The structure of the pentapeptide precursor of PG in Mtb
has been determined to be L-Ala-γ-D-Glu-meso
-DAP-D-Ala-D-Ala by direct isolation of the nucleotide-linked precursor from the cytosol (Mahapatra et al., 2005
). Analysis of monomeric stem peptides isolated from mature PG in Mtb
showed widespread amidation of the free carboxyl groups of Glu and DAP residues resulting in L-Ala-D-Glu(NH2
)-D-Ala as the most abundant monomeric peptide in Mtb
PG (Mahapatra et al., 2008
). Similarly, this tetrapeptide was shown to be the most abundant monomeric peptide in stationary phase cultures of Mtb
grown under non-shaking conditions (Lavollay et al., 2008
). Earlier reports have shown existence of DAP-DAP (3-3) cross-links in mycobacterial PG (Wietzerbin et al., 1974
), and a recent study revealed that almost 80% of dimeric peptides from stationary-phase cells were cross-linked by a 3-3 mechanism (Lavollay et al., 2008
). In this study, we also report that the majority of the monomeric stem peptides are tetrapeptides (, ). Pentapeptide (, ) was also detected albeit in very low abundance, hinting at a highly efficient carboxypeptidase activity resulting in effective processing.
Among dimeric peptides we determined the ratio of 3-3 to 4-3 cross-linked peptides to be 3:2; a ratio that did not vary significantly with growth phase or condition. It can be inferred therefore that 3-3 cross-links predominate in mycobacterial PG under all phases of growth. It was interesting to note that the α-carboxylate of Glu and ε-carboxylate of DAP were fully amidated in majority of monomeric peptides and also among majority of 4-3 cross-linked dimeric peptides, however, in 3-3 cross-linked dimeric peptides about half of donor stem peptides lacked amidation at the ε-carboxylate of DAP. The differential amidation of stem peptides suggests the intriguing possibility that the transpeptidases may selectively recognize potential substrates based on differential amidation and offers a potential regulatory mechanism for cross-linking in PG. Amidation of both glutamine and DAP in PG has also been shown to influence recognition by specific mammalian proteins such as NOD1 and NOD2 and may modulate inflammation induced during infection (Chamaillard et al., 2003
, Inohara et al., 2005
). Little attention has focused on variation in PG structure in circulating clinical isolates of Mtb
or in understanding the potential contribution of PG modification to virulence.
In many other bacteria like Bacillus subtilis
(Atrih & Foster, 1999
) and E. coli
(Driehuis & Wouters, 1987
), the PG has been shown to alter with stage of life cycle and under different growth conditions. In slowly growing E. coli
changes in PG composition have been reported showing a decrease in the major monomeric species, decreases in the average chain length of stem peptides, and a significant increase in DAP-DAP containing cross-links (Tuomanen & Cozens, 1987
). It is therefore somewhat surprising that Mtb
PG does not show any significant growth phase-dependent modulation under similar in vitro
conditions of growth. 3-3 cross-linkages in the peptidoglycan layer of Mtb
have been shown to be essential for maintenance of normal colony morphology and impacts growth of the organism in mice (Gupta et al., 2010
). The peptidoglycan layer in mycobacteria however, serves as an anchor for arabinogalactan, which is, in turn, linked to the mycolic acids of the outer lipid layer (Barry et al., 2007
). This basement role as the innermost polymer of a macromolecular complex may not lend itself to dramatic structural reorganization during growth or stress. The mycolic acids bound to arabinogalactan that form the primary permeability barrier of the mycobacterial cell do show variation in fine structure during growth in vivo
and in response to oxygen deprivation (Yuan et al., 1998
). Likewise, many other peripheral lipids and glycolipids are environmentally responsive such as phthiocerol dimycocerosates (PDIMs) and triacylglycerides (TAGs) (Flores-Valdez et al., 2009
, Barry et al., 1998
). It is worth emphasizing, however, that our sampling of the architecture of PG in Mtb
may be limited to only a subset of cross-links (approximately 70% of the total) that are accessible to muramidase digestion and analysis using HPLC as even in a relatively short time frame (several hours) nearly one-third of newly incorporated radioactive D-Ala is converted into structures that are not released by current analytical techniques (Fig. S2
and data not shown).
The essentiality of PG for cellular integrity has proven a valuable target for the development of a number of antibacterials such as β-lactams, cycloserine and vancomycin. β-lactams primarily target penicillin binding proteins (PBPs) that include D,D-transpeptidases and D,D-carboxypeptidases. It has been reported that 3-3 cross-links are installed by an ampicillin insensitive L,D-transpeptidase in Enterococcus faecium
(Mainardi et al., 2000
, Sacco et al., 2010
). The acquisition of β-lactam resistance in this organism was not associated with changes in the expression level of the corresponding transpeptidase but rather was associated with production of a D,D-carboxypeptidase leading to an increase in the amount of tetrapeptide carrying substrate for formation of 3-3 cross-links (Mainardi et al., 2002
). In Mtb
a penicillin-insensitive L,D-transpeptidase Rv0116c has also been described (Lavollay et al., 2008
), which in contrast to the E. faecium
enzyme, proved sensitive to meropenem and other carbapenems.
Our results show that meropenem treatment resulted in the accumulation of pentapeptide stems in mature PG () suggesting that meropenem may be directly inhibiting a D,D-carboxypeptidase as well as other transpeptidase targets in vivo
. Characterized L,D-transpeptidases show a preference for tetrameric substrates whose production requires D,D-carboxypeptidase activity. Inhibition of such a D,D-carboxypeptidase has recently been shown to affect L,D-transpeptidases preferentially over D,D-transpeptidase in Corynebacterium jeikeium
and these authors report a similar accumulation of pentapeptide precursors in cells treated with ampicillin (Lavollay et al., 2009
). However, unlike what these authors report in C. jeikeium,
we do not observe a significant decrease in the quantity of tetrapeptide precursors available for cross-linking in Mtb
(Lavollay et al., 2009
). This supports that both the L,D-transpeptidase which cross-links tetrapeptide stems and the D,D-carboxypeptidase that processes pentapeptide stems to substrates are both exquisitely sensitive to meropenem in Mtb.
We biochemically characterized the D,D-carboxypeptidase DacB2 for its D,D-carboxypeptidase activity () and showed that it forms a covalent adduct with meropenem () in addition to inhibiting enzymatic activity (). Our results therefore suggest that while meropenem likely has an L,D-transpeptidase target, the D,D-carboxypeptidase DacB2 is also highly sensitive. Preventing processing of pentapeptide precursors to tetrapeptides while simultaneously inhibiting the transpeptidases capable of utilizing such precursors to form 3-3 or 4-3 cross-linkages would be expected to be synergistic and may contribute to the uniquely rapid lytic activity of the carbapenems. Of note, one of the L,D-transpeptidases encoded in the Mtb
genome (Rv0116c) has been shown to form an adduct with meropenem in vitro
and to be broadly susceptible to other carbapenems (Lavollay et al., 2008
). Also consistent with the idea that meropenem simultaneously inhibits multiple targets is that we have been unable to generate spontaneously resistant mutants regardless of the number of bacteria plated, despite replenishing drug concentration daily (data not shown).
The surprising lack of structural variation of mycobacterial PG may provide a unique point of vulnerability for targeting using existing β-lactams such as meropenem. The ability of these agents to kill even non-replicating cells (Hugonnet et al., 2009
) is also surprising in this context but presumably hints that peptidoglycan synthesis, recycling and remodeling may be important processes at all stages of growth of the organism. The difficulty of parenteral administration of existing carbapenems may provide an impetus to revive attempts to make an orally bioavailable agent in this class. Because of the apparent redundancy in L,D-transpeptidase enzymes, the apparent vulnerability of the D,D-carboxypeptidase may also offer an attractive alternative target for the development of alternative chemotherapeutic approaches.