UnCoVar: a reproducible and scalable workflow for transparent and robust virus variant calling and lineage assignment using SARS-CoV-2 as an example.

BACKGROUND: At a global scale, the SARS-CoV-2 virus did not remain in its initial genotype for a long period of time, with the first global reports of variants of concern (VOCs) in late 2020. Subsequently, genome sequencing has become an indispensable tool for characterizing the ongoing pandemic, particularly for typing SARS-CoV-2 samples obtained from patients or environmental surveillance. For such SARS-CoV-2 typing, various in vitro and in silico workflows exist, yet to date, no systematic cross-platform validation has been reported. RESULTS: In this work, we present the first comprehensive cross-platform evaluation and validation of in silico SARS-CoV-2 typing workflows. The evaluation relies on a dataset of 54 patient-derived samples sequenced with several different in vitro approaches on all relevant state-of-the-art sequencing platforms. Moreover, we present UnCoVar, a robust, production-grade reproducible SARS-CoV-2 typing workflow that outperforms all other tested approaches in terms of precision and recall. CONCLUSIONS: In many ways, the SARS-CoV-2 pandemic has accelerated the development of techniques and analytical approaches. We believe that this can serve as a blueprint for dealing with future pandemics. Accordingly, UnCoVar is easily generalizable towards other viral pathogens and future pandemics. The fully automated workflow assembles virus genomes from patient samples, identifies existing lineages, and provides high-resolution insights into individual mutations. UnCoVar includes extensive quality control and automatically generates interactive visual reports. UnCoVar is implemented as a Snakemake workflow. The open-source code is available under a BSD 2-clause license at github.com/IKIM-Essen/uncovar.

Metabolic reconstruction of the genome of candidate Desulfatiglans TRIP_1 and identification of key candidate enzymes for anaerobic phenanthrene degradation.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed pollutants. As oxygen is rapidly depleted in water-saturated PAH-contaminated sites, anaerobic microorganisms are crucial for their consumption. Here, we report the metabolic pathway for anaerobic degradation of phenanthrene by a sulfate-reducing enrichment culture (TRIP) obtained from a natural asphalt lake. The dominant organism of this culture belongs to the Desulfobacteraceae family of Deltaproteobacteria and genome-resolved metagenomics led to the reconstruction of its genome along with a handful of genomes from lower abundance bacteria. Proteogenomic analyses confirmed metabolic capabilities for dissimilatory sulfate reduction and indicated the presence of the Embden-Meyerhof-Parnas pathway, a complete tricarboxylic acid cycle as well as a complete Wood-Ljungdahl pathway. Genes encoding enzymes putatively involved in the degradation of phenanthrene were identified. This includes two gene clusters encoding a multisubunit carboxylase complex likely involved in the activation of phenanthrene, as well as genes encoding reductases potentially involved in subsequent ring dearomatization and reduction steps. The predicted metabolic pathways were corroborated by transcriptome and proteome analyses, and provide the first insights into the metabolic pathway responsible for the anaerobic degradation of three-ringed PAHs.

Small-scale wastewater-based epidemiology (WBE) for infectious diseases and antibiotic resistance: A scoping review.

Wastewater analysis can serve as a source of public health information. In recent years, wastewater-based epidemiology (WBE) has emerged and proven useful for the detection of infectious diseases. However, insights from the wastewater treatment plant do not allow for the small-scale differentiation within the sewer system that is needed to analyze the target population under study in more detail. Small-scale WBE offers several advantages, but there has been no systematic overview of its application. The aim of this scoping review is to provide a comprehensive overview of the current state of knowledge on small-scale WBE for infectious diseases, including methodological considerations for its application. A systematic database search was conducted, considering only peer-reviewed articles. Data analyses included quantitative summary and qualitative narrative synthesis. Of 2130 articles, we included 278, most of which were published since 2020. The studies analyzed wastewater at the building level (n = 203), especially healthcare (n = 110) and educational facilities (n = 80), and at the neighborhood scale (n = 86). The main analytical parameters were viruses (n = 178), notably SARS-CoV-2 (n = 161), and antibiotic resistance (ABR) biomarkers (n = 99), often analyzed by polymerase chain reaction (PCR), with DNA sequencing techniques being less common. In terms of sampling techniques, active sampling dominated. The frequent lack of detailed information on the specification of selection criteria and the characterization of the small-scale sampling sites was identified as a concern. In conclusion, based on the large number of studies, we identified several methodological considerations and overarching strategic aspects for small-scale WBE. An enabling environment for small-scale WBE requires inter- and transdisciplinary knowledge sharing across countries. Promoting the adoption of small-scale WBE will benefit from a common international conceptualization of the approach, including standardized and internationally accepted terminology. In particular, the development of good WBE practices for different aspects of small-scale WBE is warranted. This includes the establishment of guidelines for a comprehensive characterization of the local sewer system and its sub-sewersheds, and transparent reporting to ensure comparability of small-scale WBE results.

Analyzing community wastewater in sub-sewersheds for the small-scale detection of SARS-CoV-2 variants in a German metropolitan area.

Wastewater surveillance of SARS-CoV-2 proved useful, including for identifying the local appearance of newly identified virus variants. Previous studies focused on wastewater treatment plants (WWTP) with sewersheds of several hundred thousand people or at single building level, representing only a small number of people. Both approaches may prove inadequate for small-scale intra-urban inferences for early detection of emerging or novel virus variants. Our study aims (i) to analyze SARS-CoV-2 single nucleotide variants (SNVs) in wastewater of sub-sewersheds and WWTP using whole genome sequencing in order to (ii) investigate the potential of small-scale detection of novel known SARS-CoV-2 variants of concern (VOC) within a metropolitan wastewater system. We selected three sub-sewershed sampling sites, based on estimated population- and built environment-related indicators, and the inlet of the receiving WWTP in the Ruhr region, Germany. Untreated wastewater was sampled weekly between October and December 2021, with a total of 22 samples collected. SARS-CoV-2 RNA was analyzed by RT-qPCR and whole genome sequencing. For all samples, genome sequences were obtained, while only 13 samples were positive for RT-qPCR. We identified multiple specific SARS-CoV-2 SNVs in the wastewater samples of the sub-sewersheds and the WWTP. Identified SNVs reflected the dominance of VOC Delta at the time of sampling. Interestingly, we could identify an Omicron-specific SNV in one sub-sewershed. A concurrent wastewater study sampling the same WWTP detected the VOC Omicron one week later. Our observations suggest that the small-scale approach may prove particularly useful for the detection and description of spatially confined emerging or existing virus variants circulating in populations. Future studies applying small-scale sampling strategies taking into account the specific features of the wastewater system will be useful to analyze temporal and spatial variance in more detail.

Immune responses in COVID-19 patients during breakthrough infection with SARS-CoV-2 variants Delta, Omicron-BA.1 and Omicron-BA.5.

Background: Breakthrough infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are increasingly observed in vaccinated individuals. Immune responses towards SARS-CoV-2 variants, particularly Omicron-BA.5, are poorly understood. We investigated the humoral and cellular immune responses of hospitalized COVID-19 patients during Delta and Omicron infection waves. Methods: The corresponding SARS-CoV-2 variant of the respective patients were identified by whole genome sequencing. Humoral immune responses were analyzed by ELISA and a cell culture-based neutralization assay against SARS-CoV-2 D614G isolate (wildtype), Alpha, Delta (AY.43) and Omicron (BA.1 and BA.5). Cellular immunity was evaluated with an IFN-γ ELISpot assay. Results: On a cellular level, patients showed a minor IFN-γ response after stimulating PBMCs with mutated regions of SARS-CoV-2 variants. Neutralizing antibody titers against Omicron-BA.1 and especially BA.5 were strongly reduced. Double-vaccinated patients with Delta breakthrough infection showed a significantly increased neutralizing antibody response against Delta compared to double-vaccinated uninfected controls (median complete neutralization titer (NT100) 640 versus 80, p<0.05). Omicron-BA.1 infection increased neutralization titers against BA.1 in double-vaccinated patients (median NT100 of 160 in patients versus 20 in controls, p=0.07) and patients that received booster vaccination (median NT100 of 50 in patients versus 20 in controls, p=0.68). For boosted patients with BA.5 breakthrough infection, we found no enhancing effect on humoral immunity against SARS-CoV-2 variants. Conclusion: Neutralizing antibody titers against Omicron-BA.1 and especially BA.5 were strongly reduced in SARS-CoV-2 breakthrough infections. Delta and Omicron-BA.1 but not Omicron-BA.5 infections boosted the humoral immunity in double-vaccinated patients and patients with booster vaccination. Despite BA.5 breakthrough infection, those patients may still be vulnerable for reinfections with BA.5 or other newly emerging variants of concern.

Bacterial Photosensory Proteins and Their Role in Plant-pathogen Interactions.

Light is an important environmental signal for almost all living organisms. The light perception is achieved by photoreceptor proteins. As can be observed from the great number of bacterial genomes sequenced, plant pathogenic bacteria encode for a large number of photoreceptor proteins. The physiological implications of these photoreceptors are still poorly characterized. However, recent studies revealed the participation of these photosensory proteins in the pathogenic process. Here, we summarize what is known about these proteins and their role during the virulence process, concluding that the light environment modulates the plant-pathogen interaction.

Functional Green-Tuned Proteorhodopsin from Modern Stromatolites.

The sequenced genome of the poly-extremophile Exiguobacterium sp. S17, isolated from modern stromatolites at Laguna Socompa (3,570 m), a High-Altitude Andean Lake (HAAL) in Argentinean Puna revealed a putative proteorhodopsin-encoding gene. The HAAL area is exposed to the highest UV irradiation on Earth, making the microbial community living in the stromatolites test cases for survival strategies under extreme conditions. The heterologous expressed protein E17R from Exiguobacterium (248 amino acids, 85% sequence identity to its ortholog ESR from E. sibiricum) was assembled with retinal displaying an absorbance maximum at 524 nm, which makes it a member of the green-absorbing PR-subfamily. Titration down to low pH values (eventually causing partial protein denaturation) indicated a pK value between two and three. Global fitting of data from laser flash-induced absorption changes gave evidence for an early red-shifted intermediate (its formation being below the experimental resolution) that decayed (τ1 = 3.5 µs) into another red-shifted intermediate. This species decayed in a two-step process (τ2 = 84 µs, τ3 = 11 ms), to which the initial state of E17-PR was reformed with a kinetics of 2 ms. Proton transport capability of the HAAL protein was determined by BLM measurements. Additional blue light irradiation reduced the proton current, clearly identifying a blue light absorbing, M-like intermediate. The apparent absence of this intermediate is explained by closely matching formation and decay kinetics.

KatG, the Bifunctional Catalase of Xanthomonas citri subsp. citri, Responds to Hydrogen Peroxide and Contributes to Epiphytic Survival on Citrus Leaves.

Xanthomonas citri subsp. citri (Xcc) is the bacterium responsible for citrus canker. This bacterium is exposed to reactive oxygen species (ROS) at different points during its life cycle, including those normally produced by aerobic respiration or upon exposition to ultraviolet (UV) radiation. Moreover, ROS are key components of the host immune response. Among enzymatic ROS-detoxifying mechanisms, catalases eliminate H2O2, avoiding the potential damage caused by this specie. Xcc genome includes four catalase genes. In this work, we studied the physiological role of KatG, the only bifunctional catalase of Xcc, through the construction and characterization of a modified strain (XcckatG), carrying an insertional mutation in the katG gene. First, we evaluated the involvement of KatG in the bacterial adaptive response to H2O2. XcckatG cultures exhibited lower catalase activity than those of the wild-type strain, and this activity was not induced upon treatment with sub-lethal doses of H2O2. Moreover, the KatG-deficient mutant exhibited decreased tolerance to H2O2 toxicity compared to wild-type cells and accumulated high intracellular levels of peroxides upon exposure to sub-lethal concentrations of H2O2. To further study the role of KatG in Xcc physiology, we evaluated bacterial survival upon exposure to UV-A or UV-B radiation. In both conditions, XcckatG showed a high mortality in comparison to Xcc wild-type. Finally, we studied the development of bacterial biofilms. While structured biofilms were observed for the Xcc wild-type, the development of these structures was impaired for XcckatG. Based on these results, we demonstrated that KatG is responsible for Xcc adaptive response to H2O2 and a key component of the bacterial response to oxidative stress. Moreover, this enzyme plays an important role during Xcc epiphytic survival, being essential for biofilm formation and UV resistance.

Functional Characterization of a LOV-Histidine Kinase Photoreceptor from Xanthomonas citri subsp. citri.

The blue-light (BL) absorbing protein Xcc-LOV from Xanthomonas citri subsp. citri is composed of a LOV-domain, a histidine kinase (HK) and a response regulator. Spectroscopic characterization of Xcc-LOV identified intermediates and kinetics of the protein's photocycle. Measurements of steady state and time-resolved fluorescence allowed determination of quantum yields for triplet (ΦT  = 0.68 ± 0.03) and photoproduct formation (Φ390  = 0.46 ± 0.05). The lifetime for triplet decay was determined as τT  = 2.4-2.8 µs. Fluorescence of tryptophan and tyrosine residues was unchanged upon light-to-dark conversion, emphasizing the absence of significant conformational changes. Photochemistry was blocked upon cysteine C76 (C76S) mutation, causing a seven-fold longer lifetime of the triplet state (τT  = 16-18.5 µs). Optoacoustic spectroscopy yielded the energy content of the triplet state. Interestingly, Xcc-LOV did not undergo the volume contraction reported for other LOV domains within the observation time window, although the back-conversion into the dark state was accompanied by a volume expansion. A radioactivity-based enzyme function assay revealed a larger HK activity in the lit than in the dark state. The C76S mutant showed a still lower enzyme function, indicating the dark state activity being corrupted by a remaining portion of the long-lived lit state.

The LOV protein of Xanthomonas citri subsp. citri plays a significant role in the counteraction of plant immune responses during citrus canker.

Pathogens interaction with a host plant starts a set of immune responses that result in complex changes in gene expression and plant physiology. Light is an important modulator of plant defense response and recent studies have evidenced the novel influence of this environmental stimulus in the virulence of several bacterial pathogens. Xanthomonas citri subsp. citri is the bacterium responsible for citrus canker disease, which affects most citrus cultivars. The ability of this bacterium to colonize host plants is influenced by bacterial blue-light sensing through a LOV-domain protein and disease symptoms are considerably altered upon deletion of this protein. In this work we aimed to unravel the role of this photoreceptor during the bacterial counteraction of plant immune responses leading to citrus canker development. We performed a transcriptomic analysis in Citrus sinensis leaves inoculated with the wild type X. citri subsp. citri and with a mutant strain lacking the LOV protein by a cDNA microarray and evaluated the differentially regulated genes corresponding to specific biological processes. A down-regulation of photosynthesis-related genes (together with a corresponding decrease in photosynthesis rates) was observed upon bacterial infection, this effect being more pronounced in plants infected with the lov-mutant bacterial strain. Infection with this strain was also accompanied with the up-regulation of several secondary metabolism- and defense response-related genes. Moreover, we found that relevant plant physiological alterations triggered by pathogen attack such as cell wall fortification and tissue disruption were amplified during the lov-mutant strain infection. These results suggest the participation of the LOV-domain protein from X. citri subsp. citri in the bacterial counteraction of host plant defense response, contributing in this way to disease development.

A LOV protein modulates the physiological attributes of Xanthomonas axonopodis pv. citri relevant for host plant colonization.

Recent studies have demonstrated that an appropriate light environment is required for the establishment of efficient vegetal resistance responses in several plant-pathogen interactions. The photoreceptors implicated in such responses are mainly those belonging to the phytochrome family. Data obtained from bacterial genome sequences revealed the presence of photosensory proteins of the BLUF (Blue Light sensing Using FAD), LOV (Light, Oxygen, Voltage) and phytochrome families with no known functions. Xanthomonas axonopodis pv. citri is a Gram-negative bacterium responsible for citrus canker. The in silico analysis of the X. axonopodis pv. citri genome sequence revealed the presence of a gene encoding a putative LOV photoreceptor, in addition to two genes encoding BLUF proteins. This suggests that blue light sensing could play a role in X. axonopodis pv. citri physiology. We obtained the recombinant Xac-LOV protein by expression in Escherichia coli and performed a spectroscopic analysis of the purified protein, which demonstrated that it has a canonical LOV photochemistry. We also constructed a mutant strain of X. axonopodis pv. citri lacking the LOV protein and found that the loss of this protein altered bacterial motility, exopolysaccharide production and biofilm formation. Moreover, we observed that the adhesion of the mutant strain to abiotic and biotic surfaces was significantly diminished compared to the wild-type. Finally, inoculation of orange (Citrus sinensis) leaves with the mutant strain of X. axonopodis pv. citri resulted in marked differences in the development of symptoms in plant tissues relative to the wild-type, suggesting a role for the Xac-LOV protein in the pathogenic process. Altogether, these results suggest the novel involvement of a photosensory system in the regulation of physiological attributes of a phytopathogenic bacterium. A functional blue light receptor in Xanthomonas spp. has been described for the first time, showing an important role in virulence during citrus canker disease.