Biology subjects

Bird, J. T.

Publications and source records attributed to Bird, J. T..

9 recordsLinked to original sources

bioRxiv2024

HIF-alpha signaling mediates the macrophage inflammatory response during Leishmania major infection

Cutaneous leishmaniasis (CL) contributes significantly to the global burden of neglected tropical diseases, with 12 million people currently infected with Leishmania parasites. CL encompasses a range of disease manifestations, from self-healing skin lesions to permanent disfigurations. Currently there is no vaccine available, and many patients are refractory to treatment, emphasizing the need for new therapeutic targets. Previous work demonstrated macrophage HIF--mediated lymphangiogenesis is necessary to achieve efficient wound resolution during murine L. major infection. Here, we investigate the role of macrophage HIF- signaling independent of lymphangiogenesis. We sought to determine the relative contributions of the parasite and the host-mediated inflammation in the lesional microenvironment to myeloid HIF- signaling. Because HIF- activation can be detected in infected and bystander macrophages in leishmanial lesions, we hypothesize it is the hosts inflammatory response and microenvironment, rather than the parasite, that triggers HIF- activation. To address this, macrophages from mice with intact HIF- signaling (LysMCreARNTf/+) or mice with deleted HIF- signaling (LysMCreARNTf/f) were subjected to RNASequencing after L. major infection and under pro-inflammatory stimulus. We report that L. major infection alone is enough to induce some minor HIF--dependent transcriptomic changes, while infection with L. major in combincation with pro-inflammatory stimuli induces numerous transcriptomic changes that are both dependent and independent of HIF- signaling. Additionally, by coupling transcriptomic analysis with several pathway analyses, we found HIF- suppresses pathways involved in protein translation during L. major infection in a pro-inflammatory environment. Together these findings show L. major induces a HIF--dependent transcriptomic program, but HIF- only suppresses protein translation in a pro-inflammatory environment. Thus, this work indicates the host inflammatory response, rather than the parasite, largely contributes to myeloid HIF- signaling during Leishmania infection.

microbiology↗

bioRxiv2024

Bacterial cross-feeding can promote gene retention by lowering gene expression costs

Cross-feeding is prevalent in microbial communities. Through time, cross-feeding is thought to enrich for loss-of-function mutations, thereby creating or reinforcing dependencies between community members. However, few studies have compared how cross-feeding affects the evolutionary trajectory of partners compared to monoculture conditions. Here we compared mutations that were differentially enriched in bacterial monocultures versus cocultures pairing phototrophic Rhodopseudomonas palustris with fermentative Escherichia coli in an obligate cross-feeding relationship based on the exchange of nitrogen and carbon for 650-800 generations. Opposite trends for the number of differentially enriched mutations were observed for each species; R. palustris accumulated more unique mutations in monoculture whereas E. coli accumulated more unique mutations in coculture. Contrary to expectations, the emergence of additional dependencies was more apparent for both species in monoculture, even though additional layers of cross-feeding involving iron and adenine were present in coculture. We reasoned that iron and adenine cross-feeding occurred at levels sufficient to repress gene expression in the recipient, thereby promoting gene retention by lowering gene cost. Thus, the influence of cross-feeding on evolutionary trajectories can vary with organisms and conditions, and there are situations where cross-feeding can limit, rather than promote, emergent dependencies. IMPORTANCEBacteria commonly engage in cross-feeding where nutrients are transferred between neighbors. Cross-feeding is thought to alleviate energy expenditures for genes whose role can be met by cross-fed nutrients, leading to eventual gene loss. However, few examples have been documented, especially in comparison to monocultures where bacteria are grown without a cross-feeding partner. Here we grew cocultures pairing phototrophic Rhodopseudomonas palustris with fermentative Escherichia coli alongside corresponding monocultures for 650-800 generations. While coculture conditions required obligate exchange of nitrogen and carbon, additional cross-feeding of adenine and iron likely occurred. Contrary to expectations, iron and uncharacterized dependencies emerged in monocultures but not in cocultures. Low expression for iron scavenging and adenine synthesis genes in cocultures suggested that cross-feeding repressed gene expression, thereby lowering gene cost. Thus, whereas there are likely cases where cross-feeding makes costly genes dispensable, there are also cases where cross-feeding lowers gene cost, thereby promoting gene retention.

microbiology↗

bioRxiv2024

Enhanced translational activity is linked to lymphatic endothelial cell activation in cutaneous leishmaniasis

Cutaneous leishmaniasis (CL) is a significant public health problem leading to permanently disfiguring skin lesions caused by Leishmania parasites. Lesion severity stems from an excessive host inflammatory response that prevents healing. Here, we characterized the transcriptional and translational responses of lymphatic endothelial cells (LECs) during murine CL using historical single-cell RNA sequencing data combined with flow cytometry and in vivo puromycin incorporation to assess translational activity. We identified upregulation of antigen presentation pathways including MHC-I, MHC-II, and immunoproteasome transcripts in dermal LECs from Leishmania major-infected mice compared to naive controls. LECs also exhibited increased expression of guanylate binding proteins and interferon-inducible genes, indicative of immune activation. Moreover, our findings demonstrate that LECs in leishmanial lesions displayed heightened translational activity relative to LECs from uninflamed ears, and LEC translational activity was highest in activated LECs. Furthermore, LEC translational activity exceeded that of other cell types within the lesion microenvironment. Validating the transcriptomic data, LECs in lesions expressed elevated MHC-II and programmed death-ligand 1 (PDL-1), supporting their potential role in antigen presentation. Functional assays using DQ-OVA confirmed that LECs from leishmanial lesions efficiently uptake and process antigens, highlighting their capability as antigen presenting cells in the inflamed dermal microenvironment. Overall, our study reveals the activation status of LECs in leishmanial lesions, shedding light on their potential role in shaping local immunity and inflammation in a variety of skin diseases.

microbiology↗

bioRxiv2023

Characterization of methionine dependence in melanoma cells

Dietary methionine restriction is associated with a reduction in tumor growth in preclinical studies and an increase in lifespan in animal models. The mechanism by which methionine restriction inhibits tumor growth while sparing normal cells is incompletely understood. We do know that normal cells can utilize methionine or homocysteine interchangeably (methionine independence) while most cancer cells are strictly dependent on methionine availability. Here, we compared a typical methionine dependent and a rare methionine independent melanoma cell line. We show that replacing methionine, a methyl donor, with its precursor homocysteine generally induced hypomethylation in gene promoters. This decrease was similar in methionine dependent and methionine independent cells. There was only a low level of pathway enrichment, suggesting that the hypomethylation is generalized rather than gene specific. Whole proteome and transcriptome were also analyzed. This analysis revealed that contrarily to the effect on methylation, the replacement of methionine with homocysteine had a much greater effect on the transcriptome and proteome of methionine dependent cells than methionine independent cells. Interestingly, methionine adenosyltransferase 2A (MAT2A), responsible for the synthesis of s-adenosylmethionine from methionine, was equally strongly upregulated in both cell lines. This suggests that the absence of methionine is equally detected but triggers different outcomes in methionine dependent versus independent cells. Our analysis reveals the importance of cell cycle control, DNA damage repair, translation, nutrient sensing, oxidative stress and immune functions in the cellular response to methionine stress in melanoma.

molecular biology↗

bioRxiv2021

In vivo reprogramming of murine host immune response genes following Leishmania major infection.

Leishmania parasites cause cutaneous leishmaniasis (CL), a pathologic disease characterized by disfiguring, ulcerative skin lesions. Both parasite and host gene expression following infection with various Leishmania species has been investigated in vitro, but global transcriptional analysis following L. major infection in vivo is lacking. Thus, we conducted a comprehensive transcriptomic profiling study combining bulk RNA sequencing (RNA-Seq) and single-cell RNA sequencing (scRNA-Seq) to identify global changes in gene expression in vivo following L. major infection. Bulk RNA-Seq analysis revealed that host immune response pathways like the antigen processing and presentation pathway were significantly enriched amongst differentially expressed genes (DEGs) upon infection, while ribosomal pathways were significantly downregulated in infected mice compared to naive controls. scRNA-Seq analyses revealed cellular heterogeneity including distinct resident and recruited cell types in the skin following murine L. major infection. Within the individual immune cell types, several DEGs indicative of many interferon induced GTPases and antigen presentation molecules were significantly enhanced in the infected ears including macrophages (Gbp2, H2-K1, H2-Aa, H2-Ab1), resident macrophages (H2-K1, H2-D1, Gbp4, Gbp8, Gbp2), and inflammatory monocytes (Gbp2, Gbp5, Gbp7, Gbp3). Ingenuity Pathway Analysis of scRNA-Seq data indicated the antigen presentation pathway was increased with infection, while EIF2 signaling is the top downregulated pathway followed by eIF4/p70S6k and mTOR signaling in multiple cell types including macrophages, BECs, and LECs. Altogether, this transcriptomic profile highlights known recruitment of myeloid cells to lesions and recognizes a previously undefined role for EIF2 signaling in murine L. major infection in vivo. Author summaryLeishmania major cause cutaneous leishmaniasis, which is characterized by disfiguring, ulcerative skin lesions. Here, we show murine L. major-directed reprogramming of the host transcriptome in vivo. Our bulk RNA-Seq analyses revealed upregulation of antigen processing and presentation pathway, while the host ribosomal pathway was downregulated following L. major infection. Similarly, scRNA-Seq analyses revealed the upregulation of transcripts responsible for antigen presentation and host defense proteins like guanylate binding proteins (GBPs) alongside the downregulation of EIF2 signalling at the site of L. major infection. Overall, our transcriptomic dataset not only provides the comprehensive list of gene expression at the single-cell resolution, and highlights a previously undefined role for EIF2 signalling during murine L. major infection in vivo.

immunology↗

bioRxiv2020

Methionine stress induces ferroptosis in methionine dependent cancer cells

Dietary methionine restriction is associated with a reduction in tumor growth in preclinical studies and an increase in lifespan in animal models. The mechanism by which methionine restriction inhibits tumor growth while sparing normal cells is incompletely understood, except for the observation that normal cells can utilize methionine or homocysteine interchangeably (methionine independence) while most cancer cells are strictly dependent on methionine availability. Here, we compared a typical methionine dependent and a rare methionine independent melanoma cell line. We found that replacing methionine with homocysteine generally induced hypomethylation in gene promoters. We isolated nuclear proteins and submitted it for tandem mass tag (TMT) proteomics. This analysis revealed that several proteins involved in the mitochondrial integrated stress response (ISR) were upregulated in response to the replacement of methionine to homocysteine in both cell lines, but to a much greater degree in the methionine dependent cell line. Consistent with the ISR signature, a proteomic analysis of a subcellular fraction enriched for mitochondrial content revealed a strong enrichment for proteins involved in oxidative phosphorylation. Analysis of cellular bioenergetics confirmed that homocysteine induces a decrease in ATP production from oxidative phosphorylation and glycolysis, but to a similar extent in methionine dependent and methionine independent cells. The mitochondrial integrated stress response shared a signature with ferroptosis. Methionine dependent cells displayed a strong ferroptotic signature, which was decreased by half in methionine independent cells. Consistent with ferroptosis, lipid peroxidation was significantly increased in methionine independent cells grown in homocysteine, and viability could be rescued partially but significantly with the inhibitor ferrostatin. Therefore, we propose that methionine stress induces ferroptotic cell death in methionine dependent cancer cells.

cancer biology↗

bioRxiv2020

Evidence for a growth zone for deep subsurface microbial clades in near-surface anoxic sediments

Global marine sediments harbor a large and highly diverse microbial biosphere, but the mechanism by which this biosphere is established during sediment burial is largely unknown. During burial in marine sediments, concentrations of easily-metabolized organic compounds and total microbial cell abundance decrease steadily. However, it is unknown whether some microbial clades increase with depth, despite the overall trend of abundance decrease. We show total population increases in 38 microbial families over 3 cm of sediment depth in the upper 7.5 cm of White Oak River (WOR) estuary sediments. Clades that increased with depth were more often anaerobic, uncultured, or common in deep marine sediments relative to those that decreased. Minimum turnover times (which are minimum in situ doubling times of growth rates) were estimated to be 2-25 years by combining sedimentation rate with either quantitative PCR (qPCR) or the product of the Fraction Read Abundance of 16S rRNA genes and total Cell counts (FRAxC). Turnover times were within an order of magnitude of each other in two adjacent cores, as well as in two laboratory enrichments of Cape Lookout Bight (CLB), NC, sediments (average difference of 28 {+/-} 19%). qPCR and FRAxC in WOR cores and FRAxC in CLB incubations produced similar turnover times for key deep subsurface uncultured clades Bathyarchaeota (8.7 {+/-} 1.9 years) and Thermoprofundales/MBG-D (4.1 {+/-} 0.7 years). We conclude that common deep subsurface microbial clades experience a narrow zone of growth in shallow sediments, offering an opportunity for natural selection of traits for long-term subsistence after resuspension events. Significance statementThe current dogma is that the deeply-branching uncultured microbes that dominate global marine sediments do not actually increase in population size as they are buried in marine sediments - rather they exist in a sort of prolonged torpor for thousands of years. This is because no evidence has ever been found that these clades actually increase population sizes, or grow, as they are gradually buried. We discovered that they actually do increase population sizes during burial, but only in the upper few centimeters. This changes our dogma about marine sediments as a vast repository of non-growing microbes, to a vast repository of non-growing microbes with a thin and relatively rapid area of growth in the upper 10 centimeters.

microbiology↗

bioRxiv2019

Enhanced nutrient uptake is sufficient to drive emergent cross-feeding between bacteria

Interactive microbial communities are ubiquitous, influencing biogeochemical cycles and host health. One widespread interaction is nutrient exchange, or cross-feeding, wherein metabolites are transferred between microbes. Some cross-fed metabolites, such as vitamins, amino acids, and ammonium (NH4+), are communally valuable and impose a cost on the producer. The mechanisms that enforce cross-feeding of communally valuable metabolites are not fully understood. Previously we engineered mutualistic cross-feeding between N2-fixing Rhodopseudomonas palustris and fermentative Escherichia coli. Engineered R. palustris excreted essential nitrogen as NH4+ to E. coli while E. coli excreted essential carbon as fermentation products to R. palustris. Here, we enriched for nascent cross-feeding in cocultures with wild-type R. palustris, not known to excrete NH4+. Emergent NH4+ cross-feeding was driven by adaptation of E. coli alone. A missense mutation in E. coli NtrC, a regulator of nitrogen scavenging, resulted in constitutive activation of an NH4+ transporter. This activity likely allowed E. coli to subsist on the small amount of leaked NH4+ and better reciprocate through elevated excretion of organic acids from a larger E. coli population. Our results indicate that enhanced nutrient uptake by recipients, rather than increased excretion by producers, is an underappreciated yet possibly prevalent mechanism by which cross-feeding can emerge.

microbiology↗

bioRxiv2016

Extracellular peptidases in subsurface sediments of the White Oak River estuary, NC, suggest microbial community adaptation to oxidize degraded organic matter

Anoxic subsurface sediments contain communities of heterotrophic microorganisms that metabolize organic carbon at extraordinarily slow rates. In order to assess the mechanisms by which subsurface microorganisms access detrital sedimentary organic matter, we measured kinetics of a range of extracellular peptidases in anoxic sediments of the White Oak River estuary, NC. Nine distinct peptidase substrates were enzymatically hydrolyzed at all depths. Potential peptidase activities (Vmax) decreased with increasing sediment depth, although Vmax expressed on a per cell basis was approximately the same at all depths. Half-saturation constants (Km) decreased with depth, indicating peptidases that functioned more efficiently at low substrate concentrations. Potential activities of extracellular peptidases acting on molecules that are enriched in degraded organic matter (D-phenylalanine and L-ornithine) increased relative to enzymes that act on L-phenylalanine, further suggesting microbial community adaptation to access degraded organic matter. Nineteen classes of predicted, exported peptidases were identified in genomic data from the same site, of which genes for class C25 (gingipain-like) peptidases represented more than 40% at each depth. Methionine aminopeptidases, zinc carboxypeptidases, and class S24-like peptidases, which are involved in single-stranded DNA repair, were also abundant. These results suggest a subsurface heterotrophic microbial community that primarily accesses low-quality detrital organic matter via a diverse suite of well-adapted extracellular enzymes.\n\nIO_SCPLOWMPORTANCEC_SCPLOWBurial of organic carbon in marine and estuarine sediments represents a long-term sink for atmospheric carbon dioxide. Globally, [~]40% of organic carbon burial occurs in anoxic estuaries and deltaic systems. However, the ultimate controls on the amount of organic matter that is buried in sediments, versus oxidized into CO2, are poorly constrained. Here we used a combination of enzyme assays and metagenomic analysis to identify how subsurface microbial communities catalyze the first step of proteinaceous organic carbon degradation. Our results show that microbial communities in deeper sediments are adapted to access molecules characteristic of degraded organic matter, suggesting that those heterotrophs are adapted to life in the subsurface.

ecology↗