Methylfolate Trap Promotes Bacterial Thymineless Death by Sulfa Drugs.

The methylfolate trap, a metabolic blockage associated with anemia, neural tube defects, Alzheimer's dementia, cardiovascular diseases, and cancer, was discovered in the 1960s, linking the metabolism of folate, vitamin B12, methionine and homocysteine. However, the existence or physiological significance of this phenomenon has been unknown in bacteria, which synthesize folate de novo. Here we identify the methylfolate trap as a novel determinant of the bacterial intrinsic death by sulfonamides, antibiotics that block de novo folate synthesis. Genetic mutagenesis, chemical complementation, and metabolomic profiling revealed trap-mediated metabolic imbalances, which induced thymineless death, a phenomenon in which rapidly growing cells succumb to thymine starvation. Restriction of B12 bioavailability, required for preventing trap formation, using an "antivitamin B12" molecule, sensitized intracellular bacteria to sulfonamides. Since boosting the bactericidal activity of sulfonamides through methylfolate trap induction can be achieved in Gram-negative bacteria and mycobacteria, it represents a novel strategy to render these pathogens more susceptible to existing sulfonamides.

Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets.

Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro ß-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the ß-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore ß-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation.

A redox regulatory system critical for mycobacterial survival in macrophages and biofilm development.

Survival of M. tuberculosis in host macrophages requires the eukaryotic-type protein kinase G, PknG, but the underlying mechanism has remained unknown. Here, we show that PknG is an integral component of a novel redox homeostatic system, RHOCS, which includes the ribosomal protein L13 and RenU, a Nudix hydrolase encoded by a gene adjacent to pknG. Studies in M. smegmatis showed that PknG expression is uniquely induced by NADH, which plays a key role in metabolism and redox homeostasis. In vitro, RenU hydrolyses FAD, ADP-ribose and NADH, but not NAD+. Absence of RHOCS activities in vivo causes NADH and FAD accumulation, and increased susceptibility to oxidative stress. We show that PknG phosphorylates L13 and promotes its cytoplasmic association with RenU, and the phosphorylated L13 accelerates the RenU-catalyzed NADH hydrolysis. Importantly, interruption of RHOCS leads to impaired mycobacterial biofilms and reduced survival of M. tuberculosis in macrophages. Thus, RHOCS represents a checkpoint in the developmental program required for mycobacterial growth in these environments.

Molecular biology of drug resistance in Mycobacterium tuberculosis.

Tuberculosis (TB) has become a curable disease, thanks to the discovery of antibiotics. However, it has remained one of the most difficult infections to treat. Most current TB regimens consist of 6-9 months of daily doses of four drugs that are highly toxic to patients. The purpose of these lengthy treatments is to completely eradicate Mycobacterium tuberculosis, notorious for its ability to resist most antibacterial agents, thereby preventing the formation of drug resistant mutants. On the contrary, the prolonged therapies have led to poor patient adherence. This, together with a severe limit of drug choices, has resulted in the emergence of strains that are increasingly resistant to the few available antibiotics. Here, we review our current understanding of molecular mechanisms underlying the profound drug resistance of M. tuberculosis. This knowledge is essential for the development of more effective antibiotics, which are not only potent against drug resistant M. tuberculosis strains but also help shorten the current treatment courses required for drug susceptible TB.

Can inhibitor-resistant substitutions in the Mycobacterium tuberculosis ß-Lactamase BlaC lead to clavulanate resistance?: a biochemical rationale for the use of ß-lactam-ß-lactamase inhibitor combinations.

The current emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis calls for novel treatment strategies. Recently, BlaC, the principal ß-lactamase of Mycobacterium tuberculosis, was recognized as a potential therapeutic target. The combination of meropenem and clavulanic acid, which inhibits BlaC, was found to be effective against even extensively drug-resistant M. tuberculosis strains when tested in vitro. Yet there is significant concern that drug resistance against this combination will also emerge. To investigate the potential of BlaC to evolve variants resistant to clavulanic acid, we introduced substitutions at important amino acid residues of M. tuberculosis BlaC (R220, A244, S130, and T237). Whereas the substitutions clearly led to in vitro clavulanic acid resistance in enzymatic assays but at the expense of catalytic activity, transformation of variant BlaCs into an M. tuberculosis H37Rv background revealed that impaired inhibition of BlaC did not affect inhibition of growth in the presence of ampicillin and clavulanate. From these data we propose that resistance to ß-lactam-ß-lactamase inhibitor combinations will likely not arise from structural alteration of BlaC, therefore establishing confidence that this therapeutic modality can be part of a successful treatment regimen against M. tuberculosis.

A lipoprotein modulates activity of the MtrAB two-component system to provide intrinsic multidrug resistance, cytokinetic control and cell wall homeostasis in Mycobacterium.

The MtrAB signal transduction system, which participates in multiple cellular processes related to growth and cell wall homeostasis, is the only two-component system known to be essential in Mycobacterium. In a screen for antibiotic resistance determinants in Mycobacterium smegmatis, we identified a multidrug-sensitive mutant with a transposon insertion in lpqB, the gene located immediately downstream of mtrA-mtrB. The lpqB mutant exhibited increased cell-cell aggregation and severe defects in surface motility and biofilm growth. lpqB cells displayed hyphal growth and polyploidism, reminiscent of the morphology of Streptomyces, a related group of filamentous Actinobacteria. Heterologous expression of M. tuberculosis LpqB restored wild-type characteristics to the lpqB mutant. LpqB interacts with the extracellular domain of MtrB, and influences MtrA phosphorylation and promoter activity of dnaA, an MtrA-regulated gene that affects cell division. Furthermore, in trans expression of the non-phosphorylated, inactive form of MtrA in wild-type M. smegmatis resulted in phenotypes similar to those of lpqB deletion, whereas expression of the constitutively active form of MtrA restored wild-type characteristics to the lpqB mutant. These results support a model in which LpqB, MtrB and MtrA form a three-component system that co-ordinates cytokinetic and cell wall homeostatic processes.

Protein kinase G is required for intrinsic antibiotic resistance in mycobacteria.

Antibiotic resistance and virulence of pathogenic mycobacteria are phenotypically associated, but the underlying genetic linkage has not been known. Here we show that PknG, a eukaryotic-type protein kinase previously found to support survival of mycobacteria in host cells, is required for the intrinsic resistance of mycobacterial species to multiple antibiotics.

Strategies for potentiation of ethionamide and folate antagonists against Mycobacterium tuberculosis.

Antifolates inhibit de novo folate biosynthesis, whereas ethionamide targets the mycolate synthetic pathway in Mycobacterium tuberculosis. These antibiotics are effective against M. tuberculosis but their use has been hampered by concerns over toxicity and low therapeutic indexes. With the increasing spread of drug-resistant forms, interest in using old drugs for tuberculosis treatment has been renewed. Specific inhibitors targeting resistance mechanisms could sensitize M. tuberculosis to these available, clinically approved drugs. This review discusses recently developed strategies to boost the antituberculous activity of ethionamide and antifolates. These approaches might help broaden the currently limited chemotherapeutic options of not only drug-resistant but also drug-susceptible tuberculosis, which still remains one of the most common infectious diseases in the developing world.