Carl J. Balibar

Disruption of a Nonribosomal Peptide Synthetase in Aspergillus fumigatus Eliminates Gliotoxin Production

ABSTRACT The fungal secondary metabolite gliotoxin produced by Aspergillus fumigatus has been hypothesized to be important in the development of invasive aspergillosis. In this study, we addressed this hypothesis by disrupting a nonribosomal peptide synthetase (NRPS) (encoded by gliP) predicted to be involved in gliotoxin production. Mutants with a disrupted gliP locus failed to produce gliotoxin, which confirmed the role of the NRPS encoded by gliP in gliotoxin biosynthesis. We found no morphological, developmental, or physiological defects in ΔgliP mutant strains. In addition, disruption of gliP resulted in down regulation of gene expression in the gliotoxin biosynthesis gene cluster, which was restored with addition of exogenous gliotoxin. This interesting result suggests a role for gliotoxin in regulating its own production. Culture filtrates from the ΔgliP mutant were unable to inhibit ionomycin-dependent degranulation of mast cells, suggesting a role for gliotoxin in suppressing mast cell degranulation and possibly in disease development. However, the ΔgliP mutant did not have an impact on survival or tissue burden in a murine inhalational model of invasive aspergillosis. This result suggests that gliotoxin is not required for virulence in an immunosuppressed host with an invasive pulmonary infection.

Selective small-molecule inhibition of an RNA structural element.

Riboswitches are non-coding RNA structures located in messenger RNAs that bind endogenous ligands, such as a specific metabolite or ion, to regulate gene expression. As such, riboswitches serve as a novel, yet largely unexploited, class of emerging drug targets. Demonstrating this potential, however, has proven difficult and is restricted to structurally similar antimetabolites and semi-synthetic analogues of their cognate ligand, thus greatly restricting the chemical space and selectivity sought for such inhibitors. Here we report the discovery and characterization of ribocil, a highly selective chemical modulator of bacterial riboflavin riboswitches, which was identified in a phenotypic screen and acts as a structurally distinct synthetic mimic of the natural ligand, flavin mononucleotide, to repress riboswitch-mediated ribB gene expression and inhibit bacterial cell growth. Our findings indicate that non-coding RNA structural elements may be more broadly targeted by synthetic small molecules than previously expected.

Terrequinone A biosynthesis through L -tryptophan oxidation, dimerization and bisprenylation

The antitumor fungal metabolite terrequinone A, identified in extracts of Aspergillus sp., is biosynthesized by the five-gene cluster tdiA–tdiE. In this work, we have overproduced all five proteins (TdiA–TdiE) in the bacterial host Escherichia coli, fully reconstituting the biosynthesis of terrequinone A. This pathway involves aminotransferase activity, head-to-tail dimerization and bisprenylation of the scaffold to yield the benzoquinone natural product. We have established that TdiD is a pyridoxal-5′-phosphate–dependent L-tryptophan aminotransferase that generates indolepyruvate for an unusual nonoxidative coupling by the tridomain nonribosomal peptide synthetase TdiA. TdiC, an NADH-dependent quinone reductase, generates the nucleophilic hydroquinone for two distinct rounds of prenylation by the single prenyltransferase TdiB. TdiE is required to shunt the benzoquinone away from an off-pathway monoprenylated species by an as yet unknown mechanism. Overall, we have biochemically characterized the complete biosynthetic pathway to terrequinone A, highlighting the nonoxidative dimerization pathway and the unique asymmetric prenylation involved in its maturation.

Identification and characterization of the Staphylococcus aureus gene cluster coding for staphyloferrin A.

Siderophores are key virulence factors that allow bacteria to grow in iron-restricted environments. The Gram-positive pathogen Staphylococcus aureus is known to produce four siderophores for which genetic and/or structural data are unknown. Here we characterize the gene cluster responsible for producing the prevalent siderophore staphyloferrin A. In addition to expressing the cluster in the heterologous host Escherichia coli, which confers the ability to synthesize the siderophore, we reconstituted staphyloferrin A biosynthesis in vitro by expressing and purifying two key enzymes in the pathway. As with other polycarboxylate siderophores, staphyloferrin A is biosynthesized using the recently described nonribosomal peptide synthetase independent siderophore (NIS) biosynthetic pathway. Two NIS synthetases condense two molecules of citric acid to d-ornithine in a stepwise ordered process with SfnaD using the delta-amine as a nucleophile to form the first amide followed by SfnaB utilizing the alpha-amine to complete staphyloferrin A synthesis.

TarO-specific inhibitors of wall teichoic acid biosynthesis restore β-lactam efficacy against methicillin-resistant staphylococci

New inhibitors of wall teichoic acid biosynthesis restore susceptibility of drug-resistant staphylococci to β-lactam antibiotics. Addressing antibiotic resistance with nonantibiotic adjuvants Coupled with the crisis in antibiotic drug resistance is a dearth of mechanistically new classes of antibacterial agents. One possible solution to this problem is to improve the efficacy of existing antibiotics against otherwise resistant bacteria using a combination agent approach. Lee et al. now describe just such a combination agent strategy to resuscitate the efficacy of β-lactam antibiotics. They identify nonantibiotic adjuvants termed tarocins that restore the killing activity of β-lactams against methicillin-resistant staphylococci, thereby enabling the application of β-lactams to treat Gram-positive bacterial infections. The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current β-lactam antibiotics and created an urgent need for new treatment options. We report an S. aureus phenotypic screening strategy involving chemical suppression of the growth inhibitory consequences of depleting late-stage wall teichoic acid biosynthesis. This enabled us to identify early-stage pathway-specific inhibitors of wall teichoic acid biosynthesis predicted to be chemically synergistic with β-lactams. We demonstrated by genetic and biochemical means that each of the new chemical series discovered, herein named tarocin A and tarocin B, inhibited the first step in wall teichoic acid biosynthesis (TarO). Tarocins do not have intrinsic bioactivity but rather demonstrated potent bactericidal synergy in combination with broad-spectrum β-lactam antibiotics against diverse clinical isolates of methicillin-resistant staphylococci as well as robust efficacy in a murine infection model of MRSA. Tarocins and other inhibitors of wall teichoic acid biosynthesis may provide a rational strategy to develop Gram-positive bactericidal β-lactam combination agents active against methicillin-resistant staphylococci.

The violacein biosynthetic enzyme vioe shares a fold with lipoprotein transporter proteins

VioE, an unusual enzyme with no characterized homologues, plays a key role in the biosynthesis of violacein, a purple pigment with antibacterial and cytotoxic properties. Without bound cofactors or metals, VioE, from the bacterium Chromobacterium violaceum, mediates a 1,2 shift of an indole ring and oxidative chemistry to generate prodeoxyviolacein, a precursor to violacein. Our 1.21 Å resolution structure of VioE shows that the enzyme shares a core fold previously described for lipoprotein transporter proteins LolA and LolB. For both LolB and VioE, a bound polyethylene glycol molecule suggests the location of the binding and/or active site of the protein. Mutations of residues near the bound polyethylene glycol molecule in VioE have identified the active site and five residues important for binding or catalysis. This structural and mutagenesis study suggests that VioE acts as a catalytic chaperone, using a fold previously associated with lipoprotein transporters to catalyze the production of its prodeoxyviolacein product.

Kibdelomycin is a bactericidal broad-spectrum aerobic antibacterial agent

ABSTRACT Bacterial resistance to antibiotics continues to grow and pose serious challenges, while the discovery rate for new antibiotics declines. Kibdelomycin is a recently discovered natural-product antibiotic that inhibits bacterial growth by inhibiting the bacterial DNA replication enzymes DNA gyrase and topoisomerase IV. It was reported to be a broad-spectrum aerobic Gram-positive agent with selective inhibition of the anaerobic bacterium Clostridium difficile. We have extended the profiling of kibdelomycin by using over 196 strains of Gram-positive and Gram-negative aerobic pathogens recovered from worldwide patient populations. We report the MIC50s, MIC90s, and bactericidal activities of kibdelomycin. We confirm the Gram-positive spectrum and report for the first time that kibdelomycin shows strong activity (MIC90, 0.125 μg/ml) against clinical strains of the Gram-negative nonfermenter Acinetobacter baumannii but only weak activity against Pseudomonas aeruginosa. We confirm that well-characterized resistant strains of Staphylococcus aureus and Streptococcus pneumoniae show no cross-resistance to kibdelomycin and quinolones and coumarin antibiotics. We also show that kibdelomycin is not subject to efflux in Pseudomonas, though it is in Escherichia coli, and it is generally affected by the outer membrane permeability entry barrier in the nonfermenters P. aeruginosa and A. baumannii, which may be addressable by structure-based chemical modification.

Mutant alleles of lptD increase the permeability of Pseudomonas aeruginosa and define determinants of intrinsic resistance to antibiotics

ABSTRACT Gram-negative bacteria provide a particular challenge to antibacterial drug discovery due to their cell envelope structure. Compound entry is impeded by the lipopolysaccharide (LPS) of the outer membrane (OM), and those molecules that overcome this barrier are often expelled by multidrug efflux pumps. Understanding how efflux and permeability affect the ability of a compound to reach its target is paramount to translating in vitro biochemical potency to cellular bioactivity. Herein, a suite of Pseudomonas aeruginosa strains were constructed in either a wild-type or efflux-null background in which mutations were engineered in LptD, the final protein involved in LPS transport to the OM. These mutants were demonstrated to be defective in LPS transport, resulting in compromised barrier function. Using isogenic strain sets harboring these newly created alleles, we were able to define the contributions of permeability and efflux to the intrinsic resistance of P. aeruginosa to a variety of antibiotics. These strains will be useful in the design and optimization of future antibiotics against Gram-negative pathogens.

From Thioesters to Amides and Back: Condensation Domain Reversibility in the Biosynthesis of Vibriobactin

Vibriobactin synthetase is a four component nonribosomal peptide synthetase (NRPS) system responsible for the biosynthesis of an iron-chelating catechol siderophore that contributes to a system of iron acquisition in Vibrio cholerae—the causative agent of cholera—during vertebrate infections. Vibriobactin synthetase contains two unusual condensation (C) domains responsible for catalyzing amide bond formation between a protein-bound substrate, tethered by thioester linkage to a thiolation (T) domain, and a soluble substrate. VibH, which is a stand alone C domain, catalyzes peptide-bond formation between a terminal amine of norspermidine (NS) and 2,3-dihydroxybenzoate (DHB) loaded on the aryl carrier protein (ArCP) domain of VibB. The second C domain of VibF (C2) then catalyzes sequential amidation of the remaining two amines of NS-DHB with dihydroxyphenyl5-methyloxazaline (DHP-mOx) loaded on the T domain of VibF. VibH and C2 of VibF differ from canonical chain elongation C domains, which catalyze nucleophilic attack of the free amine of a downstream aminoacyl-S-T domain loaded substrate on an upstream nascent peptidyl-S-T domain thioester. Recently, tailoring enzymes involved in the biosynthesis of a variety of biologically active natural products have been shown to be reversible. Glycosyltransferases from the nonribosomal peptide (NRP) vancomycin, the polyketides (PKs) calicheamicin, avermectin, and vicenistatin, and the broad specificity PK glycosyltransferase OleD from the oleandomycin producer Streptomyces antibioticus have been shown to catalyze reversible reactions that allow for sugars and aglycones to be exchanged. The acyltransferase CouN7, which catalyzes the installation of the 5-methylpyrrole pharmacophore in the penultimate step of coumermycin biosynthesis, has also been shown to be reversible, and allows for the transfer of various acyl moieties between aminocoumarin scaffolds. Herein, we demonstrate that like these tailoring enzymes, NRP scaffold generating C domains, specifically VibH and C2 of VibF, also catalyze reversible reactions, and allow for reloading of T domains with acyl moieties and exchange of downstream substrates after amide bond formation. Due to the sequential nature of the two final amidations catalyzed by C2 of VibF, it was of interest to see if the triamide ACHTUNGTRENNUNGvibriobactin could be formed from the diamide intermediate DHP-mOx-NS-DHB. If C2 was a reversible amide-to-thioester catalyst, it would be possible to do an acyl transfer from the amide intermediate to yield free NS-DHB, and DHP-mOx would be reloaded as a thioester onto the T domain of VibF. Then binding of another molecule of DHP-mOx-NS-DHB would allow forward condensation to yield vibriobactin (Scheme 1). Incuba-

Antibacterial New Target Discovery: Sentinel Examples, Strategies, and Surveying Success

Antibiotics are the bedrock of modern medicine but their efficacy is rapidly eroding due to the alarming emergence of multi-drug resistant bacteria. To begin to address this crisis, novel antibacterial agents that inhibit bacterial-specific cellular functions essential for growth, viability, and/or pathogenesis are urgently needed. Although the genomics era has contributed greatly to identifying novel antibacterial targets, it has failed to appropriately characterize, prioritize, and ultimately exploit such targets to significantly impact antibiotic discovery. Here we describe a contemporary view of new antibacterial target discovery; one which complements existing genomics strategies with a deeply rooted and fundamental understanding of target biology in the context of genetic networks and environmental conditions to rigorously identify high potential targets, and cognate inhibitors, for consideration as antibacterial leads.