Ribosomal RNA Modification and Bacterial Antibiotic Resistance
Department of Biochemistry, Emory University, Atlanta, Georgia, USA
Aminoglycosides are potent antimicrobial agents used for treatment of life-threatening infections of both gram-positive and gram-negative bacteria. However, in addition to the currently more prevalent aminoglycoside modifying enzymes, acquired aminoglycoside-resistance 16S ribosomal RNA (rRNA) methyltransferases are becoming an increasing clinical concern due to their continued emergence in major human pathogens. These methyltransferases site-specifically modify 16S rRNA to prevent aminoglycoside binding and thus confer broad and exceptionally high level resistance to this class of drug. Structural and functional analyses of 16S rRNA methyltransferases will be presented which reveal insights into the critical determinants of methyltransferase-30S subunit substrate recognition and enzyme activation. The primary focus will be on the pathogen-associated 16S rRNA methyltransferase NpmA which catalyzes m1A1408 modification, blocking the action of structurally diverse aminoglycoside antibiotics. Specifically, our most recent data support a model for NpmA action with distinct steps in 30S subunit binding and adoption of a catalytically competent state via functionally critical 30S-driven conformational changes in NpmA. This model is also consistent with catalysis being completely positional in nature, as the most significant effects on activity arise from changes that impact binding or stabilization of the flipped A1408 conformation. Studies of other aminoglycoside-resistance methyltransferases will also be described briefly to highlight features of the emerging molecular mechanism which may be common to all enzymes of this type. Together, our results provide a molecular framework for aminoglycoside-resistance methyltransferase action that may serve as a functional paradigm for related enzymes and a starting point for development of inhibitors of these resistance determinants.
Developing Novel Therapeutics against Respiratory Syncytial Virus Infection
Institute for Biomedical Sciences, Georgia State University, Atlanta, Georgia, USA
Respiratory syncytial virus (RSV) is responsible for the majority of severe lower respiratory infection (LRI) and death from viral disease among infants in the United States, and recognized as a threat to the immunocompromised and the elderly. Infection initiates in the upper respiratory epithelium, followed by gradual progression to the small airways in patients advancing to severe disease, opening a window for therapeutic intervention. No vaccine protection or effective therapeutic is currently available against RSV, and antibody immunoprophylaxis is restricted to a subset of high-risk patients. We propose that addressing the RSV problem through antiviral therapeutics will require a combination therapy approach with a pair of pathogen-directed inhibitors with distinct mechanistic profile. This notion is driven by the strict safety profile requested by a mostly pediatric patient population and the threat that resistance mutations against individual therapeutics may become fixed rapidly in circulating virus strains. Having characterized an RSV pan-resistance mechanism mediating escape from all small-molecule RSV entry inhibitors currently considered for human use, we have identified the viral RNA-dependent RNA polymerase (RdRp) complex as a premier drug target. A diverse portfolio of developmental candidates for our antiviral program was developed from three sources: i) open drug discovery campaigns using recombinant RSV reporter strains designed to predominantly yield RdRp-directed hits; ii) target hopping with an orally efficacious morbillivirus RdRp inhibitor class that we have previously developed; and iii) interrogation of specific druggable sites in the RSV polymerase complex through directed HTS. Inhibitor candidates are currently at different stages of mechanistic characterization, synthetic lead development, and small-animal pharmacokinetics and efficacy testing.
The Ins and Outs of Microbiota Transplantation Therapy to Cure a Variety of Human Diseases
Department of Soil, Water, and Climate, The BioTechnology Institute, University of Minnesota, Twin Cities, Minneapolis, Minnesota, USA
Clostridium difficile-associated disease (CDAD) is the major known cause of antibiotic-induced diarrhea and colitis, and the disease is thought to result from persistent disruption of commensal gut microbiota. Bacteriotherapy, by way of fecal transplantation (FMT) can be used to treat recurrent CDAD, which is thought to reestablish the normal colonic microflora. However, limitations of conventional microbiologic techniques have, until recently, precluded testing of this idea. We have used 16S amplicon-based rRNA gene sequencing using Illumina and PacBio Platforms to characterize the bacterial composition of the colonic microflora in a patients suffering from recurrent CDAD before and after treatment by fecal transplantation from a healthy donor. Although the patient's residual colonic microbiota, prior to therapy was deficient in members of the bacterial divisions-Firmicutes and Bacteriodetes, transplantation had a dramatic impact on the composition of the patient's gut microbiota. By 14 days post-transplantation, the fecal bacterial composition of the recipient was highly similar to that of the donor and was dominated by Bacteroides spp. strains and an uncharacterized butyrate producing bacterium. The change in bacterial composition was accompanied by resolution of the patient's symptoms. The striking similarity of the recipient's and donor's intestinal microbiota following after bacteriotherapy suggests that the donor's bacteria quickly occupied their requisite niches resulting in restoration of both the structure and function of the microbial communities present. We sought to overcome these barriers in our clinical FMT program by using frozen fecal material from a screened standard donor. Standardization of material preparation significantly simplified the practical aspects of FMT without loss of apparent efficacy in clearing recurrent CDI – the overall success rate was 96%. Current studies using large-scale 16S-based sequencing showed rapid engraftment of donor microbiota and flexibility in microbial diversity leading to healthy patients. We also now have a better understanding of the mechanism(s) allowing engraftment of fecal microbiota and curing of C. difficile disease. We have now produced and examined the efficacy of encapsulated freeze dried fecal microbiota for cure of CDAD and osymptoms associated with other disease and have an understanding of the reservoir of C. difficile in the environment. This new technology holds great promise for treating even a greater number of patients presenting a variety of disesases.
New Antibiotics from Nature’s Chemical Inventory
Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri, USA
Antibiotic drug discovery in the resistance era is a challenging venture. As new antibiotics enter large scale clinical use susceptible bacteria die off leaving behind resistant strains. The antibiotic resistance cycle in natural environments is more balanced. Antibiotics from coevolved symbiotic microbial communities maintain efficacy over millions of years. Is this same antibiotic longevity achievable in a clinical setting? A survey of nature’s antibiotic chemical inventory reveals new therapeutic strategies, novel targets, and unique molecules that show promise to meet this grand challenge.
Clinical isolates of Candida albicans impair neutrophil extracellular trap (NET) release
1 University of Wisconsin-Madison, Department of Medicine; 2 University of Wisconsin-Madison, Department of Medical Microbiology and Immunology
The immune system functions to combat foreign pathogenic species in the human body by preventing or limiting infection through complex and pervasive methods. One such antimicrobial method involves capture and digestion of microbes through neutrophil extracellular traps (NETs) that are comprised of chromatin, histones and granule proteins. Neutrophils are an integral component of the immune system to control fungal infections and release of NETs acts as defense against pathogens, including Candida albicans. Fungal species such as those of the Candida genus, however, often reside on the surfaces of human skin and mucous membranes. In an immunocompromised host, they may rapidly develop from harmless commensal organisms into invasive, life-threatening infectious agents. Past studies have found Candida albicans to form surface adherent communities called biofilms that increase resistance to the immune response due to the protected niche created by the matrices for the microorganisms. Biofilm formation results in impaired release of NETs and improved fungal survival. In this study, we examined interactions between neutrophils and the clinical isolates of C. albicans with distinctly varied morphology. These studies illustrate impaired NET release upon biofilm for a variety of C. albicans strains.
Influenza virus host range is regulated at the viral polymerase PB2:ANP32A interface where species-specific RNA synthesis defects control ribonucleoprotein assembly
Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA
Influenza A viruses rapidly adapt to their host environment and mode of transmission during infection. As a consequence, viruses can become specialized to a particular environment and restricted in their host range. This is especially true for the viral polymerase, where avian viral polymerases cannot function efficiently in mammalian cells. Rapid adaptation of avian polymerases during infection in mammalian cells selects for a single amino acid change in the polymerase PB2 subunit at position 627; the avian-signature PB2 E627 mutates to the human-signature K627 to permit replication in non-permissive cells. The pressure to select for this charge difference suggests host-specific regulation at the PB2 627 interface. Host-encoded ANP32A has been implicated in influenza replication and shown to promote vRNA synthesis in vitro. More recently, ANP32A has been identified as a species-specific host factor controlling influenza polymerase function. Avian hosts encode ANP32A that contains a unique 29-33 amino acid insertion/duplication that is necessary and sufficient to support replication of polymerases with PB2 E627. The expression of avian ANP32A in mammalian cells enabled polymerase activity and ribonucleoprotein particle assembly. We showed that ANP32A associates with the viral polymerase in cells, which is enhanced in the presence of viral genomic RNA. Moreover recombinant ANP32A directly interacts with the PB2 627 domain in vitro, together suggesting that RNA-bound polymerase conformations expose the PB2 627 domain to better support ANP32A binding. Our data suggest a checkpoint for polymerase adaptation to new hosts, where transient PB2:ANP32A interactions coordinate efficient RNA synthesis and ribonucleoprotein assembly.
The Human Gut Microbiota Metabolomic Response to Infection
1 Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA; 2 Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA
An important function of the microbes in the gastrointestinal tract, known as the gut microbiota, is to prevent pathogens from colonizing the host. The microbiota confers colonization resistance in a variety of ways: competition for nutrients, competition for niche space, and production of small molecules to inhibit pathogens. We are exploring the human gut microbiota as a source of anti-pathogenic small molecules. We searched for biosynthetic gene clusters in a collection of 90 sequenced strains isolated from human feces and found the most abundant annotated cluster is non-ribosomally synthesized peptides. To identify metabolites of interest produced in vivo, we humanized the mice by colonizing germ-free mice with these strains, and then infected the mice with Salmonella enterica Typhimurium or Candida albicans. Using liquid chromatography-mass spectrometry, we compared metabolites in the cecum and of humanized, pathogen-infected mice to ex-germ-free pathogen-infected mice, and humanized uninfected mice, in order to identify metabolites potentially made by the microbiota in response to pathogens. To target metabolites that were produced by the microbiota, we searched for metabolites that were 1.5 fold upregulated in 8/12 humanized infected group relative to the other groups, and absent in 8/12 germ-free infected samples. We found 25 metabolites upregulated in the mice infected with S. typhimurium and 11 from C. albicans. Tandem MS revealed that one metabolite from both infection groups produces a similar fragmentation pattern. We are following up with metabolomics in vitro to help further isolate compounds produced by the microbiota in response to exposure to pathogens.
The geographic mosaic of antibiotic coevolution in a bacterial symbiont of the fungus-farming ant Apterostigma dentigerum
1 Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA; 2 Department of Zoology, University of Wisconsin-Madison, Madison, Wisconsin, USA; 3. Department of Genetics, University of Wisconsin-Madison, Madison, Wisconsin, USA
Fungus-farming ants (Apterostigma) have utilized antibiotic-producing, bacterial symbionts (Pseudonocardia) to control specialized ascomycete pathogens of their basidiomycete crops for tens of millions of years. Understanding the ecological and evolutionary dynamics of this system has important implications for both human medicine and agriculture, where "super-bugs" readily evolve resistance to antibiotics and crop parasites evolve resistance to fungicides within decades. Using bioassay and population genetic approaches, we show that the Apterostigma-Pseudonocardia symbiosis conforms to a specific theoretical framework called the Geographic Mosaic of Coevolution, and we identify a coevolutionary hot spot of antibiotic adaptation on Barro Colorado Island, Panama. Genomic analysis reveals that, while the presence/absence of specific biosynthetic gene clusters (BGCs) plays an important role in shaping broad geographic mosaic patterns, evolutionary genetic changes within specific BGC clusters are likely driving local adaptation. These results suggest that the long-term maintenance of antibiotic potency against specialized parasites relies heavily on genetic changes within specific BGCs in addition to the acquisition of novel BGCs.
Marine sponge-associated bacteria as a source of biofilm inhibitors
1 Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin, USA; 2 Department of Pharmaceutical Science, College of Pharmacy, Chicago State University, Chicago, Illinois, USA; 3 Pharmacy Practice Division, School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin, USA
The aim of this project is to identify sponges with perturbations in microbial community that have caused the induction and expression of small molecules that inhibit the formation of biofilms in Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Following the identification of small molecule biofilm inhibitors, comparative metagenomics will be used to reconstruct the genome and biosynthetic pathway of these inhibitors. The genomic information will be used to identify selection media to isolate the producing bacterium and the biosynthetic pathway will be reconstructed via synthetic biology and heterologous expression in a suitable host. Screening for biofilm inhibitors of S. aureus and the identification of active metabolites from sponge FL2015-36 are presented.
Evolutionary Trends in Secondary Metabolism Reveal Insect-Associated Streptomyces as an Underexploited Antibiotic Resource
1 Laboratory of Genetics, University of Wisconsin-Madison, Madison, Wisconsin, USA; 2 Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA; 3 Pharmaceutical Sciences Division, University of Wisconsin-Madison, Madison, Wisconsin, USA; 4 Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA
Microbial secondary metabolites are an important source of complex natural molecules of high antibiotic and therapeutic value. Metabolite-mediated relationships between microbes depend on many evolutionary and ecological factors, including community composition and availability of resources. Traditionally, microbial-derived molecules, especially from Actinobacteria, have been the major source of FDA approved antibiotics. Here, we explore evolutionary trends in biosynthetic potential and antibiotic resistance broadly across Actinobacteria and within the genus Streptomyces. Through inhibition assays, genomics, and metabolomics we identified insect-associated Streptomyces as a prolific source of novel antibiotic compounds. 119 Actinomycetes were sampled from various ecologies, including insect-, plant-, fungi-associated niches. Genomes were mined for novel metabolite biosynthesis, inhibition bioactivity was assayed against a panel pathogens of clinical interest, and mass spectrometry was performed to assess compound novelty. Many novel biosynthetic gene clusters and gene cluster families were identified whose predicted products co-occur with unique pathogen inhibition and metabolomic profiles. Mouse studies highlight retained activity in vivo and serve as proof of concept examples for using evolution to guide the discovery of novel antibacterials and antifungals. A more complete understanding of the biological roles and evolutionary context of secondary metabolites offers a rational discovery strategy that can be exploited to find novel therapeutics by leveraging phylogeny and ecology.
The effect of culture media on growth and metabolic profiles of cyanobacteria: a drug discovery point of view
1 Department of Medicinal Chemistry & Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois, USA; 2 CAPES Foundation, Ministry of Education of Brazil, Brasília - DF, Brazil
Cultured cyanobacteria are an important source of natural products for drug discovery as many of them produce secondary metabolites with therapeutically relevant activities. For the purpose of drug discovery, culture conditions must provide optimum growth and maximum diversity of secondary metabolites. To investigate the effect of culture media on growth and metabolic profile of cyanobacteria, we selected strains that have been found to produce biologically active compounds and cultured them under different concentrations of phosphate and nitrate. A standardized inoculation procedure allowed for the assessment of biomass production. Dried cyanobacterial cells were extracted and analyzed by liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS), followed by metabolomics analysis on XCMS Online. Nitrate concentrations significantly impacted cell growth of all tested strains and influenced production of specialized nitrogen-fixing cells (heterocysts) in some strains. Different phosphate levels selectively increased production of a bioactive metabolite by strain Nostoc sp. UIC 10110, as well as induced production of a novel compound by strain Scytonema sp. UIC 10036. For all tested strains, high concentration of nitrate and low concentration of phosphate have showed to provide the best conditions for the purpose of drug discovery.
The influenza virus polymerase anchors PKC delta to phosphorylate nucleoprotein and control progression through the viral life cycle
1 Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA; 2 Graduate Program in Cellular & Molecular Biology, University of Wisconsin-Madison, Madison, Wisconsin, USA; 3 Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA
The influenza ribonucleoprotein (RNP) serves as the minimal unit required for viral gene expression and replication. Consequently, proper RNP formation is indispensable for influenza virus replication. The RNP is composed of the heterotrimeric polymerase bound to both ends of the nucleoprotein (NP)-coated genomic RNA. During RNP generation, monomeric NP oligomerizes to encapsidate nascent RNA produced by the viral polymerase. Perturbing NP oligomerization activity disrupts RNP assembly, leading to loss of viral gene expression and replication. We have previously shown that host-dependent phosphorylation of NP regulates its oligomerization and assembly of the RNP. Dynamic phosphorylation of NP is therefore necessary for successful replication. Yet, how NP phosphorylation is temporally regulated to enable gene expression early during infection and RNP assembly during genome replication later in infection remains poorly understood in part because the kinase responsible for NP phosphorylation is unknown. We showed that multiple protein kinase C (PKC) family members phosphorylate NP. We used knock out cell lines generated with the CRISPR/Cas9 system to demonstrate that PKC delta primarily impacts RNP activity, with minor effects on viral entry. PKC delta did not interact efficiently with its target NP, but rather is anchored by the viral polymerase to ensure phosphorylation and maintenance of monomeric NP. Together, these data shed light on how the dynamic phosphorylation of NP controls progression through the replication cycle and identify a critical virus-host interaction as an appealing target for therapeutic intervention.
Structural mechanisms of cooperative ssDNA binding by bacterial SSB
1 Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA; 2 Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, USA
Bacteria encode tetrameric single-stranded (ss) DNA-binding proteins, or SSBs, that coat and protect ssDNA intermediates formed during genome maintenance reactions. The prototypical E. coli SSB can bind ssDNA using multiple modes in vitro. In one mode, called SSB65, each SSB tetramer binds 65 nucleotides of ssDNA. Multiple SSB tetramers bind ssDNA with limited intermolecular cooperativity in this mode. In a second mode, called SSB35, 35 nucleotides of ssDNA wrap around each SSB tetramer, and DNA binding is highly positively cooperative. The SSB35 mode has been posited to function during DNA replication whereas the SSB65 mode is thought to function during recombination and repair. However, our understanding of the roles of SSB binding modes in vitro or in vivo has been hampered by the lack structural information on the interfaces that link adjacent SSB tetramers cooperatively bound to ssDNA.
We have determined a crystal structure of B. subtilis SsbA (36% identical to E. coli SSB) bound to ssDNA in which a ssDNA “bridge” is resolved between two interfacing tetramers. The structure revealed two interfaces between SSB tetramers: a symmetric interface formed between loops (called L45) in SSB that have been noted previously and a novel interface that is formed by conserved residues very near the ssDNA binding site. We have examined the roles of E. coli SSB residues from both interfaces in ensemble and single-molecule DNA binding experiments. Interestingly, SSB variants that alter key residues in the novel interface appear to bind ssDNA with altered cooperativity. Further investigation of these interfaces will provide useful tools for examining the impact of cooperative ssDNA binding by SSBs in cellular genome maintenance pathways.
Assessing the natural product biosynthethic capacity of two Lake Huron sediment samples
1 Department of Medicinal Chemistry and Pharmacognosy, University of Chicago, Chicago, Illinois, USA; 2 Center for Pharmaceutical Biotechnology, College of Pharmacy, University of Chicago, Chicago, Illinois, USA; 3 Department of Microbiology and Biotechnology, University of Tübingen, Tübingen, Germany; 4 DNA Services Facility, University of Chicago, Chicago, Illinois, USA
Despite decades of cultivating microorganisms for use in drug discovery, and several reports of a diverse, untapped array of microbial-derived natural product (NP) biosynthetic gene clusters (BGCs) in the environment, few attempts have been made to measure the extent to which common cultivation techniques are accessing this chemical space. In the current study, we employed next generation sequencing to assess the populations of 16S rRNA and conserved NP biosynthetic genes present two samples of Lake Huron sediment. Simultaneously, we applied the sediment samples to six different nutrient media in order to select for the growth of spore-forming bacteria. Our cultivation methods predominantly selected for members of the Bacillaceae, Micromonosporaceae, Paenibacillaceae, Streptomycetaceae, and Lactobacillaceae families, which represented less than 0.08% of families present in sediment. Surprisingly, despite this cultivation disparity, we calculated the recovery of 32% of estimated polyketide synthase ketosynthase operational biosynthetic units (OBUs) and 11% of estimated nonribosomal peptide synthetase adenylation OBUs, when comparing plate populations to those detected in sediment (clustering OBUs at 85%, an approximation of distinct NP classes). Most of the OBUs present in the cultivatable bacterial population are sequences not represented in commonly used BGC databases. Taking current sequencing limitations such as primer bias, low BGC concentration in sediment, and limited DNA extraction from spores into account, our study suggests that a minority of the bacterial population harbors a disproportionate share of the PKS and NRPS biosynthetic capacity in sediment, and that these taxa are readily cultivatable. Furthermore, this initial glimpse into the chemical space of Lake Huron sediment suggests that existing cultivation protocols are resulting in the presence several putatively novel NP BGCs on nutrient plates, and that efforts should be focused on accessing this diversity through more efficient frontend strain selection techniques able to highlight novel taxonomic or chemical space in situ. Further studies are required to determine whether alternate cultivation methods will access additional NP biosynthetic capacity on nutrient media, or whether this diversity will remain within the current state of NP ‘dark matter’.
Elucidating the role of a member of the MbtH-like protein superfamily in Myxococcus xanthus
Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA
One way bacteria and fungi produce bioactive natural products such as antibiotics and siderophores is through the use of nonribosomal peptide synthetase (NRPS) multimodular assembly lines. Many NRPSs in bacteria require members of the MbtH-like protein (MLP) superfamily for their solubility and function. Although MLPs are known to interact with adenylation domains of NRPSs, the role MLPs play in NRPS enzymology has yet to be elucidated. MLPs are nearly always encoded within NRPS-encoding gene clusters. To date the only exemption to this rule is found in the Myxococcales order, where genes encoding members of the MLP superfamily are commonly found independently of NRPS-encoding gene clusters. For example, Myxococcus xanthus has fifteen NRPS-encoding gene clusters and only one MLP-encoding gene (MXAN_3118), which is not encoded within any of the NRPS-encoding gene clusters. These observations led to these hypotheses that MXAN_3118 may interact with one or more NRPSs, may play some NRPS-independent role, or plays some role in both NRPS enzymology and other physiological processes in M. xanthus. To investigate potential NRPS partners for MXAN_3118 I used a comparative genomics approach, leading to the identification of two candidate NRPS-encoding gene clusters. I investigated whether the solubility of any of these NRPSs is influenced by the presence of MXAN_3118 as observed for other MLP-NRPS partners. A cryptic cluster was identified to require MXAN_3118 for solubility of its NRPS components. This finding suggests that MXAN_3118 can functionally interact with NRPSs. Preliminary data suggests that MXAN_3118 is also required for adenylation domains function. We are currently optimizing conditions to identify substrate specificity to further understand the MLP/NRPS interactions in this system. Ongoing efforts to identify the associated metabolite are being pursued. M. xanthus NRPS-associated secondary metabolism have been extensively studied, here we identify and characterize the first MLP-dependent NRPS in the Myxococcales order. These findings will allow for the development of a model for the role of MXAN_3118 plays in the physiology of M. xanthus and other members of the Myxococcales order and will provide insights into the function of MLPs.
Triggering Secondary Metabolite Production in Neosartorya fischeri
Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA
A significant bottleneck in drug discovery is the lack of new and diverse compounds for drug screening purposes. Secondary metabolism in microorganisms has the potential to generate significantly diverse natural products, yet challenges remain in eliciting robust metabolic changes due to strong regulatory control. Approaches to elicit secondary metabolism include alteration of nutrient conditions, co-cultivation, genetic modifications, and chemical elicitation. Here we describe the development of a platform to trigger the expression of new secondary metabolites in Neosartorya fischeri. We are exploring the use of chemical inhibitors that target global regulatory functions that will introduce sufficient dysregulation to favor new metabolite production. In our approach, we have chosen to challenge cells with inhibitors of various classes of phosphatases, namely tyrosine/alkaline or serine-threonine phosphatases, followed by analysis of secondary metabolite production using high resolution LC-MS. Treatment of N. fischeri with orthovanadate, a tyrosine/alkaline phosphatase inhibitor, generated both antibacterial activity, as well as a significant number of compounds that were absent in untreated controls. Our preliminary data based on LC-MS suggest that a significant new number of secondary metabolites are generated. We plan to adapt this technique to trigger new secondary metabolite production more broadly so as to increase the diversity of compounds available for drug discovery.
Identifying Genes Involved in Resistance to the Novel Polyene Antifungal Selvamicin
Department of Pediatrics, University of Wisconsin-Madison, Madison, Wisconsin, USA
Novel antibiotic compounds with the potential to be clinically viable are being discovered at very low rates. Through a large-scale screening of symbiotic microorganisms for potential antibiotic drugs, the polyene macrolide compound, selvamicin, has been identified as a potential novel antifungal drug. Unlike clinically used polyene antifungal drugs such as amphotericin B and nystatin A1, selvamicin lacks the mycosamine group, which has been shown to help bind ergosterol, and contains a second sugar group adjacent to the lactone moiety. To elucidate the mechanism of action of selvamicin, resistance was induced in Candida glabrata by serial passage with increasing concentrations of drug. Individual mutants were isolated from the resistant population by limiting dilution plating. Minimum inhibitory concentrations of the selvamicin resistant mutants were compared to strains of C. albicans with known resistance mechanism to polyene antifungals. Selvamicin resistance in C. glabrata engendered increased resistance to amphotericin B, but not natamycin, another polyene antifungal. C. glabrata mutants were more susceptible to drugs that induce osmotic stress, such as fluconazole and the HSP90 inhibitor geldanamycin, which also has been shown previously for amphotericin B resistant mutants of Candida. The genomes of C. glabrata mutants were sequenced and, through single nucleotide polymorphism analysis, we identified genes involved in cell membrane transport, ergosterol biosynthesis, and heat shock response. The identification of mutated genes in selvamicin resistant C. glabrata is an important step in elucidating the mechanism of action of selvamicin.
CRISPR/Cas9-Mediated Gene Disruption Reveals the Importance of Zinc Metabolism for the Virulence of the Dimorphic Fungal Pathogen, Blastomyces dermatitidis
1 Department of Pediatrics, University of Wisconsin-Madison, Madison, Wisconsin, USA; 2 Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA; 3 Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA
Blastomyces dermatitidis is a human fungal pathogen endemic to the waterways of the Upper Midwest (including Wisconsin), and is the causative agent of blastomycosis, a lung infection that can progress to a serious systematic disease, particularly in immunocompromised individuals. Genetic analysis of this fungus, while possible, is hampered by the relative inefficiency of traditional recombination-based gene targeting approaches. Here, we demonstrate the feasibility of applying CRISPR/Cas9-mediated gene editing to Blastomyces, including to simultaneously target multiple genes. We created targeting vectors expressing Cas9 and either single or dual guide RNAs to Pra1 and Zrt1, (two genes responsible for the scavenging and uptake of zinc from the extracellular environment, respectively) and introduced these plasmids into Blastomyces via Agrobacterium-mediated gene transfer. Single gene-targeting efficiencies varied somewhat by locus, but occurred at a frequency of approximately 25% to 75%, which is about 2 orders of magnitude greater than traditional gene disruption methods in Blastomyces. Although CRISPR/Cas9 disruption of Pra1 or Zrt1 did not impair growth under standard conditions, it resulted in a reduction in virulence in a mouse model of Blastomyces infection by a factor of ~10. These results underscore the utility of CRISPR/Cas9 for efficient gene disruption in dimorphic fungi and reveal a role for zinc metabolism in virulence in vivo.
Development of a Bacterial Host for Antibiotic Discovery and Production
Department of Medicinal Chemistry & Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois, USA
Bacterial secondary metabolites are potential resources for antibiotic drug discovery. One of the more explored phyla is Actinobacteria that is a producer of polyketides, the largest class of known bioactive compounds from microorganisms. Unfortunately, many biosynthetic gene clusters are silent under laboratory growth conditions. The option to activate biosynthetic genes in the native producer is either very time-consuming or not available due to difficulties in genetic engineering environmental isolates. An attractive option to overcome this challenge is heterologous gene expression, which is an approach to produce secondary metabolites in an appropriate and well-studied host. The goal of this project is to develop an optimal host organism that will facilitate the understanding of biosynthetic pathways and the production of natural products from Actinobacteria. We hypothesize that Burkholderia sp. FERM BP-3421 can be developed into a host for heterologous expression of secondary metabolites from Actinobacteria. Preliminary results demonstrated by Eustáquio et al. (2016) achieved high yield production of a native polyketide in FERM BP-3421. To test if FERM BP-3421 can be used as a host for heterologous polyketide production, our goals are to a) construct a yeast-E.coli-Burkholderia shuttle vector via restriction enzyme and ligation, b) clone polyketide biosynthetic gene clusters (BGCs) by transformation association recombination, and c) express BGCs in FERM BP-3421.
Reference: Eustáquio A.S., L.P., Chang, G.L. Steele & F.E. Koehn, (2016) Biosynthetic engineering and fermentation media development leads to gram-scale production of spliceostatin natural products in Burkholderia sp. Metab Eng 33: 67-75.
Intercellular Stress Signalling is Increased by Fludioxonil, and Induces the DRK1 Kinase to Dephosophorylate its Downstream Target YPD1
1 Cellular & Molecular Pathology Graduate Program, University of Wisconsin-Madison, Madison, Wisconsin, USA; 2 Department of Pediatrics, University of Wisconsin-Madison, Madison, Wisconsin, USA
Group III hybrid histidine kinases (HHKs) regulate a high osmolarity glycerol (HOG) pathway and represent an appealing antifungal drug target: HHKs are conserved in fungi, absent in humans, and required for the action of a common agricultural fungicide, fludioxonil. Fludioxonil induces unabated Group III HHK-dependent activation of the HOG pathway and cell death, but its mechanism is ill defined. In this study, we investigated fludioxonil’s mode of action. We hypothesize that fludioxonil activates the HOG pathway by converting Group III HHKs from a kinase to a phosphatase that dephosphorylates the downstream target, Ypd1. To test this, we used a model system where we engendered fludioxonil sensitivity in the normally fludioxonil-resistant Saccharomyces cerevisiae by expressing a prototypical Group III HHK, Drk1 from Blastomyces dermatitidis. Using 32P to monitor Ypd1 phosphorylation in vivo, we found that fludioxonil led to Drk1-dependent Ypd1 dephosphorylation in S. cerevisiae. These results could not be recapitulated in vitro with purified proteins, suggesting Drk1 may be an indirect target of fludioxonil. Work by us and others has implicated oxidative stress in fludioxonil’s mode of action. We found that nitrosoglutathione and glutathione, which can lead to glutathionylation or cytosolic disulphide bridging of cysteine residues, modified Drk1 cysteines and induced Drk1 to dephosphorylate Ypd1 in vitro. Mutation of Drk1 cysteines in turn enhanced drug resistance in vivo. We are currently investigating the cysteine modifications that occur on Drk1 in vivo in response to fludioxonil and how this event induces Drk1-mediated dephosphorylation of Ypd1.
Chemical inhibition of the Pseudomonas aeruginosa quorum sensing receptor LasR
Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA
Microbial resistance to antibiotics is emerging faster than new treatments are being developed, setting the stage for a public health crisis. As antibiotics become less effective, interest has arisen in targeting pathways that control virulence. Modulation of quorum sensing (QS), a mode of cell density dependent bacterial communication, has been identified as a potential anti-virulence strategy. Pseudomonas aeruginosa (PA), an important human pathogen, uses QS to regulate many of its harmful phenotypes. The PA QS regulator LasR both directly and indirectly controls the production of virulence factors. V-06-018, a small molecule first discovered in 2006, is among the most potent known LasR inhibitors. Surprisingly, despite its activity profile, this compound has seen limited scrutiny. We conducted the first systematic study of this compound's structure activity relationship (SAR) for LasR inhibition via the synthesis and biological evaluation of a library of V-06-018 derivatives. Our efforts thus far have yielded several new LasR inhibitors with IC50 values < 10 μM and identified chemical features important for activity. We have also attempted to elucidate the biological interaction between V-06-018 and LasR that explains the observed inhibition. Our experiments support the hypothesis that V-06-018 displaces the native QS signal from LasR, which leads to LasR unfolding. Our results will inform the design of the next generation of QS inhibitors.
A New 16S rRNA Cyanobacterial Specific Primer Set Used to Probe the Diversity of Soil Cyanobacteria
1 Department of Medicinal Chemistry & Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois, USA; 2 Center for Research Informatics, Research Resource Center, University of Illinois at Chicago, Chicago, Illinois, USA
Cyanobacteria have proven to be a prolific source of secondary metabolites which can be used for drug discovery. Novel 16S rRNA primers specific for cyanobacteria and amenable to next generation sequencing were developed to investigate the diversity of cyanobacteria in the environment. The primers were used to amplify cyanobacterial 16S rRNA from environmental samples with visible, macroscopic cyanobacterial growth and no visible cyanobacterial growth (soil). Amplicon sequencing analyses from samples with macroscopic growth revealed that we are able to isolate and culture the most prominent cyanobacteria in each collection. Amplicon sequencing analyses of soil samples utilizing the new primers indicated that the new primers were capable of enriching low-abundance cyanobacterial 16S rRNA. The soil amplicon sequencing analyses also revealed a greater diversity of cyanobacterial taxa than we currently have in our library. Many of these taxa are known for producing bioactive secondary metabolites. To enrich for these less frequently isolated cyanobacteria, soil samples were placed into three liquid media with cycloheximide and placed under light. The resulting "artificial blooms" were sequenced to determine if certain media enrich for specific cyanobacteria. Studies using these new primers will allow us to isolate and culture less frequently encountered cyanobacteria, which could lead to new bioactive compounds. Strains isolated from the artificial blooms are currently being evaluated for their ability to produce bioactive secondary metabolites.
Autometa: Automated extraction of microbial genomes from shotgun metagenomes
1 Division of Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA; 2 Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA
Culture-independent sequencing (metagenomics) is a powerful, high resolution technique enabling the study of microbial communities in situ. With modern sequencing technology and bioinformatics, individual genomes can be assembled and extracted directly from environmental samples containing complex microbial communities by a process known as metagenomic “binning." However, available binning programs suffer from methodological and practical shortcomings, such as the requirement of human pattern recognition, which is inherently unscalable, low-throughput, and poorly reproducible. Some methods also require the assembly of pooled samples, which can lead to poor assemblies in the case of inter-sample strain variability. We therefore devised a fully-automated pipeline, termed “Autometa," which incorporates machine learning principles to separate pure microbial genomes from single shotgun metagenomes. Autometa uses Barnes-Hut Stochastic Neighbor Embedding to analyze 5-mer frequency in the contiguous sequences (i.e., “contigs") produced by de novo metagenomic assembly. The DBSCAN algorithm is then used to identify groups of contigs (i.e., genome “bins") with congruent 5-mer frequency patterns. Unsupervised machine learning is then employed to optimize clustering for purity of genome bins, measured by the presence of gene markers known to occur as single copies in isolated strains. In preliminary tests, Autometa recovered more pure and complete genomes from simulated, synthetic, and environmental metagenomic samples as compared to available programs such as MaxBin and MetaBAT. We are actively integrating supervised machine learning to further refine the binning process and using our current implementation of Autometa to study natural product biosynthesis in marine invertebrate microbiomes.
Signatures of positive selection at Mycobacterium tuberculosis drug resistance loci
1 Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA; 2 Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA
Mycobacterium tuberculosis (M. tb) is the leading cause of death by an infectious disease. The bacterium’s complex cell wall makes treatment of M. tb infection difficult, and increasing resistance to the most commonly used drugs threatens global health. Unlike most bacterial pathogens, which may participate in lateral gene transfer, M. tb drug resistance is only acquired by de novo chromosomal mutation. Using whole genome sequencing data from two M. tb populations with high rates of drug resistance, we have characterized the population genetics of drug resistance loci using measures of nucleotide diversity, population differentiation, and convergent evolution. Despite recent reports of high rates of transmission of drug resistant strains of M. tb, drug resistant populations were equally or more diverse than susceptible populations in our sample, which is consistent with de novo mutation contributing to resistance across many genetic backgrounds. Genes associated with large genetic target size for resistance had signatures of diversifying selection and increased diversity in drug resistant populations compared to susceptible populations. Additionally, some genes had increased diversity in specific M. tb lineages, suggesting a role for epistatic interactions in the development of drug resistance. Homoplastic single nucleotide polymorphisms were significantly enriched in drug resistance loci and showed evidence of population differentiation between resistant and susceptible isolates. In addition to describing the effects of selection on M. tb drug resistance loci, we have identified methods to characterize positively selected loci in other clonal bacterial populations.
An Oxylipin Signal Mediates Hyphal Branching in Pathogenic Aspergilli
1 Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA; 2 Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA
Oxylipins are a group of diverse oxygenated polyunsaturated fatty acids found in all eukaryotes that modulate growth, development, and cellular communication. Three oxylipin generating oxygenases, PpoA (Psi Producing Oxygenase A), PpoB, and PpoC, mediate development and stress responses in several Aspergillus species. However, the cellular targets of these oxylipin metabolites, their cellular functions, and signal transduction pathway(s) transmitting the signal are yet to be investigated. Our laboratory has recently identified that exogenous treatment of 5(S),8(R)-dihydroxide octadecadienoic acid (5,8-diHODE), the final oxylipin product of PpoA, resulted in stunted apical growth, increased lateral growth or hyper-branching, and decreased septal distance in the human fungal pathogen A. fumigatus and plant pathogen A. flavus. Our results suggested that the observed hyper-branching phenotype is specific to C18 diol-oxylipin acids with specific structural features yet to be identified. In addition, the branching phenotype induced by 5,8-diHODE treatment was remediated by high amount of Ca2+ to the wildtype level, suggesting that Ca2+ is involved in oxylipin signal transduction in the pathogenic Aspergilli species.
Exploring Small Molecule Specificity and Receptor Homology in Pseudomonas aeruginosa Quorum Sensing
Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that employs quorum sensing (QS) for cell-cell communication. QS allows P. aeruginosa to coordinate group behaviors at high cell densities and controls up to 10% of the genome of P. aeruginosa, including critical virulence factors. Our laboratory uses chemical strategies to regulate QS. P. aeruginosa contains three homologous and interconnected receptors, LasR, RhlR, and QscR. LasR is traditionally considered to be at the top of the quorum sensing hierarchy in P. aeruginosa. In a study recently undertaken our lab, we tested the most potent reported LasR modulators in a single system. Of these 22 compounds, we found a wide variety of modulators that have diverse effects on LasR. In this work, we wish to determine if these modulators are specific for LasR and use the knowledge gained to help us elucidate differences between LasR, RhlR, and QscR.
The Chemistry and Biology of Small Molecule Modulators of Quorum Sensing in Pseudomonas aeruginosa
1 Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA; 2 Department of Biochemistry, University of Illinois at Urbana-Champaign, Champaign, Illinois, USA; 3 Department of Chemistry, SUNY Cortland, Cortland, New York, USA
Many bacterial pathogens utilize a communication system called quorum sensing to coordinate group behaviors and initiate virulence at high cell densities. The use of small molecules to block quorum sensing via LuxR-type receptor inhibition provides a means of abrogating pathogenic phenotypes, but many known quorum sensing modulators have limitations, including hydrolytic instability and non-classical antagonism dose-curves (indicative of additional targets and/or modes-of-action). To address these issues, we systematically evaluated the structure activity relationships (SAR) of the triphenyl (TP) scaffold, a compound class discovered in a high-throughput screen to agonize LasR, a LuxR-type receptor found in the clinically relevant pathogen Pseudomonas aeruginosa. By improving the synthetic route toward this compound type, we prepared TP derived agonists of varying potency, and these compounds were used to elucidate functional aspects of the LasR structure via protein crystallography. Additionally, by combining chemical motifs known for LasR antagonism with TP components essential for activity, we effectively mode switched the TP scaffold to antagonize LasR. Importantly, many of these LasR antagonists were potent and hydrolytically stable. Furthermore, they did not exhibit non-classical antagonism dose-curve effects, providing the field with probes that inhibit LasR across a wider range of assay conditions relative to known lactone-based ligands.
Production of the essential cell wall precursor UDP-GlcNAc is affected by YvcK and is important for the virulence of Listeria monocytogenes
1 Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA; 2 Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA
The cytosol of host cells is restrictive to the growth of bacteria that are not adapted for that niche. In addition to traditional cytosolic pathogens such as Listeria monocytogenes, some canonic extracellular and vacuolar pathogens such as Staphylococcus aureus and Mycobacterium tuberculosis spend part of their time in the cytosol during infection. Thus it is important to determine how adaptations to this environment affect the outcome of infection. Previously a protein of unknown function, YvcK, has been shown to be required for cytosolic survival, virulence, and resistance to host antimicrobial proteins in L. monocytogenes. A suppressor selection for transposon mutants which restore resistance to the cell wall degrading enzyme lysozyme identified a number of genes in cell wall synthesis and central metabolism. To determine whether YvcK plays a role in central metabolism or in the metabolism of cell wall precursors we performed a non-targeted metabolomics approach. Using this approach we determined that the yvcK mutant is impaired in the production of the essential peptidoglycan precursor UDP-GlcNAc through the GlmSMU pathway. Furthermore, we hypothesized that a suppressor mutation which inactivates the gene gtcA would prevent alternative use of UDP-GlcNAc and increase levels of the metabolite available for peptidoglycan synthesis. We found that the Tn:gtcA mutant indeed increases levels of UDP-GlcNAc and also significantly reduces the virulence attenuation of the yvcK mutant. These results suggest that either regulation of the GlmSMU pathway or an enzymatic activity contributing to the production of UDP-GlcNAc is an important function of YvcK in L. monocytogenes.
Utilizing whole genome sequencing to revitalize a forward genetic screen for mutants deficient in the production of sterigmatocystin in Aspergillus nidulans
1 Department of Genetics, University of Wisconsin-Madison, Madison, Wisconsin, USA; 2 Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA; 3 Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas, USA; 4 Genome Sequencing and Analysis Program, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA; 5 Center for Forest Mycology Research, Northern Research Station, Newtown Square, Pennsylvania, USA; 6 Department of Plant Pathology, University of Wisconsin-Madison, Madison, Wisconsin, USA; 7. Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA
The study of aflatoxin in Aspergillus spp. has garnered the attention of many researchers due to aflatoxin's carcinogenic properties and frequency as a food and feed contaminant. Significant progress has been made by utilizing the model organism Aspergillus nidulans to characterize the regulation of sterigmatocystin (ST), the penultimate precursor of aflatoxin. A previous chemical screen identified 23 A. nidulans mutants involved in regulating ST production. Only two of the loci were characterized from this screen using classical mapping (five mutations in mcsA) and complementation with a cosmid library (one mutation in laeA). Recently the remaining mutants were backcrossed and sequenced using Illumina and Ion Torrent sequencing platforms. All but one mutant contained one or more sequence variants in predicted open reading frames. Deletion of these genes resulted in identification of mutant alleles responsible for the loss of ST production in 12 out of the 17 remaining mutants. Eight of these mutations were in genes already known to affect ST synthesis (laeA, mcsA, fluG, and stcA), while the remaining four mutations (laeB, sntB and hamI) were previously uncharacterized genes not known to be involved in ST production. SntB, and LaeB under certain growth conditions, appear to regulate the ST cluster transcriptionally through the cluster-specific transcription factor aflR. Based on protein domains and homologs, HamI has predicted roles in plasma membrane fusion. This study highlights the intricate regulatory mechanisms of secondary metabolism in A. nidulans.
Elucidation of the PASTA Kinase Stk1's Role in Antibiotic Resistance in Staphylococcus aureus
1 Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA; 2 Molecular & Cellular Pharmacology Doctoral Training Program, University of Wisconsin-Madison, Madison, Wisconsin, USA; 3 Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA
The Penicillin-binding-protein And Ser/Thr-Associated (PASTA) kinases are eukaryotic-like ser/thr kinases found in the Actinobacteria and Firmicutes such as Staphylococcus aureus. PASTA kinases play important roles in processes such as biofilm formation and virulence as well as resistance to beta-lactam antibiotics; as such, they have been proposed as a potential target for the development of novel antimicrobials. It has been well established that deletion of the PASTA kinase Stk1 sensitizes Methicillin-Resistant Staphylococcus aureus (MRSA) to beta-lactams; however, the mechanism for this sensitivity remains unknown. We propose a strategy that combines bacterial genetics and pharmacologic inhibition of Stk1 to elucidate Stk1's role in beta-lactam resistance. To this end, we have previously identified the kinase inhibitor GW779439X (GW) as an inhibitor that sensitized MRSA to beta-lactams in an Stk1-dependent manner. Furthermore, we have performed a preliminary screen of an ordered transposon mutant library to identify MRSA mutants with increased sensitivity to beta-lactams. 42 mutants were identified, 5 of which are also suggested to be Stk1 substrates by mass spectrometry. Of these 5, we selected MurZ for further analysis due to its role in muropeptide synthesis. Finally, we have established that pharmacologic inhibition of Stk1 masks the phenotype of a murZ::tn mutant, suggesting an epistatic relationship. Future work will aim to characterize Stk1’s influence on the function of MurZ. Upon completion of this work, we will have identified an Stk1 signaling axis responsible for beta-lactam resistance, elaborated on GW's mechanism of action, and identified potential novel targets for future antibiotic development.
Siderophore Biosynthesis as a Model System for Dissecting the Role of MbtH-like Proteins in Natural Product Biosynthesis
1 Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA; 2 Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
Nonribosomal peptides (NRPs) are a structurally diverse class of natural products that comprise a significant portion of our current medicinal arsenal. In nature, microorganisms have evolved to use these molecules for a wide range of physiological roles from nutrient acquisition to virulence. NRPs are constructed by nonribosomal peptide synthetases (NRPSs) that use a mechanistically repetitive and modular enzymology. NRPS enzymology has been extensively studied, but until recently it was unappreciated that many NRPSs are dependent on the presence of an MbtH-like protein (MLP) for function and solubility. This incomplete understanding of the role of MLPs in NRP biosynthesis implies there is more to be learned about the basic enzymology of NRPSs. Further investigation of MLPs may lead to an understanding of how to disrupt their function, opening up the possibility of using MLPs as new drug targets. Structural and genetic studies indicate that MLPs do not have a catalytic site, leading to the hypothesis that a complex protein-protein interaction between the NRPS and MLP is required. Using enterobactin biosynthesis in E. coli as a model system, we have identified several residues in the cognate MLP, YbdZ, required for in vivo metabolite production. Analysis of YbdZ variants and other noncognate MLPs in vitro and in vivo will be presented here. These data have led to the generation of hypotheses for how MLPs interact with NRPSs and enable optimal NRPS enzymology. A detailed understanding of what dictates functional MLP/NRPS interaction is essential to the successful engineering of NRPs for medicinal or other applications.
Development of small molecule agonists and antagonists of SdiA in Salmonella typhimurium
1 Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA; 2 Biotechnology Training Program, University of Wisconsin-Madison, Madison, Wisconsin, USA
Many common bacteria use chemical signals to communicate and coordinate group behavior in a process called quorum sensing (QS). In most Gram-negative bacteria QS system, a synthase produces an N-acyl-homoserine lactone (AHL) and a receptor protein recognizes the AHL signal, as in the canonical case of LuxI and LuxR originally characterized in V. fischeri. This cell-cell signaling system allows bacteria to regulate myriad phenotypes based on population density, many of which are connected to the development of infection or in mediating host beneficial symbioses. SdiA is a LuxR-type receptor found in and S. typhimurium; however, SdiA does not have a corresponding synthase or native AHL ligand, and is thus an "orphan". SdiA is thought to eavesdrop on other bacterial species in its environment and regulate several aspects of pathogenesis. Prior studies have identified several characteristics of AHL ligands that can activate SdiA; however, no chemical inhibitors have been identified, nor have significant modifications to the AHL structure been probed. In this study, the activity of a wider range of AHL analogs have been evaluated using a cell-based SdiA reporter strain to develop a thorough understanding of the structure-activity-relationships that belay AHL-mediated SdiA activation and inhibition. Several highly potent compounds have been discovered so far. These compounds represent useful probes for studying the molecular mechanisms by which SdiA acts and the feasibility of targeting SdiA in anti-infective or anti-virulence therapies.
Discovery of NDM-1 Inhibitors
1 Division of Pharmaceutical Sciences Department, School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin, USA; 2 Department of Bacteriology, University Wisconsin-Madison, Madison, Wisconsin, USA
The threat of antibiotic resistance is a growing concern. Carbapenem-Resistant Enterobacteriaceae is a class of pathogens designated by the CDC as an "urgent" threat with potentially serious consequences if not addressed in the near future. New-Delhi Metallo beta-lactamase 1 (NDM-1) is one of the enzymes responsible for the carbapenem resistance in these pathogens. Inhibitors of this enzyme could reinvigorate beta-lactam antibiotic treatment options against these dangerously resistant bacteria. We developed a cell-based screening method to identify new inhibitors of NDM-1. 480 fractionated extracts from 120 Actinomycete strains collected from marine invertebrates were screened in this assay. A secondary cell-free screen was used to confirm hits. Bioassay-guided fractionation led to the discovery of three inhibitors of NDM-1. Further characterization of these inhibitors is underway.
Structural and Biosynthetic Analysis of Fabrubactin, a Siderophore from Agrobacterium fabrum C58
1 Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA; 2 Division of Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA; 3 Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA; 4 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA
The bioavailability of iron in aerobic environments at neutral pH is low due to the formation of insoluble oxidized iron complexes. To overcome this challenge, many bacteria produce siderophores, which are soluble high-affinity iron-chelating metabolites. Members of our lab previously identified a siderophore and the associated biosynthetic gene cluster from Agrobacterium fabrum strain C58. Here we present conditions for large-scale production and purification of this metabolite that allow for structure determination. NMR data suggest the siderophore contains a heterocyclic moiety characteristic of the anachelin family of siderophores, 1,1-dimethyl-3-amino-1,2,3,4-tetrahydro-7,8-dihydroxyquinolin (Dmaq). We propose fabrubactin (FBB) as the name for this siderophore. Our data suggest fabrubactin is made via hybrid polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) enzymology. In addition to structural studies, the PKS and NRPS biosynthetic components were purified in order to assess substrate specificity. The enzymology and structure of fabrubactin provide insights into the formation of the heterocyclic moiety.
Towards a Small Molecule Inhibitor of Bacterial SSB Protein-Protein Interactions
Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA
Bacterial antibiotic resistance is rapidly increasing in prevalence and hindering the treatment of once routine infections. Resistance has led to the routine use of antibiotics of last resort and resistance to even these bulwarks has emerged. Few new antibiotics have entered the market in recent years, making the discovery of novel classes of antibiotics capable of overcoming the existing resistance mechanisms an urgent need. Bacteria rely on a set of protein-protein interactions (PPI) between the single-stranded DNA binding protein (SSB) to recruit enzymes critical for DNA replication, DNA damage repair and replication restart. Because loss of the interaction domain of SSB is lethal to cells, we hypothesized that small molecules capable of disrupting these protein-SSB interactions could function as antibacterial agents. To test this hypothesis, we developed and validated a high-throughput screening strategy to identify inhibitors of the Klebsiella pneumonia PriA-SSB interaction. We have completed a pilot primary screen of 75,000 compounds using a primary AlphaScreen assay and confirmed the activity of hits in a secondary fluorescence polarization assay. Non-specific inhibitors were eliminated by screening against a fluorescence polarization assay targeted against unrelated eukaryotic PPI. Using this screening platform, we identified a small set of specific inhibitors of the PriA-SSB PPI with low micromolar IC50 values in our AlphaScreen assay. We are currently seeking to confirm direct binding of the inhibitors to PriA, evaluate the activity of these inhibitors against other SSB PPIs and assay the antibacterial capabilities of each of our lead compounds.
Repurposed kinase inhibitors and b-lactams as a novel therapy for antibiotic resistant bacteria
1 Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA; 2 Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA; 3 Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA; 4 UNC Eshelman School of Pharmacy, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina, USA; 5 William S. Middleton Veterans Affairs Hospital, Madison, Wisconsin, USA
Antibiotic resistant bacteria are an increasing global problem, and pathogenic actinomycetes and firmicutes are particularly challenging obstacles. These pathogens share several eukaryotic like kinases that present antibiotic development opportunities. We used computational modelling to identify human kinase inhibitors that we could repurpose towards bacteria as part of a novel combination therapy. A family of inhibitors, the imidazopyridine aminofurazans (IPAs), were predicted to bind PknB with high affinity. We found that these inhibitors biochemically inhibit PknB, and a novel x ray structure confirms that the inhibitors bind as predicted. These inhibitors have antimicrobial activity towards mycobacteria and nocardia, and normally ineffective b-lactams can potentiate IPAs to more efficiently inhibit growth of these pathogens. Collectively, our data show that in silico modeling can be used as a tool to discover promising drug leads, and the inhibitors we discovered can synergize with clinically relevant antibiotics to restore their efficacy against bacteria with limited treatment options.
Phenotypic variability and community interactions via chemicals released by germinating Streptomyces spores
1 Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA; and 2 Wisconsin Institute for Discovery, Madison, Wisconsin, USA
While many types of bacterial interactions have been studied, little is known about how the germination decisions of spores are affected by their microbial neighbors. Either promotional or inhibitory germination interactions, both within and between species, may be used as germination strategies in uncertain environments. But there is little systematic data on the specificity and diversity of these interactions and how they interplay with the stochastic properties of germination. Focusing on Streptomycetes, a high-throughput platform was developed for automatic quantification of germination and early growth within communities of spores at the single cell level. We found that the germination process is stochastic at three different levels: spores vary in their germination times, emergent mycelium networks grow exponentially at different rates, and a fraction of germlings stall their growth shortly after germination. Furthermore, by monitoring how the stochastic properties of germination are affected by spore density and chemicals released from spores, germination interactions were quantified for four species. Germination was frequently promoted or inhibited by compounds released by spores from the same or different species, and all species had distinct interaction profiles. Such interactions could also weakly affected initial mycelium growth rates in an independent manner from the germination. The spatial distribution patterns were important, and clusters of spores behave differently than individual spores. Aged spores exhibited higher dormancy but could efficiently geminate in the presence of chemicals released during germination. This work suggests that stochastic germination is commonly affected by the community context and species have adapted diverse germination strategies.