Antibiotic drug-target interactions, and their respective direct effects, are generally well characterized. By contrast, the bacterial responses to antibiotic drug treatments that contribute to cell death are not as well understood and have proven to be complex as they involve many genetic and biochemical pathways. In this Review, we discuss the multilayered effects of drug-target interactions, including the essential cellular processes that are inhibited by bactericidal antibiotics and the associated cellular response mechanisms that contribute to killing. We also discuss new insights into these mechanisms that have been revealed through the study of biological networks, and describe how these insights, together with related developments in synthetic biology, could be exploited to create new antibacterial therapies.

How antibiotics kill bacteria: from targets to networks

The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins.

Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope

To withstand the high intracellular pressure, the cell wall of most bacteria is stabilized by a unique cross-linked biopolymer called murein or peptidoglycan. It is made of glycan strands [poly-(GlcNAc-MurNAc)], which are linked by short peptides to form a covalently closed net. Completely surrounding the cell, the murein represents a kind of bacterial exoskeleton known as the murein sacculus. Not only does the sacculus endow bacteria with mechanical stability, but in addition it maintains the specific shape of the cell. Enlargement and division of the murein sacculus is a prerequisite for growth of the bacterium. Two groups of enzymes, hydrolases and synthases, have to cooperate to allow the insertion of new subunits into the murein net. The action of these enzymes must be well coordinated to guarantee growth of the stress-bearing sacculus without risking bacteriolysis. Protein-protein interaction studies suggest that this is accomplished by the formation of a multienzyme complex, a murein-synthesizing machinery combining murein hydrolases and synthases. Enlargement of both the multilayered murein of gram-positive and the thin, single-layered murein of gram-negative bacteria seems to follow an inside-to-outside growth strategy. New material is hooked in a relaxed state underneath the stress-bearing sacculus before it becomes inserted upon cleavage of covalent bonds in the layer(s) under tension. A model is presented that postulates that maintenance of bacterial shape is achieved by the enzyme complex copying the preexisting murein sacculus that plays the role of a template.

Growth of the Stress-Bearing and Shape-Maintaining Murein Sacculus of Escherichia coli

4Crawford, L. V., R. Dulbecco, M. Fried, L. Montagnier, and M. G. P. Stoker, these PNoCEEDINGS, 52, 148 (1964). Bourgaux, P., D. Bourgaux-Ramoisy, and M. G. P. Stoker, Virology, 25, 364 (1965). 6Black, P. H., and W. P. Rowe, these PROCEEDINGS, 50, 606 (1963). 7Diderholm, H., B. Stenkvist, J. Ponten, and T. Wesslen, Exptl. Cell Res., 37, 452 (1965). 8 Black, P. H., and W. P. Rowe, J. Nati. Cancer Inst., 32, 253 (1964). 9 Hopps, H. E., B. C. Bernheim, A. Nisalak, J. H. Tjio, and J. E. Smadel, J. Immunol., 91, 416 (1963). 10Black, P. H., E. M. Crawford, and L. V. Crawford, Virology, 24, 381 (1964). 11 Weil, R., Virology, 14 (1961). 12 Black, P. H., and W. P. Rowe, Virology, in press. 13 A clone of fibroblasts (C13), derived from the BHK-21 line,' was obtained from Professor M. Stoker, Giasgow, Scotland, and was used at the 80th-lOOth generations since cloning. 14 Pope, J. H., and W. P. Rowe, J. Exptl. Med., 120, 121 (1964). 16 Black, P. H., W. P. Rowe, H. C. Turner, and R. J. Huebner, these PROCEEDINGS, 50, 1148 (1963). 16 Defendi, V., J. Lehman, and P. Kraemer, Virology, 19, 592 (1963). 17 Black, P. H., and W. P. Rowe, Proc. Soc. Exptl. Biol. Med., 114, 721 (1963). 18 Gotlieb-Stematsky, T., and R. Shilo, Virology, 22, 314 (1964). 19 Dulbecco, R., personal communication. 20 Le Bouvier, G., personal communication. 21 Crawford, L. V., and P. H. Black, Virology, 24, 388 (1964). 22 Mayor, H. D., R. M. Jamison, and L. E. Jordan, Virology, 19, 359 (1963). 23Stoker, M., and P. Abel, in Cold Spring Harbor Symposia on Quantitative Biology, vol. 17, (1962), p. 375. 24 Black, P. H., unpublished data. 25 Vinograd, J., R. Bruner, R. Kent, and J. Weigle, these PROCEEDINGS, 49, 902 (1963).

https://www.pnas.org/content/pnas/54/4/1133.full.pdf

Mechanism of action of penicillins: a proposal based on their structural similarity to acyl-D-alanyl-D-alanine.

As the decade progresses this prediction is rapidly being realized, and microbial enzymes are becoming increasingly important in such diverse fields as medicine, brewing, and timber preservation. The genus Bacillus has played a major role in this development as evidenced by the distribution of the papers read at the Fifth International Fermentation Symposium, 1976 (64). Of 23 papers in the session devoted to "Microbial Enzymes of Industrial Interest" no less than ten were concerned with enzymes from bacilli. Reasons for the predominance of these bacteria in this area of study are several. First, they comprise a group of chemoorganotrophs that can be easily maintained and cultivated and yet are markedly heterogeneous in character. Psychrophiles, mesophiles, and thermophiles, in addition to alkalophilic, neutrophilic, and acidophilic species are well represented. Furthermore, virtually all 48 species of the genus listed in Bergey's Manual ofDeterminative Bacteriology (92) secrete a variety of soluble extracellular enzymes, which reflects the diversity of the parental habitats. Amylases that can liquefy starch under pressure at 11000 (194) and proteases that are stable and active at pH 12.0 (6) are extreme examples of enzyme adaption. This article will attempt to review the recent literature concerned with the characterization and properties of the exoenzymes synthesized by the bacilli and the control and mechanisms of their synthesis. It is restricted to this genus because the commercial importance of extracellular enzymes and academic interest in the process of sporulation have prompted a considerable amount of research into this general area. Nevertheless, in the final section I have attempted to equate our present knowledge of exoenzyme synthesis in procaryotes other than

Extracellular enzyme synthesis in the genus Bacillus.

https://www.pnas.org/content/pnas/54/1/75.full.pdf

Penicillin: its basic site of action as an inhibitor of a peptide cross-linking reaction in cell wall mucopeptide synthesis.

Several natural isolate E. coli strains highly resistant to sulfonamides and antibiotics are shown to contain a sulfonamide-resistant dihydropteroate synthase (2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine-diphosphate:4-aminobenzoate 2-amino-4-hydroxydihydropteridine-6-methenyltransferase, EC 2.5.1.15) in addition to the normal sensitive enzyme. The resistant dihydropteroate synthases examined are determined by an R plasmid and are smaller and less heat stable than the normal sulfonamide-sensitive enzyme. One synthase resistant to any sulfonamide tested, and to sulfanilic and arsanilic acids, was still inhibited by several non-sulfonamide analogs of p-aminobenzoate. Citrobacter and Klebsiella pneumoniae strains also show similar mechanisms of sulfonamide resistance.

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Sulfonamide resistance mechanism in Escherichia coli: R plasmids can determine sulfonamide-resistant dihydropteroate synthases.

Adenosine 3′:5′-cyclic monophosphate added to exponentially growing cells of Hemophilus influenzae strain Rd increases competence for transformation 100- to 10,000-fold. Cyclic AMP added to near-stationary or stationary cells does not increase competence over the high level normally found in the early stationary phase.

https://www.pnas.org/content/pnas/70/2/471.full.pdf

Adenosine 3':5'-cyclic monophosphate as a regulator of bacterial transformation.

The principal autolytic enzyme activity of the cell sap of Staphylococcus aureus H has been purified 400-fold. It is an N-acetylmuramyl-l-alanine amidase. This enzyme has a molecular weight of 8 to 10 × 105, a pH optimum of 7.3, an ionic strength optimum of 0.16 m and a Km of 10−3m murein repeating units.

Properties and Purification of N-Acetylmuramyl-l-Alanine Amidase from Staphylococcus aureus H

Staphylococcus aureus strain H has an autolytic activity which can be found in the cell wall but is most easily obtained from high-speed supernatant fractions of broken-cell preparations. As measured either turbidimetrically or radioactively, this activity is much greater on murein extracted from penicillin-treated cells than on murein extracted from normal cells.

Edmund M. Wise

Papers

Penicillin: its basic site of action as an inhibitor of a peptide cross-linking reaction in cell wall mucopeptide synthesis.

Sulfonamide resistance mechanism in Escherichia coli: R plasmids can determine sulfonamide-resistant dihydropteroate synthases.

Adenosine 3':5'-cyclic monophosphate as a regulator of bacterial transformation.

Properties and Purification of N-Acetylmuramyl-l-Alanine Amidase from Staphylococcus aureus H

Staphylococcus aureus H Autolytic Activity: General Properties