Ashootosh Tripathi

Baulamycins A and B, broad-spectrum antibiotics identified as inhibitors of siderophore biosynthesis in Staphylococcus aureus and Bacillus anthracis.

Siderophores are high-affinity iron chelators produced by microorganisms and frequently contribute to the virulence of human pathogens. Targeted inhibition of the biosynthesis of siderophores staphyloferrin B of Staphylococcus aureus and petrobactin of Bacillus anthracis hold considerable potential as a single or combined treatment for methicillin-resistant S. aureus (MRSA) and anthrax infection, respectively. The biosynthetic pathways for both siderophores involve a nonribosomal peptide synthetase independent siderophore (NIS) synthetase, including SbnE in staphyloferrin B and AsbA in petrobactin. In this study, we developed a biochemical assay specific for NIS synthetases to screen for inhibitors of SbnE and AsbA against a library of marine microbial-derived natural product extracts (NPEs). Analysis of the NPE derived from Streptomyces tempisquensis led to the isolation of the novel antibiotics baulamycins A (BmcA, 6) and B (BmcB, 7). BmcA and BmcB displayed in vitro activity with IC50 values of 4.8 μM and 19 μM against SbnE and 180 μM and 200 μM against AsbA, respectively. Kinetic analysis showed that the compounds function as reversible competitive enzyme inhibitors. Liquid culture studies with S. aureus , B. anthracis , E. coli , and several other bacterial pathogens demonstrated the capacity of these natural products to penetrate bacterial barriers and inhibit growth of both Gram-positive and Gram-negative species. These studies provide proof-of-concept that natural product inhibitors targeting siderophore virulence factors can provide access to novel broad-spectrum antibiotics, which may serve as important leads for the development of potent anti-infective agents.

Discovery of cahuitamycins as biofilm inhibitors derived from a convergent biosynthetic pathway

Pathogenic microorganisms often have the ability to attach to a surface, building a complex matrix where they colonize to form a biofilm. This cellular superstructure can display increased resistance to antibiotics and cause serious, persistent health problems in humans. Here we describe a high-throughput in vitro screen to identify inhibitors of Acinetobacter baumannii biofilms using a library of natural product extracts derived from marine microbes. Analysis of extracts derived from Streptomyces gandocaensis results in the discovery of three peptidic metabolites (cahuitamycins A–C), with cahuitamycin C being the most effective inhibitor (IC50=14.5 μM). Biosynthesis of cahuitamycin C proceeds via a convergent biosynthetic pathway, with one of the steps apparently being catalysed by an unlinked gene encoding a 6-methylsalicylate synthase. Efforts to assess starter unit diversification through selective mutasynthesis lead to production of unnatural analogues cahuitamycins D and E of increased potency (IC50=8.4 and 10.5 μM).

Evolution of Efficient Modular Polyketide Synthases by Homologous Recombination

The structural scaffolds of many complex natural products are produced by multifunctional type I polyketide synthase (PKS) enzymes that operate as biosynthetic assembly lines. The modular nature of these mega-enzymes presents an opportunity to construct custom biocatalysts built in a lego-like fashion by inserting, deleting, or exchanging native or foreign domains to produce targeted variants of natural polyketides. However, previously engineered PKS enzymes are often impaired resulting in limited production compared to native systems. Here, we show a versatile method for generating and identifying functional chimeric PKS enzymes for synthesizing custom macrolactones and macrolides. PKS genes from the pikromycin and erythromycin pathways were hybridized in Saccharomyces cerevisiae to generate hybrid libraries. We used a 96-well plate format for plasmid purification, transformations, sequencing, protein expression, in vitro reactions and analysis of metabolite formation. Active chimeric enzymes were identified with new functionality. Streptomyces venezuelae strains that expressed these PKS chimeras were capable of producing engineered macrolactones. Furthermore, a macrolactone generated from selected PKS chimeras was fully functionalized into a novel macrolide analogue. This method permits the engineering of PKS pathways as modular building blocks for the production of new antibiotic-like molecules.

Structural Basis for Cyclopropanation by a Unique Enoyl-Acyl Carrier Protein Reductase

The natural product curacin A, a potent anticancer agent, contains a rare cyclopropane group. The five enzymes for cyclopropane biosynthesis are highly similar to enzymes that generate a vinyl chloride moiety in the jamaicamide natural product. The structural biology of this remarkable catalytic adaptability is probed with high-resolution crystal structures of the curacin cyclopropanase (CurF ER), an in vitro enoyl reductase (JamJ ER), and a canonical curacin enoyl reductase (CurK ER). The JamJ and CurK ERs catalyze NADPH-dependent double bond reductions typical of enoyl reductases (ERs) of the medium-chain dehydrogenase reductase (MDR) superfamily. Cyclopropane formation by CurF ER is specified by a short loop which, when transplanted to JamJ ER, confers cyclopropanase activity on the chimeric enzyme. Detection of an adduct of NADPH with the model substrate crotonyl-CoA provides indirect support for a recent proposal of a C2-ene intermediate on the reaction pathway of MDR enoyl-thioester reductases.

Borrelidin Induces the Unfolded Protein Response in Oral Cancer Cells and Chop-Dependent Apoptosis

Oral squamous cell carcinoma (OSCC) is the most common cancer affecting the oral cavity, and US clinics will register about 30,000 new patients in 2015. Current treatment modalities include chemotherapy, surgery, and radiotherapy, which often result in astonishing disfigurement. Cancers of the head and neck display enhanced levels of glucose-regulated proteins and translation initiation factors associated with endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Previous work demonstrated that chemically enforced UPR could overwhelm these adaptive features and selectively kill malignant cells. The threonyl-tRNA synthetase (ThRS) inhibitor borrelidin and two congeners were discovered in a cell-based chemical genomic screen. Borrelidin increased XBP1 splicing and led to accumulation of phosphorylated eIF2α and UPR-associated genes, prior to death in panel of OSCC cells. Murine embryonic fibroblasts (MEFs) null for GCN2 and PERK were less able to accumulate UPR markers and were resistant to borrelidin. This study demonstrates that UPR induction is a feature of ThRS inhibition and adds to a growing body of literature suggesting ThRS inhibitors might selectively target cancer cells.

OxaD: A Versatile Indolic Nitrone Synthase from the Marine-Derived Fungus Penicillium oxalicum F30

Indole alkaloids are a diverse class of natural products known for their wide range of biological activities and complex chemical structures. Rarely observed in this class are indolic nitrones, such as avrainvillamide and waikialoid, which possess potent bioactivities. Herein the oxa gene cluster from the marine-derived fungus Penicillium oxalicum F30 is described along with the characterization of OxaD, a flavin-dependent oxidase that generates roquefortine L, a nitrone-bearing intermediate in the biosynthesis of oxaline. Nitrone functionality in roquefortine L was confirmed by spectroscopic methods and 1,3-dipolar cycloaddition with methyl acrylate. OxaD is a versatile biocatalyst that converts an array of semisynthetic roquefortine C derivatives bearing indoline systems to their respective nitrones. This work describes the first implementation of a nitrone synthase as a biocatalyst and establishes a novel platform for late-stage diversification of a range of complex natural products.

Thioesterase domain swapping of a linear polyketide tautomycetin with a macrocyclic polyketide pikromycin in Streptomyces sp. CK4412

Tautomycetin (TMC) is a linear polyketide metabolite produced by Streptomyces sp. CK4412 that has been reported to possess multiple biological functions including T cell-specific immunosuppressive and anticancer activities that occur through a mechanism of differential inhibition of protein phosphatases such as PP1, PP2A, and SHP2. We previously reported the characterization of the entire TMC biosynthetic gene cluster constituted by multifunctional type I polyketide synthase (PKS) assembly and suggested that the linear form of TMC could be generated via free acid chain termination by a narrow TMC thioesterase (TE) pocket. The modular nature of the assembly presents a unique opportunity to alter or interchange the native biosynthetic domains to produce targeted variants of TMC. Herein, we report swapping of the TMC TE domain sequence with the exact counterpart of the macrocyclic polyketide pikromycin (PIK) TE. PIK TE-swapped Streptomyces sp. CK4412 mutant produced not only TMC, but also a cyclized form of TMC, implying that the bioengineering based in vivo custom construct can be exploited to produce engineered macrolactones with new structural functionality.

Petrobactin protects against oxidative stress and enhances sporulation efficiency in Bacillus anthracis Sterne

Bacillus anthracis is a gram-positive bacillus that under conditions of environmental stress, such as low nutrients, can convert from a vegetative bacillus to a highly durable spore that enables long-term survival. The sporulation process is regulated by a sequential cascade of dedicated transcription factors but requires key nutrients to complete, one of which is iron. Iron acquisition by the iron-scavenging siderophore petrobactin is the only such system known to be required for vegetative growth of B. anthracis in iron-depleted conditions, e.g., in the host. However, the extent to which petrobactin is involved in spore formation is unknown. This work shows that efficient in vitro sporulation of B. anthracis requires petrobactin, that the petrobactin biosynthesis operon (asbA-F) is induced prior to sporulation, and that petrobactin itself is associated with spores. Petrobactin is also required for both oxidative stress protection during late stage growth and wild-type levels of sporulation in sporulation medium. When considered with the petrobactin-dependent sporulation in bovine blood also described in this work, these effects on in vitro growth and sporulation suggest that petrobactin is required for B. anthracis transmission via the spore during natural infections in addition to its key functions during active anthrax infections. Importance Bacillus anthracis causes the disease anthrax, which is transmitted via its dormant, spore phase. However, converting from bacilli to spore is a complex, energetically costly process that requires many nutrients including iron. B. anthracis requires the siderophore petrobactin to scavenge iron from host environments. We show that in the Sterne strain, petrobactin is required also for efficient sporulation, even when ample iron is available. The petrobactin biosynthesis operon is expressed during sporulation, and petrobactin is biosynthesized during growth in high iron sporulation medium but instead of being exported, the petrobactin remains intracellular to protect against oxidative stress and improve sporulation. It is also required for full growth and sporulation in blood (bovine), an essential step for anthrax transmission between mammalian hosts.

Chemoenzymatic Dissection of Polyketide β-Branching in the Bryostatin Pathway

β-Branching is an expansion upon canonical polyketide synthase extension that allows for the installation of diverse chemical moieties in several natural products. Several of these moieties are unique among natural products, including the two vinyl methylesters found in the core structure of bryostatins. This family of molecules is derived from an obligate bacterial symbiont of a sessile marine bryozoan, Bugula neritina. Within this family, bryostatin 1 has been investigated as an anticancer, neuroprotective, and immunomodulatory compound. We have turned to the biosynthetic gene cluster within the bacterial symbiont to investigate the biosynthesis of bryostatins. Recent sequencing efforts resulted in the annotation of two missing genes: bryT and bryU. Using novel chemoenzymatic techniques, we have validated these as the missing enoyl-CoA hydratase and donor acyl carrier protein, essential components of the β-branching cassette of the bryostatin pathway. Together, this cassette installs the vinyl methylester moieties essential to the activity of bryostatins.

PD12-01 A NOVEL FAMILY OF NATURAL PRODUCTS THAT TARGETS UROPATHOGENIC ESCHERISCHIA COLI IRON ACQUISITION

and are associated with poor oncologic outcomes. These data, however, are based on single institution studies and it is unknown if abnormal preoperative lab values are associated with postoperative complication rates. We sought to characterize abnormal lab values suggestive of paraneoplastic syndromes in patients undergoing surgery for RCC and evaluate their association with postoperative complications. METHODS: Using the National Surgical Quality Improvement Program (NSQIP) database, we identified all patients having surgery for RCC from 2005-2014. We queried Participant User Files with ICD-9 codes for RCC (189.00) and CPT codes for partial or radical nephrectomy (50220, 50225, 50230, 50240, 50543, 50545, 50546) selecting localized disease. We identified abnormal preoperative lab values consistent with paraneoplastic syndromes and categorized renal function by chronic kidney disease (CKD) stage. Normal lab reference ranges were defined using data from the National Library of Medicine where ranges delimit 2 standard deviations (SD) of the mean value. We used logistic regression to test if abnormal preoperative lab values were associated with postoperative complications, including postoperative transfusion, readmission and death. We extended the normal range of lab values to 3SD of the mean for a sensitivity analysis. RESULTS: A total of 12,986 patients were identified. At least 1 lab abnormality suggestive of a paraneoplastic syndrome was identified in 46% of patients (Table 1) and CKD stage >3 was found in 27% of patients. On multivariate analysis, the presence of a preoperative lab abnormality was associated with an increased odds of postoperative transfusion (OR [95% CI]: 2.84 [2.47-3.25], p<0.01), readmission (1.31 [1.09-1.58], p<0.01) and death (2.42 [1.25-4.70], p<0.01) controlling for patient and surgical factors. When we extended the range of normal to 3SD, 29% had abnormal labs and our conclusions were unchanged. CONCLUSIONS: About one half of patients presenting for surgery for RCC have abnormal lab values suggestive of paraneoplastic syndromes. These patients may be at increased risk of postoperative events including bleeding, readmission, and death.