Biology subjects

Gomer, R. H.

Publications and source records attributed to Gomer, R. H..

11 recordsLinked to original sources

bioRxiv2025

Phosphoproteomic Profiling Reveals Overlapping and Distinct Signaling Pathways in Dictyostelium discoideum in Response to two different chemorepellents

Chemorepulsion causes cells to move away from the source of a signal, but the underlying mechanisms for eukaryotic cells are poorly understood. We performed proteomics and phosphoproteomics to elucidate how Dictyostelium discoideum responds to its two endogenous chemorepellent signals, the protein AprA and inorganic polyphosphate (polyP). At 60 minutes, AprA increased levels of 211 proteins and reduced levels of 57 proteins while polyP increased levels of 152 proteins and reduced levels of 168 proteins. Surprisingly, many of the AprA- and polyP-regulated proteins are associated with RNA metabolism and ribosomes. AprA and polyP both upregulated 19 proteins, one protein was downregulated by both, and one was upregulated by AprA and downregulated by polyP. AprA increased phosphorylation of 12 proteins and decreased phosphorylation of 60 proteins. PolyP increased phosphorylation of 7 proteins and decreased phosphorylation of 18 proteins. As expected, the two chemorepellents affected phosphorylation of signal transduction/ motility proteins, but unexpectedly affected phosphorylation of RNA-associated proteins. Both AprA and polyP decreased phosphorylation of six proteins including the Ras-interacting protein RipA and guanine nucleotide exchange factors (GEFs) such as the RacGEF GxcT. Mutants lacking RipA or GxcT were unresponsive to both AprA and polyP chemorepulsion. Together, this work supports the idea that rather than activating the same chemorepulsion mechanism, AprA and polyP activate only partially overlapping chemorepulsion mechanisms, and identifies two new components that are used by both chemorepellents.

cell biology↗

bioRxiv2025

Differences between human male and female neutrophils with respect to which RNAs are present in polysomes

BackgroundHuman males and females show differences in the incidence of neutrophil-associated diseases such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis, and differences in neutrophil physiological responses such as responses to infection, tissue damage, and chemotactic factors. However, little is known about the basis of sex-based differences in human neutrophils. MethodsStarting with human neutrophils from healthy donors, we used RNA-seq to examine total RNA profiles, RNAs not associated with ribosomes and thus not being translated, RNAs in monosomes, and RNAs in polysomes and thus heavily translated. ResultsThere were sex-based differences in the levels of RNAs across free RNA, monosome, and polysome fractions. Male neutrophils had increased levels of mRNAs encoding mitochondrial proteins in the free RNA fractions, indicating low levels of translation. The polysomes of male neutrophils were enriched for mRNAs encoding cytoskeletal organization, cell motility, and cell activation. The polysomes of female neutrophils were enriched for mRNAs associated the regulation of metabolic processes, cytokine responses, and mitochondrial proteins. ConclusionsThese data indicate that male and female neutrophils have different expression patterns and different translation efficiency of some mRNAs. This may contribute to the observed sex-based differences in neutrophil behavior and neutrophil-associated disease incidence and severity.

immunology↗

bioRxiv2025

Pharmacological inhibition of host pathways enhances macrophage killing of intracellular bacterial pathogens

After ingestion into macrophage phagosomes, some bacterial pathogens such as Mycobacterium tuberculosis (Mtb) evade killing by preventing phagosome acidification and fusion of the phagosome with a lysosome. Mtb accumulates extracellular polyphosphate (polyP), and polyP inhibits macrophage phagosome acidification and bacterial killing. In Dictyostelium discoideum, polyP also inhibits bacterial killing, and we identified some proteins in D. discoideum that polyP requires to suppress the killing of ingested bacteria. Here, we find that pharmacological inhibition of human orthologues of the D. discoideum proteins, including P2Y1 receptors, mammalian Target of Rapamycin (mTOR), and inositol hexakisphosphate kinase, enhances the killing of Mtb, Legionella pneumophila, and Listeria monocytogenes by human macrophages. Mtb inhibits phagosome acidification, expression of the proinflammatory marker CD54, and autophagy, and increases expression of the anti-inflammatory marker CD206. In Mtb-infected macrophages, the polyP-degrading enzyme polyphosphatase (ScPPX) and inhibitors reversed these effects, with ScPPX increasing CD54 expression more in female macrophages compared to male macrophages. In addition, Mtb inhibits proteasome activity, and some, but not all, inhibitors reversed these effects. While the existence of a dedicated polyP signaling pathway remains uncertain, our findings suggest that pharmacological inhibition of select host proteins can restore macrophage function and enhances the killing of intracellular pathogens. ImportanceHuman macrophages engulf bacteria into phagosomes, which then fuse with lysosomes to kill the bacteria. However, after engulfment, pathogenic bacteria such as Mycobacterium tuberculosis, Legionella pneumophila, and Listeria monocytogenes can block phagosome-lysosome fusion, allowing their survival. Here, we show that pharmacological inhibition of specific macrophage proteins reverses these effects and enhances bacterial killing. These findings suggest that targeting host factors involved in these processes may provide a therapeutic strategy to improve macrophage function against infections such as tuberculosis, Legionnaires disease, and listeriosis.

microbiology↗

bioRxiv2024

Gallein and isoniazid act synergistically to attenuate Mycobacterium tuberculosis growth in human macrophages

Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis (TB), can be difficult to treat because of drug resistance. Increased intracellular polyphosphate (polyP) in Mtb enhances resistance to antibiotics, and capsular polyP in Neisseria gonorrhoeae potentiates resistance to antimicrobials. The mechanism by which bacteria utilize polyP to adapt to antimicrobial pressure is not known. In this study, we found that Mtb adapts to the TB frontline antibiotic isoniazid (INH) by enhancing the accumulation of cellular, extracellular, and cell surface polyP. Gallein, a broad-spectrum inhibitor of the polyphosphate kinase that synthesizes polyP, prevents this INH-induced increase in extracellular and cell surface polyP levels. Gallein and INH work synergistically to attenuate Mtbs ability to grow in in vitro culture and within human macrophages. Mtb when exposed to INH, and in the presence of INH, gallein inhibits cell envelope formation in most but not all Mtb cells. Metabolomics indicated that INH or gallein have a modest impact on levels of Mtb metabolites, but when used in combination, they significantly reduce levels of metabolites involved in cell envelope synthesis and amino acid, carbohydrate, and nucleoside metabolism, revealing a synergistic effect. These data suggest that gallein represents a promising avenue to potentiate the treatment of TB. Author summaryMycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), which is responsible for more deaths than any other infectious disease. The alarming prevalence of drug-resistant Mtb strains has further exacerbated this global health crisis. Some pathogenic bacteria such as Mtb appear to increase levels of polyphosphate as a defense against antibiotics. We found that gallein, a small molecule inhibitor of bacterial polyphosphate kinases, strongly potentiates the ability of the frontline anti-tuberculosis drug isoniazid to inhibit the growth of Mtb both alone and in human macrophages. This has unveiled vulnerabilities in Mtb that could be strategically leveraged to reverse INH resistance.

microbiology↗

bioRxiv2023

Polyphosphate uses mTOR, pyrophosphate, and Rho GTPase components to potentiate bacterial survival in Dictyostelium

Human macrophages and the eukaryotic microbe Dictyostelium discoideum ingest bacteria by phagocytosis, and then kill the ingested bacteria. Some pathogenic bacteria secrete linear chains of phosphate residues (polyphosphate; polyP), and the polyP causes the phagocytes to not kill the ingested bacteria. In D. discoideum, the effect of polyP requires the G protein-coupled receptor GrlD, suggesting that polyP uses a signal transduction pathway to inhibit killing of ingested bacteria. Here we show that in addition to GrlD, the D. discoideum polyP signaling pathway requires the GPCR interacting arrestin-like protein AdcB, inositol hexakisphosphate kinase A (I6kA), the Rho GTPase RacE, and the TOR component Lst8. D. discoideum also secretes polyP, and at high concentrations polyP inhibits D. discoideum cytokinesis. The polyP inhibition of bacterial killing pathway does not appear to involve many of the polyP inhibition of cytokinesis pathway components. These data suggest the intriguing possibility that if there is a similar polyP inhibition of bacterial killing pathway in macrophages, pharmacologically blocking this pathway could potentiate macrophage killing of pathogenic bacteria. ImportanceAlthough most bacteria are quickly killed after phagocytosis by a eukaryotic cell, some pathogenic bacteria prevent their killing after phagocytosis. Pathogenic Mycobacterium species secrete polyP, and the polyP is necessary for the bacteria to prevent their killing after phagocytosis. Conversely, exogenous polyP prevents the killing of ingested bacteria that are normally killed after phagocytosis by human macrophages and the eukaryotic microbe Dictyostelium discoideum. This suggests the possibility that in these cells, a signal transduction pathway is used to sense polyP and prevent killing of ingested bacteria. In this report, we identify key components of the polyP signal transduction pathway in D. discoideum. In cells lacking these components, polyP is unable to inhibit killing of ingested bacteria. The pathway components have orthologues in human cells, and an exciting possibility is that pharmacologically blocking this pathway in human macrophages would cause them to kill ingested pathogens such as M. tuberculosis.

microbiology↗

bioRxiv2023

Starvation induces extracellular accumulation of polyphosphate in Dictyostelium discoideum to inhibit macropinocytosis, phagocytosis, and exocytosis

Dictyostelium discoideum is a soil-dwelling unicellular eukaryote that accumulates extracellular polyphosphate (polyP). At high cell densities, when the cells are about to overgrow their food supply and starve, the corresponding high extracellular concentrations of polyP allow the cells to preemptively anticipate starvation, inhibit proliferation, and prime themselves to begin development. In this report, we show that starved D. discoideum cells accumulate cell surface and extracellular polyP. Starvation reduces macropinocytosis, exocytosis, and phagocytosis, and we find that these effects require the G protein-coupled polyP receptor (GrlD) and two enzymes, Polyphosphate kinase 1 (Ppk1), which is required for synthesizing intracellular polyP, cell surface polyP, and some of the extracellular polyP, and Inositol hexakisphosphate kinase (I6kA), which is required for cell surface polyP and polyP binding to cells, and some of the extracellular polyP. PolyP reduces membrane fluidity, and we find that starvation reduces membrane fluidity, and this effect requires GrlD and Ppk1 but not I6kA. Together, these data suggest that in starved cells, extracellular polyP decreases membrane fluidity, possibly as a protective measure. In the starved cells, sensing polyP appears to decrease energy expenditure from ingestion, and decrease exocytosis, to both decrease energy expenditures and retain nutrients.

cell biology↗

bioRxiv2022

Inhibiting a mRNA motif binding protein that mediates TGF-β1 upregulation of translation attenuates pulmonary fibrosis in mice

In human lung cells, the profibrotic cytokine TGF-{beta}1 increases sialidase 3 (NEU3) protein by increasing NEU3 translation without increasing levels of NEU3 mRNA. To elucidate how TGF-{beta}1 regulates translation, we treated human lung fibroblasts (HLF) with TGF-{beta}1 and used proteomics and RNA-seq to determine the effect of TGF-{beta}1 on proteins, mRNAs, and mRNA polysome/monosome ratios. We identified 181 mRNAs where TGF-{beta}1 also increases translation to increase protein levels without significantly affecting mRNA levels. These mRNAs share a common 20 nucleotide motif. Deletion or insertion of this motif in mRNAs eliminates or induces the TGF-{beta}1 regulation of translation. At least 5 RNA-binding proteins including DDX3 bind the RNA motif, and TGF-{beta}1 regulates their protein levels and/or binding to the motif. Inhibiting DDX3, either by siRNA or small molecule inhibitors, reduced TGF-{beta}1 induced NEU3 levels. In the mouse bleomycin model of pulmonary fibrosis, injections of the DDX3 inhibitor RK-33 starting 10 days after bleomycin potentiated survival and reduced lung inflammation, fibrosis, and lung tissue levels of DDX3, TGF-{beta}1, and NEU3. Together, these results suggest that TGF-{beta}1 regulates RNA-binding proteins that interact with a mRNA motif that is necessary and sufficient for TGF-{beta}1 to regulate mRNA translation, and that blocking this effect can reduce fibrosis.

immunology↗

bioRxiv2022

Changes in lung sialidases in male and female mice after bleomycin aspiration

Aim of the studySialidases, also called neuraminidases, are enzymes that cleave terminal sialic acids from glycoconjugates. In humans and mice, lung fibrosis is associated with desialylation of glycoconjugates and upregulation of sialidases. There are four mammalian sialidases, and it is unclear when the four mammalian sialidases are elevated over the course of inflammatory and fibrotic responses, whether tissue resident and inflammatory cells express different sialidases, and if sialidases are differentially expressed in male and females. Materials and MethodsTo determine the time course of sialidase expression and the identity of sialidase expressing cells, we used the bleomycin model of pulmonary fibrosis in mice to examine levels of sialidases during inflammation (days 3 - 10) and fibrosis (days 10 - 21). ResultsBleomycin aspiration increased sialidase NEU1 at days 14 and 21 in male mice and day 10 in female mice. NEU2 levels increased at day 7 in male and day 10 in female mice. NEU3 appears to have a biphasic response in male mice with increased levels at day 7 and then at days 14 and 21, whereas in female mice NEU3 levels increased over 21 days. In control mice, the sialidases were mainly expressed by EpCAM positive epithelial cells, but after bleomycin, epithelial cells, CD45 positive immune cells, and alveolar cells expressed NEU1, NEU2, and NEU3. Sialidase expression was higher in male compared to female mice. There was little expression of NEU4 in murine lung tissue. ConclusionsThese results suggest that sialidases are dynamically expressed following bleomycin, that sialidases are differentially expressed in male and females, and that of the four sialidases only NEU3 upregulation is associated with fibrosis in both male and female mice.

immunology↗

bioRxiv2022

The extracellular sialidase NEU3 induces neutrophil priming

Some extracellular glycoconjugates have sialic acid as the terminal sugar, and sialidases are enzymes that remove this sugar. Mammals have four sialidases, but their biological functions are unclear. In this report, we show that incubation of human neutrophils with the human sialidase NEU3, but not NEU1, NEU2 or NEU4, inducess human male and female neutrophils to change from a round to a more amoeboid morphology, causes the primed neutrophil markers CD66, CD11B, and CD18 to localize to the cell cortex, and decreases the localization of the unprimed neutrophil markers CD43 and CD62L at the cell cortex. NEU3, but not the other 3 sialidases, also causes human male and female neutrophils to increase their F-actin content. The inhibition of NEU3 by the NEU3 inhibitor 2-acetylpyridine attenuated the NEU3 effect on neutrophil morphology, indicating that the effect of NEU3 is dependent on its enzymatic activity. Together, these results indicate that NEU3 can prime human male and female neutrophils, and that NEU3 is a potential regulator of inflammation.

immunology↗

bioRxiv2022

The sialidase NEU3 promotes pulmonary fibrosis in mice

Sialic acid is often the distal sugar on glycoconjugates, and sialidases are enzymes that remove this sugar. In fibrotic lesions in human and mouse lungs, there is extensive desialylation of glycoconjugates, and upregulation of sialidases including the extracellular sialidase NEU3. In the bleomycin model of pulmonary fibrosis, mice lacking NEU3 (Neu3-/-) showed strongly attenuated bleomycin-induced weight loss, lung damage, inflammation, upregulation of TGF-{beta}1, and fibrosis. This indicates that NEU3 is necessary for the full spectrum of bleomycin-induced pulmonary fibrosis. To determine if NEU3 is sufficient for fibrosis, mice not treated with bleomycin were treated with recombinant murine NEU3 or inactive NEU3. Aspiration of NEU3 caused inflammation and fibrosis in the lungs, while inactive NEU3 caused inflammation but not fibrosis. Mice were also treated with NEU3 starting 10 days after bleomycin. In male but not female mice, NEU3 increased inflammation and fibrosis. Inactive NEU3 did not enhance bleomycin-induced lung fibrosis. These results suggest that NEU3 is sufficient to induce fibrosis in the lungs, and that this effect is mediated by NEU3s enzymic activity.

immunology↗

bioRxiv2020

Serum Amyloid P inhibits single stranded RNA-induced lung inflammation, lung damage, and cytokine storm in mice

SARS-CoV-2 is a single stranded RNA (ssRNA) virus and contains GU-rich sequences distributed abundantly in the genome. In COVID-19, the infection and immune hyperactivation causes accumulation of inflammatory immune cells, blood clots, and protein aggregates in lung fluid, increased lung alveolar wall thickness, and upregulation of serum cytokine levels. A serum protein called serum amyloid P (SAP) has a calming effect on the innate immune system and shows efficacy as a therapeutic for fibrosis in animal models and clinical trials. In this report, we show that aspiration of the GU-rich ssRNA oligonucleotide ORN06 into mouse lungs induces all of the above COVID-19-like symptoms. Men tend to have more severe COVID-19 symptoms than women, and in the aspirated ORN06 model, male mice tended to have more severe symptoms than female mice. Intraperitoneal injections of SAP starting from day 1 post ORN06 aspiration attenuated the ORN06-induced increase in the number of inflammatory cells and formation of clot-like aggregates in the mouse lung fluid, reduced ORN06-increased alveolar wall thickness and accumulation of exudates in the alveolar airspace, and attenuated an ORN06-induced upregulation of the inflammatory cytokines IL-1{beta}, IL-6, IL-12p70, IL-23, and IL-27 in serum. Together, these results suggest that aspiration of ORN06 is a simple model for both COVID-19 as well as cytokine storm in general, and that SAP is a potential therapeutic for diseases with COVID-19-like symptoms as well as diseases that generate a cytokine storm.

immunology↗