Life in the Balance: Cell Walls and Antibiotic Resistance

As a result of the advent of antibiotics, we have been largely victorious in fighting bacterial infectious disease. However, the rapid development and spread of mechanisms of bacterial resistance are making virtually all antibiotics obsolete. One wonders if a return to the preantibiotic era is in our immediate future.

Since the discovery of penicillin, b-lactam antibiotics have been the most important family of antibacterial agents. How b-lactam antibiotics kill bacteria is still something of a mystery. They all inhibit the final stage of murein synthesis, which somehow triggers the autolytic activities of murein hydrolases. Murein is an essential heteropolymer that protects the bacterium from osmotic rupture, dictates cell shape, and is intimately involved in cell growth and division. To maintain cell integrity and viability, the murein must remain physically continuous during the bacterial cell cycle. Yet, bonds have to be broken to allow insertion of new material during cell growth. This must be done in a manner that does not endanger the osmotic stability of the cell. Temporal and topological controls must exist that keep wall assembly and turnover in balance and ensure synchronization of cell wall growth and the cell cycle. This view suggests a bidirectional communication between the exterior wall and the transcriptional machinery. The fact that general inhibition of murein synthesis caused by the presence of b-lactam antibiotics results in cell lysis suggests that these drugs disrupt the delicate balance between murein synthetic and degradative activities. Therefore, the elucidation of the mechanisms that upset the control of the murein hydrolases should help identify the control devices themselves, which in turn has important consequences for the discovery of new antibacterial drugs.

The major mechanism of resistance to b-lactam antibiotics is the synthesis of a bacterial enzyme, b-lactamase, that cleaves the b-lactam ring and renders the antibiotic inactive. Some bacterial species have refined this defense mechanism by making the b-lactamase synthesis inducible in the presence of b-lactam antibiotic. In many Gram-negative bacteria, the inducible b-lactamase gene ampC is transcriptionally controlled by a regulator encoded by ampR, which belongs to the LysR family of transcriptional regulators (1, 2). Mutations in another locus, ampD, result in constitutive hyperproduction of the AmpC b-lactamase even in the absence of b-lactam antibiotics. The ampD mutants are therefore highly resistant to b-lactam antibiotics (3). Another gene required for induction of b-lactamase is ampG, encoding the AmpG transmembrane protein (4). Interestingly, the ampG and ampD genes are also found in noninducible b-lactamase strains (3). We are therefore left with the following questions: What are the cellular functions of AmpG and AmpD? And how is the b-lactam antibiotic attack on the exterior cell wall signaled to the genetic apparatus?

Our first breakthrough came from the demonstration of a direct link between b-lactamase induction and cell wall metabolism. We discovered that ampG and ampD, genes essential for b-lactamase induction, were also required for cell wall recycling (5). Escherichia coli degrades up to 50% of its murein per generation, but most of the liberated murein fragments (muropeptides) are transported from the periplasm into the cytoplasm and recycled for further murein biosynthesis (6). Mutations in ampG and ampD dramatically decrease the ability of the cell to recycle its murein (5). An ampG mutant releases muropeptides into the external medium, whereas an ampD mutant accumulates a novel muropeptide species in its cytoplasm, the anhMurNAc-tripeptide (anhydro-N-acetylmuramyl-L-Ala-D-Glu-m-, A₂pm being diaminopimelic acid). We further showed in vitro that purified AmpD had N-acetylmuramyl amidase activity with a strict requirement for the presence of an anhydro function on the muramic acid residue (7). In that way, de novo cell wall precursors, which lack the anhydro bond, will not be degraded by the enzyme.

On the basis of these findings, a new recycling scheme was proposed (see figure). The first step is the degradation of murein by specific cell wall hydrolases to yield GlcNAc-anhMurNAc-tripeptide (N-acetylglucosamyl-anhMurNAc-tripeptide). This muropeptide is transported into the cytoplasm by the permease AmpG. It is then either directly cleaved by AmpD or first converted into anhMurNAc-tripeptide by a cytosolic b-N-acetylglucosaminidase (Gmase) and then hydrolyzed by AmpD. In both cases, the resulting tripeptide is reintroduced into the murein biosynthetic pathway by direct addition to the peptidoglycan precursor uridine 5¢-diphosphate (UDP)-MurNAc, as previously proposed (6). The gene encoding the tripeptide-adding enzyme responsible for this last step was recently identified as mpl (8).

Subsequently, using in vitro transcription studies, we showed that purified AmpR, in the absence of any effector, directed ampC transcription. This observation was somewhat surprising since other members of the LysR family usually depend on a ligand to activate transcription. Instead, the main murein precursor, UDP-MurNAc-pentapeptide (UDP-MurNAc-L-Ala-D-Glu-m-diaminopimelate-D-Ala-D-Ala), was found to inhibit transcription, and this inhibition was reversed by an intermediate in murein recycling, the anhMurNAc-tripeptide (9). These results led us to propose that b-lactamase expression is regulated by sensing the relative levels of the two endogenous murein intermediates (see figure).

During normal growth in the absence of a b-lactam antibiotic inducer, the AmpR regulator expressed is maintained in an inactive form by the murein precursor UDP-MurNAc-pentapeptide. This inactivation of AmpR can be relieved by both "knockout" mutations in the ampD gene and the presence of b-lactam antibiotics in the culture medium. Inactivation of ampD, which encodes the cytosolic amidase specific for the recycling of muropeptides, results in a drastic accumulation of its substrate, the anhMurNAc-tripeptide (5). The high concentration of this muropeptide inside the cell is sufficient to displace the UDP-MurNAc-pentapeptide from its AmpR-binding site, thereby reactivating AmpR. In wild-type cells, by impairing cell wall synthesis, the presence of b-lactam antibiotics results in a decrease of the UDP-MurNAc-pentapeptide concentration and an increase in the anhMurNac-tripeptide concentration in the cytoplasm (5). These two effects are likely to be additive, displacing the UDP-MurNAc-pentapeptide repressor from AmpR, resulting in activation of b-lactamase expression.

Because the relative levels of the cytosolic intermediates of murein metabolism are altered by exposure to b-lactam antibiotics, the net result is the sensitive control of a bacterial defense mechanism. In addition, the presence (and maintenance) of this network in strains with noninducible b-lactamase (like E. coli) strongly suggests that this system functions as a means of monitoring cell wall integrity and maintaining a proper balance between murein synthesis and degradation during bacterial growth.

References

1. S. Normark et al., New Compr. Biochem. 27, 485 (1994).
2. F. Lindberg, L. Westman, S. Normark, Proc. Natl. Acad. Sci. USA. 82, 4620 (1985) [Medline].
3. F. Lindberg, S. Lindquist, S. Normark, J. Bacteriol. 169, 1923 (1987) [Medline].
4. G. Korfmann and C. C. Sanders, Antimicrob. Agents Chemother. 33, 1946 (1989) [Medline].
5. C. Jacobs, L.-J. Huang, E. Bartowsky, S. Normark, J. T. Park, EMBO J. 13, 4684 (1994) [Medline].
6. E. W. Goodell, J. Bacteriol. 163, 305 (1985) [Medline].
7. C. Jacobs et al., Mol. Microbiol. 15, 553 (1995) [Medline].
8. D. Mengin-Lecreulx, J. van Heijenoort, J. T. Park, J. Bacteriol. 178, 5347 (1996) [Medline].
9. C. Jacobs, J.-M. Frère, S. Normark, Cell 88, 823 (1997) [Medline].

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