Biology subjects

Robinson, C. M.

Publications and source records attributed to Robinson, C. M..

18 recordsLinked to original sources

bioRxiv2026

Evidence for lanthanide and PQQ dependent dehydrogenases in Eukarya

Lanthanides function as enzyme cofactors in bacteria, where they are widely distributed in pyrroloquinoline quinone-dependent 8-bladed beta-propeller dehydrogenases. No lanthanide-dependent enzymes, however, have been described outside prokaryotes. Here, we combined structural bioinformatics, phylogenetics, AlphaFold3 co-folding, coordination-sphere comparison, and quantum-mechanical cluster modeling to search for and rank putative lanthanide-coordinating 8-bladed beta-propeller enzymes in Eukarya. We identified candidate lanthanide-coordinating proteins in a diverse range of eukaryotes, predominantly plants and fungi, including species of clear industrial and agricultural relevance. A high-confidence subset matched validated bacterial Ln-binders based on both geometric similarity to canonical Ln-binding sites and on predicted Ln3+ versus Ca2+ selectivity. Our findings indicate that lanthanide biology likely extends beyond bacteria, with implications for plant, fungal, and broader eukaryotic metabolism, and warrant targeted biochemical investigation.

bioinformatics↗

bioRxiv2026

Diverse and novel lanthanide-binding PQQ-dependent enzymes

First described pyrroloquinoline quinone-dependent (PQQ) eight-bladed {beta}-propeller proteins are calcium (Ca)-dependent, but many homologous bacterial enzymes are lanthanide (Ln)-dependent. Discovery of Ca-dependent six-bladed {beta}-propeller PQQ-dependent dehydrogenases motivated the search for Ln-dependent six-bladed {beta}-propeller PQQ-dependent enzymes in bacteria. Using in silico structural prediction of sequences from weathered rock, we identified [~]22,000 PQQ six-bladed {beta}-propeller proteins in bacteria from 77 phyla, of which 63% of sequences have the active site residues needed to bind Ln. PQQ and La binding was biochemically confirmed for enzymes from uncultivated Chloroflexi and Acidobacteria. In structural models, Ln-binding periplasmic proteins interact with TonB-dependent transporters that may enable Ln uptake. Most genomes also encode predicted Ln-and PQQ-dependent eight-bladed dehydrogenases that clade with diverse alcohol and sugar dehydrogenases. Thus, Ln-dependent six and eight-bladed PQQ-dependent {beta}-propeller proteins are implicated in diverse carbon substrate metabolisms in weathering rock and soil. We predict that many PQQ-dependent microbial enzymes are lanthanide dependent.

microbiology↗

bioRxiv2024

Targeting GOF p53 and c-MYC through LZK Inhibition or Degradation Suppresses Head and Neck Tumor Growth

The worldwide frequency of head and neck squamous cell carcinoma (HNSCC) is approximately 800,000 new cases, with 430,000 deaths annually. We determined that LZK (encoded by MAP3K13) is a therapeutic target in HNSCC and showed that inhibition with small molecule inhibitors decreases the viability of HNSCC cells with amplified MAP3K13. A drug-resistant mutant of LZK blocks decreases in cell viability due to LZK inhibition, indicating on-target activity by two separate small molecules. Inhibition of LZK catalytic activity suppressed tumor growth in HNSCC PDX models with amplified MAP3K13. We found that the kinase activity of LZK stabilized c-MYC and that LZK stabilized gain-of-function (GOF) p53 through a kinase-independent mechanism. Therefore, we designed proteolysis-targeting chimeras (PROTACs) and demonstrate that our lead PROTAC promotes LZK degradation and suppresses expression of GOF p53 and c-MYC leading to impaired viability of HNSCC cell lines. This research provides a strong basis for development of therapeutics targeting LZK in HNSCCs with amplification of the gene. One Sentence SummaryThis study establishes the kinase LZK as a therapeutic target for HNSCC through regulation of c-MYC expression.

cancer biology↗

bioRxiv2024

A reconstruction of the mammalian secretory pathway identifies mechanisms regulating antibody production

The secretory pathway processes >30% of mammalian proteins, orchestrating their synthesis, modification, trafficking, and quality control. However, its complexity-- spanning multiple organelles and dependent on coordinated protein interactions--limits our ability to decipher how protein secretion is controlled in biomedical and biotechnological applications. To advance such research, we present secRecon--a comprehensive reconstruction of the mammalian secretory pathway, comprising 1,127 manually curated genes organized within an ontology of 77 secretory process terms, annotated with functional roles, subcellular localization, protein interactions, and complex composition. Using secRecon to integrate multi-omics data, we identified distinct secretory topologies in antibody-producing plasma cells compared to CHO cells. Genes within proteostasis, translocation, and N-glycosylation are deficient in CHO cells, highlighting them as potential engineering targets to boost secretion capacity. Applying secRecon to single-cell transcriptomics and SEC-seq data, we uncovered secretory pathway signatures underlying secretion diversity among IgG-secreting plasma cells. Different transcriptomic clusters had unique secretory phenotypes characterized by variations in the unfolded protein response (UPR), endoplasmic reticulum-associated degradation (ERAD), and vesicle trafficking pathways. Additionally, we discovered specific secretory machinery genes as new markers for plasma cell differentiation. These findings demonstrate secRecon can identify mechanisms regulating protein secretion and guide diverse studies in biomedical research and biotechnology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/623668v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@f9f865org.highwire.dtl.DTLVardef@122db7forg.highwire.dtl.DTLVardef@1788a1aorg.highwire.dtl.DTLVardef@b2b2bd_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2024

Convergent evolution of viral-like Borg archaeal extrachromosomal elements and giant eukaryotic viruses

Borgs are huge extrachromosomal elements of anaerobic methane-oxidizing archaea. They exist in exceedingly complex microbiomes, lack cultivated hosts and have few protein functional annotations, precluding their classification as plasmids, viruses or other. Here, we used in silico structure prediction methods to investigate potential roles for [~]10,000 Borg proteins. Prioritizing analysis of multicopy genes that could signal importance for Borg lifestyles, we uncovered highly represented de-ubiquitination-like Zn-metalloproteases that may counter host targeting of Borg proteins for proteolysis. Also prevalent are clusters of multicopy genes for production of diverse glycoconjugates that could contribute to decoration of the host cell surface, or of putative capsid proteins that we predict multimerize into hexagonal arrays. Features including megabase-scale linear genomes with inverted terminal repeats, genomic repertoires for energy metabolism, central carbon compound transformations and translation, and pervasive direct repeat regions are shared with giant viruses of eukaryotes, although analyses suggest that these parallels arose via convergent evolution. If Borgs are giant archaeal viruses they would fill the gap in the tri(um)virate of giant viruses of all three domains of life. One Sentence SummaryProtein analyses, informed by in silico protein structure prediction, revealed that Borgs share numerous features with giant eukaryotic viruses, suggesting that Borgs have a viral-like lifestyle and evolutionary convergence of large extrachromosomal elements across the Domains of Life.

microbiology↗

bioRxiv2024

Imaging of live bacterial whole-cell biosensors illuminates spatial sialic acid availability within the inflamed mammalian gut

Host mucin-derived sialic acids are key drivers of microbial colonisation, growth and pathogenicity within the mammalian gut. However, their study is hindered by complex spatiotemporal dynamics: Many gut metabolites, including sialic acids, are rapidly consumed, transformed or absorbed by the microbiota or host, meaning that conventional measurements of faeces or bulk digesta often fail to capture their true local availability. In contrast, engineered bacterial-whole cell biosensors provide an in situ read-out of metabolite exposure at the point and time of use, before these molecules are depleted. Here, we demonstrate increases in the bioavailability of sialic acid in the inflamed mouse gut using an engineered Escherichia coli biosensor that reports on sialic acid exposure via NanR-regulated transcriptional circuit. The biosensor robustly colonises the mouse gut and remains functional for at least six weeks. Through organ-scale imaging at single bacterial resolution we observe strong correlations between disease status and biosensor response in two inflammation models. Profiling along the length of the gut uncovers regional variations between the maximal sialic acid sensing and the peak inflammatory response within host tissues in a murine colitis model. Simultaneous tracking of host and microbial markers of inflammation informs the therapeutic response to sialidase inhibition, which accelerates disease recovery. Together, these data illuminate the complex spatial dynamics involved in shared host-microbiome metabolism and demonstrate the broader power of using engineered bacterial biosensors to monitor in situ bioavailability of rapidly turned-over metabolites within the gut.

synthetic biology↗

bioRxiv2023

A viral-specific CD4+ T cell response protects female mice from Coxsackievirus B3 infection

Biological sex plays an integral role in the immune response to various pathogens. The underlying basis for these sex differences is still not well defined. Here, we show that Coxsackievirus B3 (CVB3) induces a viral-specific CD4+ T cell response that can protect female mice from mortality. We found that CVB3 can induce expansion of CD62Llo CD4+ T cells in the mesenteric lymph node and spleen of female but not male mice as early as 5 days post-inoculation, indicative of activation. Using a recombinant CVB3 virus expressing a model CD4+ T cell epitope, we found that this response is due to viral antigen and not bystander activation. Finally, the depletion of CD4+ T cells before infection increased mortality in female mice, indicating that CD4+ T cells play a protective role against CVB3 in our model. Overall, these data demonstrated that CVB3 can induce an early CD4 response in female but not male mice and further emphasize how sex differences in immune responses to pathogens affect disease outcomes.

immunology↗

bioRxiv2023

A discovery platform to identify inducible synthetic circuitry from varied microbial sources

Gut microbes encode a variety of systems for molecular sensing and controlling conditional gene expression within the mammalian gut. Synthetic biology approaches such as whole-cell biosensing and sense-and-respond therapeutics aim to tap into this vast sensing repertoire to drive clinical and pre-clinical applications. An ongoing constraint is the limited number of well-characterized inducible circuit components to specifically sense in vivo conditions of interest, such as disease. Here, we extend the flexibility and power of a biosensor screening platform using bacterial memory circuits encoded in a gut commensal E. coli. We construct libraries driven by potential sensory components derived from a combination of E. coli promoters or bacterial two-component systems (TCSs) sourced from diverse gut bacteria. Each is tagged with unique DNA barcodes using a pooled construction method. Using our pipeline, we evaluate sensor activity and performance heterogeneity across in vitro and in vivo conditions including using a mouse inflammation model. We demonstrate the methods ability to identify biosensors of interest, including the identification of unannotated TCSs. Following the optimisation of library construction, analysis, and delivery to account for the challenges of working with engineered bacteria within a conventional mammalian gut microbiome, we identify and validate several further biosensors of interest responding to the murine gut environment, and specifically during inflammatory conditions. This approach can be applied to transcriptionally activated sensing elements of any type and will allow for rapid development of new biosensors that can advance synthetic biology approaches for complex environments.

synthetic biology↗

bioRxiv2023

Inferring secretory and metabolic pathway activity from omic data with secCellFie

Understanding protein secretion has considerable importance in the biotechnology industry and important implications in a broad range of normal and pathological conditions including development, immunology, and tissue function. While great progress has been made in studying individual proteins in the secretory pathway, measuring and quantifying mechanistic changes in the pathways activity remains challenging due to the complexity of the biomolecular systems involved. Systems biology has begun to address this issue with the development of algorithmic tools for analyzing biological pathways; however most of these tools remain accessible only to experts in systems biology with extensive computational experience. Here, we expand upon the user-friendly CellFie tool which quantifies metabolic activity from omic data to include secretory pathway functions, allowing any scientist to infer protein secretion capabilities from omic data. We demonstrate how the secretory expansion of CellFie (secCellFie) can be used to predict metabolic and secretory functions across diverse immune cells, hepatokine secretion in a cell model of NAFLD, and antibody production in Chinese Hamster Ovary cells.

systems biology↗

bioRxiv2022

Coxsackievirus B3 elicits a sex-specific CD8+ T cell response in female mice.

Sex is a significant contributor to the outcome of human infections. Males are frequently more susceptible to viral, bacterial, and fungal infections, which is often attributed to a weaker immune response. In contrast, a heightened immune response in females enables better pathogen elimination but leaves females more predisposed to autoimmune diseases. Unfortunately, the underlying basis for sex-specific immune responses remains poorly understood. Here, we show a sex-specific difference in the CD8+ T cell response to an enteric virus, Coxsackievirus B3 (CVB3). We found that CVB3 induced expansion of CD8+ T cells in female mice but not in male mice. CVB3 also increased the proportion and number of CD11ahiCD62Llo CD8+ T cells in female mice, indicative of activation. Further, this response was independent of the inoculation route and type I interferon. Using a recombinant CVB3 virus expressing a model CD8+ T cell epitope, we found that the expansion of CD8+ T cells is viral-specific and not due to bystander activation. These data demonstrate that CVB3 induces a sex-dependent CD8+ T cell response and highlight the importance of sex-specific immune responses to viral pathogens.

immunology↗

bioRxiv2021

Testosterone promotes Coxsackievirus B3 pathogenesis in an oral inoculation mouse model

Enteroviruses initiate infection in the gastrointestinal tract, and sex is often a biological variable that impacts pathogenesis. Previous data suggest that sex hormones can influence intestinal replication of Coxsackievirus B3 (CVB3), an enterovirus in the Picornaviridae family. However, the specific sex hormone(s) that regulate intestinal CVB3 replication is poorly understood. To determine if testosterone promotes intestinal CVB3 replication, we orally inoculated male and female Ifnar-/- mice that were treated with either placebo or testosterone-filled capsules. Following oral inoculation, we found that testosterone-treated male and female mice shed significantly more CVB3 in the feces than placebo-treated mice indicating that testosterone enhances intestinal replication. Similarly, testosterone enhanced viral dissemination in both sexes as we observed higher viral loads in peripheral tissues following infection. Further, male mice treated with testosterone also had a higher mortality rate than testosterone-depleted male mice. Finally, we observed that testosterone significantly affected the immune response to CVB3. We found that testosterone broadly increased pro-inflammatory cytokines and chemokines while decreasing the number of splenic B cells and dendritic cells following CVB3 infection. Moreover, while testosterone did not affect the early CD4 T cell response to CVB3, testosterone reduced the activation of CD8 T cells. These data indicate that testosterone can promote intestinal CVB3 replication and dissemination while impacting the subsequent viral immune response. ImportanceBiological sex plays a significant role in the outcome of various infections and diseases. The impact of sex hormones on intestinal replication and dissemination of Coxsackievirus B3 remains poorly understood. Using an oral inoculation model, we found that testosterone enhances CVB3 shedding and dissemination in male and female mice. Further, testosterone can alter the immune response to CVB3. This work highlights the role of testosterone in CVB3 pathogenesis and suggests that sex hormones can impact the replication and dissemination of enteric viruses.

microbiology↗

bioRxiv2021

Specific bacterial cell wall components influence the stability of Coxsackievirus B3

Enteric viruses infect the mammalian gastrointestinal tract and lead to significant morbidity and mortality worldwide. Data indicate that enteric viruses can utilize intestinal bacteria to promote viral replication and pathogenesis. However, the precise interactions between enteric viruses and bacteria are unknown. Here we examined the interaction between bacteria and Coxsackievirus B3, an enteric virus from the picornavirus family. We found that bacteria enhance the infectivity of Coxsackievirus B3 (CVB3) in vitro. Notably, specific bacteria are required as gram-negative Salmonella enterica, but not Escherichia coli, enhanced CVB3 infectivity and stability. Investigating the cell wall components of both S. enterica and E. coli revealed that structures in the O-antigen or core of lipopolysaccharide, a major component of the gram-negative bacterial cell wall, were required for S. enterica to enhance CVB3. To determine if these requirements were necessary for similar enteric viruses, we investigated if S. enterica and E. coli enhanced infectivity of poliovirus, another enteric virus in the picornavirus family. We found that, in contrast to CVB3, these bacteria enhanced the infectivity of poliovirus in vitro. Overall, these data indicate that distinct bacteria enhance CVB3 infectivity and stability, and specific enteric viruses may have differing requirements for their interactions with specific bacterial species. ImportancePrevious data indicate that several enteric viruses utilize bacteria to promote intestinal infection and viral stability. Here we show that specific bacteria and bacterial cell wall components are required to enhance infectivity and stability of Coxsackievirus B3 in vitro. These requirements are likely enteric virus-specific as the bacteria for CVB3 differs from poliovirus, a closely related virus. Therefore, these data indicate that specific bacteria and their cell wall components dictate the interaction with various enteric viruses in distinct mechanisms.

microbiology↗

bioRxiv2020

FlopR: An open source software package for calibration and normalization of plate reader and flow cytometry data

The measurement of gene expression using fluorescence markers has been a cornerstone of synthetic biology for the last two decades. However, the use of arbitrary units has limited the usefulness of this data for many quantitative purposes. Calibration of fluorescence measurements from flow cytometry and plate reader spectrophotometry has been implemented previously but the tools are disjointed. Here we pull together, and in some cases improve, extant methods into a single software tool, written as a package in the R statistical framework. The workflow is validated using Escherichia coli engineered to express GFP from a set of commonly used constitutive promoters. We then demonstrate its power by identifying the time evolution of distinct subpopulations of bacteria from bulk plate reader data, a task previously reliant on laborious flow cytometry experiments. Along with standardized parts and experimental methods, the development and dissemination of usable tools for quantitative measurement and data analysis will benefit the synthetic biology community by improving interoperability.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2020

In situ detection of protein interactions for recombinant therapeutic enzymes

Despite their therapeutic potential, many protein drugs remain inaccessible to patients since they are difficult to secrete. Each recombinant protein has unique physicochemical properties and requires different machinery for proper folding, assembly, and post-translational modifications (PTMs). Here we aimed to identify the machinery supporting recombinant protein secretion by measuring the protein-protein interaction (PPI) networks of four different recombinant proteins (SERPINA1, SERPINC1, SERPING1 and SeAP) with various PTMs and structural motifs using the proximity-dependent biotin identification (BioID) method. We identified PPIs associated with specific features of the secreted proteins using a Bayesian statistical model, and found proteins involved in protein folding, disulfide bond formation and N-glycosylation were positively correlated with the corresponding features of the four model proteins. Among others, oxidative folding enzymes showed the strongest association with disulfide bond formation, supporting their critical roles in proper folding and maintaining the ER stability. Knockdown of disulfide-isomerase PDIA4, a measured interactor with significance for SERPINC1 but not SERPINA1, led to the decreased secretion of SERPINC1, which relies on its extensive disulfide bonds, compared to SERPINA1, which has no disulfide bonds. Proximity-dependent labeling successfully identified the transient interactions supporting synthesis of secreted recombinant proteins and refined our understanding of key molecular mechanisms of the secretory pathway during recombinant protein production.

synthetic biology↗

bioRxiv2019

Neonatal granulocytic MDSCs possess phagocytic properties during bacterial infection.

Myeloid-derived suppressor cells (MDSCs) are an immunosuppressive cell type found in high abundance in early life. Currently, there has been limited mechanistic understanding of MDSC phagocytosis of bacteria and the corresponding consequences in the context of acute infection. We set out to determine whether human granulocytic MDSCs have phagocytic capacity that is comparable to other professional phagocytes. To investigate these properties, we utilized fluorescent confocal microscopy, flow cytometry, and bacterial burden assays. We demonstrate that human granulocytic MDSCs phagocytose E. coli O1:K1:H7, and subsequently traffic the bacteria into acidic compartments similar to other phagocytes. However, MDSCs were significantly less efficient at bacterial uptake and killing compared to monocytes. This activity is associated with an inflammatory response, but the amount of TNF gene and protein expression was reduced in infected MDSCs compared to monocytes. Interestingly, we also found that MDSCs release DNA (MeDNA) into the extracellular space that resembles neutrophil extracellular traps. We found that MeDNA had some impact on bacterial viability in single cultures, with an increase in bacterial recovery in MDSCs treated with DNAse. However, MeDNA did not impact the ability of monocytes to eliminate bacteria in co-cultures, suggesting that MDSC extracellular DNA does not compromise monocyte function. Overall, our data reveals mechanistic insight into MDSC activity during infection that includes the kinetics and efficiency of bacterial uptake, elimination through trafficking to acidified compartments, and inflammatory contributions relative to primary human monocytes. These results enhance our understanding of MDSC contributions during acute bacterial infection and identify host-directed targets for immune intervention to improve outcomes and reduce susceptibility to infection early in life.

immunology↗

bioRxiv2019

Elevated levels of interleukin-27 in early life compromise protective immunity during neonatal sepsis

Neonates are at increased risk for bacterial sepsis as a result of immature immunity. We established that the immune suppressive cytokine interleukin (IL)-27 is elevated in early life. In the present work, we hypothesized that increased levels of IL-27 may predispose the neonatal population to more severe infection during sepsis. In a neonatal sepsis model, systemic IL-27 levels continued to rise during infection. Peripheral tissue analysis revealed systemic IL-27 expression, while myeloid cell profiling identified Gr-1 and F4/80-expressing cells as the most abundant producers of IL-27 during infection. Increased IL-27 levels were consistent with increased mortality that was improved in WSX-1-/- mice that lack a functional IL-27 receptor. Infected WSX-1-/- pups exhibited improved weight gain and reduced morbidity. IL-27 signaling in WT mice promoted increased bacterial burdens and systemic inflammation compared to WSX-1-/- neonates. This was consistent with more efficient bacterial killing by Ly6B.2+ myeloid cells and macrophages from WSX-1-deficient compared to wild-type neonates. Live animal imaging further supported a more severe and disseminated infection in WT neonates. This is the first report to describe the impact of elevated early life IL-27 on the host response in neonates while also defining the cell and tissue sources of cytokine. IL-27 is frequently associated with suppressed inflammation. In contrast, our findings demonstrate that IL-27 promotes inflammation during neonatal sepsis by directly compromising control of bacteria that drive the inflammatory response. Collectively, our results suggest that IL-27 represents a therapeutic target to limit susceptibility and improve infectious outcomes in neonatal sepsis.\n\nIMPORTANCEA number of differences in the neonatal immune response compared with adults have been well described. However, a mechanistic understanding of what needs to be overcome in the neonate to generate a more protective immune response during acute bacterial infection has been limited. The work described here helps fill the gap of what is necessary to overcome in order to achieve improved host response to infection. To further the novelty, IL-27 has not previously been attributed to dysfunction or deficiency in neonatal immunity. Our results enhance the understanding of IL-27 biology in the neonatal population while providing evidence that elevated IL-27 levels limit a protective immune response and are detrimental during neonatal sepsis. Strategies aimed at targeting circulating IL-27 concentrations early in life have the potential to improve control of bacterial infection in neonates.

immunology↗

bioRxiv2019

Related enteric viruses have different requirements for host microbiota in mice

Accumulating evidence suggests that intestinal bacteria promote enteric virus infection in mice. For example, previous work demonstrated that antibiotic treatment of mice prior to oral infection with poliovirus reduced viral replication and pathogenesis. Here we examined the effect of antibiotic treatment on infection with coxsackievirus B3 (CVB3), a picornavirus closely related to poliovirus. We treated mice with a mixture of five antibiotics to deplete host microbiota and examined CVB3 replication and pathogenesis following oral inoculation. We found that, like poliovirus, CVB3 shedding and pathogenesis were reduced in antibiotic-treated mice. While treatment with just two antibiotics, vancomycin and ampicillin, was sufficient to reduce CVB3 replication and pathogenesis, this treatment had no effect on poliovirus. Quantity and composition of bacterial communities were altered by treatment with the five antibiotic cocktail and by treatment with vancomycin and ampicillin. To determine whether more subtle changes in bacterial populations impact viral replication, we examined viral infection in mice treated with milder antibiotic regimens. Mice treated with one-tenth the concentration of the normal antibiotic cocktail supported replication of poliovirus but not CVB3. Importantly, a single dose of one antibiotic, streptomycin, was sufficient to reduce CVB3 shedding and pathogenesis, while having no effect on poliovirus shedding and pathogenesis. Overall, replication and pathogenesis of CVB3 is more sensitive to antibiotic treatment than poliovirus, indicating that closely related viruses may differ in their reliance on microbiota.\n\nImportanceRecent data indicate that intestinal bacteria promote intestinal infection of several enteric viruses. Here we show that coxsackievirus, an enteric virus in the picornavirus family, also relies on microbiota for intestinal replication and pathogenesis. Relatively minor depletion of the microbiota was sufficient to decrease coxsackievirus infection, while poliovirus infection was unaffected. Surprisingly, a single dose of one antibiotic was sufficient to reduce coxsackievirus infection. Therefore, these data indicate that microbiota can influence enteric virus infection through distinct mechanisms, even for closely related viruses.

microbiology↗

bioRxiv2017

Plaques formed by mutagenized viral populations have elevated co-infection frequencies

The plaque assay is a common technique used to measure virus concentrations and is based upon the principle that each plaque represents a single infectious unit. As such, plaque number is expected to correlate linearly with the virus dilution plated and each plaque should be formed by a single founder virus. Here, we examined whether more than one virus can contribute to plaque formation. By using genetic and phenotypic assays with genetically marked polioviruses, we found that multiple parental viruses are present in 5-7% of plaques, even at an extremely low multiplicity of infection. We demonstrated through visual and biophysical assays that, like many viral stocks, our viral stocks contain both single particles and aggregates. These data suggest that aggregated virions are capable of inducing co-infection and chimeric plaque formation. In fact, inducing virion aggregation via exposure to low pH increased co-infection in a flow cytometry-based assay. We hypothesized that plaques generated by viruses with high mutation loads may have higher co-infection frequencies due to fitness restoring processes such as complementation and recombination. Indeed, we found that co-infection frequency correlated with mutation load, with 17% chimeric plaque formation for heavily mutagenized viruses. Importantly, the frequency of chimeric plaques may be underestimated by up to three-fold, since co-infection with the same parental virus cannot be scored in our assay. This work indicates that more than one virus can contribute to plaque formation and that co-infection may assist plaque formation in situations where the amount of genome damage is high.

microbiology↗