Our understanding of the mechanisms used byIn spite of a stable decline in the incidence of tuberculosis in countries participating in control surveys, there were an estimated 8.8 million new cases and 1.6 million deaths in 2005 (28). The treatment of Mycobacterium tuberculosis infections requires at least 6 months of antimycobacterial therapy with the use of multiple drugs. This long duration of treatment is justified by the poor efficacy of available antibiotics, including the main drugs isoniazid and rifampin, against the dormant M. tuberculosis bacilli (10, 26) that are thought to persist in particular environments such as the granuloma or caseum (6,21). In vitro models that mimic the persistent state have been developed based on nutrient starvation (4, 12), oxygen deprivation (25), and exposure to nitric oxide (23). These models showed that nonreplicative and low metabolic states of the bacteria could be responsible for the poor in vivo response to currently available drugs. The adaptive response of M. tuberculosis during the transition from aerobic growth to stationary phase results in the activation of a "dormancy" regulon (4, 22, 24). The regulon includes genes that are likely to play an essential role in the long-term survival of the bacteria and therefore encode potential targets for the development of sterilizing drugs.The "dormancy" regulon of M. tuberculosis was not previously reported to include genes involved in peptidoglycan metabolism, although changes in the structure of this cell wall polymer are known to be associated with the transition to the stationary phase in other bacteria. In Escherichia coli, the transition is associated with an increase (1.8 to 5%) in the content of 333 cross-links to the detriment of the classical 433 crosslinks formed by the D,D-transpeptidase activity of penicillinbinding proteins (PBPs) (Fig. 1) (11). We have previously identified the L,D-transpeptidases (Ldt) that catalyze the formation of 333 peptidoglycan cross-links as members of a novel family of active-site cysteine peptidases that have various cellular functions (5,14,15,18). In E. coli, these functions include the anchoring of a lipoprotein to the peptidoglycan in addition to the formation of 333 cross-links (14,15). In a mutant of Enterococcus faecium, an L,D-transpeptidase (Ldt fm ) is the key enzyme of an adaptive response to -lactam antibiotics since it bypasses the D,D-transpeptidase activity of PBPs, leading to high-level resistance to the drugs (18,20).Examination of the microarray data published by Betts et al. showed that an M. tuberculosis gene encoding a member of the active-site cysteine peptidase family, referred to as Rv0116c, of unknown function, was upregulated 17-fold under nutrient starvation (4). We have therefore investigated here the structure of peptidoglycan from M. tuberculosis to evaluate whether formation of 333 peptidoglycan cross-links could be part of
fThe Mycobacterium tuberculosis peptidoglycan is cross-linked mainly by L,D-transpeptidases (LDTs), which are efficiently inactivated by a single -lactam class, the carbapenems. Development of carbapenems for tuberculosis treatment has recently raised considerable interest since these drugs, in association with the -lactamase inhibitor clavulanic acid, are uniformly active against extensively drug-resistant M. tuberculosis and kill both exponentially growing and dormant forms of the bacilli. We have purified the five L,D-transpeptidase paralogues of M. tuberculosis (Mt1 to -5) and compared their activities with those of peptidoglycan fragments and carbapenems. The five LDTs were functional in vitro since they were active in assays of peptidoglycan cross-linking (Mt5), -lactam acylation (Mt3), or both (Mt1, Mt2, and Mt4). Mt3 was the only LDT that was inactive in the crosslinking assay, suggesting that this enzyme might be involved in other cellular functions such as the anchoring of proteins to peptidoglycan, as shown in Escherichia coli. Inactivation of LDTs by carbapenems is a two-step reaction comprising reversible formation of a tetrahedral intermediate, the oxyanion, followed by irreversible rupture of the -lactam ring that leads to formation of a stable acyl enzyme. Determination of the rate constants for these two steps revealed important differences (up to 460-fold) between carbapenems, which affected the velocity of oxyanion and acyl enzyme formation. Imipenem inactivated LDTs more rapidly than ertapenem, and both drugs were more efficient than meropenem and doripenem, indicating that modification of the carbapenem side chain could be used to optimize their antimycobacterial activity.
Three active-site cysteine L,D-transpeptidases can individually anchor the Braun lipoprotein to the Escherichia coli peptidoglycan. We show here that two additional enzymes of the same family form peptide bonds between the third residues of peptidoglycan stems, generating meso-DAP 3 3meso-DAP 3 unusual cross-links. This activity partially replaces the D,D-transpeptidase activity of penicillin-binding proteins.
BackgroundThe formation of the molluscan shell is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell forming tissue, the mantle. This so called "calcifying matrix" is a complex mixture of proteins and glycoproteins that is assembled and occluded within the mineral phase during the calcification process. While the importance of the calcifying matrix to shell formation has long been appreciated, most of its protein components remain uncharacterised.ResultsRecent expressed sequence tag (EST) investigations of the mantle tissue from the tropical abalone (Haliotis asinina) provide an opportunity to further characterise the proteins in the shell by a proteomic approach. In this study, we have identified a total of 14 proteins from distinct calcified layers of the shell. Only two of these proteins have been previously characterised from abalone shells. Among the novel proteins are several glutamine- and methionine-rich motifs and hydrophobic glycine-, alanine- and acidic aspartate-rich domains. In addition, two of the new proteins contained Kunitz-like and WAP (whey acidic protein) protease inhibitor domains.ConclusionThis is one of the first comprehensive proteomic study of a molluscan shell, and should provide a platform for further characterization of matrix protein functions and interactions.
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