs of trimethoprim (53, 61). Sulfonamides had similar effects on hemagglutination and epithelial cell adhesion (49, 55, 61). Sulfamethoxazole and trimethoprim used together acted synergistically on type 1 fimbrial subunit synthesis (49). How sulfamethoxazole alone affected hemagglutination but not fimbriation and fimbrial subunit synthesis remains unclear (49). It is possible that this drug is more effective in inhibiting the synthesis of the adhesin, a minor protein of type 1 fimbriae, than that of the major structural subunit (29, 32, 33).

Bacterial adhesion: modulation by antibiotics which perturb protein synthesis.

It has been known for a long time that antibiotics are capable of altering bacterial surfaces, resulting in morphological changes that can be detected by electron microscopy (2); however, more subtle alterations at the molecular level may be undetectable at the ultrastructural level. Certain antibiotics disturb the metabolism and processing of bacterial surface components (59, 71), whereas others disorganize the bacterial surface architecture (36, 39). Regardless of the mechanism of action, the induced surface changes can influence the strength of the attractive and repulsive forces responsible for bacterial surface interactions with molecules and cells in the environment. These interactions are important in the early stages of bacterial pathogenesis, that is, attachment to mucosal surfaces and invasion, and perhaps also during subsequent steps of the infectious process (5, 14, 15, 18, 38). Adhesins are ligand molecules that are located on the surfaces of pathogenic bacteria and that endow the organisms with the ability to bind specifically to complementary receptors on the mucosal surfaces of the susceptible host. Many studies have provided evidence that the expression and specific function of adhesins can be affected by concentrations of antibiotics that are unable to completely inhibit bacterial growth in vitro (sub-MICs; reviewed in references 13 and 66). These observations raise the possibility that sub-MICs of antibiotics may prevent the infectious process by inhibiting the mucosal attachment step (8, 64). Although many in vitro experiments have shown an effect of antibiotics on adhesion, only a few have been designed to define the molecular events involved. Because of the complexity of the microbe-drug-host interaction, interpretations concerning the effect of antibiotics on bacterial adhesion have been difficult. In this article, we review this topic, relating the known sites of action of various classes of antibiotics which perturb protein synthesis (31, 71) with what is known about the molecular mechanism of bacterial adhesion. Other antimicrobial agents are reviewed elsewhere (65). The concepts described in this review will be highlighted by examples referring only to the better-characterized adhesin-receptor systems, the paradigm being the type 1 fimbria-mediated adhesion of Escherichia coli to mannosylated receptors on eucaryotic cells. We emphasize technical aspects when necessary to reconcile apparent contradictions between different studies. A synopsis of the reviewed studies is shown in Table 1. The effectiveness of antibiotics that inhibit protein synthe-

Collagen-Induced Platelet Aggregation: Involvement of an Active Glycopeptide Fragment (α1-CB5)

It is widely held that the tertiary structure of collagen is essential for induction of platelet aggregation. However, we have found that the purified α1 chain prepared from denatured chick skin collagen aggregates platelets. This activity appears to be confined to a distinct region of the molecule representing less than 4 percent of the length of the α1 chain. Of all of the cyanogen bromide peptides of the α1 chain tested, only one (α1-CB5) was active. This glycopeptide, devoid of any ordered tertiary structure, contains only 36 amino acids and one residue of O-α-D-glucopyranosyl-(1 → 2)-O-β-D-galactopyranosyloxy-(1 → 5)-lysine (Glc-Gal-Hyl). Blocking experiments strongly suggest that the Glc-Gal-Hyl is one of the structural determinants involved in collagen-induced platelet aggregation.

Bacterial Adherence of Group A Streptococci to Mucosal Surfaces

It is now recognized that bacteria bind to and colonize mucosal surfaces in a highly selective manner. After the organisms penetrate the nonspecific mechanical and cleansing forces, ligands (or adhesins) on the surface of the bacteria interact in a lock-and-key (or induced fit) fashion with complementary receptors on mucosal surfaces of the host. The adhesins are usually composed of proteins in the form of fimbriae or fibrillae and the receptors of glycolipids or glycoproteins. In group-A streptococci the adhesin, lipoteichoic acid (LTA), is anchored to a protein(s) on the surface of the bacterial cells and interacts through its lipid moiety with fibronectin molecules deposited on and bound to the epithelial cells. In an attempt to locate the region of fibronectin recognized by LTA and group-A streptococci, fibronectin was cleaved with thermolysin and the fragment mixture absorbed with Staphylococcus aureus or Streptococcus pyogenes. Staphylococci adsorbed several high molecular weight fragments as well as a 28- and a 23-kdalton fragment, whereas S. pyogenes cells absorbed only the 28-kdalton fragment completely. The adsorbtion of the fragments by S. pyogenes was blocked by LTA. Antibodies raised against a synthetic peptide copying the NH2 terminus of fibronectin reacted in a Western blot with the 28-kdalton fragment, indicating that S. pyogenes and its LTA react with the NH2-terminal region of fibronectin at a site distinct from that of S. aureus. Our findings are consistent with the idea that LTA mediates the attachment of group-A streptococci to fatty acid-binding sites of fibronectin deposited on mucosal epithelial cells.

Antigenic conservation of primary structural regions of S-adenosylmethionine synthetase

Although the physical and kinetic properties of S-adenosylmethionine (AdoMet) synthetases from different sources are quite different, it appears that these enzymes have structurally or antigenically conserved regions as demonstrated by studies with AdoMet synthetase specific antibodies. Polyclonal anti-human lymphocyte AdoMet synthetase crossreacted with enzyme from rat liver (beta isozyme), Escherichia coli and yeast. In addition, polyclonal anti-E. coli enzyme and antibodies to synthetic peptides copying several regions of the yeast enzyme reacted with the human gamma and rat beta isozymes. Antibodies to yeast SAM1 encoded protein residues 6-21, 87-113 and 87-124 inhibited the activity of human lymphocyte AdoMet synthetase, while antibodies to residues 272-287 had no effect on the enzyme activity. Our results suggest that these conserved regions may be important in enzyme activity.

Prospects for Group A Streptococcal VaCCIne

Although the attack rate of acute rheumatic fever has declined dramatically in the industrialized nations of the world, it remains a rampant disease in third world countries and continues to be the major cause of heart disease in children around the world. Indeed, the prevalence rates of rheumatic heart disease in school-aged children is as high as 33 per thousand in the urban slums of some developing countries (Kholy et al., 1978). A number of programmes have been undertaken for the prevention and control of rheumatic fever and rheumatic heart disease in various regions around the world. The major emphasis of these programmes has been on secondary prevention, including long-term antibiotic prophylaxis to prevent recurrent attacks of acute rheumatic fever. Primary prevention programmes directed toward prevention of the initial attack of rheumatic fever also have been instituted, but with less success. These have emphasized early diagnosis of streptococcal infections with prompt antibiotic treatment of the acute infections. Inasmuch as the diagnosis of streptococcal infections is often difficult to make in many parts of the world, and mass antibiotic prophylaxis is impractical if not impossible, the primary prevention programmes have been far from successful.