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Bacterial glycoconjugates, such as cell surface polysaccharides and glycoproteins, play important roles in cellular interactions and survival. Enzymes called nucleotidyltransferases use sugar-1-phosphates and nucleoside triphosphates (NTPs) to produce nucleoside diphosphate sugars (NDP-sugars), which serve as building blocks for most glycoconjugates. Research spanning several decades has shown that some bacterial nucleotidyltransferases have broad substrate tolerance and can be exploited to produce a variety of NDP-sugars in vitro . While these enzymes are known to be allosterically regulated by NDP-sugars and their fragments, much work has focused on the effect of active site mutations alone. Here, we show that rational mutations in the allosteric site of the nucleotidyltransferase RmlA lead to expanded substrate tolerance and improvements in catalytic activity that can be explained by subtle changes in quaternary structure and interactions with ligands. These observations will help inform future studies on the directed biosynthesis of diverse bacterial NDP-sugars and downstream glycoconjugates.

Bacterial DnaK is an ATP-dependent molecular chaperone important for maintaining cellular proteostasis in concert with cofactor proteins. The cofactor DnaJ delivers non-native client proteins to DnaK and activates its ATPase activity, which is required for protein folding. In the bacterial pathogen Mycobacterium tuberculosis , DnaK is assisted by two DnaJs, DnaJ1 and DnaJ2. Functional protein-protein interactions (PPIs) between DnaK and at least one DnaJ are essential for survival of mycobacteria; hence, these PPIs represent untapped antibacterial targets. Here, we synthesize peptide-based mimetics of DnaJ1 and DnaJ2 N-terminal domains as rational inhibitors of DnaK-cofactor interactions. We find that covalently stabilized DnaJ mimetics are capable of disrupting DnaK-cofactor activity in vitro and prevent mycobacterial recovery from proteotoxic stress in vivo, leading to cell death. Since chaperones and cofactors are highly conserved, we anticipate these results will inform the design of other mimetics to modulate chaperone function across cell types.

The rifamycins are broad-spectrum antibiotics that are primarily utilized to treat infections caused by mycobacteria, including tuberculosis. Interestingly, various species of bacteria are known to contain an enzyme called Arr that catalyzes ADP-ribosylation of rifamycin antibiotics as a mechanism of resistance. Here, we study Arr modulation in relevant Gram-positive and -negative species. We show that a C-terminal truncation of Arr (Arr C ), encoded in the genome of Mycobacterium smegmatis , activates Arr-mediated rifamycin modification. Through structural comparisons of mycobacterial Arr and human poly(ADP-ribose) polymerases (PARPs), we identify a known small molecule PARP inhibitor that can act as an adjuvant to sensitize M. smegmatis to the rifamycin antibiotic rifampin via inhibition of Arr, even in the presence of Arr C . Finally, we demonstrate that this rifampin/adjuvant combination treatment is effective at inhibiting growth of the multidrug-resistant (MDR) nontuberculosis pathogen Mycobacterium abscessus , which has become a growing cause of human infections in the clinic.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains one of the world's deadliest infectious diseases and urgently requires new antibiotics to treat drug-resistant strains and to decrease the duration of therapy. During infection, Mtb encounters numerous stresses associated with host immunity, including hypoxia, reactive oxygen and nitrogen species, mild acidity, nutrient starvation, and metal sequestration and intoxication. The Mtb proteostasis network, composed of chaperones, proteases, and a eukaryotic-like proteasome, provides protection from stresses and chemistries of host immunity by maintaining the integrity of the mycobacterial proteome. In this Review, we explore the proteostasis network as a noncanonical target for antibacterial drug discovery.