Publications by authors named "W H Gera Hol"

370 Publications

Structure-guided discovery of selective methionyl-tRNA synthetase inhibitors with potent activity against .

RSC Med Chem 2020 Aug 18;11(8):885-895. Epub 2020 May 18.

Department of Biochemistry , University of Washington , Seattle , WA 98195 , USA . Email:

Based on crystal structures of methionyl-tRNA synthetase (MetRS) bound to inhibitors, we designed, synthesized, and evaluated two series of novel MetRS inhibitors targeting this parasite enzyme. One series has a 1,3-dihydro-imidazol-2-one containing linker, the other has a rigid fused aromatic ring in the linker. For both series of compounds, potent inhibition of parasite growth was achieved with EC < 10 nM and most compounds exhibited low general toxicity to mammalian cells with CCs > 20 000 nM. Selectivity over human mitochondrial methionyl tRNA synthetase was also evaluated, using a cell-based mitochondrial protein synthesis assay, and selectivity in a range of 20-200-fold was achieved. The inhibitors exhibited poor permeability across the blood brain barrier, necessitating future efforts to optimize the compounds for use in late stage human African trypanosomiasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/d0md00057dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7649832PMC
August 2020

Suppressor Mutations in Type II Secretion Mutants of Vibrio cholerae: Inactivation of the VesC Protease.

mSphere 2020 12 16;5(6). Epub 2020 Dec 16.

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, USA

The type II secretion system (T2SS) is a conserved transport pathway responsible for the secretion of a range of virulence factors by many pathogens, including Disruption of the T2SS genes in results in loss of secretion, changes in cell envelope function, and growth defects. While T2SS mutants are viable, high-throughput genomic analyses have listed these genes among essential genes. To investigate whether secondary mutations arise as a consequence of T2SS inactivation, we sequenced the genomes of six T2SS mutants with deletions or insertions in either the , , or genes and identified secondary mutations in all mutants. Two of the six T2SS mutants contain distinct mutations in the gene encoding the T2SS-secreted protease VesC. Other mutations were found in genes coding for cell envelope proteins. Subsequent sequence analysis of the gene in 92 additional T2SS mutant isolates identified another 19 unique mutations including insertions or deletions, sequence duplications, and single-nucleotide changes resulting in amino acid substitutions in the VesC protein. Analysis of VesC variants and the X-ray crystallographic structure of wild-type VesC suggested that all mutations lead to loss of VesC production and/or function. One possible mechanism by which T2SS mutagenesis can be tolerated is through selection of -inactivating mutations, which may, in part, suppress cell envelope damage, establishing permissive conditions for the disruption of the T2SS. Other mutations may have been acquired in genes encoding essential cell envelope proteins to prevent proteolysis by VesC. Genome-wide transposon mutagenesis has identified the genes encoding the T2SS in as essential for viability, but the reason for this is unclear. Mutants with deletions or insertions in these genes can be isolated, suggesting that they have acquired secondary mutations that suppress their growth defect. Through whole-genome sequencing and phenotypic analysis of T2SS mutants, we show that one means by which the growth defect can be suppressed is through mutations in the gene encoding the T2SS substrate VesC. VesC homologues are present in other species and close relatives, and this may be why inactivation of the T2SS in species such as , sp. strain 60, and also results in a pleiotropic effect on their outer membrane assembly and integrity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mSphere.01125-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7771236PMC
December 2020

Competition and predation as possible causes of bacterial rarity.

Environ Microbiol 2019 04 18;21(4):1356-1368. Epub 2019 Mar 18.

Department of Terrestrial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), P.O. Box 50, 6700 AB, Wageningen, The Netherlands.

We assembled communities of bacteria and exposed them to different nutrient concentrations with or without predation by protists. Taxa that were rare in the field were less abundant at low nutrient concentrations than common taxa, independent of predation. However, some taxa that were rare in the field became highly abundant in the assembled communities, especially under ample nutrient availability. This high abundance points at a possible competitive advantage of some rare bacterial taxa under nutrient-rich conditions. In contrast, the abundance of most rare bacterial taxa decreased at low resource availability. Since low resource availability will be the prevailing situation in most soils, our data suggests that under those conditions poor competitiveness for limiting resources may contribute to bacterial rarity. Interestingly, taxa that were rare in the field and most successful under predator-free conditions in the lab also tended to be more reduced by predation than common taxa. This suggests that predation contributes to rarity of bacterial taxa in the field. We further discuss whether there may be a trade-off between competitiveness and predation resistance. The substantial variability among taxa in their responses to competition and predation suggests that other factors, for example abiotic conditions and dispersal ability, also influence the local abundance of soil bacteria.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1462-2920.14569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6850713PMC
April 2019

Optimization of Methionyl tRNA-Synthetase Inhibitors for Treatment of Infection.

Antimicrob Agents Chemother 2019 04 27;63(4). Epub 2019 Mar 27.

Department of Biochemistry, University of Washington, Seattle, Washington, USA

Cryptosporidiosis is one of the leading causes of moderate to severe diarrhea in children in low-resource settings. The therapeutic options for cryptosporidiosis are limited to one drug, nitazoxanide, which unfortunately has poor activity in the most needy populations of malnourished children and HIV-infected persons. We describe here the discovery and early optimization of a class of imidazopyridine-containing compounds with potential for treating infections. The compounds target the methionyl-tRNA synthetase (MetRS), an enzyme that is essential for protein synthesis. The most potent compounds inhibited the enzyme with values in the low picomolar range. cells in culture were potently inhibited with 50% effective concentrations as low as 7 nM and >1,000-fold selectivity over mammalian cells. A parasite persistence assay indicates that the compounds act by a parasiticidal mechanism. Several compounds were demonstrated to control infection in two murine models of cryptosporidiosis without evidence of toxicity. Pharmacological and physicochemical characteristics of compounds were investigated to determine properties that were associated with higher efficacy. The results indicate that MetRS inhibitors are excellent candidates for development for anticryptosporidiosis therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.02061-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6437504PMC
April 2019

Cultivation-success of rare soil bacteria is not influenced by incubation time and growth medium.

PLoS One 2019 10;14(1):e0210073. Epub 2019 Jan 10.

Department of Terrestrial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Wageningen, The Netherlands.

Rare bacterial species have recently attracted interest due to their many potential beneficial functions. However, only little is known about their cultivability. In this study we test the hypotheses that the use of flow cell-sorting for cultivation results in a high proportion of rare soil bacterial isolates relative to bacterial taxa that are abundant in soil. Moreover, we investigate whether different oligotrophic cultivation media and a prolonged incubation time increase the number of cultivated rare species. In a cultivation study we used flow cell sorting to select for small cells and to separate single cells, and grew bacteria on different oligotrophic media with prolonged incubation times. The abundance of the isolates in the field was assessed by comparing them to a 454-sequencing dataset from the same soil. Consequentially, all bacterial isolates were classified as either rare (<0.01% relative abundance) or abundant (>0.01% relative abundance) in the field soil. We found more bacterial taxa among the isolates that were abundant in soil than would be expected by the proportion of abundant species in the field. Neither incubation time nor growth medium had an influence on the recovery of rare species. However, we did find differences in time until visible growth on the plate between different phylogenetic classes of the isolates. These results indicate that rare cultivable species are active and not more likely to be dormant than abundant species, as has been suggested as a reason for their rarity. Moreover, future studies should be aware of the influence incubation time might have on the phylogenetic composition of the isolate collection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0210073PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328151PMC
September 2019

The crystal structure of the drug target Mycobacterium tuberculosis methionyl-tRNA synthetase in complex with a catalytic intermediate.

Acta Crystallogr F Struct Biol Commun 2018 04 28;74(Pt 4):245-254. Epub 2018 Mar 28.

Department of Biochemistry, University of Washington, Seattle, Washington, USA.

Mycobacterium tuberculosis is a pathogenic bacterial infectious agent that is responsible for approximately 1.5 million human deaths annually. Current treatment requires the long-term administration of multiple medicines with substantial side effects. Lack of compliance, together with other factors, has resulted in a worrisome increase in resistance. New treatment options are therefore urgently needed. Here, the crystal structure of methionyl-tRNA synthetase (MetRS), an enzyme critical for protein biosynthesis and therefore a drug target, in complex with its catalytic intermediate methionyl adenylate is reported. Phenylalanine 292 of the M. tuberculosis enzyme is in an `out' conformation and barely contacts the adenine ring, in contrast to other MetRS structures where ring stacking occurs between the adenine and a protein side-chain ring in the `in' conformation. A comparison with human cytosolic MetRS reveals substantial differences in the active site as well as regarding the position of the connective peptide subdomain 1 (CP1) near the active site, which bodes well for arriving at selective inhibitors. Comparison with the human mitochondrial enzyme at the amino-acid sequence level suggests that arriving at inhibitors with higher affinity for the mycobacterial enzyme than for the mitochondrial enzyme might be achievable.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2053230X18003151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5893993PMC
April 2018

Fragment-Based Screening of a Natural Product Library against 62 Potential Malaria Drug Targets Employing Native Mass Spectrometry.

ACS Infect Dis 2018 04 3;4(4):431-444. Epub 2018 Mar 3.

Griffith Institute for Drug Discovery , Griffith University , 170 Kessels Road , Nathan , Queensland 4111 , Australia.

Natural products are well known for their biological relevance, high degree of three-dimensionality, and access to areas of largely unexplored chemical space. To shape our understanding of the interaction between natural products and protein targets in the postgenomic era, we have used native mass spectrometry to investigate 62 potential protein targets for malaria using a natural-product-based fragment library. We reveal here 96 low-molecular-weight natural products identified as binding partners of 32 of the putative malarial targets. Seventy-nine (79) fragments have direct growth inhibition on Plasmodium falciparum at concentrations that are promising for the development of fragment hits against these protein targets. This adds a fragment library to the published HTS active libraries in the public domain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsinfecdis.7b00197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5902791PMC
April 2018

Soil microbial species loss affects plant biomass and survival of an introduced bacterial strain, but not inducible plant defences.

Ann Bot 2018 02;121(2):311-319

Department of Terrestrial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Wageningen, The Netherlands.

Background And Aims: Plant growth-promoting rhizobacteria (PGPR) strains can influence plant-insect interactions. However, little is known about the effect of changes in the soil bacterial community in general and especially the loss of rare soil microbes on these interactions. Here, the influence of rare soil microbe reduction on induced systemic resistance (ISR) in a wild ecotype of Arabidopsis thaliana against the aphid Myzus persicae was investigated.

Methods: To create a gradient of microbial abundances, soil was inoculated with a serial dilution of a microbial community and responses of Arabidopsis plants that originated from the same site as the soil microbes were tested. Plant biomass, transcription of genes involved in plant defences, and insect performance were measured. In addition, the effects of the PGPR strain Pseudomonas fluorescens SS101 on plant and insect performance were tested under the influence of the various soil dilution treatments.

Key Results: Plant biomass showed a hump-shaped relationship with soil microbial community dilution, independent of aphid or Pseudomonas treatments. Both aphid infestation and inoculation with Pseudomonas reduced plant biomass, and led to downregulation of PR1 (salicylic acid-responsive gene) and CYP79B3 (involved in synthesis of glucosinolates). Aphid performance and gene transcription were unaffected by soil dilution.

Conclusions: Neither the loss of rare microbial species, as caused by soil dilution, nor Pseudomonas affect the resistance of A. thaliana against M. persicae. However, both Pseudomonas survival and plant biomass respond to rare species loss. Thus, loss of rare soil microbial species can have a significant impact on both above- and below-ground organisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/aob/mcx162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5808785PMC
February 2018

Development of Methionyl-tRNA Synthetase Inhibitors as Antibiotics for Gram-Positive Bacterial Infections.

Antimicrob Agents Chemother 2017 11 24;61(11). Epub 2017 Oct 24.

Department of Medicine, University of Washington, Seattle, Washington, USA

Antibiotic-resistant bacteria are widespread and pose a growing threat to human health. New antibiotics acting by novel mechanisms of action are needed to address this challenge. The bacterial methionyl-tRNA synthetase (MetRS) enzyme is essential for protein synthesis, and the type found in Gram-positive bacteria is substantially different from its counterpart found in the mammalian cytoplasm. Both previously published and new selective inhibitors were shown to be highly active against Gram-positive bacteria with MICs of ≤1.3 μg/ml against , , and strains. Incorporation of radioactive precursors demonstrated that the mechanism of activity was due to the inhibition of protein synthesis. Little activity against Gram-negative bacteria was observed, consistent with the fact that Gram-negative bacterial species contain a different type of MetRS enzyme. The ratio of the MIC to the minimum bactericidal concentration (MBC) was consistent with a bacteriostatic mechanism. The level of protein binding of the compounds was high (>95%), and this translated to a substantial increase in MICs when the compounds were tested in the presence of serum. Despite this, the compounds were very active when they were tested in a murine thigh infection model. Compounds 1717 and 2144, given by oral gavage, resulted in 3- to 4-log decreases in the bacterial load compared to that in vehicle-treated mice, which was comparable to the results observed with the comparator drugs, vancomycin and linezolid. In summary, the research describes MetRS inhibitors with oral bioavailability that represent a class of compounds acting by a novel mechanism with excellent potential for clinical development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.00999-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655057PMC
November 2017

Optimization of a binding fragment targeting the "enlarged methionine pocket" leads to potent Trypanosoma brucei methionyl-tRNA synthetase inhibitors.

Bioorg Med Chem Lett 2017 06 17;27(12):2702-2707. Epub 2017 Apr 17.

Department of Biochemistry, University of Washington, Seattle, WA 98195, United States. Electronic address:

Potent inhibitors of Trypanosoma brucei methionyl-tRNA synthetase were previously designed using a structure-guided approach. Compounds 1 and 2 were the most active compounds in the cyclic and linear linker series, respectively. To further improve cellular potency, SAR investigation of a binding fragment targeting the "enlarged methionine pocket" (EMP) was performed. The optimization led to the identification of a 6,8-dichloro-tetrahydroquinoline ring as a favorable fragment to bind the EMP. Replacement of 3,5-dichloro-benzyl group (the EMP binding fragment) of inhibitor 2 using this tetrahydroquinoline fragment resulted in compound 13, that exhibited an EC of 4nM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmcl.2017.04.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5542777PMC
June 2017

Leishmania donovani tyrosyl-tRNA synthetase structure in complex with a tyrosyl adenylate analog and comparisons with human and protozoan counterparts.

Biochimie 2017 Jul 18;138:124-136. Epub 2017 Apr 18.

Department of Biochemistry, University of Washington, Seattle, WA, USA. Electronic address:

The crystal structure of Leishmania donovani tyrosyl-tRNA synthetase (LdTyrRS) in complex with a nanobody and the tyrosyl adenylate analog TyrSA was determined at 2.75 Å resolution. Nanobodies are the variable domains of camelid heavy chain-only antibodies. The nanobody makes numerous crystal contacts and in addition reduces the flexibility of a loop of LdTyrRS. TyrSA is engaged in many interactions with active site residues occupying the tyrosine and adenine binding pockets. The LdTyrRS polypeptide chain consists of two pseudo-monomers, each consisting of two domains. Comparing the two independent chains in the asymmetric unit reveals that the two pseudo-monomers of LdTyrRS can bend with respect to each other essentially as rigid bodies. This flexibility might be useful in the positioning of tRNA for catalysis since both pseudo-monomers in the LdTyrRS chain are needed for charging tRNA. An "extra pocket" (EP) appears to be present near the adenine binding region of LdTyrRS. Since this pocket is absent in the two human homologous enzymes, the EP provides interesting opportunities for obtaining selective drugs for treating infections caused by L. donovani, a unicellular parasite causing visceral leishmaniasis, or kala azar, which claims 20,000 to 30,000 deaths per year. Sequence and structural comparisons indicate that the EP is a characteristic which also occurs in the active site of several other important pathogenic protozoa. Therefore, the structure of LdTyrRS could inspire the design of compounds useful for treating several different parasitic diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biochi.2017.04.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5484532PMC
July 2017

From Cells to Mice to Target: Characterization of NEU-1053 (SB-443342) and Its Analogues for Treatment of Human African Trypanosomiasis.

ACS Infect Dis 2017 03 8;3(3):225-236. Epub 2017 Feb 8.

Department of Chemistry & Chemical Biology, Northeastern University 360 Huntington Avenue, Boston, Massachusetts, United States.

Human African trypanosomiasis is a neglected tropical disease that is lethal if left untreated. Existing therapeutics have limited efficacy and severe associated toxicities. 2-(2-(((3-((1H-Benzo[d]imidazol-2-yl)amino)propyl)amino)methyl)-4,6-dichloro-1H-indol-1-yl)ethan-1-ol (NEU-1053) has recently been identified from a high-throughput screen of >42,000 compounds as a highly potent and fast-acting trypanocidal agent capable of curing a bloodstream infection of Trypanosoma brucei in mice. We have designed a library of analogues to probe the structure-activity relationship and improve the predicted central nervous system (CNS) exposure of NEU-1053. We report the activity of these inhibitors of T. brucei, the efficacy of NEU-1053 in a murine CNS model of infection, and identification of the target of NEU-1053 via X-ray crystallography.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsinfecdis.6b00202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5346068PMC
March 2017

Where less may be more: how the rare biosphere pulls ecosystems strings.

ISME J 2017 04 10;11(4):853-862. Epub 2017 Jan 10.

Netherlands Institute of Ecology, Department of Terrestrial Ecology, Wageningen, The Netherlands.

Rare species are increasingly recognized as crucial, yet vulnerable components of Earth's ecosystems. This is also true for microbial communities, which are typically composed of a high number of relatively rare species. Recent studies have demonstrated that rare species can have an over-proportional role in biogeochemical cycles and may be a hidden driver of microbiome function. In this review, we provide an ecological overview of the rare microbial biosphere, including causes of rarity and the impacts of rare species on ecosystem functioning. We discuss how rare species can have a preponderant role for local biodiversity and species turnover with rarity potentially bound to phylogenetically conserved features. Rare microbes may therefore be overlooked keystone species regulating the functioning of host-associated, terrestrial and aquatic environments. We conclude this review with recommendations to guide scientists interested in investigating this rapidly emerging research area.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ismej.2016.174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5364357PMC
April 2017

Low abundant soil bacteria can be metabolically versatile and fast growing.

Ecology 2017 Feb;98(2):555-564

Department of Terrestrial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), P.O. Box 50, 6700 AB, Wageningen, The Netherlands.

The abundance of species is assumed to depend on their life history traits, such as growth rate and resource specialization. However, this assumption has not been tested for bacteria. Here we investigate how abundance of soil bacteria relates to slow growth and substrate specialization (oligotrophy) vs. fast growth and substrate generalization (copiotrophy). We collected 47 saprotrophic soil bacterial isolates of differing abundances and measured their growth rate and the ability to use a variety of single carbon sources. Opposite to our expectation, there was no relationship between abundance in soil and the measured growth rate or substrate utilization profile (SUP). However, isolates with lower growth rates used fewer substrates than faster growing ones supporting the assumption that growth rate may relate to substrate specialization. Interestingly, growth rate and SUP were correlated with phylogeny, rather than with abundance in soil. Most markedly, Gammaproteobacteria on average grew significantly faster and were able to use more substrates than other bacterial classes, whereas Alphaproteobacteria were growing relatively slowly and used fewer substrates. This finding suggests that growth and substrate utilization are phylogenetically deeply conserved. We conclude that growth rate and substrate utilization of soil bacteria are not general determinants of their abundance. Future studies on explaining bacterial abundance need to determine how other factors, such as competition, predation and abiotic factors may contribute to rarity or abundance in soil bacteria.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ecy.1670DOI Listing
February 2017

Structure-guided design of novel Trypanosoma brucei Methionyl-tRNA synthetase inhibitors.

Eur J Med Chem 2016 Nov 14;124:1081-1092. Epub 2016 Oct 14.

Department of Biochemistry, University of Washington, Seattle, WA 98195, United States. Electronic address:

A screening hit 1 against Trypanosoma brucei methionyl-tRNA synthetase was optimized using a structure-guided approach. The optimization led to the identification of two novel series of potent inhibitors, the cyclic linker and linear linker series. Compounds of both series were potent in a T. brucei growth inhibition assay while showing low toxicity to mammalian cells. The best compound of each series, 16 and 31, exhibited ECs of 39 and 22 nM, respectively. Compounds 16 and 31 also exhibited promising PK properties after oral dosing in mice. Moreover, compound 31 had moderately good brain permeability, with a brain/plasma ratio of 0.27 at 60 min after IP injection. This study provides new lead compounds for arriving at new treatments of human African trypanosomiasis (HAT).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejmech.2016.10.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5120670PMC
November 2016

5-Fluoroimidazo[4,5-b]pyridine Is a Privileged Fragment That Conveys Bioavailability to Potent Trypanosomal Methionyl-tRNA Synthetase Inhibitors.

ACS Infect Dis 2016 06 11;2(6):399-404. Epub 2016 Apr 11.

Department of Biochemistry, University of Washington , 1705 N.E. Pacific Street, Seattle, Washington 98195, United States.

Fluorination is a well-known strategy for improving the bioavailability of drug molecules. However, its impact on efficacy is not easily predicted. On the basis of inhibitor-bound protein crystal structures, we found a beneficial fluorination spot for inhibitors targeting methionyl-tRNA synthetase of Trypanosoma brucei. In particular, incorporating 5-fluoroimidazo[4,5-b]pyridine into inhibitors leads to central nervous system bioavailability and maintained or even improved efficacy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsinfecdis.6b00036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5108244PMC
June 2016

The Potential of Hyperspectral Patterns of Winter Wheat to Detect Changes in Soil Microbial Community Composition.

Front Plant Sci 2016 9;7:759. Epub 2016 Jun 9.

Department of Terrestrial Ecology, NIOO-KNAW, Netherlands Institute of Ecology Wageningen, Netherlands.

Reliable information on soil status and crop health is crucial for detecting and mitigating disasters like pollution or minimizing impact from soil-borne diseases. While infestation with an aggressive soil pathogen can be detected via reflected light spectra, it is unknown to what extent hyperspectral reflectance could be used to detect overall changes in soil biodiversity. We tested the hypotheses that spectra can be used to (1) separate plants growing with microbial communities from different farms; (2) to separate plants growing in different microbial communities due to different land use; and (3) separate plants according to microbial species loss. We measured hyperspectral reflectance patterns of winter wheat plants growing in sterilized soils inoculated with microbial suspensions under controlled conditions. Microbial communities varied due to geographical distance, land use and microbial species loss caused by serial dilution. After 3 months of growth in the presence of microbes from the two different farms plant hyperspectral reflectance patterns differed significantly from each other, while within farms the effects of land use via microbes on plant reflectance spectra were weak. Species loss via dilution on the other hand affected a number of spectral indices for some of the soils. Spectral reflectance can be indicative of differences in microbial communities, with the Renormalized Difference Vegetation Index the most common responding index. Also, a positive correlation was found between the Normalized Difference Vegetation Index and the bacterial species richness, which suggests that plants perform better with higher microbial diversity. There is considerable variation between the soil origins and currently it is not possible yet to make sufficient reliable predictions about the soil microbial community based on the spectral reflectance. We conclude that measuring plant hyperspectral reflectance has potential for detecting changes in microbial communities yet due to its sensitivity high replication is necessary and a strict sampling design to exclude other 'noise' factors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fpls.2016.00759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4899463PMC
July 2016

Development of an Orally Available and Central Nervous System (CNS) Penetrant Toxoplasma gondii Calcium-Dependent Protein Kinase 1 (TgCDPK1) Inhibitor with Minimal Human Ether-a-go-go-Related Gene (hERG) Activity for the Treatment of Toxoplasmosis.

J Med Chem 2016 07 1;59(13):6531-46. Epub 2016 Jul 1.

Department of Biochemistry, University of Washington , Seattle, Washington 98195, United States.

New therapies are needed for the treatment of toxoplasmosis, which is a disease caused by the protozoan parasite Toxoplasma gondii. To this end, we previously developed a potent and selective inhibitor (compound 1) of Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) that possesses antitoxoplasmosis activity in vitro and in vivo. Unfortunately, 1 has potent human ether-a-go-go-related gene (hERG) inhibitory activity, associated with long Q-T syndrome, and consequently presents a cardiotoxicity risk. Here, we describe the identification of an optimized TgCDPK1 inhibitor 32, which does not have a hERG liability and possesses a favorable pharmacokinetic profile in small and large animals. 32 is CNS-penetrant and highly effective in acute and latent mouse models of T. gondii infection, significantly reducing the amount of parasite in the brain, spleen, and peritoneal fluid and reducing brain cysts by >85%. These properties make 32 a promising lead for the development of a new antitoxoplasmosis therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.6b00760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5100899PMC
July 2016

Zinc coordination is essential for the function and activity of the type II secretion ATPase EpsE.

Microbiologyopen 2016 Oct 10;5(5):870-882. Epub 2016 May 10.

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan.

The type II secretion system Eps in Vibrio cholerae promotes the extracellular transport of cholera toxin and several hydrolytic enzymes and is a major virulence system in many Gram-negative pathogens which is structurally related to the type IV pilus system. The cytoplasmic ATPase EpsE provides the energy for exoprotein secretion through ATP hydrolysis. EpsE contains a unique metal-binding domain that coordinates zinc through a tetracysteine motif (CXXCX CXXC), which is also present in type IV pilus assembly but not retraction ATPases. Deletion of the entire domain or substitution of any of the cysteine residues that coordinate zinc completely abrogates secretion in an EpsE-deficient strain and has a dominant negative effect on secretion in the presence of wild-type EpsE. Consistent with the in vivo data, chemical depletion of zinc from purified EpsE hexamers results in loss of in vitro ATPase activity. In contrast, exchanging the residues between the two dicysteines with those from the homologous ATPase XcpR from Pseudomonas aeruginosa does not have a significant impact on EpsE. These results indicate that, although the individual residues in the metal-binding domain are generally interchangeable, zinc coordination is essential for the activity and function of EpsE.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/mbo3.376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5061722PMC
October 2016

Root-Lesion Nematodes Suppress Cabbage Aphid Population Development by Reducing Aphid Daily Reproduction.

Front Plant Sci 2016 10;7:111. Epub 2016 Feb 10.

German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-LeipzigLeipzig, Germany; Institute of Ecology, Friedrich Schiller University JenaJena Germany; Molecular Interaction Ecology, Institute of Water and Wetland Research, Radboud UniversityNijmegen Netherlands.

Empirical studies have shown that belowground feeding herbivores can affect the performance of aboveground herbivores in different ways. Often the critical life-history parameters underlying the observed performance effects remain unexplored. In order to better understand the cause for the observed effects on aboveground herbivores, these ecological mechanisms must be better understood. In this study we combined empirical experiments with a modeling approach to analyze the effect of two root feeding endoparasitic nematodes with different feeding strategies on the population growth of the aboveground feeding specialist aphid Brevicoryne brassicae on Brassica nigra. The aim was to test whether emerging differences in life history characteristics (days until reproduction, daily reproduction) would be sufficient to explain observed differences in aphid population development on plants with and without two species of nematodes. Aphid numbers were lower on plants with Pratylenchus penetrans in comparison to aphid numbers on plants with Meloidogyne spp. A dedicated experiment showed that aphid daily reproduction was lower on plants with P. penetrans (3.08 offspring female(-1) day(-1)) in comparison to both uninfested plants and plants with Meloidogyne spp. (3.50 offspring female(-1) day(-1)). The species-specific reduction of aphid reproduction appeared independent of changes in amino acids, soluble sugars or the glucosinolate sinigrin in the phloem. An individual-based model revealed that relatively small differences in reproduction rate per female were sufficient to yield a similar difference in aphid populations as was found in the empirical experiments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fpls.2016.00111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4748742PMC
February 2016

SAR Studies of 5-Aminopyrazole-4-carboxamide Analogues as Potent and Selective Inhibitors of CDPK1.

ACS Med Chem Lett 2015 Dec 22;6(12):1184-1189. Epub 2015 Oct 22.

Department of Biochemistry, University of Washington , Seattle, Washington 98195, United States.

We previously discovered compounds based on a 5-aminopyrazole-4-carboxamide scaffold to be potent and selective inhibitors of CDPK1 from . The current work, through structure-activity relationship studies, led to the discovery of compounds ( and ) with improved characteristics over the starting inhibitor in terms of solubility, plasma exposure after oral administration in mice, or efficacy on parasite growth inhibition. Compounds and were further demonstrated to be more effective than in a mouse infection model and markedly reduced the amount of in the brain, spleen, and peritoneal fluid, and given at 20 mg/kg eliminated from the peritoneal fluid.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsmedchemlett.5b00319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4677665PMC
December 2015

Non-random species loss in bacterial communities reduces antifungal volatile production.

Ecology 2015 Aug;96(8):2042-8

The contribution of low-abundance microbial species to soil ecosystems is easily overlooked because there is considerable overlap between metabolic abilities (functional redundancy) of dominant and subordinate microbial species. Here we studied how loss of less abundant soil bacteria affected the production of antifungal volatiles, an important factor in the natural control of soil-borne pathogenic fungi. We provide novel empirical evidence that the loss of soil bacterial species leads to a decline in the production of volatiles that suppress root pathogens. By using dilution-to-extinction for seven different soils we created bacterial communities with a decreasing number of species and grew them under carbon-limited conditions. Communities with high bacterial species richness produced volatiles that strongly reduced the hyphal growth of the pathogen Fusarium oxysporum. For most soil origins loss of bacterial species resulted in loss of antifungal volatile production. Analysis of the volatiles revealed that several known antifungal compounds were only produced in the more diverse bacterial communities. Our results suggest that less abundant bacterial species play an important role in antifungal volatile production by soil bacterial communities and, consequently, in the natural suppression of soil-borne pathogens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1890/14-2359.1DOI Listing
August 2015

Inhibitors of methionyl-tRNA synthetase have potent activity against Giardia intestinalis trophozoites.

Antimicrob Agents Chemother 2015 Nov 31;59(11):7128-31. Epub 2015 Aug 31.

Department of Medicine, University of Washington, Seattle, Washington, USA

The methionyl-tRNA synthetase (MetRS) is a novel drug target for the protozoan pathogen Giardia intestinalis. This protist contains a single MetRS that is distinct from the human cytoplasmic MetRS. A panel of MetRS inhibitors was tested against recombinant Giardia MetRS, Giardia trophozoites, and mammalian cell lines. The best compounds inhibited trophozoite growth at 500 nM (metronidazole did so at ∼5,000 nM) and had low cytotoxicity against mammalian cells, indicating excellent potential for further development as anti-Giardia drugs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.01573-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4604383PMC
November 2015

A binding hotspot in Trypanosoma cruzi histidyl-tRNA synthetase revealed by fragment-based crystallographic cocktail screens.

Acta Crystallogr D Biol Crystallogr 2015 Aug 31;71(Pt 8):1684-98. Epub 2015 Jul 31.

Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.

American trypanosomiasis, commonly known as Chagas disease, is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. The chronic form of the infection often causes debilitating morbidity and mortality. However, the current treatment for the disease is typically inadequate owing to drug toxicity and poor efficacy, necessitating a continual effort to discover and develop new antiparasitic therapeutic agents. The structure of T. cruzi histidyl-tRNA synthetase (HisRS), a validated drug target, has previously been reported. Based on this structure and those of human cytosolic HisRS, opportunities for the development of specific inhibitors were identified. Here, efforts are reported to identify small molecules that bind to T. cruzi HisRS through fragment-based crystallographic screening in order to arrive at chemical starting points for the development of specific inhibitors. T. cruzi HisRS was soaked into 68 different cocktails from the Medical Structural Genomics of Pathogenic Protozoa (MSGPP) fragment library and diffraction data were collected to identify bound fragments after soaking. A total of 15 fragments were identified, all bound to the same site on the protein, revealing a fragment-binding hotspot adjacent to the ATP-binding pocket. On the basis of the initial hits, the design of reactive fragments targeting the hotspot which would be simultaneously covalently linked to a cysteine residue present only in trypanosomatid HisRS was initiated. Inhibition of T. cruzi HisRS was observed with the resultant reactive fragments and the anticipated binding mode was confirmed crystallographically. These results form a platform for the development of future generations of selective inhibitors for trypanosomatid HisRS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S1399004715007683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4528801PMC
August 2015

Legacy effects of anaerobic soil disinfestation on soil bacterial community composition and production of pathogen-suppressing volatiles.

Front Microbiol 2015 10;6:701. Epub 2015 Jul 10.

Department of Microbial Ecology, Netherlands Institute of Ecology, NIOO-KNAW Wageningen, Netherlands ; Department of Soil Quality, Wageningen University and Research Centre Wageningen, Netherlands.

There is increasing evidence that microbial volatiles (VOCs) play an important role in natural suppression of soil-borne diseases, but little is known on the factors that influence production of suppressing VOCs. In the current study we examined whether a stress-induced change in soil microbial community composition would affect the production by soils of VOCs suppressing the plant-pathogenic oomycete Pythium. Using pyrosequencing of 16S ribosomal gene fragments we compared the composition of bacterial communities in sandy soils that had been exposed to anaerobic disinfestation (AD), a treatment used to kill harmful soil organisms, with the composition in untreated soils. Three months after the AD treatment had been finished, there was still a clear legacy effect of the former anaerobic stress on bacterial community composition with a strong increase in relative abundance of the phylum Bacteroidetes and a significant decrease of the phyla Acidobacteria, Planctomycetes, Nitrospirae, Chloroflexi, and Chlorobi. This change in bacterial community composition coincided with loss of production of Pythium suppressing soil volatiles (VOCs) and of suppression of Pythium impacts on Hyacinth root development. One year later, the composition of the bacterial community in the AD soils was reflecting that of the untreated soils. In addition, both production of Pythium-suppressing VOCs and suppression of Pythium in Hyacinth bioassays had returned to the levels of the untreated soil. GC/MS analysis identified several VOCs, among which compounds known to be antifungal, that were produced in the untreated soils but not in the AD soils. These compounds were again produced 15 months after the AD treatment. Our data indicate that soils exposed to a drastic stress can temporarily lose pathogen suppressive characteristics and that both loss and return of these suppressive characteristics coincides with shifts in the soil bacterial community composition. Our data are supporting the suggested importance of microbial VOCs in the natural buffer of soils against diseases caused by soil-borne pathogens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2015.00701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4498103PMC
July 2015

Context dependency and saturating effects of loss of rare soil microbes on plant productivity.

Front Plant Sci 2015 30;6:485. Epub 2015 Jun 30.

Department of Terrestrial Ecology, Netherlands Institute of Ecology, Wageningen Netherlands ; Laboratory of Nematology, Wageningen University, Wageningen Netherlands.

Land use intensification is associated with loss of biodiversity and altered ecosystem functioning. Until now most studies on the relationship between biodiversity and ecosystem functioning focused on random loss of species, while loss of rare species that usually are the first to disappear received less attention. Here we test if the effect of rare microbial species loss on plant productivity depends on the origin of the microbial soil community. Soils were sampled from three land use types at two farms. Microbial communities with increasing loss of rare species were created by inoculating sterilized soils with serially diluted soil suspensions. After 8 months of incubation, the effects of the different soil communities on abiotic soil properties, soil processes, microbial community composition, and plant productivity was measured. Dilution treatments resulted in increasing species loss, which was in relation to abundance of bacteria in the original field soil, without affecting most of the other soil parameters and processes. Microbial species loss affected plant biomass positively, negatively or not at all, depending on soil origin, but not on land use history. Even within fields the effects of dilution on plant biomass varied between replicates, suggesting heterogeneity in microbial community composition. The effects of medium and severe species loss on plant biomass were similar, pointing toward a saturating effect of species loss. We conclude that changes in the composition of the soil microbial community, including rare species loss, can affect plant productivity, depending on the composition of the initial microbial community. Future work on the relation between function and species loss effects should address this variation by including multiple sampling origins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fpls.2015.00485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4485053PMC
July 2015

Three-dimensional structures in the design of therapeutics targeting parasitic protozoa: reflections on the past, present and future.

Authors:
Wim G J Hol

Acta Crystallogr F Struct Biol Commun 2015 May 16;71(Pt 5):485-99. Epub 2015 Apr 16.

Department of Biochemistry and Biomolecular Structure Center, University of Washington, Seattle, WA 98195, USA.

Parasitic protozoa cause a range of diseases which threaten billions of human beings. They are responsible for tremendous mortality and morbidity in the least-developed areas of the world. Presented here is an overview of the evolution over the last three to four decades of structure-guided design of inhibitors, leads and drug candidates aiming at targets from parasitic protozoa. Target selection is a crucial and multi-faceted aspect of structure-guided drug design. The major impact of advances in molecular biology, genome sequencing and high-throughput screening is touched upon. The most advanced crystallographic techniques, including XFEL, have already been applied to structure determinations of drug targets from parasitic protozoa. Even cryo-electron microscopy is contributing to our understanding of the mode of binding of inhibitors to parasite ribosomes. A number of projects have been selected to illustrate how structural information has assisted in arriving at promising compounds that are currently being evaluated by pharmacological, pharmacodynamic and safety tests to assess their suitability as pharmaceutical agents. Structure-guided approaches are also applied to incorporate properties into compounds such that they are less likely to become the victim of resistance mechanisms. A great increase in the number of novel antiparasitic compounds will be needed in the future. These should then be combined into various multi-compound therapeutics to circumvent the diverse resistance mechanisms that render single-compound, or even multi-compound, drugs ineffective. The future should also see (i) an increase in the number of projects with a tight integration of structural biology, medicinal chemistry, parasitology and pharmaceutical sciences; (ii) the education of more `medicinal structural biologists' who are familiar with the properties that compounds need to have for a high probability of success in the later steps of the drug-development process; and (iii) the expansion of drug-development capabilities in middle- and low-income countries.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S2053230X15004987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4427157PMC
May 2015

Identification of potent inhibitors of the Trypanosoma brucei methionyl-tRNA synthetase via high-throughput orthogonal screening.

J Biomol Screen 2015 Jan 27;20(1):122-30. Epub 2014 Aug 27.

The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA Amgen, Inc., Thousand Oaks, CA.

Improved therapies for the treatment of Trypanosoma brucei, the etiological agent of the neglected tropical disease human African trypanosomiasis, are urgently needed. We targeted T. brucei methionyl-tRNA synthetase (MetRS), an aminoacyl-tRNA synthase (aaRS), which is considered an important drug target due to its role in protein synthesis, cell survival, and its significant differences in structure from its mammalian ortholog. Previous work using RNA interference of MetRS demonstrated growth inhibition of T. brucei, further validating it as an attractive target. We report the development and implementation of two orthogonal high-throughput screening assays to identify inhibitors of T. brucei MetRS. First, a chemiluminescence assay was implemented in a 1536-well plate format and used to monitor adenosine triphosphate depletion during the aminoacylation reaction. Hit confirmation then used a counterscreen in which adenosine monophosphate production was assessed using fluorescence polarization technology. In addition, a miniaturized cell viability assay was used to triage cytotoxic compounds. Finally, lower throughput assays involving whole parasite growth inhibition of both human and parasite MetRS were used to analyze compound selectivity and efficacy. The outcome of this high-throughput screening campaign has led to the discovery of 19 potent and selective T. brucei MetRS inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1087057114548832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4378865PMC
January 2015

Comparison of histidine recognition in human and trypanosomatid histidyl-tRNA synthetases.

Biochimie 2014 Nov 20;106:111-20. Epub 2014 Aug 20.

Department of Biochemistry, University of Washington, Seattle, WA 98195, USA. Electronic address:

As part of a project aimed at obtaining selective inhibitors and drug-like compounds targeting tRNA synthetases from trypanosomatids, we have elucidated the crystal structure of human cytosolic histidyl-tRNA synthetase (Hs-cHisRS) in complex with histidine in order to be able to compare human and parasite enzymes. The resultant structure of Hs-cHisRS•His represents the substrate-bound state (H-state) of the enzyme. It provides an interesting opportunity to compare with ligand-free and imidazole-bound structures Hs-cHisRS published recently, both of which represent the ligand-free state (F-state) of the enzyme. The H-state Hs-cHisRS undergoes conformational changes in active site residues and several conserved motif of HisRS, compared to F-state structures. The histidine forms eight hydrogen bonds with HisRS of which six engage the amino and carboxylate groups of this amino acid. The availability of published imidazole-bound structure provides a unique opportunity to dissect the structural roles of individual chemical groups of histidine. The analysis revealed the importance of the amino and carboxylate groups, of the histidine in leading to these dramatic conformational changes of the H-state. Further, comparison with previously published trypanosomatid HisRS structures reveals a pocket in the F-state of the parasite enzyme that may provide opportunities for developing specific inhibitors of Trypanosoma brucei HisRS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biochi.2014.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4250371PMC
November 2014

Crystal structure of the full-length ATPase GspE from the Vibrio vulnificus type II secretion system in complex with the cytoplasmic domain of GspL.

J Struct Biol 2014 Sep 1;187(3):223-235. Epub 2014 Aug 1.

Department of Biochemistry and Biomolecular Structure Center, University of Washington, Seattle, WA 98195, United States. Electronic address:

The type II secretion system (T2SS) is present in many Gram-negative bacteria and is responsible for secreting a large number of folded proteins, including major virulence factors, across the outer membrane. The T2SS consists of 11-15 different proteins most of which are present in multiple copies in the assembled secretion machinery. The ATPase GspE, essential for the functioning of the T2SS, contains three domains (N1E, N2E and CTE) of which the N1E domain is associated with the cytoplasmic domain of the inner membrane protein GspL. Here we describe and analyze the structure of the GspE•cyto-GspL complex from Vibrio vulnificus in the presence of an ATP analog, AMPPNP. There are three such ∼83 kDa complexes per asymmetric unit with essentially the same structure. The N2E and CTE domains of a single V. vulnificus GspE subunit adopt a mutual orientation that has not been seen before in any of the previous GspE structures, neither in structures of related ATPases from other secretion systems. This underlines the tremendous conformational flexibility of the T2SS secretion ATPase. Cyto-GspL interacts not only with the N1E domain, but also with the CTE domain and is even in contact with AMPPNP. Moreover, the cyto-GspL domains engage in two types of mutual interactions, resulting in two essentially identical, but crystallographically independent, "cyto-GspL rods" that run throughout the crystal. Very similar rods are present in previous crystals of cyto-GspL and of the N1E•cyto-GspL complex. This arrangement, now seen four times in three entirely different crystal forms, involves contacts between highly conserved residues suggesting a role in the biogenesis or the secretion mechanism or both of the T2SS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jsb.2014.07.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4150747PMC
September 2014