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

More than 60 years after the introduction of penicillin, the class of ␤-lactam antibiotics remains the most commonly used to treat severe infections despite the emergence of various resistance mechanisms, including drug detoxification by production of ␤-lactamases (1), decreased affinity of the target (2), and decreased permeability (3). The success story of ␤-lactams involves the development of several generations of ␤-lactams to defeat the resistance mechanisms and their association with inhibitors of ␤-lactamases (4, 5). All ␤-lactams have a common mechanism of action, as they share structural similarity with bacterial cell wall precursors. These drugs act as suicide substrates of the DD-transpeptidase catalytic domain of the penicillin-binding proteins (PBPs) 2 responsible for the last cross-linking step of cell wall assembly (Fig. 1A) (2, 6, 7).Among the DD-transpeptidases of Enterococcus faecium, the low affinity PBP5 is unessential for growth but responsible for intrinsic low level resistance to ampicillin (8, 9), the first-line drug used at high dosage to treat severe enterococcal infections. In clinical isolates, higher levels of resistance to ampicillin are commonly associated with multiple amino acid substitutions in the transpeptidase domain of PBP5 (10, 11). We have shown previously that even higher levels of resistance to ampicillin can be achieved by a novel mechanism in the absence of PBP5 (12 (Fig. 1B). In the presence of ampicillin, the novel L-Lys 3 3D-iAsn-L-Lys 3 cross-links were exclusively found in the peptidoglycan of E. faecium M512, indicating that the DD-transpeptidase activity of the PBPs did not participate in the formation of the cross-links. Selection of E. faecium M512 from E. faecium D344S in five consecutive steps on increasing concentrations of ampicillin led to the gradual activation of the LD-transpeptidation pathway because the proportion of L-Lys 3 3D-iAsn-L-Lys 3 was 3.1% for D344S and increased at each selection step (13). Activation of the resistance pathway was associated with the production of a DD-carboxypeptidase that generated precursors containing a tetrapeptide stem in the cytoplasm of E. faecium M512 (13). Based on these observations, we have speculated that precursors containing a tetrapeptide stem are essential for the formation of the L-Lys 3 3D-iAsn-L-Lys 3 cross-links. In the present work, we have identified in E. faecium an LD-transpeptidase as the key enzyme of this alternate transpeptidation pathway and have shown that its substrate does not bear structural similarity to ␤-lactams. The bypass mechanism could invalidate the ␤-lactams as the major class of antibiotics. EXPERIMENTAL PROCEDURES Purification of the LD-Transpeptidase and N-terminal Sequencing-The LD-transpeptidase from E. faecium (Ldt fm ) was partially purified in four chromatographic steps using a radioactive exchange assay (13) to detect active fractions. Briefly, E. faecium M512 (12) was grown to an A 650 of 0.7 in 24 liters of brain heart infusion broth (Difco), harvested by centrifugati...

No abstract