The importance of the cell wall to the viability of the bacterium is underscored by the breadth of antibiotic structures that act by blocking key enzymes that are tasked with cell-wall creation, ...preservation, and regulation. The interplay between cell-wall integrity, and the summoning forth of resistance mechanisms to deactivate cell-wall-targeting antibiotics, involves exquisite orchestration among cell-wall synthesis and remodeling and the detection of and response to the antibiotics through modulation of gene regulation by specific effectors. Given the profound importance of antibiotics to the practice of medicine, the assertion that understanding this interplay is among the most fundamentally important questions in bacterial physiology is credible. The enigmatic regulation of the expression of the AmpC β-lactamase, a clinically significant and highly regulated resistance response of certain Gram-negative bacteria to the β-lactam antibiotics, is the exemplar of this challenge. This review gives a current perspective to this compelling, and still not fully solved, 35-year enigma.
Bacterial cell-wall recycling Johnson, Jarrod W.; Fisher, Jed F.; Mobashery, Shahriar
Annals of the New York Academy of Sciences,
January 2013, Volume:
1277, Issue:
1
Journal Article
Peer reviewed
Open access
Many Gram‐negative and Gram‐positive bacteria recycle a significant proportion of the peptidoglycan components of their cell walls during their growth and septation. In many—and quite possibly ...all—bacteria, the peptidoglycan fragments are recovered and recycled. Although cell‐wall recycling is beneficial for the recovery of resources, it also serves as a mechanism to detect cell‐wall–targeting antibiotics and to regulate resistance mechanisms. In several Gram‐negative pathogens, anhydro‐MurNAc‐peptide cell‐wall fragments regulate AmpC β‐lactamase induction. In some Gram‐positive organisms, short peptides derived from the cell wall regulate the induction of both β‐lactamase and β‐lactam–resistant penicillin‐binding proteins. The involvement of peptidoglycan recycling with resistance regulation suggests that inhibitors of the enzymes involved in the recycling might synergize with cell‐wall–targeted antibiotics. Indeed, such inhibitors improve the potency of β‐lactams in vitro against inducible AmpC β‐lactamase–producing bacteria. We describe the key steps of cell‐wall remodeling and recycling, the regulation of resistance mechanisms by cell‐wall recycling, and recent advances toward the discovery of cell‐wall–recycling inhibitors.
We recently reported on the discovery of a novel antibacterial (2) with a 4(3H)-quinazolinone core. This discovery was made by in silico screening of 1.2 million compounds for binding to a ...penicillin-binding protein and the subsequent demonstration of antibacterial activity against Staphylococcus aureus. The first structure–activity relationship for this antibacterial scaffold is explored in this report with evaluation of 77 variants of the structural class. Eleven promising compounds were further evaluated for in vitro toxicity, pharmacokinetics, and efficacy in a mouse peritonitis model of infection, which led to the discovery of compound 27. This new quinazolinone has potent activity against methicillin-resistant (MRSA) strains, low clearance, oral bioavailability and shows efficacy in a mouse neutropenic thigh infection model.
β-Lactams from the Ocean Fisher, Jed F; Mobashery, Shahriar
Marine drugs,
01/2023, Volume:
21, Issue:
2
Journal Article
Peer reviewed
Open access
The title of this essay is as much a question as it is a statement. The discovery of the β-lactam antibiotics-including penicillins, cephalosporins, and carbapenems-as largely (if not exclusively) ...secondary metabolites of terrestrial fungi and bacteria, transformed modern medicine. The antibiotic β-lactams inactivate essential enzymes of bacterial cell-wall biosynthesis. Moreover, the ability of the β-lactams to function as enzyme inhibitors is of such great medical value, that inhibitors of the enzymes which degrade hydrolytically the β-lactams, the β-lactamases, have equal value. Given this privileged status for the β-lactam ring, it is therefore a disappointment that the exemplification of this ring in marine secondary metabolites is sparse. It may be that biologically active marine β-lactams are there, and simply have yet to be encountered. In this report, we posit a second explanation: that the value of the β-lactam to secure an ecological advantage in the marine environment might be compromised by its close structural similarity to the β-lactones of quorum sensing. The steric and reactivity similarities between the β-lactams and the β-lactones represent an outside-of-the-box opportunity for correlating new structures and new enzyme targets for the discovery of compelling biological activities.
Nonhealing chronic wounds are major complications of diabetes resulting in >70,000 annual lower-limb amputations in the United States alone. The reasons the diabetic wound is recalcitrant to healing ...are not fully understood, and there are limited therapeutic agents that could accelerate or facilitate its repair. We previously identified two active forms of matrix metalloproteinases (MMPs), MMP-8 and MMP-9, in the wounds ofdb/dbmice. We argued that the former might play a role in the body’s response to wound healing and that the latter is the pathological consequence of the disease with detrimental effects. Here we demonstrate that the use of compound ND-336, a novel highly selective inhibitor of gelatinases (MMP-2 and MMP-9) and MMP-14, accelerates diabetic wound healing by lowering inflammation and by enhancing angiogenesis and re-epithelialization of the wound, thereby reversing the pathological condition. The detrimental role of MMP-9 in the pathology of diabetic wounds was confirmed further by the study of diabetic MMP-9–knockout mice, which exhibited wounds more prone to healing. Furthermore, topical administration of active recombinant MMP-8 also accelerated diabetic wound healing as a consequence of complete re-epithelialization, diminished inflammation, and enhanced angiogenesis. The combined topical application of ND-336 (a small molecule) and the active recombinant MMP-8 (an enzyme) enhanced healing even more, in a strategy that holds considerable promise in healing of diabetic wounds.
The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the β-lactam antibiotics by methicillin-resistant Staphylococcus aureus (MRSA). The ...high-molecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell wall. In bacteria susceptible to β-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of irreversible acylation of an active site serine by the β-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to β-lactam acylation and successfully catalyzes the dd -transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with β-lactam antibiotics is a continuing public health concern. We report herein the identification of an allosteric binding domain—a remarkable 60 Å distant from the dd -transpeptidase active site—discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry. This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA β-lactam antibiotic. The ability of an anti-MRSA β-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second β-lactam molecule, opens an unprecedented realm for β-lactam antibiotic structure-based design.
Assembly of the peptidoglycan is crucial in maintaining viability of bacteria and in defining bacterial cell shapes, both of which are important for existence in the ecological niche that the ...organism occupies. Here, eight crystal structures for a member of the cell-shape-determining class of Campylobacter jejuni, the peptidoglycan peptidase 3 (Pgp3), are reported. Characterization of the turnover chemistry of Pgp3 reveals cell wall D,D-endopeptidase and D,D-carboxypeptidase activities. Catalysis is accompanied by large conformational changes upon peptidoglycan binding, whereby a loop regulates access to the active site. Furthermore, prior hydrolysis of the crosslinked peptide stem from the saccharide backbone of the peptidoglycan on one side is a pre-requisite for its recognition and turnover by Pgp3. These analyses reveal the noncanonical nature of the transformations at the core of the events that define the morphological shape for C. jejuni as an intestinal pathogen.
ATP-binding cassette (ABC) transporters represent one of the largest protein superfamilies. Functionally diverse, ABC transporters have been implicated in many aspects of microbial physiology. The ...genome of the human fungal pathogen Cryptococcus neoformans encodes 54 putative ABC transporters and most of them remain uncharacterized. In a previous genetic screen for fungal regulators of phagocytosis, we identified an uncharacterized gene, CNAG_06909, that modulates host interactions. This gene encoded a half-size ABC transporter of the PDR-type, and phenotypic studies of a strain with this gene deleted revealed an altered antifungal susceptibility profile, including hypersensitivity to fluconazole (FLC). This gene, which we named
, localized to the endoplasmic reticulum (ER) and plasma membrane (PM), and when absent, less ergosterol was observed in the PM. Additionally, we observed that the
Δ strain displayed a reduction in secreted polysaccharide capsular material. These changes to the cellular surface may explain the observed increased uptake by macrophages and the reduced intracellular survival. Finally, studies in mice demonstrated that Pdr6 function was required for the normal progression of cryptococcal infection. Taken together, this study demonstrates a novel dual role for PDR transporters in C. neoformans, which could represent a potential target for antifungal therapeutics. Furthermore, the atypical half-size transporter encoded by
is conserved in many fungal pathogens, but absent in model nonpathogenic fungi. Hence, this study provided a function for this unique group of fungal half-size PDR transporters that, although conserved, remain largely understudied.
Conserved across all kingdoms of life, ABC transporters comprise one of the largest protein families. They are associated with multidrug resistance, affecting aspects such as resistance to antimicrobials or anti-cancer drugs. Despite their importance, they are understudied in fungal pathogens. In the environmental fungus Cryptococcus neoformans, a leading cause of fungal infections, only a few ABC transporters have been studied. Here, we characterized an atypical, half-size, ABC transporter of the PDR-type, that affected both antifungal resistance and host-pathogen interactions. PDR-type transporters are only present in fungi and plants, and this subgroup of half-size transporters was conserved in fungal pathogens, yet their function was completely unknown. Because the current treatments for cryptococcal infection are suboptimal, understanding the mechanisms of antifungal resistance and the host interactions that drive the infection is critical to improving the management of this disease. Here, we provide insights into these important aspects of cryptococcal pathogenesis.
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