Biology subjects

Rijal, R.

Publications and source records attributed to Rijal, R..

8 recordsLinked to original sources

bioRxiv2026

Identification of novel inhibitors of Mycobacterium smegmatis growth through genome-wide overexpression of Cluster P3 mycobacteriophage Xavia genes

We examined whether genes encoded by the mycobacteriophage Xavia disrupt growth of Mycobacterium smegmatis, a widely used mycobacterial model. Seventy-one Xavia genes were individually expressed using an inducible plasmid system and assessed for effects on colony formation. Two genes were lethal even without induction, indicating toxicity under basal expression. Induction of sixteen additional genes reduced bacterial growth, spanning structural proteins, lysogeny regulators, DNA-associated enzymes, a lysis protein, and several genes with no known function. These findings expand functional insights into mycobacteriophage gene repertoires and identify candidates for future mechanistic studies. AbstractBacteriophage genomes encode large numbers of genes with no known function, and many of these genes affect essential host processes when expressed in a heterologous system. For mycobacteriophages, genome-wide overexpression in Mycobacterium smegmatis provides a direct way to identify proteins that impair growth and to determine which mycobacterial pathways are sensitive to phage gene products. To evaluate the cytotoxic potential of the Cluster P3 phage Xavia, a lineage that has not undergone functional screening, we constructed an arrayed pExTra library containing 71 predicted Xavia genes under control of the anhydrotetracycline inducible promoter pTet. All constructs were sequence-verified and transformed into M. smegmatis, and induction allowed measurement of gene-specific effects on growth. Two genes prevented recovery of transformants, suggesting toxicity under basal promoter leakiness. Inducible expression of 16 additional genes impaired growth, and these inhibitory proteins include structural components, regulators of lysogeny, enzymes of DNA metabolism, a lysis factor, and several proteins with no known function. Four of the strongest inhibitors were genes with no known function. These results extend functional screening into the previously untested P3 branch of Actinobacteriophages and identify new proteins that require mechanistic analysis.

microbiology↗

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

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

PTEN and the PTEN-like phosphatase CnrN have both distinct and overlapping roles in a Dictyostelium chemorepulsion pathway

The directed movement of eukaryotic cells is crucial for processes such as embryogenesis and immune cell trafficking. The enzyme Phosphatase and tensin homolog (PTEN) dephosphorylates phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Dictyostelium discoideum cells require both PTEN and the PTEN-like phosphatase CnrN to locally inhibit Ras activation to induce biased movement of cells away from the secreted chemorepellent protein AprA. Both PTEN and CnrN decrease basal levels of PI(3,4,5)P3 and increase basal numbers of macropinosomes, and AprA prevents this increase. AprA requires both PTEN and CnrN to increase PI(4,5)P2 levels, decrease PI(3,4,5)P3 levels, inhibit proliferation, decrease myosin II phosphorylation, and increase filopod sizes. AprA causes PTEN, similar to CnrN, to localize to the side of the cell towards AprA in an AprA gradient. However, PTEN and CnrN also have distinct roles in some signaling pathways. PTEN, but not CnrN, decreases basal levels of PI(4,5)P2, AprA requires PTEN, but not CnrN, to induce cell roundness, and CnrN and PTEN have different effects on the number of filopods and pseudopods, and the sizes of filopods. Together, our results suggest that CnrN and PTEN play unique roles in D. discoideum signaling pathways, and possibly dephosphorylate PI(3,4,5)P3 in different membrane domains, to mediate chemorepulsion away from AprA.

cell biology↗

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

Dictyostelium discoideum cells sense their local density and retain nutrients when the cells are about to overgrow their food source

Dictyostelium discoideum is a unicellular eukaryote that eats bacteria, and eventually overgrows the bacteria. D. discoideum cells accumulate extracellular polyphosphate (polyP), and the polyP concentration increases as the local cell density increases. At high cell densities, the correspondingly high extracellular polyP concentrations allow cells to sense that they are about to overgrow their food supply and starve, causing the D. discoideum cells to inhibt their proliferation. In this report, we show that high extracellular polyP inhibits exocytosis of undigested or partially digested nutrients. PolyP decreases cell membrane fluidity and plasma membrane recycling, and this requires the G protein-coupled polyP receptor GrlD, the polyphosphate kinase Ppk1, and the inositol hexakisphosphate kinase I6kA. PolyP did not affect random cell motility, cell speed, or F-actin levels. PolyP decreased membrane saturated fatty acids and altered lipid and protein contents in detergent-insoluble lipid microdomains. Together, these data suggest that D. discoideum cells use polyP as a signal to sense their local cell density and reduce cell membrane fluidity and membrane recycling, perhaps as a mechanism to retain ingested food when the cells are about to starve.

cell biology↗