Biology subjects

Goodlett, D. R.

Publications and source records attributed to Goodlett, D. R..

10 recordsLinked to original sources

bioRxiv2026

Informatics Methods to Enhance Bacterial Peptide Detection

Detecting low-abundance bacterial peptides in samples dominated by human proteins using liquid chromatography tandem mass spectrometry (LC MS/MS) is challenging. Bacterial peptides are typically far less abundant than host-derived peptides, and sequence similarity between bacterial and human peptides can complicate confident attribution to a bacterial origin. To address this problem, we developed and benchmarked an in silico spectral library protein identification workflow for detecting Staphylococcus aureus USA300 peptides against a background of A375 cells. Peptide and protein identifications obtained at a 1% peptide-level false discovery rate were compared between the in silico spectral library search and a conventional sequence database search across a defined bacterial dilution series containing 0, 1%, 2%, 10%, 25%, 50%, and 100% bacterial protein. A human sequence similarity filter was applied to identifications from both search strategies to reduce the likelihood of retaining human derived or human like peptide sequences. Both search strategies showed concentration dependent bacterial detection and shared a practical detection limit of approximately 1 to 2% bacterial protein but exhibited distinct performance profiles. The in silico spectral library search produced fewer bacterial-like background identifications in the human-only control, whereas MS2Rescore substantially increased spectral library identifications at higher bacterial concentrations, exceeding database search at 100% bacterial protein. The partially overlapping peptide sets identified by the two strategies indicate that they provide complementary coverage of the bacterial proteome.

biochemistry↗

bioRxiv2025

Methionine regulates antitumor function of CD8⁺ T cells through polyamine synthesis

Methionine is an essential amino acid critical for T cell activation. While methionine restriction (MR) combined with immune checkpoint blockade has been shown to enhance T cell function, the impact of methionine on adoptive T cell therapies is largely unexplored. Here, we examined the functionality of T cells under MR and pharmaceutical inhibition of the methionine cycle (MAT2Ai), using primary T cells and a murine adoptive T cell therapy model. In vitro, transient MR or MAT2Ai treatment increased interferon gamma (IFN{gamma}) expression in CD8+ T cells, whereas sustained MR led to the upregulation of T cell exhaustion-associated markers. Mechanistically, transient MR suppressed the polyamine synthesis pathway, and supplementation with polyamines reversed MR-induced IFN{gamma} expression. Genetic ablation of s-adenosylmethionine decarboxylase, an enzyme in the polyamine synthesis pathway, recapitulated the effect of MR, indicating that transient MR enhances T cell function by inhibiting polyamine synthesis. Despite this, transient MR treatment of ovalbumin (OVA)-specific (OT-I) CD8+ T cells prior to adoptive transfer did not improve antitumor efficacy against EG7-OVA tumors in vivo. In contrast, sustained dietary MR accelerated EG7-OVA tumor growth in mice treated with OT-I T cells, demonstrating that methionine availability is essential for the activity of adoptively transferred T cells. These findings suggest that enhancing methionine availability in the tumor microenvironment may improve the efficacy of adoptive T cell therapies.

immunology↗

bioRxiv2025

DMEM and EMEM are suitable surrogate media to mimic host environment and expand leptospiral pathogenesis studies using in vitro tools

Pathogenic Leptospira species can survive and thrive in a wide range of environments. Distinct environments expose the bacteria to different temperatures, osmolarities, and amounts and sources of nutrition. However, leptospires are mostly cultured, in a laboratory setting under in vitro conditions that do not reflect natural environments. This constraint on laboratory cultures limits the applicability of in vitro studies to the understanding of even simple pathogenic processes. Here we report, investigate, and identify a medium and conditions that mimic the host environment during leptospirosis infection, expanding the available in vitro tools to evaluate leptospiral pathogenesis. We quantified genome-wide gene expression of pathogenic Leptospira interrogans cultured in different in vitro media compositions (EMJH, DMEM, EMEM, and HAN). Using EMJH as standard, we compared gene expression in these compositions to genome-wide gene expression gathered in a host environment: whole blood (WB) of hamsters after infection with pathogenic leptospires. Leptospires cultured in DMEM and EMEM media shared 40% and 47% of all differentially expressed genes (DEGs) of leptospires present within WB (FDR<0.01), while leptospires cultured in HAN media only shared 20% of DEGs with those from WB. Furthermore, gene and pathway expression of leptospires cultured on DMEM and EMEM media exhibited a better correlation with leptospires grown in WB, including promoting expression of a similar leptospiral lipid A profile to the one identified directly in host tissues. Taken together, these results indicate that commercial cell-culture media EMEM or DMEM are better surrogates for in vivo pathogenic studies than EMJH or HAN media in Leptospira. These alternative culture conditions, using media that are a standard supply worldwide, provide a reproducible and cost-effective approach that can accelerate research investigation and reduce the number of animal infections necessary for basic research of leptospirosis.

microbiology↗

bioRxiv2024

A Lipidomics-Based Method to Eliminate Negative Urine Culture in General Population

Urinary tract infections (UTIs) pose a significant challenge to human health. Accurate and timely detection remains pivotal for effective intervention. Current urine culture techniques, while essential, often encounter challenges where urinalysis yields positive results, but subsequent culture testing produces a negative result. This highlights potential discrepancies between the two methods and emphasizes the need for improved correlation in urinary tract infection (UTI) detection. Employing advanced lipidomics techniques, we deployed the fast lipid analysis technique (FLAT) on a clinical cohort suspected of having UTIs. Lipid fingerprinting by MALDI-TOF MS, directly from urine samples without ex vivo growth, correctly identified the common uropathogens within a 1-hour timeframe when compared to urine culture. FLAT analysis also identified urine samples without culturable pathogens (negative UTIs) with 99% agreement, whereas urinalysis showed 37% agreement with the gold standard urine culture. In 402 urine samples suspected for UTI from out-patients, FLAT assay rapidly ruled out negative urines without the need for culture in 77% of all cases. The potential impact of this innovative lipidomic-based approach extends beyond conventional diagnostic limitations, offering new avenues for early detection and targeted management of urinary tract infections. This research marks a paradigm shift in urine culture methodology, paving the way for improved clinical outcomes and public health interventions.

microbiology↗

bioRxiv2024

Lipid A double bond position determination using ozone and laser-induced dissociation

Ozone coupled with tandem MS is a traditional tool to assign double bond position in lipids. However, few studies were reported on the use of ozone for double bond determination of bacterial lipids. Most MS-based double bond localization methods used in bacterial lipidomics focused on low molecular weight phospholipids, but never tested for assigning double bond position in complex glycolipids such as Lipid A. Here, we present a simple approach to identify unsaturated lipid A double bond position using on-MALDI plate ozone treatment of intact Lipid A.

microbiology↗

bioRxiv2023

Structural Elucidation of Intact Rough-Type Lipopolysaccharides using Field Asymmetric Ion Mobility Spectrometry and Kendrick Mass Defect Plots

Lipopolysaccharide (LPS) is a hallmark virulence factor of Gram-negative bacteria. It is a complex, structurally heterogeneous mixture due to variations in number, type, and position of its simplest units: fatty acids and monosaccharides. Thus, LPS structural characterization by traditional mass spectrometry (MS) methods is challenging. Here, we describe the benefits of field asymmetric ion mobility spectrometry (FAIMS) for analysis of intact R-type lipopolysaccharide complex mixture (lipooligosaccharide; LOS). Structural characterization was performed using Escherichia coli J5 (Rc mutant) LOS, a TLR4 agonist widely used in glycoconjugate vaccine research. FAIMS gas phase fractionation improved the (S/N) ratio and number of detected LOS species. Additionally, FAIMS allowed the separation of overlapping isobars facilitating their tandem MS characterization and unequivocal structural assignments. In addition to FAIMS gas phase fractionation benefits, extra sorting of the structurally related LOS molecules was further accomplished using Kendrick mass defect (KMD) plots. Notably, a custom KMD base unit of [Na-H] created a highly organized KMD plot that allowed identification of interesting and novel structural differences across the different LOS ion families; i.e., ions with different acylation degrees, oligosaccharides composition, and chemical modifications. Defining the composition of a single LOS ion by tandem MS along with the organized KMD plot structural network was sufficient to deduce the composition of 179 LOS species out of 321 species present in the mixture. The combination of FAIMS and KMD plots allowed in-depth characterization of the complex LOS mixture and uncovered a wealth of novel information about its structural variations.

biochemistry↗

bioRxiv2022

Rickettsia typhi peptidoglycan mapping with data-dependent tandem mass spectrometry

Rickettsia species are diverse Gram-negative obligate intracellular bacteria often pervasive in numerous invertebrates, as well as fungal, nematode and microeukaryotic hosts. Certain species are etiological agents for well-known arthropod-borne illnesses; e.g., R. rickettsii (Rocky Mountain Spotted Fever), R. prowazekii (Epidemic Typhus), and R. typhi, (Endemic Typhus). Living freely in eukaryotic cytosol presumably exposes rickettsiae to host cell immune receptors, particularly those recognizing bacterial cell envelope glycoconjugates. However, the mechanics of host recognition of rickettsiae remain poorly defined. As rickettsiae synthesize a canonical Gram-negative cell envelope that includes peptidoglycan (PGN) and lipopolysaccharide (LPS), structural insight on these macromolecules is important for deciphering host responses to these pathogens. In this work, PGN from R. typhi was digested and the resultant subunits were analyzed by two different, albeit complementary, sample preparation methods. Both approaches were subsequently subjected to liquid chromatography/mass spectrometry analysis to infer PGN structure. R. typhi PGN was determined to be similar to most other Gram-negative bacteria, with mDAP-type muropeptide subunits. However, additional alanine residues were observed elongating the muropeptide stems, rather than the glycine residues usually observed in Gram-negative bacterial PGN. Despite this deviation, R. typhi contains a murein layer that is predicted to agonize host cellular PGN receptors and be susceptible to PGN-targeting antimicrobials. This same structure is likely synthesized by all Rickettsia species, as bioinformatics and comparative genomics analyses indicate the biosynthesis of PGN is highly conserved. Determining how host cells process this canonical glycoconjugate during infection is crucial for identifying factors behind rickettsial pathogenesis, including immunoavoidance or proinflammatory mechanisms possibly employed by rickettsiae with varying pathogenic potential.

microbiology↗

bioRxiv2022

Caulobacter requires anionic sphingolipids and deactivation of fur to lose lipid A

Lipid A, the membrane-anchored portion of lipopolysaccharide, is an essential component of the outer membrane (OM) of nearly all Gram-negative bacteria. Here, we identify regulatory and structural factors that together permit Caulobacter crescentus to eliminate lipid A from its OM. Mutations in the ferric uptake regulator fur allow Caulobacter to survive in the absence of either LpxC, which catalyzes an early step of lipid A synthesis, or CtpA, a tyrosine phosphatase homolog which we find is needed for wild-type lipid A structure and abundance. Alterations in Fur-regulated processes, rather than iron status per se, underlie the ability to eliminate lipid A. Fitness of lipid A-deficient Caulobacter requires a previously uncharacterized anionic sphingolipid, ceramide phosphoglycerate (CPG), which also mediates sensitivity to the antibiotic colistin. Our results demonstrate that, in an altered regulatory landscape, anionic sphingolipids can support the integrity of a lipid A-deficient OM.

microbiology↗

bioRxiv2022

A comprehensive library of canonical and non-canonical MHC class I antigens for cancer vaccine development.

A longstanding disconnect between the growing number of MHC Class I immunopeptidomic studies and genomic medicine hinders cancer vaccine design. We develop COD-dipp to genomically map the full spectrum of detected canonical and non-canonical (non-exonic) MHC Class I antigens from 26 cancer studies. We demonstrate that patient mutations in regions overlapping physically identified antigens better predict immunotherapy response when compared to neoantigen predictions. We suggest a vaccine design approach using 140,966 highly immune-visible regions of the genome annotated by their expression and haplotype frequency in the human population. These regions tend to be highly conserved, mutated in cancer and harbor 7.8 times more immunogenicity. Intersecting pan-cancer mutations with these immune surveilled regions revealed a potential to create off-the-shelf multi-epitope vaccines against public neoantigens. Here we release COD-dipp, a cancer vaccine toolkit as a web-application (https://www.proteogenomics.ca/COD-dipp) and open-source high-throughput resource.

bioinformatics↗

bioRxiv2021

Enhanced longitudinal differential expression detection in proteomics with robust reproducibility optimization regression

Quantitative proteomics has matured into an established tool and longitudinal proteomic experiments have begun to emerge. However, no effective, simple-to-use differential expression method for longitudinal proteomics data has been released. Typically, such data is noisy, contains missing values, has only few time points and biological replicates. To address this need, we provide a comprehensive evaluation of several existing differential expression methods for high-throughput longitudinal omics data and introduce a new method, Robust longitudinal Differential Expression (RolDE). The methods were evaluated using nearly 2000 semi-simulated spike-in proteomic datasets and a large experimental dataset. The RolDE method performed overall best; it was most tolerant to missing values, displayed good reproducibility and was the top method in ranking the results in a biologically meaningful way. Furthermore, contrary to many approaches, the open source RolDE does not require prior knowledge concerning the types of differences searched, but can easily be applied even by non-experienced users.

bioinformatics↗