Biology subjects

Edwards, R. A.

Publications and source records attributed to Edwards, R. A..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

The sensor of the bacterial histidine kinase CpxA is a novel dimer of extracytoplasmic Per-ARNT-Sim (PAS) domains

Histidine kinases are key bacterial sensors that recognize diverse environmental stimuli. While mechanisms of phosphorylation and phosphotransfer by cytoplasmic kinase domains are relatively well-characterized, the ways in which extracytoplasmic sensor domains regulate activation remain mysterious. The Cpx envelope stress response is a conserved Gram-negative two-component system which is controlled by the sensor kinase CpxA. We report the structure of the Escherichia coli CpxA sensor domain (CpxA-SD) as a globular Per-ARNT-Sim (PAS)-like fold highly similar to that of Vibrio parahaemolyticus CpxA as determined by X-ray crystallography. Because sensor kinase dimerization is important for signaling, we used AlphaFold2 to model CpxA-SD in the context of its connected transmembrane domains, which yielded a novel dimer of PAS domains possessing a distinct dimer organization compared to previously characterized sensor domains. Gain of function cpxA* alleles map to the dimer interface, and mutation of other residues in this region also leads to constitutive activation. CpxA activation can be suppressed by mutations that restore inter-monomer interactions, suggesting that inhibitory interactions between CpxA-SD monomers are the major point of control for CpxA activation and signaling. Searching through hundreds of structural homologues revealed the sensor domain of Pseudomonas aeruginosa sensor kinase PfeS as the only PAS structure in the same novel dimer orientation as CpxA, suggesting that our dimer orientation may be utilized by other extracytoplasmic PAS domains. Overall, our findings provide insight into the diversity of the organization of PAS sensory domains and how they regulate sensor kinase activation. SignificanceBacterial two-component systems play an essential role in sensing environmental cues, mitigating stress, and regulating virulence. We approach the study of a key Gram-negative sensor kinase CpxA with both classical methods in structural biology and genetic analysis and emerging protein-folding prediction software. This approach provides a wholistic perspective on the structure and function of histidine kinases as proteins with modular and cellular compartment-spanning domain architectures. We report a novel organization of PAS domains in CpxA, highlighting the versatility and diversity of this sensory fold. Ultimately, these studies will facilitate the continued development of novel antimicrobials against sensor kinases, including CpxA, which is a previously studied target for antimicrobials.

molecular biology↗

bioRxiv2023

Temperate gut phages are prevalent, diverse, and predominantly inactive

Large-scale metagenomic and data mining efforts have uncovered an expansive diversity of bacteriophages (phages) within the human gut1-3. These insights include broader phage populational dynamics such as temporal stability4, interindividual uniqueness5,6 and potential associations to specific disease states7,8. However, the functional understanding of phage-host interactions and their impacts within this complex ecosystem have been limited due to a lack of cultured isolates for experimental validation. Here we characterise 125 active prophages originating from 252 diverse human gut bacterial isolates using seven different induction conditions to substantially expand the experimentally validated temperate phage-host pairs originating from the human gut. Importantly, only 17% of computationally predicted prophages were induced with common induction agents and these exhibited distinct gene patterns compared to non-induced predictions. Active Bacteroidota prophages were among the most prevalent members of the gut virome, with extensive use of diversity generating retroelements and exhibiting broad host ranges. Moreover, active polylysogeny was present in 52% of studied gut lysogens and led to coordinated prophage induction across diverse conditions. This study represents a substantial expansion of experimentally validated gut prophages, providing key insights into their diversity and genetics, including a genetic pathway for prophage domestication and demonstration that differential induction was complex and influenced by divergent prophage integration sites. More broadly, it highlights the importance of experimental validation alongside genomic based computational prediction to enable further functional understanding of these commensal viruses within the human gut.

microbiology↗

bioRxiv2023

Anti-Viral and Anti-Inflammatory Therapeutic Effect of RAGE-Ig Protein Against Multiple SARS-CoV-2 Variants of Concern Demonstrated in K18-hACE2 Mouse and Syrian Golden Hamster Models

SignificanceSARS-CoV-2 Variants of Concern (VOCs) continue to evolve and re-emerge with chronic inflammatory long-COVID sequelae necessitating the development of anti-inflammatory therapeutic molecules. Therapeutic effects of the Receptor for Advanced Glycation End products (RAGE) were reported in many inflammatory diseases. However, a therapeutic effect of the RAGE in COVID-19 has not been reported. In the present study, we investigated whether and how the RAGE-Ig fusion protein would have an anti-viral and anti-inflammatory therapeutic effect in the COVID-19 system. MethodsThe protective therapeutic effect of RAGE-Ig was determined in vitro in K18-hACE2 transgenic mice and Syrian golden hamsters infected with six various VOCs of SARS-CoV-2. The underlying anti-viral mechanism of RAGE-Ig was determined in vitro in SARS-CoV-2-infected human lung epithelial cells (BEAS-2B). ResultsFollowing treatment of K18-hACE2 mice and hamsters infected with various SARS-CoV-2 VOCs with RAGE-Ig, we demonstrated: (i) significant dose-dependent protection (i.e. greater survival, less weight loss, lower virus replication in the lungs); (ii) a reduction of inflammatory macrophages (F4/80+/Ly6C+) and neutrophils (CD11b+/Ly6G+) infiltrating the infected lungs; (iii) a RAGE-Ig dose-dependent increase in the expression of type I interferons (IFN-, and IFN-{beta}) and type III interferon (IFN{lambda}2) and a decrease in the inflammatory cytokines (IL-6 and IL-8) in SARS-CoV-2-infected human lung epithelial cells; and (iv) a dose-dependent decrease in the expression of CD64 (FcgR1) on monocytes and lung epithelial cells from symptomatic COVID-19 patients. ConclusionOur pre-clinical findings revealed type I and III interferons-mediated anti-viral and anti-inflammatory therapeutic effects of RAGE-Ig protein against COVID-19 caused by multiple SARS-CoV-2 VOCs.

immunology↗

bioRxiv2023

Cross-Protection Induced by Highly Conserved Human B, CD4+, and CD8+ T Cell Epitopes-Based Coronavirus Vaccine Against Severe Infection, Disease, and Death Caused by Multiple SARS-CoV-2 Variants of Concern

BackgroundThe Coronavirus disease 2019 (COVID-19) pandemic has created one of the largest global health crises in almost a century. Although the current rate of SARS-CoV-2 infections has decreased significantly; the long-term outlook of COVID-19 remains a serious cause of high death worldwide; with the mortality rate still surpassing even the worst mortality rates recorded for the influenza viruses. The continuous emergence of SARS-CoV-2 variants of concern (VOCs), including multiple heavily mutated Omicron sub-variants, have prolonged the COVID-19 pandemic and outlines the urgent need for a next-generation vaccine that will protect from multiple SARS-CoV-2 VOCs. MethodsIn the present study, we designed a multi-epitope-based Coronavirus vaccine that incorporated B, CD4+, and CD8+ T cell epitopes conserved among all known SARS-CoV-2 VOCs and selectively recognized by CD8+ and CD4+ T-cells from asymptomatic COVID-19 patients irrespective of VOC infection. The safety, immunogenicity, and cross-protective immunity of this pan-Coronavirus vaccine were studied against six VOCs using an innovative triple transgenic h-ACE-2-HLA-A2/DR mouse model. ResultsThe Pan-Coronavirus vaccine: (i) is safe; (ii) induces high frequencies of lung-resident functional CD8+ and CD4+ TEM and TRM cells; and (iii) provides robust protection against virus replication and COVID-19-related lung pathology and death caused by six SARS-CoV-2 VOCs: Alpha (B.1.1.7), Beta (B.1.351), Gamma or P1 (B.1.1.28.1), Delta (lineage B.1.617.2) and Omicron (B.1.1.529). Conclusions: A multi-epitope pan-Coronavirus vaccine bearing conserved human B and T cell epitopes from structural and non-structural SARS-CoV-2 antigens induced cross-protective immunity that cleared the virus, and reduced COVID-19-related lung pathology and death caused by multiple SARS-CoV-2 VOCs.

immunology↗

bioRxiv2023

Prophage rates in the human microbiome vary by body site and host health.

Phages integrated into a bacterial genome-called prophages-continuously monitor the health of the host bacteria to determine when to escape the genome, protect their host from other phage infections, and may provide genes that promote bacterial growth. Prophages are essential to almost all microbiomes, including the human microbiome. However, most human microbiome studies focus on bacteria, ignoring free and integrated phages, so we know little about how these prophages affect the human microbiome. We compared the prophages identified in 11,513 bacterial genomes isolated from human body sites to characterise prophage DNA in the human microbiome. Here, we show that prophage DNA comprised an average of 1-5% of each bacterial genome. The prophage content per genome varies with the isolation site on the human body, the health of the human, and whether the disease was symptomatic. The presence of prophages promotes bacterial growth and sculpts the microbiome. However, the disparities caused by prophages vary throughout the body.

genomics↗

bioRxiv2023

The Promise and Pitfalls of Prophages

Phages dominate every ecosystem on the planet. While virulent phages sculpt the microbiome by killing their bacterial hosts, temperate phages provide unique growth advantages to their hosts through lysogenic conversion. Many prophages benefit their host, and prophages are responsible for genotypic and phenotypic differences that separate individual microbial strains. However, the microbes also endure a cost to maintain those phages: additional DNA to replicate and proteins to transcribe and translate. We have never quantified those benefits and costs. Here, we analysed over two and a half million prophages from over half a million bacterial genome assemblies. Analysis of the whole dataset and a representative subset of taxonomically diverse bacterial genomes demonstrated that the normalised prophage density was uniform across all bacterial genomes above 2 Mbp. We identified a constant carrying capacity of phage DNA per bacterial DNA. We estimated that each prophage provides cellular services equivalent to approximately 2.4 % of the cells energy or 0.9 ATP per bp per hour. We demonstrate analytical, taxonomic, geographic, and temporal disparities in identifying prophages in bacterial genomes that provide novel targets for identifying new phages. We anticipate that the benefits bacteria accrue from the presence of prophages balance the energetics involved in supporting prophages. Furthermore, our data will provide a new framework for identifying phages in environmental datasets, diverse bacterial phyla, and from different locations.

bioinformatics↗

bioRxiv2023

Programmed ribosomal frameshifts, and how to find them

One of the stranger phenomena that can occur during gene translation is where, as a ribosome reads along the mRNA, various cellular and molecular properties contribute to stalling the ribosome on a slippery sequence, shifting the ribosome into one of the other two alternate reading frames. The alternate frame has different codons, so different amino acids are added to the peptide chain, but more importantly, the original stop codon is no longer in-frame, so the ribosome can bypass the stop codon and continue to translate the codons past it. This produces a longer version of the protein, a fusion of the original in-frame amino acids, followed by all the alternate frame amino acids. There is currently no automated software to predict the occurrence of these programmed ribosomal frameshifts (PRF), and they are currently only identified by manual curation. Here we present the first machine-learning based method to detect and predict the presence of PRFs in all types of coding genes and taxa with an accuracy exceding 90%.

bioinformatics↗

bioRxiv2023

Phables: from fragmented assemblies to high-quality bacteriophage genomes

MotivationMicrobial communities influence both human health and different environments. Viruses infecting bacteria, known as bacteriophages or phages, play a key role in modulating bacterial communities within environments. High-quality phage genome sequences are essential for advancing our understanding of phage biology, enabling comparative genomics studies, and developing phage-based diagnostic tools. Most available viral identification tools consider individual sequences to determine whether they are of viral origin. As a result of the challenges in viral assembly, fragmentation of genomes can occur, leading to the need for new approaches in viral identification. Therefore, the identification and characterisation of novel phages remain a challenge. ResultsWe introduce Phables, a new computational method to resolve phage genomes from fragmented viral metagenome assemblies. Phables identifies phage-like components in the assembly graph, models each component as a flow network, and uses graph algorithms and flow decomposition techniques to identify genomic paths. Experimental results of viral metagenomic samples obtained from different environments show that Phables recovers on average over 49% more high-quality phage genomes compared to existing viral identification tools. Furthermore, Phables can resolve variant phage genomes with over 99% average nucleotide identity, a distinction that existing tools are unable to make. Availability and ImplementationPhables is available on GitHub at https://github.com/Vini2/phables. Contactvijini.mallawaarachchi@flinders.edu.au

bioinformatics↗

bioRxiv2023

Novel crAssphage isolates exhibit conserved gene order and purifying selection of the host specificity protein

Bacteroides, the prominent bacteria in the human gut, play a crucial role in degrading complex polysaccharides. Their abundance is influenced by phages belonging to the Crassvirales order. Despite identifying over 600 Crassvirales genomes computationally, only few have been successfully isolated. Continued efforts in isolation of more Crassvirales genomes can provide insights into phage-host-evolution and infection mechanisms. We focused on wastewater samples, as potential sources of phages infecting various Bacteroides hosts. Sequencing, assembly, and characterization of isolated phages revealed 14 complete genomes belonging to three novel Crassvirales species infecting Bacteroides cellulosilyticus WH2. These species, Kehishuvirus sp. tikkala strain Bc01, Kolpuevirus sp. frurule strain Bc03, and Rudgehvirus jaberico strain Bc11, spanned two families, and three genera, displaying a broad range of virion productions. Upon testing all successfully cultured Crassvirales species and their respective bacterial hosts, we discovered that they do not exhibit co-evolutionary patterns with their bacterial hosts. Furthermore, we observed variations in gene similarity, with greater shared similarity observed within genera. However, despite belonging to different genera, the three novel species shared a unique structural gene that encodes the tail spike protein. When investigating the relationship between this gene and host interaction, we discovered evidence of purifying selection, indicating its functional importance. Moreover, our analysis demonstrated that this tail spike protein binds to the TonB-dependent receptors present on the bacterial host surface. Combining these observations, our findings provide insights into phage-host interactions and present three Crassvirales species as an ideal system for controlled infectivity experiments on one of the most dominant members of the human enteric virome. Impact statementBacteriophages play a crucial role in shaping microbial communities within the human gut. Among the most dominant bacteriophages in the human gut microbiome are Crassvirales phages, which infect Bacteroides. Despite being widely distributed, only a few Crassvirales genomes have been isolated, leading to a limited understanding of their biology, ecology, and evolution. This study isolated and characterized three novel Crassvirales genomes belonging to two different families, and three genera, but infecting one bacterial host, Bacteroides cellulosilyticus WH2. Notably, the observation confirmed the phages are not co-evolving with their bacterial hosts, rather have a shared ability to exploit similar features in their bacterial host. Additionally, the identification of a critical viral protein undergoing purifying selection and interacting with the bacterial receptors opens doors to targeted therapies against bacterial infections. Given Bacteroides role in polysaccharide degradation in the human gut, our findings advance our understanding of the phage-host interactions and could have important implications for the development of phage-based therapies. These discoveries may hold implications for improving gut health and metabolism to support overall well-being. Data summaryThe genomes used in this research are available on Sequence Read Archive (SRA) within the project, PRJNA737576. Bacteroides cellulosilyticus WH2, Kehishuvirus sp. tikkala strain Bc01, Kolpuevirus sp. frurule strain Bc03, and Rudgehvirus jaberico strain Bc11 are all available on GenBank with accessions NZ_CP072251.1 (B. cellulosilyticus WH2), QQ198717 (Bc01), QQ198718 (Bc03), and QQ198719 (Bc11), and we are working on making the strains available through ATCC. The 3D protein structures for the three Crassvirales genomes are available to download at doi.org/10.25451/flinders.21946034.

bioinformatics↗

bioRxiv2022

Biogeography of human-health associated butyrate-producing bacteria

AimButyrate-producing bacteria are found in many outdoor ecosystems and host organisms, including humans, and are vital to ecosystem functionality and human health. These bacteria ferment organic matter, producing the short-chain fatty acid butyrate. However, few (if any) studies have examined the macroecological influences on their large-scale biogeographical distribution. Here we aimed to characterise their global biogeography together with key explanatory climatic, geographic, and physicochemical variables. LocationGlobal, and the Australian continent Time period2005-2020 Major taxa studiedButyrate-producing bacteria MethodsWe developed new normalised butyrate production capacity (BPC) indices derived from global metagenomic (n=13,078) and Australia-wide soil 16S rRNA (n=1,331) data, using Geographic Information System (GIS) and modelling techniques to detail their ecological and biogeographical associations. ResultsThe highest BPC scores were found in anoxic and fermentative environments, including the human and non-human animal gut, and in some plant-soil systems. Within plant-soil systems, roots and rhizospheres had the highest BPC scores. Among soil samples, geographic and climatic variables had the strongest overall influence on BPC scores, with human influence also making key contributions. Higher BPC scores were in soils from seasonally productive sandy rangelands, temperate rural residential areas, and sites with moderate-to-high soil iron concentrations. Main conclusionsAbundances of butyrate-producing bacteria in outdoor soils follow complex ecological patterns influenced by geography, climate, soil chemistry, and hydrological fluctuations. Human population density and soil iron also play substantial roles, and their effects are dependent on a combination of ecological variables. These new biogeographical insights further our understanding of the global ecology patterns of butyrate-producing bacteria, with implications for emerging microbially-focussed ecological and human health policies.

ecology↗

bioRxiv2022

High Frequencies of PD-1+TIM3+TIGIT+CTLA4+ Functionally Exhausted SARS-CoV-2-Specific CD4+ and CD8+ T Cells Associated with Severe Disease in Critically ill COVID-19 Patients

SARS-CoV-2-specific memory T cells that cross-react with common cold coronaviruses (CCCs) are present in both healthy donors and COVID-19 patients. However, whether these cross-reactive T cells play a role in COVID-19 pathogenesis versus protection remain to be fully elucidated. In this study, we characterized cross-reactive SARS-CoV-2-specific CD4+ and CD8+ T cells, targeting genome-wide conserved epitopes in a cohort of 147 non-vaccinated COVID-19 patients, divided into six groups based on the degrees of disease severity. We compared the frequency, phenotype, and function of these SARS-CoV-2-specific CD4+ and CD8+ T cells between severely ill and asymptomatic COVID-19 patients and correlated this with -CCCs and {beta}-CCCs co-infection status. Compared with asymptomatic COVID-19 patients, the severely ill COVID-19 patients and patients with fatal outcomes: (i) Presented a broad leukocytosis and a broad CD4+ and CD8+ T cell lymphopenia; (ii) Developed low frequencies of functional IFN-{gamma}-producing CD134+CD138+CD4+ and CD134+CD138+CD8+ T cells directed toward conserved epitopes from structural, non-structural and regulatory SARS-CoV-2 proteins; (iii) Displayed high frequencies of SARS-CoV-2-specific functionally exhausted PD-1+TIM3+TIGIT+CTLA4+CD4+ and PD-1+TIM3+TIGIT+CTLA4+CD8+ T cells; and (iv) Displayed similar frequencies of co-infections with {beta}-CCCs strains but significantly fewer co-infections with -CCCs strains. Interestingly, the cross-reactive SARS-CoV-2 epitopes that recalled the strongest CD4+ and CD8+ T cell responses in unexposed healthy donors (HD) were the most strongly associated with better disease outcome seen in asymptomatic COVID-19 patients. Our results demonstrate that, the critically ill COVID-19 patients displayed fewer co-infection with -CCCs strain, presented broad T cell lymphopenia and higher frequencies of cross-reactive exhausted SARS-CoV-2-specific CD4+ and CD8+ T cells. In contrast, the asymptomatic COVID-19 patients, appeared to present more co-infections with -CCCs strains, associated with higher frequencies of functional cross-reactive SARS-CoV-2-specific CD4+ and CD8+ T cells. These findings support the development of broadly protective, T-cell-based, multi-antigen universal pan-Coronavirus vaccines. KEY POINTSO_LIA broad lymphopenia and lower frequencies of SARS-CoV-2-specific CD4+ and CD8+ T-cells were associated with severe disease onset in COVID-19 patients. C_LIO_LIHigh frequencies of phenotypically and functionally exhausted SARS-CoV-2-specific CD4+ and CD8+ T cells, co-expressing multiple exhaustion markers, and targeting multiple structural, non-structural, and regulatory SARS-CoV-2 protein antigens, were detected in severely ill COVID-19 patients. C_LIO_LICompared to severely ill COVID-19 patients and to patients with fatal outcomes, the (non-vaccinated) asymptomatic COVID-19 patients presented more functional cross-reactive CD4+ and CD8+ T cells targeting conserved epitopes from structural, non-structural, and regulatory SARS-CoV-2 protein antigens. C_LIO_LIThe cross-reactive SARS-CoV-2 epitopes that recalled the strongest CD4+ and CD8+ T cell responses in unexposed healthy donors (HD) were the most strongly associated with better disease outcomes seen in asymptomatic COVID-19 patients. C_LIO_LICompared to severely ill COVID-19 patients and to patients with fatal outcomes, the (non-vaccinated) asymptomatic COVID-19 patients presented higher rates of co-infection with the -CCCs strains. C_LIO_LICompared to patients with mild or asymptomatic COVID-19, severely ill symptomatic patients and patients with fatal outcomes had more exhausted SARS-CoV-2-speccific CD4+ and CD8+ T cells that preferentially target cross-reactive epitopes that share high identity and similarity with the {beta}-CCCs strains. C_LI

immunology↗

bioRxiv2021

Microglia do not restrict SARS-CoV-2 replication following infection of the central nervous system of K18-hACE2 transgenic mice

Unlike SARS-CoV-1 and MERS-CoV, infection with SARS-CoV-2, the viral pathogen responsible for COVID-19, is often associated with neurologic symptoms that range from mild to severe, yet increasing evidence argues the virus does not exhibit extensive neuroinvasive properties. We demonstrate SARS-CoV-2 can infect and replicate in human iPSC-derived neurons and that infection shows limited anti-viral and inflammatory responses but increased activation of EIF2 signaling following infection as determined by RNA sequencing. Intranasal infection of K18 human ACE2 transgenic mice (K18-hACE2) with SARS-CoV-2 resulted in lung pathology associated with viral replication and immune cell infiltration. In addition, [~]50% of infected mice exhibited CNS infection characterized by wide-spread viral replication in neurons accompanied by increased expression of chemokine (Cxcl9, Cxcl10, Ccl2, Ccl5 and Ccl19) and cytokine (Ifn-{lambda} and Tnf-) transcripts associated with microgliosis and a neuroinflammatory response consisting primarily of monocytes/macrophages. Microglia depletion via administration of colony-stimulating factor 1 receptor inhibitor, PLX5622, in SARS-CoV-2 infected mice did not affect survival or viral replication but did result in dampened expression of proinflammatory cytokine/chemokine transcripts and a reduction in monocyte/macrophage infiltration. These results argue that microglia are dispensable in terms of controlling SARS-CoV-2 replication in in the K18-hACE2 model but do contribute to an inflammatory response through expression of pro-inflammatory genes. Collectively, these findings contribute to previous work demonstrating the ability of SARS-CoV-2 to infect neurons as well as emphasizing the potential use of the K18-hACE2 model to study immunological and neuropathological aspects related to SARS-CoV-2-induced neurologic disease. ImportanceUnderstanding the immunological mechanisms contributing to both host defense and disease following viral infection of the CNS is of critical importance given the increasing number of viruses that are capable of infecting and replicating within the nervous system. With this in mind, the present study was undertaken to evaluate the role of microglia in aiding in host defense following experimental infection of the central nervous system (CNS) of K18-hACE2 with SARS-CoV-2, the causative agent of COVID-19. Neurologic symptoms that range in severity are common in COVID-19 patients and understanding immune responses that contribute to restricting neurologic disease can provide important insight into better understanding consequences associated with SARS-CoV-2 infection of the CNS.

microbiology↗

bioRxiv2021

Deep immune profiling of the maternal-fetal interface with mild SARS-CoV-2 infection

Pregnant women are an at-risk group for severe COVID-19, though the majority experience mild/asymptomatic disease. Although severe COVID-19 has been shown to be associated with immune activation at the maternal-fetal interface even in the absence of active viral replication, the immune response to asymptomatic/mild COVID-19 remains unknown. Here, we assessed immunological adaptations in both blood and term decidua from 9 SARS-exposed pregnant women with asymptomatic/mild disease and 15 pregnant SARS-naive women. In addition to selective loss of tissue-resident decidual macrophages, we report attenuation of antigen presentation and type I IFN signaling but upregulation of inflammatory cytokines and chemokines in blood monocyte derived decidual macrophages. On the other hand, infection was associated with remodeling of the T cell compartment with increased frequencies of activated CD69+ tissue-resident T cells and decreased abundance of Tregs. Interestingly, frequencies of cytotoxic CD4 and CD8 T cells increased only in the blood, while CD8 effector memory T cells were expanded in the decidua. In contrast to decidual macrophages, signatures of type I IFN signaling were increased in decidual T cells. Finally, T cell receptor diversity was significantly reduced with infection in both compartments, albeit to a much greater extent in the blood. The resulting aberrant immune activation in the placenta, even with asymptomatic disease may alter the exquisitely sensitive developing fetal immune system, leading to long-term adverse outcomes for offspring.

immunology↗

bioRxiv2021

hafeZ: Active prophage identification through read mapping

SummaryBacteriophages that have integrated their genomes into bacterial chromosomes, termed prophages, are widespread across bacteria. Prophages are key components of bacterial genomes, with their integration often contributing novel, beneficial, characteristics to the infected host. Likewise, their induction--through the production and release of progeny virions into the surrounding environment--can have considerable ramifications on bacterial communities. Yet, not all prophages can excise following integration, due to genetic degradation by their host bacterium. Here, we present hafeZ, a tool able to identify active prophages (i.e. those undergoing induction) within bacterial genomes through genomic read mapping. We demonstrate its use by applying hafeZ to publicly available sequencing data from bacterial genomes known to contain active prophages and show that hafeZ can accurately identify their presence and location in the host chromosomes. Availability and ImplementationhafeZ is implemented in Python 3.7 and freely available under an open-source GPL-3.0 license from https://github.com/Chrisjrt/hafeZ. Bugs and issues may be reported by submitting them via the hafeZ github issues page. Contactcturkington@ucmerced.edu or chrisjrt1@gmail.com

bioinformatics↗

bioRxiv2021

Philympics 2021: Prophage Predictions Perplex Programs

Most bacterial genomes contain integrated bacteriophages--prophages--in various states of decay. Many are active and able to excise from the genome and replicate, while others are cryptic prophages, remnants of their former selves. Over the last two decades, many computational tools have been developed to identify the prophage components of bacterial genomes, and it is a particularly active area for the application of machine learning approaches. However, progress is hindered and comparisons thwarted because there are no manually curated bacterial genomes that can be used to test new prophage prediction algorithms. Here, we present a library of gold-standard bacterial genome annotations that include manually curated prophage annotations, and a computational framework to compare the predictions from different algorithms. We use this suite to compare all extant stand-alone prophage prediction algorithms to identify their strengths and weaknesses. We provide a FAIR dataset for prophage identification, and demonstrate the accuracy, precision, recall, and f1 score from the analysis of seven different algorithms for the prediction of prophages. We discuss caveats and concerns in this analysis and how those concerns may be mitigated.

bioinformatics↗

bioRxiv2020

Antibody Response to a Nucleocapsid Epitope as a Marker for COVID-19 Disease Severity

Effective methods for predicting COVID-19 disease trajectories are urgently needed. Here, ELISA and coronavirus antigen microarray (COVAM) analysis mapped antibody epitopes in the plasma of COVID-19 patients (n = 86) experiencing a wide-range of disease states. The experiments identified antibodies to a 21-residue epitope from nucleocapsid (termed Ep9) associated with severe disease, including admission to the ICU, requirement for ventilators, or death. Importantly, anti-Ep9 antibodies can be detected within six days post-symptom onset and sometimes within one day. Furthermore, anti-Ep9 antibodies correlate with various comorbidities and hallmarks of immune hyperactivity. We introduce a simple-to-calculate, disease risk factor score to quantitate each patients comorbidities and age. For patients with anti-Ep9 antibodies, scores above 3.0 predict more severe disease outcomes with a 13.42 Likelihood Ratio (96.7% specificity). The results lay the groundwork for a new type of COVID-19 prognostic to allow early identification and triage of high-risk patients. Such information could guide more effective therapeutic intervention. Significance statementThe COVID-19 pandemic has resulted in over two million deaths worldwide. Despite efforts to fight the virus, the disease continues to overwhelm hospitals with severely ill patients. Diagnosis of COVID-19 is readily accomplished through a multitude of reliable testing platforms; however, prognostic prediction remains elusive. To this end, we identified a short epitope from the SARS-CoV-2 nucleocapsid protein and also a disease risk factor score based upon comorbidities and age. The presence of antibodies specifically binding to this epitope plus a score cutoff can predict severe COVID-19 outcomes with 96.7% specificity.

immunology↗

bioRxiv2020

MultiPhATE2: Code for Functional Annotation and Comparison of Bacteriophage Genomes

To address the need for improved tools for annotation and comparative genomics of bacteriophage genomes, we developed multiPhATE2. As an extension of the multiPhATE code, multiPhATE2 performs gene finding and functional sequence annotation of predicted gene and protein sequences, and additional search algorithms and databases extend the search space of the original functional annotation subsystem. MultiPhATE2 includes comparative genomics codes for gene matching among sets of input bacteriophage genomes, and scales well to large input data sets with the incorporation of multiprocessing in the functional annotation and comparative genomics subsystems. MultiPhATE2 was implemented in Python 3.7 and runs as a command-line code under Linux or MAC-OS. MultiPhATE2 is freely available under an open-source GPL-3 license at https://github.com/carolzhou/multiPhATE2. Instructions for acquiring the databases and third party codes used by multiPhATE2 are found in the README file included with the distribution. Users may report bugs by submitting issues to the project GitHub repository webpage. Contact: zhou4@llnl.gov or multiphate@gmail.com. Supplementary materials, which demonstrate the outputs of multiPhATE2, are available in a GitHub repository, at https://github.com/carolzhou/multiPhATE2_supplementaryData/.

bioinformatics↗

bioRxiv2020

Genome-Wide Asymptomatic B-Cell, CD4+ and CD8+ T-Cell Epitopes, that are Highly Conserved Between Human and Animal Coronaviruses, Identified from SARS-CoV-2 as Immune Targets for Pre-Emptive Pan-Coronavirus Vaccines

Over the last two decades, there have been three deadly human outbreaks of Coronaviruses (CoVs) caused by emerging zoonotic CoVs: SARS-CoV, MERS-CoV, and the latest highly transmissible and deadly SARS-CoV-2, which has caused the current COVID-19 global pandemic. All three deadly CoVs originated from bats, the natural hosts, and transmitted to humans via various intermediate animal reservoirs. Because there is currently no universal pan-Coronavirus vaccine available, two worst-case scenarios remain highly possible: (1) SARS-CoV-2 mutates and transforms into a seasonal "flu-like" global pandemic; and/or (2) Other global COVID-like pandemics will emerge in the coming years, caused by yet another spillover of an unknown zoonotic bat-derived SARS-like Coronavirus (SL-CoV) into an unvaccinated human population. Determining the antigen and epitope landscapes that are conserved among human and animal Coronaviruses as well as the repertoire, phenotype and function of B cells and CD4+ and CD8+ T cells that correlate with resistance seen in asymptomatic COVID-19 patients should inform in the development of pan-Coronavirus vaccines 1. In the present study, using several immuno-informatics and sequence alignment approaches, we identified several human B-cell, CD4+ and CD8+ T cell epitopes that are highly conserved in: (i) greater than 81,000 SARS-CoV-2 human strains identified to date in 190 countries on six continents; (ii) six circulating CoVs that caused previous human outbreaks of the "Common Cold"; (iii) five SL-CoVs isolated from bats; (iv) five SL-CoV isolated from pangolins; (v) three SL-CoVs isolated from Civet Cats; and (vi) four MERS strains isolated from camels. Furthermore, we identified cross-reactive asymptomatic epitopes that: (i) recalled B cell, CD4+ and CD8+ T cell responses from both asymptomatic COVID-19 patients and healthy individuals who were never exposed to SARS-CoV-2; and (ii) induced strong B cell and T cell responses in "humanized" Human Leukocyte Antigen (HLA)-DR/HLA-A*02:01 double transgenic mice. The findings herein pave the way to develop a pre-emptive multi-epitope pan-Coronavirus vaccine to protect against past, current, and potential future outbreaks.

immunology↗