Biology subjects

Tomlinson, B.

Publications and source records attributed to Tomlinson, B..

6 recordsLinked to original sources

bioRxiv2025

Lack of consistent effect of dietary fiber on immune checkpoint blockade efficacy across diverse murine tumor models

Immune checkpoint blockade (ICB) has transformed cancer treatment, but success rates remain low in most cancers. Recent research suggest that dietary fiber enhances ICB response in melanoma patients and murine preclinical models through microbiome-dependent mechanisms. Yet, the robustness of this effect across cancer types and dietary contexts remains unclear. Specifically, prior literature compared grain-based chow (high fiber) to low-fiber purified diet, but these diets differ also on other dimensions including phytochemicals. Here we investigated, in mice fed grain-based chow or purified diets with differing quantities of isolated fibers (cellulose and inulin), metabolite levels and ICB activity in multiple tumor models. The blood and fecal metabolome were relatively similar between mice fed high- and low-fiber purified diets, but differed massively between mice fed purified diets or chow, identifying the factor as diet type, independent of fiber. Tumor growth studies in three implantable and two spontaneous genetically engineered tumor models revealed that fiber has a weaker impact on ICB (anti-PD-1) efficacy than previously reported. In some models, dietary modulation impacted ICB activity, but not in a consistent direction across models. In none of the models did we observe the pattern expected if fiber controlled ICB efficacy: strong efficacy in both chow and high-fiber purified diet but low efficacy in low-fiber purified diet. Thus, dietary fiber appears to have limited or inconsistent effect on ICB efficacy in mouse models, and other dietary factors that correlate with fiber intake may underlie the clinical correlations between fiber consumption and immunotherapy outcomes.

cancer biology↗

bioRxiv2022

Staphylococcus aureus SigS induces expression of a regulatory protein pair that modulate its mRNA stability

SigS is the sole extracytoplasmic function sigma in S. aureus and is necessary for virulence, immune evasion, as well as surviving exposure to toxic chemicals and environmental stressors. Despite the contribution of SigS to a myriad of critical phenotypes, the downstream effectors of the SigS-dependent S. aureus pathogenesis, immune evasion, and stress response remain elusive. To address this knowledge gap, we analyzed the S. aureus transcriptome following transient over-expression of SigS. We identified a bi-cistronic transcript, up-regulated by 1000-fold, containing two mid-sized genes each containing single domains of unknown function (DUF). We renamed these genes sroA (SigS regulated orfA) and sroB (SigS regulated orfB). We demonstrated that the SigS regulation of the sroAB operon is direct using in vitro transcription analysis. Using northern blot analysis, we also demonstrated that SroA and SroB have opposing auto- regulatory functions on the transcriptional architecture of the sigS locus; with SroA stimulated SigS mRNA levels and SroB stimulating s750 (SigS antisense) levels. We hypothesized that these this opposing regulatory effects were due to a direct interaction. We demonstrated an interaction between SroA and SroB using an in-vivo surrogate genetics approach via Bacterial Two Hybrid. We demonstrated that the SroA effect on SigS is at the post-transcriptional level of mRNA stability, highlighting a mechanism likely used by S. aureus to tightly control SigS levels. Finally, we demonstrate that the sroAB locus promotes virulence in a female murine pneumonia model of infection.

microbiology↗

bioRxiv2021

Phenogenomic characterization of a newly domesticated and novel species from the genus Verrucosispora

The concept of bacterial dark matter stems from our inability to culture most microbes and represents a fundamental hole in our knowledge of microbial diversity. Herein we present the domestication of such an organism: a previously uncultured, novel species from the rare-Actinomycetes genus Verrucosispora. Although initial recovery took >4 months, isolation of phenotypically distinct, domesticated generations occurred within weeks. Two isolates were subjected to phenogenomic analyses, revealing domestication correlated with enhanced growth rates in nutrient-rich media, but diminished capacity to metabolize diverse amino acids. This is seemingly mediated by genomic decay through the pseudogenization of amino acids metabolism genes. Conversely, later generational strains had enhanced spore germination rates, potentially through the reversion of a sporulation-associated kinase from pseudogene to true gene status. We observed that our most wild-type isolate had the greatest potential for antibacterial activity, which correlated with extensive mutational attrition of biosynthetic gene clusters in domesticated strains. Comparative analyses revealed wholesale genomic reordering in strains, with widespread SNP, indel and pseudogene mutations observed. We hypothesize that domestication of this previously unculturable organism resulted from the shedding of genomic flexibility required for life in a dynamic marine environment, parsing out genetic redundancy to allow for a newfound cultivable amenability.

microbiology↗

bioRxiv2021

Assessing the role of cold-shock protein C: A novel regulator of Acinetobacter baumannii biofilm formation and virulence

Acinetobacter baumannii is a formidable opportunistic pathogen that is notoriously difficult to eradicate from hospital settings and can spread quickly via healthcare personnel despite preventive measures. This resilience is often attributed to a proclivity for biofilm formation, which grants A. baumannii a higher tolerance towards external stress, desiccation, and antimicrobials. Despite this, little is known regarding the mechanisms orchestrating A. baumannii biofilm formation. Herein, we performed RNA-seq on biofilm and planktonic populations for the multidrug resistant isolate, AB5075, and identified 438 genes with altered expression. To assess the potential role of genes upregulated within biofilms, we tested the biofilm forming capacity of their respective mutants from an A. baumannii transposon library. In so doing, we uncovered 24 genes whose disruption led to reduced biofilm formation. One such element, cold shock protein C (cspC), produced a mucoidal, non-mucoviscous colony phenotype. RNA-sequencing of this mutant revealed the down regulation of pili and fimbriae in the cspC mutant, which would explain the decreased biofilm observed. Using MIC assays, we note that the mutant displayed increased antibiotic tolerance that we hypothesize is mediated by overexpression of multi-drug efflux pumps and altered mRNA stability of their corresponding transcriptional repressor. Finally, we show that CspC is required for survival during oxidative stress and challenge by the human immune system, and plays a pivotal role during systemic infection. Collectively, our work identifies a cadre of new biofilm associated genes within A. baumannii and provides insight into the global regulatory network of this emerging human pathogen.

microbiology↗

bioRxiv2020

Resistance to decitabine and 5-azacytidine emerges from adaptive responses of the pyrimidine metabolism network

Mechanisms-of-resistance to decitabine and 5-azacytidine, mainstay treatments for myeloid malignancies, require investigation and countermeasures. Both are nucleoside analog pro-drugs processed by pyrimidine metabolism into a nucleotide analog that depletes the key epigenetic regulator DNA methyltranseferase 1 (DNMT1). We report here that DNMT1 protein, although substantially depleted (~50%) in patients bone marrows at response, rebounded at relapse, and explaining this, we found pyrimidine metabolism gene expression shifts averse to the processing of each pro-drug. The same metabolic shifts observed clinically were rapidly recapitulated in leukemia cells exposed to the pro-drugs in vitro. Pyrimidine metabolism is a network that senses and preserves nucleotide balances: Decitabine, a deoxycytidine analog, and 5-azacytidine, a cytidine analog, caused acute and distinct nucleotide imbalances, by off-target inhibition of thymidylate synthase and ribonucleotide reductase respectively. Resulting expression changes in key pyrimidine metabolism enzymes peaked 72-96 hours later. Continuous pro-drug exposure stabilized metabolic shifts generated acutely, preventing DNMT1-depletion and permitting exponential leukemia out-growth as soon as day 40. Although dampening to activity of the pro-drug initially applied, adaptive metabolic responses primed for activity of the other. Hence, in xenotransplant models of chemorefractory AML, alternating decitabine with 5-azacytidine, timed to exploit compensating metabolic shifts, and addition of an inhibitor of a catabolic enzyme induced by decitabine/5-azacytidine, extended DNMT1-depletion and time-to-distress by several months versus either pro-drug alone. In sum, resistance to decitabine and 5-azacytidine emerges from adaptive responses of the pyrimidine metabolism network; these responses can be anticipated and thus exploited. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/958405v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@e3f77aorg.highwire.dtl.DTLVardef@8caaa4org.highwire.dtl.DTLVardef@117236dorg.highwire.dtl.DTLVardef@16ee98_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2019

MroQ is a Novel Abi-domain Protein That Influences Virulence Gene Expression in Staphylococcus aureus via Modulation of Agr Activity

Numerous factors have to date been identified as playing a role in the regulation of Agr activity in S. aureus, including transcription factors, antisense RNAs, and host elements. Herein we investigate the product of SAUSA300_1984 (termed MroQ), a transmembrane Abi-domain/M79 protease-family protein, as a novel effector of this system. Using a USA300 mroQ mutant we observed a drastic reduction in proteolysis, hemolysis and pigmentation that was fully complementable. This appears to result from diminished agr activity, as transcriptional analysis revealed significant decreases in expression of both RNAII and RNAIII in the mroQ mutant. Such effects appear to be direct, rather than indirect, as known agr effectors demonstrated limited alterations in their activity upon mroQ disruption. A comparison of RNA-sequencing datasets for both mroQ and agr mutants reveal a profound overlap in their regulomes, with the majority of factors affected being known virulence determinants. Importantly, the preponderance of alterations in expression were more striking in the agr mutant, indicating that MroQ is necessary, but not sufficient, for Agr function. Mechanism profiling revealed that putative residues for metalloprotease activity within MroQ are required for its Agr controlling effect, however this is not wielded at the level of AgrD processing. Virulence assessment demonstrated that mroQ and agr mutants both exhibited increased formation of renal abscesses, but decreased skin abscess formation, alongside diminished dermonecrosis. Collectively, we present the characterization of a novel agr effector in S. aureus, which would appear to be a direct regulator, potentially functioning via interaction with the AgrC histidine kinase.

microbiology↗