Commercial AHAS-inhibiting herbicides are promising drug leads for the treatment of human fungal pathogenic infections

The increased prevalence of drug-resistant human pathogenic fungal diseases poses a major threat to global human health. Thus, new drugs are urgently required to combat these infections. Here, we demonstrate that acetohydroxyacid synthase (AHAS), the first enzyme in the branched-chain amino acid biosynthesis pathway, is a promising new target for antifungal drug discovery. First, we show that several AHAS inhibitors developed as commercial herbicides are powerful accumulative inhibitors of Candida albicans AHAS (K-i values as low as 800 pM) and have determined high-resolution crystal structures of this enzyme in complex with several of these herbicides. In addition, we have demonstrated that chlorimuron ethyl (CE), a member of the sulfonylurea herbicide family, has potent antifungal activity against five different Candida species and Cryptococcus neoformans (with minimum inhibitory concentration, 50% values as low as 7 nM). Furthermore, in these assays, we have shown CE and itraconazole (a P450 inhibitor) can act synergistically to further improve potency. Finally, we show in Candida albicans-infected mice that CE is highly effective in clearing pathogenic fungal burden in the lungs, liver, and spleen, thus reducing overall mortality rates. Therefore, in view of their low toxicity to human cells, AHAS inhibitors represent a new class of antifungal drug candidates

MiDAS 4: A global catalogue of full-length 16S rRNA gene sequences and taxonomy for studies of bacterial communities in wastewater treatment plants

Microbial communities are responsible for biological wastewater treatment, but our knowledge of their diversity and function is still poor. Here, we sequence more than 5 million high-quality, full-length 16S rRNA gene sequences from 740 wastewater treatment plants (WWTPs) across the world and use the sequences to construct the ‘MiDAS 4’ database. MiDAS 4 is an amplicon sequence variant resolved, full-length 16S rRNA gene reference database with a comprehensive taxonomy from domain to species level for all sequences. We use an independent dataset (269 WWTPs) to show that MiDAS 4, compared to commonly used universal reference databases, provides a better coverage for WWTP bacteria and an improved rate of genus and species level classification. Taking advantage of MiDAS 4, we carry out an amplicon-based, global-scale microbial community profiling of activated sludge plants using two common sets of primers targeting regions of the 16S rRNA gene, revealing how environmental conditions and biogeography shape the activated sludge microbiota. We also identify core and conditionally rare or abundant taxa, encompassing 966 genera and 1530 species that represent approximately 80% and 50% of the accumulated read abundance, respectively. Finally, we show that for well-studied functional guilds, such as nitrifiers or polyphosphate-accumulating organisms, the same genera are prevalent worldwide, with only a few abundant species in each genus.Fil: Dueholm, Morten Kam Dahl. Aalborg University; DinamarcaFil: Nierychlo, Marta. Aalborg University; DinamarcaFil: Andersen, Kasper Skytte. Aalborg University; DinamarcaFil: Rudkjøbing, Vibeke. Aalborg University; DinamarcaFil: Knutsson, Simon. Aalborg University; DinamarcaFil: Arriaga, Sonia. Instituto Potosino de Investigación Científica y Tecnológica; MéxicoFil: Bakke, Rune. University College of Southeast Norway; NoruegaFil: Boon, Nico. University of Ghent; BélgicaFil: Bux, Faizal. Durban University of Technology; SudáfricaFil: Christensson, Magnus. Veolia Water Technologies Ab; SueciaFil: Chua, Adeline Seak May. University Malaya; MalasiaFil: Curtis, Thomas P.. University of Newcastle; Reino UnidoFil: Cytryn, Eddie. Agricultural Research Organization Of Israel; IsraelFil: Erijman, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires; ArgentinaFil: Etchebehere, Claudia. Instituto de Investigaciones Biológicas "Clemente Estable"; UruguayFil: Fatta Kassinos, Despo. University of Cyprus; ChipreFil: Frigon, Dominic. McGill University; CanadáFil: Garcia Chaves, Maria Carolina. Universidad de Antioquia; ColombiaFil: Gu, April Z.. Cornell University; Estados UnidosFil: Horn, Harald. Karlsruher Institut Für Technologie; AlemaniaFil: Jenkins, David. David Jenkins & Associates Inc; Estados UnidosFil: Kreuzinger, Norbert. Tu Wien; AustriaFil: Kumari, Sheena. Durban University of Technology; SudáfricaFil: Lanham, Ana. University of Bath; Reino UnidoFil: Law, Yingyu. Singapore Centre For Environmental Life Sciences Engineering; SingapurFil: Leiknes, TorOve. King Abdullah University of Science and Technology; Arabia SauditaFil: Morgenroth, Eberhard. Eth Zürich; SuizaFil: Muszyński, Adam. Politechnika Warszawska; PoloniaFil: Petrovski, Steve. La Trobe University; AustraliaFil: Pijuan, Maite. Catalan Institute For Water Research; EspañaFil: Pillai, Suraj Babu. Va Tech Wabag Ltd; IndiaFil: Reis, Maria A. M.. Universidade Nova de Lisboa; PortugalFil: Rong, Qi. Chinese Academy of Sciences; ChinaFil: Rossetti, Simona. Istituto Di Ricerca Sulle Acque (irsa) ; Consiglio Nazionale Delle Ricerche;Fil: Seviour, Robert. La Trobe University; AustraliaFil: Tooker, Nick. University of Massachussets; Estados UnidosFil: Vainio, Pirjo. Espoo R&D Center; FinlandiaFil: van Loosdrecht, Mark. Delft University of Technology; Países BajosFil: Vikraman, R.. VA Tech Wabag, Philippines Inc; FilipinasFil: Wanner, Jiří. University of Chemistry And Technology; República ChecaFil: Weissbrodt, David. Delft University of Technology; Países BajosFil: Wen, Xianghua. Tsinghua University; ChinaFil: Zhang, Tong. The University of Hong Kong; Hong KongFil: Nielsen, Per H.. Aalborg University; DinamarcaFil: Albertsen, Mads. Aalborg University; DinamarcaFil: Nielsen, Per Halkjær. Aalborg University; Dinamarc

Targeted genome editing via CRISPR in the pathogen Cryptococcus neoformans

Low rates of homologous integration have hindered molecular genetic studies in Cryptococcus neoformans over the past 20 years, and new tools that facilitate genome manipulation in this important pathogen are greatly needed. To this end, we have investigated the use of a Class 2 CRISPR system in C. neoformans (formerly C. neoformans var. grubii). We first expressed a derivative of the Streptococcus pyogenes Cas9 nuclease in C. neoformans, and showed that it has no effect on growth, production of virulence factors in vitro, or virulence in a murine inhalation model. In proof of principle experiments, we tested the CAS9 construct in combination with multiple self-cleaving guide RNAs targeting the wellcharacterized phosphoribosylaminoamidazole carboxylase-encoding ADE2 gene. Utilizing combinations of transient and stable expression of our constructs, we revealed that functionality of our CRISPR constructs in C. neoformans is dependent upon the CAS9 construct being stably integrated into the genome, whilst transient expression of the guide RNA is sufficient to enhance rates of homologous recombination in the CAS9 genetic background. Given that the presence of the CRISPR nuclease does not influence virulence in a murine inhalation model, we have successfully demonstrated that this system is compatible with studies of C. neoformans pathogenesis and represents a powerful tool that can be exploited by researchers in the field

Deleting <i>ADE2</i> with the aid of a CRISPR-Cas9 system.

<p><b>A.</b> Transformations where only one part of the CRISPR system was included to determine if constitutive expression of individual components had any effect on the rate of homologous integration at the <i>ADE2</i> locus. <b>B.</b> Transformations where the recipient strain is stably expressing <i>CAS9</i> at the Safe Haven site, with gRNA plasmids co-transformed with the <i>ade2Δ</i> construct. <b>C.</b> Transformations where the recipient strain is stably expressing a gRNA construct at the Safe Haven site, with the <i>CAS9</i> plasmid co-transformed with the <i>ade2Δ</i> construct. For all graphs, values show mean, error bars show S.E.M and * = P<0.05.</p

Virulence of H99_CAS9 is indistinguishable from wild-type in a mouse model.

<p><b>A.</b> No significant difference was found between H99 and H99_<i>CAS9</i> in a murine inhalation model of virulence. <b>B.</b> No significant difference was observed in fungal organ burden of mice infected with H99 and H99_<i>CAS9</i>.</p

The <i>C</i>. <i>neoformans</i> Cas9 strain is indistinguishable from wild-type in growth or virulence production assays.

<p>H99 and H99_<i>CAS9</i> were compared for growth on YPD and YNB (<b>A</b>), melanin production on L-DOPA (<b>B</b>), urease production on Christensen’s agar, phospholipase production on egg yolk agar, protease production BSA agar (<b>C</b>) and capsule production in RPMI media (<b>D</b>). All assays were visualized at 48 hr at 30 and 37°C excluding growth on egg yolk agar, which was visualized at 96 hr, and capsule, visualized at 24 hr.</p

gRNA constructs targeting <i>C</i>. <i>neoformans ADE2</i>.

<p>The target sites for gRNA1 and gRNA2 within the <i>ADE2</i> gene are indicated; the gRNA construct shown is for gRNA2. HH, Hammerhead ribozyme; HDV, Hepatitis Delta Virus ribozyme. Ribozyme cleavage sites are represented as scissors.</p

Co-transformations combining the <i>ade2</i>::<i>NEO</i> deletion construct with plasmid-borne Cas9 and gRNA constructs does not enhance the frequency of <i>ADE2</i> deletion.

<p>No increase in the rate of homologous recombination was observed between transformations with a deletion construct only and transformations with the deletion construct, Cas9 plasmid and gRNA plasmid present. Values show mean, error bars show S.E.M.</p

Expressing Cas9 in <i>C</i>. <i>neoformans</i>.

<p><b>A.</b> Comparison of the <i>CAS9</i> expression constructs from human [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164322#pone.0164322.ref037" target="_blank">37</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164322#pone.0164322.ref047" target="_blank">47</a>] and <i>C</i>. <i>neoformans</i>. <b>B.</b> Transcript abundance of <i>CAS9</i> in H99 and H99_<i>CAS9</i> relative to <i>ACT1</i>. Values show mean, error bars show S.E.M.</p