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Asset Tag: HGAP

April 21, 2020

Infection mechanisms and putative effector repertoire of the mosquito pathogenic oomycete Pythium guiyangense uncovered by genomic analysis.

Pythium guiyangense, an oomycete from a genus of mostly plant pathogens, is an effective biological control agent that has wide potential to manage diverse mosquitoes. However, its mosquito-killing mechanisms are almost unknown. In this study, we observed that P. guiyangense could utilize cuticle penetration and ingestion of mycelia into the digestive system to infect mosquito larvae. To explore pathogenic mechanisms, a high-quality genome sequence with 239 contigs and an N50 contig length of 1,009 kb was generated. The genome assembly is approximately 110 Mb, which is almost twice the size of other sequenced Pythium genomes. Further genome analysis suggests that P. guiyangense may arise from a hybridization of two related but distinct parental species. Phylogenetic analysis demonstrated that P. guiyangense likely evolved from common ancestors shared with plant pathogens. Comparative genome analysis coupled with transcriptome sequencing data suggested that P. guiyangense may employ multiple virulence mechanisms to infect mosquitoes, including secreted proteases and kazal-type protease inhibitors. It also shares intracellular Crinkler (CRN) effectors used by plant pathogenic oomycetes to facilitate the colonization of plant hosts. Our experimental evidence demonstrates that CRN effectors of P. guiyangense can be toxic to insect cells. The infection mechanisms and putative virulence effectors of P. guiyangense uncovered by this study provide the basis to develop improved mosquito control strategies. These data also provide useful knowledge on host adaptation and evolution of the entomopathogenic lifestyle within the oomycete lineage. A deeper understanding of the biology of P. guiyangense effectors might also be useful for management of other important agricultural pests.

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April 21, 2020

Chromulinavorax destructans, a pathogen of microzooplankton that provides a window into the enigmatic candidate phylum Dependentiae.

Members of the major candidate phylum Dependentiae (a.k.a. TM6) are widespread across diverse environments from showerheads to peat bogs; yet, with the exception of two isolates infecting amoebae, they are only known from metagenomic data. The limited knowledge of their biology indicates that they have a long evolutionary history of parasitism. Here, we present Chromulinavorax destructans (Strain SeV1) the first isolate of this phylum to infect a representative from a widespread and ecologically significant group of heterotrophic flagellates, the microzooplankter Spumella elongata (Strain CCAP 955/1). Chromulinavorax destructans has a reduced 1.2 Mb genome that is so specialized for infection that it shows no evidence of complete metabolic pathways, but encodes an extensive transporter system for importing nutrients and energy in the form of ATP from the host. Its replication causes extensive reorganization and expansion of the mitochondrion, effectively surrounding the pathogen, consistent with its dependency on the host for energy. Nearly half (44%) of the inferred proteins contain signal sequences for secretion, including many without recognizable similarity to proteins of known function, as well as 98 copies of proteins with an ankyrin-repeat domain; ankyrin-repeats are known effectors of host modulation, suggesting the presence of an extensive host-manipulation apparatus. These observations help to cement members of this phylum as widespread and diverse parasites infecting a broad range of eukaryotic microbes.

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April 21, 2020

One Health Genomic Surveillance of Escherichia coli Demonstrates Distinct Lineages and Mobile Genetic Elements in Isolates from Humans versus Livestock.

Livestock have been proposed as a reservoir for drug-resistant Escherichia coli that infect humans. We isolated and sequenced 431 E. coli isolates (including 155 extended-spectrum ß-lactamase [ESBL]-producing isolates) from cross-sectional surveys of livestock farms and retail meat in the East of England. These were compared with the genomes of 1,517 E. coli bacteria associated with bloodstream infection in the United Kingdom. Phylogenetic core genome comparisons demonstrated that livestock and patient isolates were genetically distinct, suggesting that E. coli causing serious human infection had not directly originated from livestock. In contrast, we observed highly related isolates from the same animal species on different farms. Screening all 1,948 isolates for accessory genes encoding antibiotic resistance revealed 41 different genes present in variable proportions in human and livestock isolates. Overall, we identified a low prevalence of shared antimicrobial resistance genes between livestock and humans based on analysis of mobile genetic elements and long-read sequencing. We conclude that within the confines of our sampling framework, there was limited evidence that antimicrobial-resistant pathogens associated with serious human infection had originated from livestock in our region. IMPORTANCE The increasing prevalence of E. coli bloodstream infections is a serious public health problem. We used genomic epidemiology in a One Health study conducted in the East of England to examine putative sources of E. coli associated with serious human disease. E. coli from 1,517 patients with bloodstream infections were compared with 431 isolates from livestock farms and meat. Livestock-associated and bloodstream isolates were genetically distinct populations based on core genome and accessory genome analyses. Identical antimicrobial resistance genes were found in livestock and human isolates, but there was limited overlap in the mobile elements carrying these genes. Within the limitations of sampling, our findings do not support the idea that E. coli causing invasive disease or their resistance genes are commonly acquired from livestock in our region. Copyright © 2019 Ludden et al.

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April 21, 2020

Potentially mobile denitrification genes identified in Azospirillum sp. strain TSH58.

Denitrification ability is sporadically distributed among diverse bacteria, archaea, and fungi. In addition, disagreement has been found between denitrification gene phylogenies and the 16S rRNA gene phylogeny. These facts have suggested potential occurrences of horizontal gene transfer (HGT) for the denitrification genes. However, evidence of HGT has not been clearly presented thus far. In this study, we identified the sequences and the localization of the nitrite reductase genes in the genomes of 41 denitrifying Azospirillum sp. strains and searched for mobile genetic elements that contain denitrification genes. All Azospirillum sp. strains examined in this study possessed multiple replicons (4 to 11 replicons), with their sizes ranging from 7 to 1,031 kbp. Among those, the nitrite reductase gene nirK was located on large replicons (549 to 941 kbp). Genome sequencing showed that Azospirillum strains that had similar nirK sequences also shared similar nir-nor gene arrangements, especially between the TSH58, Sp7T, and Sp245 strains. In addition to the high similarity between nir-nor gene clusters among the three Azospirillum strains, a composite transposon structure was identified in the genome of strain TSH58, which contains the nir-nor gene cluster and the novel IS6 family insertion sequences (ISAz581 and ISAz582). The nirK gene within the composite transposon system was actively transcribed under denitrification-inducing conditions. Although not experimentally verified in this study, the composite transposon system containing the nir-nor gene cluster could be transferred to other cells if it is moved to a prophage region and the phage becomes activated and released outside the cells. Taken together, strain TSH58 most likely acquired its denitrification ability by HGT from closely related Azospirillum sp. denitrifiers.IMPORTANCE The evolutionary history of denitrification is complex. While the occurrence of horizontal gene transfer has been suggested for denitrification genes, most studies report circumstantial evidences, such as disagreement between denitrification gene phylogenies and the 16S rRNA gene phylogeny. Based on the comparative genome analyses of Azospirillum sp. denitrifiers, we identified denitrification genes, including nirK and norCBQD, located on a mobile genetic element in the genome of Azospirillum sp. strain TSH58. The nirK was actively transcribed under denitrification-inducing conditions. Since this gene was the sole nitrite reductase gene in strain TSH58, this strain most likely benefitted by acquiring denitrification genes via horizontal gene transfer. This finding will significantly advance our scientific knowledge regarding the ecology and evolution of denitrification. Copyright © 2019 American Society for Microbiology.

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April 21, 2020

Genetic characterisation of variants of the virulence plasmid, pSLT, in Salmonella enterica serovar Typhimurium provides evidence of a variety of evolutionary directions consistent with vertical rather than horizontal transmission.

The virulence plasmid pSLT as exemplified by the 94 Kb plasmid in Salmonella Typhimurium strain LT2 is only found in isolates of serovar Typhimurium. While it occurs commonly among such isolates recent genotyping methods have shown that it is mostly confined to certain genotypes. Although pSLT plasmids are capable of self-transmissibility under experimental conditions their confinement to certain host genotypes suggests that in practice they are maintained by vertical rather than by horizontal transmission. This would imply that evolution of the pSLT plasmid proceeds in parallel with evolution of its host. The development of a phylogenetic evolutionary framework for genotypes of S. Typhimurium based on single-nucleotide-polymorphism (SNPs) typing provided an opportunity to test whether the pSLT plasmid coevolves with its host genotype. Accordingly SNPs analysis was applied to the pSLT plasmids from 71 strains S. Typhimurium of Australian and international origins representing most of the genotypes which commonly have a pSLT. The phylogenetic tree showed that pSLT sequences clustered into almost the same groups as the host chromosomes so that each pSLT genotype was associated with a single host genotype. A search for tandem repeats in pSLT plasmids showed that a 9 bp VNTR in the traD gene occurred in the pSLT from all isolates belonging to Clade II but not from isolates belonging to Clade I. Another 9 bp repeat occurred only in three Clade I genotypes with a recent common ancestor. The evidence relating to both of these VNTRs supports the proposition that the pSLT plasmid is only transmitted vertically. Some isolates belonging to one S. Typhimurium genotype were found to have pSLTs which have lost a large block of genes when a resistance gene cassette has been acquired. Examples were found of pSLT plasmids which have recombined with other plasmids to form fusion plasmids sometimes with loss of some pSLT genes. In all cases the underlying genotype of the modified pSLT was the same as the genotype of regular pSLTs with the same host genotype implying that these changes have occurred within the host cell of the pSLT plasmid.

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April 21, 2020

Genomic characterization of Kerstersia gyiorum SWMUKG01, an isolate from a patient with respiratory infection in China.

The Gram-negative bacterium Kerstersia gyiorum, a potential etiological agent of clinical infections, was isolated from several human patients presenting clinical symptoms. Its significance as a possible pathogen has been previously overlooked as no disease has thus far been definitively associated with this bacterium. To better understand how the organism contributes to the infectious disease, we determined the complete genomic sequence of K. gyiorum SWMUKG01, the first clinical isolate from southwest China.The genomic data obtained displayed a single circular chromosome of 3, 945, 801 base pairs in length, which contains 3, 441 protein-coding genes, 55 tRNA genes and 9 rRNA genes. Analysis on the full spectrum of protein coding genes for cellular structures, two-component regulatory systems and iron uptake pathways that may be important for the success of the bacterial survival, colonization and establishment in the host conferred new insights into the virulence characteristics of K. gyiorum. Phylogenomic comparisons with Alcaligenaceae species indicated that K. gyiorum SWMUKG01 had a close evolutionary relationships with Alcaligenes aquatilis and Alcaligenes faecalis.The comprehensive analysis presented in this work determinates for the first time a complete genome sequence of K. gyiorum, which is expected to provide useful information for subsequent studies on pathogenesis of this species.

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April 21, 2020

Integrated transcriptomic and proteomic analysis of pathogenic mycobacteria and their esx-1 mutants reveal secretion-dependent regulation of ESX-1 substrates and WhiB6 as a transcriptional regulator.

The mycobacterial type VII secretion system ESX-1 is responsible for the secretion of a number of proteins that play important roles during host infection. The regulation of the expression of secreted proteins is often essential to establish successful infection. Using transcriptome sequencing, we found that the abrogation of ESX-1 function in Mycobacterium marinum leads to a pronounced increase in gene expression levels of the espA operon during the infection of macrophages. In addition, the disruption of ESX-1-mediated protein secretion also leads to a specific down-regulation of the ESX-1 substrates, but not of the structural components of this system, during growth in culture medium. This effect is observed in both M. marinum and M. tuberculosis. We established that down-regulation of ESX-1 substrates is the result of a regulatory process that is influenced by the putative transcriptional regulator whib6, which is located adjacent to the esx-1 locus. In addition, the overexpression of the ESX-1-associated PE35/PPE68 protein pair resulted in a significantly increased secretion of the ESX-1 substrate EsxA, demonstrating a functional link between these proteins. Taken together, these data show that WhiB6 is required for the secretion-dependent regulation of ESX-1 substrates and that ESX-1 substrates are regulated independently from the structural components, both during infection and as a result of active secretion.

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April 21, 2020

Genome Sequence of the Black Yeast Exophiala lecanii-corni.

The genome sequence of Exophiala lecanii-corni, a melanized dimorphic fungus with the capability of degrading several volatile organic compounds, was sequenced using PacBio single-molecule real-time (SMRT) sequencing to assist with understanding the molecular basis of its uncommon morphological and metabolic characteristics. The assembled draft genome is presented here.

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