Biology subjects

Rijal, R.

Publications and source records attributed to Rijal, R..

4 recordsLinked to original sources

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↗