Search

Bacteriophages such as γ and AP50c have been shown to infect strains of Bacillus anthracis with high specificity, and this feature has been exploited in the development of bacterial detection assays. To better understand the emergence of phage resistance, and thus the potential failure of such assays, it is important to identify the host and phage receptors necessary for attachment and entry. Using genetic approaches, the bacterial receptors of AP50c and γ have been identified as sap and GamR, respectively. A second AP50c-like phage, Wip1, also appears to use sap as a receptor. In parallel with this work, the cognate phage-encoded receptor binding proteins (RBPs) have also been identified (Gp14 for γ, P28 for AP50c, and P23 for Wip1); however, the strength of evidence supporting these protein–protein interactions varies, necessitating additional investigation. Here, we present genetic evidence further supporting the interaction between sap and the RBPs of AP50c and Wip1 using fluorescently tagged proteins and a panel of B. anthracis mutants. These results showed that the deletion of the sap gene, as well as the deletion of csaB, whose encoded protein anchors sap to the bacterial S-layer, resulted in the loss of RBP binding. Binding could then be rescued by expressing these genes in trans. We also found that the RBP of the γ-like prophage λBa03 relied on csaB activity for binding, possibly by a different mechanism. RBPλBa03 binding to B. anthracis cells was also unique in that it was not ablated by heat inactivation of vegetative cells, suggesting that its receptor is still functional following incubation at 98°C. These results extend our understanding of the diverse attachment and entry strategies used by B. anthracis phages, enabling future assay development. [ABSTRACT FROM AUTHOR]

Simple Summary: The deployment of working dogs to environments containing potentially pathogenic biological agents necessitates a straightforward and fieldable decontamination protocol. This work describes the effectiveness of a wipe-based method using common veterinary cleaners on various surfaces contaminated with infectious virus. Surface characteristics, especially porosity, have a marked effect on the success of any decontamination protocol; however, in general, wiping with 70% isopropyl alcohol or 0.5% chlorhexidine proved to be most efficient at decontaminating surfaces common to the working dog community. Given the increased deployment of working dogs to settings with pathogenic biological agents, a safe, effective, and logistically feasible surface decontamination protocol is essential to protect both the animals and their human handlers. Our group previously found that superficial contamination on surfaces relevant to the working dog community, including leashes and toys, could be significantly reduced using a standardized wiping protocol with various cleansing products. To expand upon this work, we analyzed the ability of this protocol to decontaminate surface-deposited bovine coronavirus, which was used as a BSL2 surrogate for SARS-CoV-2. Unsurprisingly, the physical characteristics of a given surface, including porosity and texture, had a significant effect on the ability to recover viable virus remaining on the surface post treatment. After correcting for these differences, however, wiping with 70% isopropyl alcohol (IPA) and 0.5% chlorhexidine performed best, reducing viral titers by >3 log on plastic bumper toys and nylon collars, and by >2 log on rubber toys and tennis balls. Leather leashes and Velcro proved more difficult to decontaminate, but both still showed significant loss of viral contamination following wiping with IPA or chlorhexidine. This work (i) validates the utility of a simple protocol for the neutralization of viruses on several surfaces, (ii) identifies materials that are more difficult to decontaminate, which should, thus, be considered for removal from field use, and (iii) highlights the need for further development of protocols testing porous or textured surfaces. [ABSTRACT FROM AUTHOR]

Odor detection canines are a valuable resource used by multiple agencies for the sensitive detection of explosives, narcotics, firearms, agricultural products, and even human bodies. These canines and their handlers are frequently deployed to pathogen-contaminated environments or to work in close proximity with potentially sick individuals. Appropriate decontamination protocols must be established to mitigate both canine and handler exposure in these scenarios. Despite this potential risk, extremely limited guidance is available on routine canine decontamination from pathogenic biological materials. In this article, we evaluate the ability of several commercial off-the-shelf cleansing products, used in wipe form, to remove superficial contamination from fur, canine equipment, and toys. Using Glo Germ MIST as a proxy for biological contamination, our analysis demonstrated more than a 90% average reduction in contamination after wiping with a Nolvasan scrub solution, 0.5% chlorhexidine solution, or 70% isopropyl alcohol. Wiping with nondisinfectant baby wipes or water yielded an almost 80% average removal of contaminant from all surfaces. Additionally, researchers used Gwet's AC2 measurement to assess interrater reliability, which demonstrated substantial agreement (P < .001). These data provide key insights toward the development of a rapid, convenient, and fieldable alternative to traditional water-intensive bathing of working canines. [ABSTRACT FROM AUTHOR]

Background: Examination of complex biological systems has long been achieved through methodical investigation of the system's individual components. While informative, this strategy often leads to inappropriate conclusions about the system as a whole. With the advent of high-throughput "omic" technologies, however, researchers can now simultaneously analyze an entire system at the level of molecule (DNA, RNA, protein, metabolite) and process (transcription, translation, enzyme catalysis). This strategy reduces the likelihood of improper conclusions, provides a framework for elucidation of genotype-phenotype relationships, and brings finer resolution to comparative genomic experiments. Here, we apply a multi-omic approach to analyze the gene expression profiles of two closely related Pseudomonas aeruginosa strains grown in n-alkanes or glycerol. Results: The environmental P. aeruginosa isolate ATCC 33988 consumed medium-length (C10-C16) n-alkanes more rapidly than the laboratory strain PAO1, despite high genome sequence identity (average nucleotide identity >99%). Our data shows that ATCC 33988 induces a characteristic set of genes at the transcriptional, translational and post-translational levels during growth on alkanes, many of which differ from those expressed by PAO1. Of particular interest was the lack of expression from the rhl operon of the quorum sensing (QS) system, resulting in no measurable rhamnolipid production by ATCC 33988. Further examination showed that ATCC 33988 lacked the entire lasI/lasR arm of the QS response. Instead of promoting expression of QS genes, ATCC 33988 up-regulates a small subset of its genome, including operons responsible for specific alkaline proteases and sphingosine metabolism. Conclusion: This work represents the first time results from RNA-seq, microarray, ribosome footprinting, proteomics, and small molecule LC-MS experiments have been integrated to compare gene expression in bacteria. Together, these data provide insights as to why strain ATCC 33988 is better adapted for growth and survival on n-alkanes. [ABSTRACT FROM AUTHOR]

Viruses rely on the metabolic network of the host cell to provide energy and macromolecular precursors to fuel viral replication. Here we used mass spectrometry to examine the impact of two related herpesviruses, human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1), on the metabolism of fibroblast and epithelial host cells. Each virus triggered strong metabolic changes that were conserved across different host cell types. The metabolic effects of the two viruses were, however, largely distinct. HCMV but not HSV-1 increased glycolytic flux. HCMV profoundly increased TCA compound levels and flow of two carbon units required for TCA cycle turning and fatty acid synthesis. HSV-1 increased anapleurotic influx to the TCA cycle through pyruvate carboxylase, feeding pyrimidine biosynthesis. Thus, these two related herpesviruses drive diverse host cells to execute distinct, virus-specific metabolic programs. Current drugs target nucleotide metabolism for treatment of both viruses. Although our results confirm that this is a robust target for HSV-1, therapeutic interventions at other points in metabolism might prove more effective for treatment of HCMV. [ABSTRACT FROM AUTHOR]

The 2014–2016 Zika virus (ZIKV) epidemic in the Americas resulted in large deposits of next-generation sequencing data from clinical samples. This resource was mined to identify emerging mutations and trends in mutations as the outbreak progressed over time. Information on transmission dynamics, prevalence, and persistence of intra-host mutants, and the position of a mutation on a protein were then used to prioritize 544 reported mutations based on their ability to impact ZIKV phenotype. Using this criteria, six mutants (representing naturally occurring mutations) were generated as synthetic infectious clones using a 2015 Puerto Rican epidemic strain PRVABC59 as the parental backbone. The phenotypes of these naturally occurring variants were examined using both cell culture and murine model systems. Mutants had distinct phenotypes, including changes in replication rate, embryo death, and decreased head size. In particular, a NS2B mutant previously detected during in vivo studies in rhesus macaques was found to cause lethal infections in adult mice, abortions in pregnant females, and increased viral genome copies in both brain tissue and blood of female mice. Additionally, mutants with changes in the region of NS3 that interfaces with NS5 during replication displayed reduced replication in the blood of adult mice. This analytical pathway, integrating both bioinformatic and wet lab experiments, provides a foundation for understanding how naturally occurring single mutations affect disease outcome and can be used to predict the of severity of future ZIKV outbreaks. To determine if naturally occurring individual mutations in the Zika virus epidemic genotype affect viral virulence or replication rate in vitro or in vivo, we generated an infectious clone representing the epidemic genotype of stain Puerto Rico, 2015. Using this clone, six mutants were created by changing nucleotides in the genome to cause one to two amino acid substitutions in the encoded proteins. The six mutants we generated represent mutations that differentiated the early epidemic genotype from genotypes that were either ancestral or that occurred later in the epidemic. We assayed each mutant for changes in growth rate, and for virulence in adult mice and pregnant mice. Three of the mutants caused catastrophic embryo effects including increased embryonic death or significant decrease in head diameter. Three other mutants that had mutations in a genome region associated with replication resulted in changes in in vitro and in vivo replication rates. These results illustrate the potential impact of individual mutations in viral phenotype. [ABSTRACT FROM AUTHOR]

Herpes simplex virus 1 infection triggers multiple changes in the metabolism of host cells, including a dramatic decrease in the levels of NAD+. In addition to its role as a cofactor in reduction-oxidation reactions, NAD+ is required for certain posttranslational modifications. Members of the poly(ADP-ribose) polymerase (PARP) family of enzymes are major consumers of NAD+, which they utilize to form poly(ADP-ribose) (PAR) chains on protein substrates in response to DNA damage. PAR chains can subsequently be removed by the enzyme poly(ADP-ribose) glycohydrolase (PARG). We report here that the HSV-1 infection-induced drop in NAD+ levels required viral DNA replication, was associated with an increase in protein poly(ADP-ribosyl)ation (PARylation), and was blocked by pharmacological inhibition of PARP-1/PARP-2 (PARP-1/2). Neither virus yield nor the cellular metabolic reprogramming observed during HSV-1 infection was altered by the rescue or further depletion of NAD+ levels. Expression of the viral protein ICP0, which possesses E3 ubiquitin ligase activity, was both necessary and sufficient for the degradation of the 111-kDa PARG isoform. This work demonstrates that HSV-1 infection results in changes to NAD+ metabolism by PARP-1/2 and PARG, and as PAR chain accumulation can induce caspase-independent apoptosis, we speculate that the decrease in PARG levels enhances the auto-PARylation-mediated inhibition of PARP, thereby avoiding premature death of the infected cell. [ABSTRACT FROM AUTHOR]