Bacterial survival requires an intact peptidoglycan layer, a 3-dimensional exoskeleton that encapsulates the cytoplasmic membrane. Historically, the final steps of peptidoglycan synthesis are known to be carried out by d,d-transpeptidases, enzymes that are inhibited by the β-lactams which constitute >50% of all antibacterials in clinical use. Here, we show that the carbapenem subclass of β-lactams is distinctly effective not only because they inhibit d,d-transpeptidases and are poor substrates for β-lactamases, but primarily because they also inhibit non-classical transpeptidases, namely the l,d-transpeptidases, that generate the majority of linkages in the peptidoglycan of mycobacteria. We have characterized the molecular mechanisms responsible for inhibition of l,d-transpeptidases of M. tuberculosis and a range of bacteria, including ESKAPE pathogens, and utilized this information to design, synthesize and test simplified carbapenems with potent antibacterial activity.

Mycobacterium tuberculosis utilizes many mechanisms to establish itself within the macrophage, and bacterially derived cAMP is important in modulating the host cellular response. Although the genome of M. tuberculosis is endowed with a number of mammalian-like adenylyl cyclases, only a single cAMP phosphodiesterase has been identified that can decrease levels of cAMP produced by the bacterium. We present the crystal structure of the full-length and sole cAMP phosphodiesterase, Rv0805, found in M. tuberculosis, whose orthologs are present only in the genomes of slow growing and pathogenic mycobacteria. The dimeric core catalytic domain of Rv0805 adopts a metallophosphoesterase-fold, and the C-terminal region builds the active site and contributes to multiple substrate utilization. Localization of Rv0805 to the cell wall is dependent on its C terminus, and expression of either wild type or mutationally inactivated Rv0805 in M. smegmatis alters cell permeability to hydrophobic cytotoxic compounds. Rv0805 may therefore play a key role in the pathogenicity of mycobacteria, not only by hydrolyzing bacterial cAMP, but also by moonlighting as a protein that can alter cell wall functioning.Mycobacterium tuberculosis is probably one of the most successful human pathogens known so far, being singly responsible for the largest number of deaths worldwide due to an infectious disease. M. tuberculosis is phagocytosed by the macrophage and is able to subvert the defenses of the host by a number of mechanisms. These include the presence of a complex cell wall that prevents free passage of potentially toxic material, the ability of the bacteria to withstand the acidic environment of the phagolysosome, and to neutralize reactive oxygen and nitrogen species produced by the activated macrophage (1). An increased understanding of mechanisms utilized by this pathogen to evade the host immune system and continue to reside in the hostile environment of the macrophage, would no doubt provide avenues for the development of drugs to novel targets in the bacterium.Cross-communication between the pathogen and host could involve the utilization of signaling molecules that are conserved evolutionarily. Cyclic AMP is found in all kingdoms of life, and proteins that synthesize and degrade the cyclic nucleotide have been well characterized. The genome of M. tuberculosis H37Rv encodes 16 mammalian-like nucleotide cyclase-like genes (2), and intracellular and extracellular levels of cAMP are very high in both pathogenic and non-pathogenic (e.g. Mycobacterium smegmatis) mycobacteria (2, 3). Recent evidence has highlighted the importance of cAMP in modulating the host macrophage response to M. tuberculosis infection (4). A single adenylyl cyclase was shown to be responsible for the burst of cAMP that is seen in the macrophage following phagocytosis of M. tuberculosis, and this bacterially derived increase in cAMP was essential to attenuate the response of the macrophage to the pathogen.The regulated degradation of cAMP is as important as its synthesi...

Mycobacterial genomes are endowed with many eukaryote-like nucleotide cyclase genes encoding proteins that can synthesize 3,5-cyclic AMP (cAMP). However, the roles of cAMP and the need for such redundancy in terms of adenylyl cyclase genes remain unknown. We measured cAMP levels in Mycobacterium smegmatis during growth and under various stress conditions and report the first biochemical and functional characterization of the MSMEG_3780 adenylyl cyclase, whose orthologs in Mycobacterium tuberculosis (Rv1647) and Mycobacterium leprae (ML1399) have been recently characterized in vitro. MSMEG_3780 was important for producing cAMP levels in the logarithmic phase of growth, since the ⌬MSMEG_3780 strain showed lower intracellular cAMP levels at this stage of growth. cAMP levels decreased in wild-type M. smegmatis under conditions of acid stress but not in the ⌬MSMEG_3780 strain. This was correlated with a reduction in MSMEG_3780 promoter activity, indicating that the effect of the reduction in cAMP levels on acid stress was caused by a decrease in the transcription of MSMEG_3780. Complementation of the ⌬MSMEG_3780 strain with the genomic integration of MSMEG_3780 or the Rv1647 gene could restore cAMP levels during logarithmic growth. The Rv1647 promoter was also acid sensitive, emphasizing the biochemical and functional similarities in these two adenylyl cyclases. This study therefore represents the first detailed biochemical and functional analysis of an adenylyl cyclase that is important for maintaining cAMP levels in mycobacteria and underscores the subtle roles that these genes may play in the physiology of the organism.Cyclic AMP (cAMP) is an important intracellular second messenger that is involved in several signaling pathways in both bacteria and eukaryotes (2, 5). Studies have shown a role for cAMP not only in physiological processes such as catabolite repression and sporulation (16) but also in the regulation of several virulence pathways, e.g., in Pseudomonas, Vibrio, and Candida species (1,17,19,40,41). The synthesis of cAMP is dependent on the activity of adenylyl cyclases, which can be membrane-bound or soluble proteins (7). The largely differing amino acid sequences of nucleotide cyclases allow their classification into six classes, among which the class III nucleotide cyclases have the widest phyletic distribution (7, 37).Adenylyl and guanylyl cyclases of the class III family are proteins that form head-to-tail dimers and generate either two (homodimeric enzymes) or one (heterodimeric enzymes) active site at the dimer interface (20). Nucleotide cyclases catalyze the conversion of ATP or GTP to cAMP or GMP, respectively, in a metal-dependent manner, and biochemical and structural studies have identified specific sequence motifs that are involved in substrate (ATP or GTP) or metal (Mg 2ϩ ) binding (20). In contrast to the limited domain compositions of mammalian nucleotide cyclases, the bacterial counterparts are often fused to a variety of domains, highlighting their ability to be regulated allostericall...

BackgroundThe carbapenem subclass of β-lactams is among the most potent antibiotics available today. Emerging evidence shows that, unlike other subclasses of β-lactams, carbapenems bind to and inhibit non-classical transpeptidases (L,D-transpeptidases) that generate 3 → 3 linkages in bacterial peptidoglycan. The carbapenems biapenem and tebipenem exhibit therapeutically valuable potencies against Mycobacterium tuberculosis (Mtb).ResultsHere, we report the X-ray crystal structures of Mtb L,D-transpeptidase-2 (LdtMt2) complexed with biapenem or tebipenem. Despite significant variations in carbapenem sulfur side chains, biapenem and tebipenem ultimately form an identical adduct that docks to the outer cavity of LdtMt2. We propose that this common adduct is an enzyme catalyzed decomposition of the carbapenem adduct by a mechanism similar to S-conjugate elimination by β-lyases.ConclusionThe results presented here demonstrate biapenem and tebipenem bind to the outer cavity of LdtMt2, covalently inactivate the enzyme, and subsequently degrade via an S-conjugate elimination mechanism. We discuss structure based drug design based on the findings and propose that the S-conjugate elimination can be leveraged to design novel agents to deliver and locally release antimicrobial factors to act synergistically with the carbapenem carrier.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-017-0082-4) contains supplementary material, which is available to authorized users.