Biology subjects

Russell, D. A.

Publications and source records attributed to Russell, D. A..

6 recordsLinked to original sources

bioRxiv2023

Mycobacterium trehalose polyphleates are required for mycobacteriophage infection

Mycobacteriophages are good model systems for understanding their bacterial hosts and show promise as therapeutic agents for nontuberculous mycobacterium infections. However, little is known about phage recognition of Mycobacterium cell surfaces, or mechanisms of phage resistance. We show here that surface-exposed trehalose polyphleates (TPPs) are required for infection of Mycobacterium abscessus and Mycobacterium smegmatis by clinically useful phages BPs and Muddy, and that TPP loss leads to defects in adsorption, infection, and confers resistance. Transposon mutagenesis indicates that TPP loss is the primary mechanism for phage resistance. Spontaneous phage resistance occurs through TPP loss, and some M. abscessus clinical isolates are phage-insensitive due to TPP absence. Both BPs and Muddy become TPP-independent through single amino acid substitutions in their tail spike proteins, and M. abscessus mutants resistant to TPP-independent phages reveal additional resistance mechanisms. Clinical use of BPs and Muddy TPP-independent mutants should preempt phage resistance caused by TPP loss.

microbiology↗

bioRxiv2022

Internal Validation of the ForenSeq Kintelligence Kit for Application to Forensic Genetic Genealogy

Forensic Genetic Genealogy (FGG) requires high density single nucleotide polymorphism (SNP) profiles to infer distant relationships. The ForenSeq Kintelligence kit is a recently developed method targeting approximately 10,000 SNPs that were selected to be compatible with genetic genealogy databases, while avoiding medically relevant SNPs. The targeted PCR method is ideal for low input and degraded samples, which are particularly challenging for microarray analysis. We made modifications to library preparation and sample pooling to further enhance the performance of low input samples. To align this FGG method with forensic laboratory standards, we performed an internal validation study in accordance with the Scientific Working Group on DNA Analysis Methods Validation Guidelines for DNA Analysis Methods. The sensitivity and precision and accuracy studies produced accurate and reliable profiles down to 0.05 ng of DNA. The mixture and contamination studies demonstrated that the method could detect the presence of a minor contributor down to 0.02 ng, while system wide contamination was negligible. Nonprobative bone, fired shell casing, and adhesive tape samples performed well with inputs ranging from 0.05 to 1.0 ng of DNA. A bone with a high degradation index and tested at 0.1 ng DNA input resulted in a call rate of 92.9% and a heterozygosity rate of 0.387. We demonstrated the importance of both call rate and heterozygosity rate to assess profile performance. Validating FGG methods is critical to ensuring the reliability and utility of SNP profiles that will be uploaded to genetic genealogy databases for the purpose of generating investigative leads.

genomics↗

bioRxiv2022

Developmental Validation of the Illumina Infinium Assay using the Global Screening Array (GSA) on the iScan System for use in Forensic Laboratories

Microarray processing, which interrogates hundreds of thousands of single nucleotide polymorphisms (SNPs) across the human genome, has recently gained traction in forensics due to its use in forensic genetic genealogy, which is based on analysis using SNPs to compare distant relatives in publicly curated databases for the purposes of developing investigative leads or identifying human remains. To date, there has been no published developmental validation of microarray processing using the Scientific Working Group on DNA Analysis Methods (SWGDAM) Validation Guidelines for DNA Analysis Methods and Federal Bureau of Investigation Quality Assurance Standards. Validation of these methods are warranted to identify samples suitable for microarray analysis and to assess the quality of the data obtained prior to upload to genealogical databases. In this study, we validated the Global Screening Array (GSA) for use in forensic investigations according to SWGDAM guidelines, including the following studies: precision and accuracy, sensitivity, contamination, degradation, species specificity, mock case-type samples, mixtures, repeatability and reproducibility, and stability. Results indicated accurate genotype calls with SNP call rates >95% at DNA input as low as 0.20 ng. In addition to SNP call rate, we developed interpretation thresholds for signal intensity and heterozygosity to allow for sample quality assessment and identification of highly degraded and/or non-human DNA. This study demonstrates that high quality data can be generated from multiple sample types, including mock forensic evidence that simulated the challenges that are often encountered in forensic cold cases.

genomics↗

bioRxiv2022

METTL7A and METTL7B are responsible for S-thiol methyl transferase activity in liver

S-Methylation of drugs containing thiol-moieties often alters their activity and results in detoxification. Historically, scientists attributed methylation of exogenous aliphatic and phenolic thiols to a putative S-adenosyl-L-methionine dependent membrane-associated phase II enzyme known as thiol methyltransferase (TMT). TMT has a broad substrate specificity and methylates the thiol metabolite of spironolactone, mertansine, ziprasidone, captopril, and the active metabolites of the thienopyridine pro-drugs, clopidogrel, and prasugrel. Despite TMTs role in the S-methylation of clinically relevant drugs, the enzyme(s) responsible for this activity remained unknown. We recently identified methyltransferase-like protein 7B (METTL7B) as an alkyl thiol-methyltransferase. METTL7B is an endoplasmic-reticulum-associated protein with similar biochemical properties and substrate specificity to TMT. Yet, the historic TMT inhibitor, 2,3-dichloro--methylbenzylamine (DCMB), has no effect on the activity of METTL7B, indicating that multiple enzymes contribute to TMT activity. Here we report that methyltransferase-like protein 7A (METTL7A), an uncharacterized member of the METTL7 family, also acts as a thiolmethyltransferase. METTL7A exhibits similar biochemical properties to TMT, including inhibition by DCMB (IC50 1.2 {micro}M). Applying quantitative proteomics to human liver microsomes and gene modulation experiments in HepG2 and HeLa cells, we determined that TMT activity correlates closely with METTL7A and METTL7B protein levels. Furthermore, purification of a novel His-GST-tagged recombinant protein and subsequent activity experiments prove that METTL7A can selectively methylate exogenous thiol-containing substrates, including 7-thiospironolactone, dithiothreitol, 4-chlorothiophenol, and mertansine. We conclude that the METTL7 family encodes for two enzymes, METTL7A and METTL7B, which we have renamed TMT1A1 and TMT1B1, respectively, that are responsible for TMT activity in liver microsomes. Significance StatementWe identified METTL7A (TMT1A1) and METTL7B (TMT1B1) as the enzymes responsible for the microsomal alkyl thiol methyltransferase activity. These are the first two enzymes directly associated with microsomal TMT activity. S-Methylation of many commonly prescribed thiol-containing drugs alters their pharmacological activity and/or toxicity, and identifying the enzymes responsible, will improve our understanding of the DMPK properties of alkyl- or phenolic-thiol-containing therapeutics. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/511968v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@2419f9org.highwire.dtl.DTLVardef@254c5borg.highwire.dtl.DTLVardef@b81378org.highwire.dtl.DTLVardef@1064c79_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Genomic diversity of bacteriophages infecting Rhodobacter capsulatus and their relatedness to its gene transfer agent RcGTA

The diversity of bacteriophages is likely unparalleled in the biome due to the immense variety of hosts and the multitude of viruses that infect them. Recent efforts have led to description at the genomic level of numerous bacteriophages that infect the Actinobacteria, but relatively little is known about those infecting other prokaryotic phyla, such as the purple non-sulfur photosynthetic -proteobacterium Rhodobacter capsulatus. This species is a common inhabitant of freshwater ecosystems and has been an important model system for the study of photosynthesis. Additionally, it is notable for its utilization of a unique form of horizontal gene transfer via a bacteriophage-like element known as the gene transfer agent (RcGTA). Only three bacteriophages of R. capsulatus had been sequenced prior to this report. Isolation and characterization at the genomic level of 26 new bacteriophages infecting this host advances the understanding of bacteriophage diversity and the origins of RcGTA. These newly discovered isolates can be grouped along with three that were previously sequenced to form six clusters with four remaining as single representatives. These bacteriophages share genes with RcGTA that seem to be related to host recognition. One isolate was found to cause lysis of a marine bacterium when exposed to high titer lysate. Although some clusters are more highly represented in the sequenced genomes, it is evident that many more bacteriophage types that infect R. capsulatus are likely to be found in the future.

microbiology↗

bioRxiv2020

Bacteroides thetaiotaomicron-infecting bacteriophage isolates inform sequence-based host range prediction

Our emerging view of the gut microbiome largely focuses on bacteria and less is known about other microbial components such as of bacteriophages (phages). Though phages are abundant in the gut, very few phages have been isolated from this ecosystem. Here, we report the genomes of 27 phages from the United States and Bangladesh that infect the prevalent human gut bacterium Bacteroides thetaiotaomicron. These phages are mostly distinct from previously sequenced phages with the exception of two, which are crAss-like phages. We compare these isolates to existing human gut metagenomes, revealing similarities to previously inferred phages and additional unexplored phage diversity. Finally, we use host tropisms of these phages to identify alleles of phage structural genes associated with infectivity. This work provides a detailed view of the guts "viral dark matter" and a framework for future efforts to further integrate isolation- and sequencing-focused efforts to understand gut-resident phages.

microbiology↗