Longitudinal host transcriptional responses to SARS-CoV-2 infection in adults with extremely high viral load.

Current understanding of viral dynamics of SARS-CoV-2 and host responses driving the pathogenic mechanisms in COVID-19 is rapidly evolving. Here, we conducted a longitudinal study to investigate gene expression patterns during acute SARS-CoV-2 illness. Cases included SARS-CoV-2 infected individuals with extremely high viral loads early in their illness, individuals having low SARS-CoV-2 viral loads early in their infection, and individuals testing negative for SARS-CoV-2. We could identify widespread transcriptional host responses to SARS-CoV-2 infection that were initially most strongly manifested in patients with extremely high initial viral loads, then attenuating within the patient over time as viral loads decreased. Genes correlated with SARS-CoV-2 viral load over time were similarly differentially expressed across independent datasets of SARS-CoV-2 infected lung and upper airway cells, from both in vitro systems and patient samples. We also generated expression data on the human nose organoid model during SARS-CoV-2 infection. The human nose organoid-generated host transcriptional response captured many aspects of responses observed in the above patient samples, while suggesting the existence of distinct host responses to SARS-CoV-2 depending on the cellular context, involving both epithelial and cellular immune responses. Our findings provide a catalog of SARS-CoV-2 host response genes changing over time.

Longitudinal host transcriptional responses to SARS-CoV-2 infection in adults with extremely high viral load.

Current understanding of viral dynamics of SARS-CoV-2 and host responses driving the pathogenic mechanisms in COVID-19 is rapidly evolving. Here, we conducted a longitudinal study to investigate gene expression patterns during acute SARS-CoV-2 illness. Cases included SARS-CoV-2 infected individuals with extremely high viral loads early in their illness, individuals having low SARS-CoV-2 viral loads early in their infection, and individuals testing negative for SARS-CoV-2. We could identify widespread transcriptional host responses to SARS-CoV-2 infection that were initially most strongly manifested in patients with extremely high initial viral loads, then attenuating within the patient over time as viral loads decreased. Genes correlated with SARS-CoV-2 viral load over time were similarly differentially expressed across independent datasets of SARS-CoV-2 infected lung and upper airway cells, from both in vitro systems and patient samples. We also generated expression data on the human nose organoid model during SARS-CoV-2 infection. The human nose organoid-generated host transcriptional response captured many aspects of responses observed in the above patient samples, while suggesting the existence of distinct host responses to SARS-CoV-2 depending on the cellular context, involving both epithelial and cellular immune responses. Our findings provide a catalog of SARS-CoV-2 host response genes changing over time.

Mortality difference from Klebsiella aerogenes vs Enterobacter cloacae bloodstream infections.

Members of the order Enterobacterales, including Escherichia coli , Klebsiella species and Enterobacter species, are important pathogens in healthcare-associated infections. Higher mortality has been reported from infections due to Klebsiella pneumoniae than from E. coli , but prior studies comparing Enterobacter aerogenes (recently renamed Klebsiella aerogenes ) bacteraemia and Enterobacter cloacae complex bacteraemia have yielded conflicting results regarding whether clinical outcomes differ. We found bacteraemia with K. aerogenes was independently associated with greater risk of 30-day mortality than bacteraemia with Enterobacter cloacae complex.

Oligonucleotide capture sequencing of the SARS-CoV-2 genome and subgenomic fragments from COVID-19 individuals.

The newly emerged and rapidly spreading SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). To facilitate a deeper understanding of the viral biology we developed a capture sequencing methodology to generate SARS-CoV-2 genomic and transcriptome sequences from infected patients. We utilized an oligonucleotide probe-set representing the full-length genome to obtain both genomic and transcriptome (subgenomic open reading frames [ORFs]) sequences from 45 SARS-CoV-2 clinical samples with varying viral titers. For samples with higher viral loads (cycle threshold value under 33, based on the CDC qPCR assay) complete genomes were generated. Analysis of junction reads revealed regions of differential transcriptional activity among samples. Mixed allelic frequencies along the 20kb ORF1ab gene in one sample, suggested the presence of a defective viral RNA species subpopulation maintained in mixture with functional RNA in one sample. The associated workflow is straightforward, and hybridization-based capture offers an effective and scalable approach for sequencing SARS-CoV-2 from patient samples.

Meta-analysis of host transcriptional responses to SARS-CoV-2 infection reveals their manifestation in human tumors.

A deeper understanding of the molecular biology of SARS-CoV-2 infection, including the host response to the virus, is urgently needed. Commonalities exist between the host immune response to viral infections and cancer. Here, we defined transcriptional signatures of SARS-CoV-2 infection involving hundreds of genes common across lung adenocarcinoma cell lines (A549, Calu-3) and normal human bronchial epithelial cells (NHBE), with additional signatures being specific to one or both adenocarcinoma lines. Cross-examining eight transcriptomic databases, we found that host transcriptional responses of lung adenocarcinoma cells to SARS-CoV-2 infection shared broad similarities with host responses to multiple viruses across different model systems and patient samples. Furthermore, these SARS-CoV-2 transcriptional signatures were manifested within specific subsets of human cancer, involving ~ 20% of cases across a wide range of histopathological types. These cancer subsets show immune cell infiltration and inflammation and involve pathways linked to the SARS-CoV-2 response, such as immune checkpoint, IL-6, type II interferon signaling, and NF-κB. The cell line data represented immune responses activated specifically within the cancer cells of the tumor. Common genes and pathways implicated as part of the viral host response point to therapeutic strategies that may apply to both SARS-CoV-2 and cancer.

Oligonucleotide Capture Sequencing of the SARS-CoV-2 Genome and Subgenomic Fragments from COVID-19 Individuals.

The newly emerged and rapidly spreading SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). To facilitate a deeper understanding of the viral biology we developed a capture sequencing methodology to generate SARS-CoV-2 genomic and transcriptome sequences from infected patients. We utilized an oligonucleotide probe-set representing the full-length genome to obtain both genomic and transcriptome (subgenomic open reading frames [ORFs]) sequences from 45 SARS-CoV-2 clinical samples with varying viral titers. For samples with higher viral loads (cycle threshold value under 33, based on the CDC qPCR assay) complete genomes were generated. Analysis of junction reads revealed regions of differential transcriptional activity and provided evidence of expression of ORF10. Heterogeneous allelic frequencies along the 20kb ORF1ab gene suggested the presence of a defective interfering viral RNA species subpopulation in one sample. The associated workflow is straightforward, and hybridization-based capture offers an effective and scalable approach for sequencing SARS-CoV-2 from patient samples.

Oligonucleotide capture sequencing of the SARS-CoV-2 genome and subgenomic fragments from COVID-19 individuals.

The newly emerged and rapidly spreading SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). To facilitate a deeper understanding of the viral biology we developed a capture sequencing methodology to generate SARS-CoV-2 genomic and transcriptome sequences from infected patients. We utilized an oligonucleotide probe-set representing the full-length genome to obtain both genomic and transcriptome (subgenomic open reading frames [ORFs]) sequences from 45 SARS-CoV-2 clinical samples with varying viral titers. For samples with higher viral loads (cycle threshold value under 33, based on the CDC qPCR assay) complete genomes were generated. Analysis of junction reads revealed regions of differential transcriptional activity and provided evidence of expression of ORF10. Heterogeneous allelic frequencies along the 20kb ORF1ab gene suggested the presence of a defective interfering viral RNA species subpopulation in one sample. The associated workflow is straightforward, and hybridization-based capture offers an effective and scalable approach for sequencing SARS-CoV-2 from patient samples.

Loss of the Polyketide Synthase StlB Results in Stalk Cell Overproduction in Polysphondylium violaceum.

Major phenotypic innovations in social amoeba evolution occurred at the transition between the Polysphondylia and group 4 Dictyostelia, which comprise the model organism Dictyostelium discoideum, such as the formation of a new structure, the basal disk. Basal disk differentiation and robust stalk formation require the morphogen DIF-1, synthesized by the polyketide synthase StlB, the des-methyl-DIF-1 methyltransferase DmtA, and the chlorinase ChlA, which are conserved throughout Dictyostelia. To understand how the basal disk and other innovations evolved in group 4, we sequenced and annotated the Polysphondylium violaceum (Pvio) genome, performed cell type-specific transcriptomics to identify cell-type marker genes, and developed transformation and gene knock-out procedures for Pvio. We used the novel methods to delete the Pvio stlB gene. The Pvio stlB- mutants formed misshapen curly sorogens with thick and irregular stalks. As fruiting body formation continued, the upper stalks became more regular, but structures contained 40% less spores. The stlB- sorogens overexpressed a stalk gene and underexpressed a (pre)spore gene. Normal fruiting body formation and sporulation were restored in Pvio stlB- by including DIF-1 in the supporting agar. These data indicate that, although conserved, stlB and its product(s) acquired both a novel role in the group 4 Dictyostelia and a role opposite to that in its sister group.

Correction: Repurposed Transcriptomic Data Reveal Small Viral RNA Produced by Influenza Virus during Infection in Mice.

[This corrects the article DOI: 10.1371/journal.pone.0165729.].

Escherichia coli DNA ligase B may mitigate damage from oxidative stress.

Escherichia coli encodes two DNA ligases, ligase A, which is essential under normal laboratory growth conditions, and ligase B, which is not. Here we report potential functions of ligase B. We found that across the entire Enterobacteriaceae family, ligase B is highly conserved in both amino acid identity and synteny with genes associated with oxidative stress. Deletion of ligB sensitized E. coli to specific DNA damaging agents and antibiotics resulted in a weak mutator phenotype, and decreased biofilm formation. Overexpression of ligB caused a dramatic extension of lag phase that eventually resumed normal growth. The ligase function of ligase B was not required to mediate the extended lag phase, as overexpression of a ligase-deficient ligB mutant also blocked growth. Overexpression of ligB during logarithmic growth caused an immediate block of cell growth and DNA replication, and death of about half of cells. These data support a potential role for ligase B in the base excision repair pathway or the mismatch repair pathway.

Repurposed Transcriptomic Data Reveal Small Viral RNA Produced by Influenza Virus during Infection in Mice.

Influenza virus, a highly infectious ssRNA virus, replicates in the nucleus of host cells. This unusual feature brings the possibility that the virus may hijack host small noncoding RNA metabolism. Influenza small viral RNA production has been examined in vitro but has not yet been studied in an in vivo setting. We assessed small RNA species from influenza virus during mouse infection by mining publicly available mouse small RNA transcriptome data. We uncovered 26 nt reads corresponding to svRNA, a small viral RNA previously detected in vitro that regulates the transition from transcription to replication during infection, and found a strong positive correlation between svRNA production and host susceptibility to influenza virus infection. We also detected significant overrepresentation of a non-coding 23 nt sequence that we speculate may behave like a miRNA and work with influenza protein NS1 to prevent the transcription and maturation of interferon-stimulated mRNAs.

Emergence of Klebsiella pneumoniae ST273 Carrying blaNDM-7 and ST656 Carrying blaNDM-1 in Manila, Philippines.

We sought to determine the epidemiology of carbapenem-resistant Enterobacteriaceae and to investigate the emergence of carbapenem-resistant Klebsiella pneumoniae in two teaching hospitals in Manila, Philippines. We screened 364 Enterobacteriaceae for carbapenem resistance between 2012 and 2013 and detected four carbapenem-resistant K. pneumoniae isolates from three different patients. We used whole genome sequencing to determine the antibiotic resistance profiles and confirmed the presence of carbapenemase genes by multiplex PCR. We used multilocus sequence typing and PCR-based replicon typing to genetically characterize the carbapenem-resistant isolates. The carbapenemase gene blaNDM was detected in K. pneumoniae isolates from two patients. The first patient had ventilator-associated pneumonia and lumbar shunt infection from K. pneumoniae ST273 carrying blaNDM-7. The second patient had asymptomatic genitourinary colonization with K. pneumoniae ST656 carrying blaNDM-1. The third patient had a gluteal abscess with K. pneumoniae ST1 that did not carry a carbapenemase gene, but did carry blaDHA-1, blaOXA-1, and blaSHV-1. In this study, we report the first cases of blaNDM-carrying pathogens in the Philippines and add to the growing evidence of the worldwide spread of ST273 and NDM-7, a more efficient carbapenem hydrolyzer than NDM-1.

Genomic signatures of cooperation and conflict in the social amoeba.

Cooperative systems are susceptible to invasion by selfish individuals that profit from receiving the social benefits but fail to contribute. These so-called "cheaters" can have a fitness advantage in the laboratory, but it is unclear whether cheating provides an important selective advantage in nature. We used a population genomic approach to examine the history of genes involved in cheating behaviors in the social amoeba Dictyostelium discoideum, testing whether these genes experience rapid evolutionary change as a result of conflict over spore-stalk fate. Candidate genes and surrounding regions showed elevated polymorphism, unusual patterns of linkage disequilibrium, and lower levels of population differentiation, but they did not show greater between-species divergence. The signatures were most consistent with frequency-dependent selection acting to maintain multiple alleles, suggesting that conflict may lead to stalemate rather than an escalating arms race. Our results reveal the evolutionary dynamics of cooperation and cheating and underscore how sequence-based approaches can be used to elucidate the history of conflicts that are difficult to observe directly.

Novel Conserved Genotypes Correspond to Antibiotic Resistance Phenotypes of E. coli Clinical Isolates.

Current efforts to understand antibiotic resistance on the whole genome scale tend to focus on known genes even as high throughput sequencing strategies uncover novel mechanisms. To identify genomic variations associated with antibiotic resistance, we employed a modified genome-wide association study; we sequenced genomic DNA from pools of E. coli clinical isolates with similar antibiotic resistance phenotypes using SOLiD technology to uncover single nucleotide polymorphisms (SNPs) unanimously conserved in each pool. The multidrug-resistant pools were genotypically similar to SMS-3-5, a previously sequenced multidrug-resistant isolate from a polluted environment. The similarity was evenly spread across the entire genome and not limited to plasmid or pathogenicity island loci. Among the pools of clinical isolates, genomic variation was concentrated adjacent to previously reported inversion and duplication differences between the SMS-3-5 isolate and the drug-susceptible laboratory strain, DH10B. SNPs that result in non-synonymous changes in gyrA (encoding the well-known S83L allele associated with fluoroquinolone resistance), mutM, ligB, and recG were unanimously conserved in every fluoroquinolone-resistant pool. Alleles of the latter three genes are tightly linked among most sequenced E. coli genomes, and had not been implicated in antibiotic resistance previously. The changes in these genes map to amino acid positions in alpha helices that are involved in DNA binding. Plasmid-encoded complementation of null strains with either allelic variant of mutM or ligB resulted in variable responses to ultraviolet light or hydrogen peroxide treatment as markers of induced DNA damage, indicating their importance in DNA metabolism and revealing a potential mechanism for fluoroquinolone resistance. Our approach uncovered evidence that additional DNA binding enzymes may contribute to fluoroquinolone resistance and further implicate environmental bacteria as a reservoir for antibiotic resistance.

A functional variomics tool for discovering drug-resistance genes and drug targets.

Comprehensive discovery of genetic mechanisms of drug resistance and identification of in vivo drug targets represent significant challenges. Here we present a functional variomics technology in the model organism Saccharomyces cerevisiae. This tool analyzes numerous genetic variants and effectively tackles both problems simultaneously. Using this tool, we discovered almost all genes that, due to mutations or modest overexpression, confer resistance to rapamycin, cycloheximide, and amphotericin B. Most significant among the resistance genes were drug targets, including multiple targets of a given drug. With amphotericin B, we discovered the highly conserved membrane protein Pmp3 as a potent resistance factor and a possible target. Widespread application of this tool should allow rapid identification of conserved resistance mechanisms and targets of many more compounds. New genes and alleles that confer resistance to other stresses can also be discovered. Similar tools in other systems, such as human cell lines, will also be useful.

Comparative genomics of the social amoebae Dictyostelium discoideum and Dictyostelium purpureum.

BACKGROUND: The social amoebae (Dictyostelia) are a diverse group of Amoebozoa that achieve multicellularity by aggregation and undergo morphogenesis into fruiting bodies with terminally differentiated spores and stalk cells. There are four groups of dictyostelids, with the most derived being a group that contains the model species Dictyostelium discoideum. RESULTS: We have produced a draft genome sequence of another group dictyostelid, Dictyostelium purpureum, and compare it to the D. discoideum genome. The assembly (8.41 × coverage) comprises 799 scaffolds totaling 33.0 Mb, comparable to the D. discoideum genome size. Sequence comparisons suggest that these two dictyostelids shared a common ancestor approximately 400 million years ago. In spite of this divergence, most orthologs reside in small clusters of conserved synteny. Comparative analyses revealed a core set of orthologous genes that illuminate dictyostelid physiology, as well as differences in gene family content. Interesting patterns of gene conservation and divergence are also evident, suggesting function differences; some protein families, such as the histidine kinases, have undergone little functional change, whereas others, such as the polyketide synthases, have undergone extensive diversification. The abundant amino acid homopolymers encoded in both genomes are generally not found in homologous positions within proteins, so they are unlikely to derive from ancestral DNA triplet repeats. Genes involved in the social stage evolved more rapidly than others, consistent with either relaxed selection or accelerated evolution due to social conflict. CONCLUSIONS: The findings from this new genome sequence and comparative analysis shed light on the biology and evolution of the Dictyostelia.

Conserved developmental transcriptomes in evolutionarily divergent species.

BACKGROUND: Evolutionarily divergent organisms often share developmental anatomies despite vast differences between their genome sequences. The social amoebae Dictyostelium discoideum and Dictyostelium purpureum have similar developmental morphologies although their genomes are as divergent as those of man and jawed fish. RESULTS: Here we show that the anatomical similarities are accompanied by extensive transcriptome conservation. Using RNA sequencing we compared the abundance and developmental regulation of all the transcripts in the two species. In both species, most genes are developmentally regulated and the greatest expression changes occur during the transition from unicellularity to multicellularity. The developmental regulation of transcription is highly conserved between orthologs in the two species. In addition to timing of expression, the level of mRNA production is also conserved between orthologs and is consistent with the intuitive notion that transcript abundance correlates with the amount of protein required. Furthermore, the conservation of transcriptomes extends to cell-type specific expression. CONCLUSIONS: These findings suggest that developmental programs are remarkably conserved at the transcriptome level, considering the great evolutionary distance between the genomes. Moreover, this transcriptional conservation may be responsible for the similar developmental anatomies of Dictyostelium discoideum and Dictyostelium purpureum.

Polymorphic members of the lag gene family mediate kin discrimination in Dictyostelium.

Self and kin discrimination are observed in most kingdoms of life and are mediated by highly polymorphic plasma membrane proteins. Sequence polymorphism, which is essential for effective recognition, is maintained by balancing selection. Dictyostelium discoideum are social amoebas that propagate as unicellular organisms but aggregate upon starvation and form fruiting bodies with viable spores and dead stalk cells. Aggregative development exposes Dictyostelium to the perils of chimerism, including cheating, which raises questions about how the victims survive in nature and how social cooperation persists. Dictyostelids can minimize the cost of chimerism by preferential cooperation with kin, but the mechanisms of kin discrimination are largely unknown. Dictyostelium lag genes encode transmembrane proteins with multiple immunoglobulin (Ig) repeats that participate in cell adhesion and signaling. Here, we describe their role in kin discrimination. We show that lagB1 and lagC1 are highly polymorphic in natural populations and that their sequence dissimilarity correlates well with wild-strain segregation. Deleting lagB1 and lagC1 results in strain segregation in chimeras with wild-type cells, whereas elimination of the nearly invariant homolog lagD1 has no such consequences. These findings reveal an early evolutionary origin of kin discrimination and provide insight into the mechanism of social recognition and immunity.

Relationships among ciprofloxacin, gatifloxacin, levofloxacin, and norfloxacin MICs for fluoroquinolone-resistant Escherichia coli clinical isolates.

Fluoroquinolones are some of the most prescribed antibiotics in the United States. Previously, we and others showed that the fluoroquinolones exhibit a class effect with regard to the CLSI-established breakpoints for resistance, such that decreased susceptibility (i.e., an increased MIC) to one fluoroquinolone means a simultaneously decreased susceptibility to all. For defined strains, however, clear differences exist in the pharmacodynamic properties of each fluoroquinolone and the extent to which resistance-associated genotypes affect the MICs of each fluoroquinolone. In a pilot study of 920 clinical Escherichia coli isolates, we uncovered tremendous variation in norfloxacin MICs. The MICs for all of the fluoroquinolone-resistant isolates exceeded the resistance breakpoint, reaching 1,000 microg/ml. Approximately 25% of the isolates (n = 214), representing the full range of resistant norfloxacin MICs, were selected for the simultaneous determinations of ciprofloxacin, gatifloxacin, levofloxacin, and norfloxacin MICs. We found that (i) great MIC variation existed for all four fluoroquinolones, (ii) the ciprofloxacin and levofloxacin MICs of >90% of the fluoroquinolone-resistant isolates were higher than the resistance breakpoints, (iii) ciprofloxacin and levofloxacin MICs were distributed into two distinct groups, (iv) the MICs of two drug pairs (ciprofloxacin and norfloxacin by Kendall's Tau-b test and gatifloxacin and levofloxacin by paired t test) were similar with statistical significance but were different from each other, and (v) approximately 2% of isolates had unprecedented fluoroquinolone MIC relationships. Thus, although the fluoroquinolones can be considered equivalent with regard to clinical susceptibility or resistance, fluoroquinolone MICs differ dramatically for fluoroquinolone-resistant clinical isolates, likely because of differences in drug structure.

Facultative cheater mutants reveal the genetic complexity of cooperation in social amoebae.

Cooperation is central to many major transitions in evolution, including the emergence of eukaryotic cells, multicellularity and eusociality. Cooperation can be destroyed by the spread of cheater mutants that do not cooperate but gain the benefits of cooperation from others. However, cooperation can be preserved if cheaters are facultative, cheating others but cooperating among themselves. Several cheater mutants have been studied before, but no study has attempted a genome-scale investigation of the genetic opportunities for cheating. Here we describe such a screen in a social amoeba and show that cheating is multifaceted by revealing cheater mutations in well over 100 genes of diverse types. Many of these mutants cheat facultatively, producing more than their fair share of spores in chimaeras, but cooperating normally when clonal. These findings indicate that phenotypically stable cooperative systems may nevertheless harbour genetic conflicts. The opportunities for evolutionary moves and countermoves in such conflicts may select for the involvement of multiple pathways and numerous genes.