Biology subjects

Limper, A. H.

Publications and source records attributed to Limper, A. H..

4 recordsLinked to original sources

bioRxiv2026

Pneumocystis phosphoglucomutase links glucose-phosphate metabolism to β-1,3-glucan synthesis and cell-wall integrity

The fungal cell wall is a dynamic, carbohydrate-rich structure whose composition is closely coordinated with cellular metabolism. Phosphoglucomutase (PGM) interconverts glucose-1-phosphate and glucose-6-phosphate, linking carbon metabolism with UDP-glucose-dependent cell-wall biosynthesis. We previously identified functional Pgm2 homologs from Pneumocystis jirovecii and Pneumocystis murina. Here, using a Saccharomyces cerevisiae pgm2{Delta} complementation model, we examined the relationship between Pgm2 activity, {beta}-1,3-glucan, and cell-wall integrity. Pgm2 deficiency reduced {beta}-1,3-glucan content and altered zymolyase sensitivity without major changes in cell-wall thickness. Bulk RNA sequencing revealed that the pgm2{Delta} mutant exhibited coordinated metabolic and cell-wall transcriptional responses, including increased SLT2/MPK1, RLM1, FKS2/GSC2, GFA1, CHS1, CRH1, SED1, and CWP1 expression and decreased FKS1, GAS1, GAS3, and CHS2 expression. Expression of either Pjpgm2 or Pmpgm2 broadly shifted these responses toward wild-type levels. Selected transcriptional changes were further evaluated by qRT-PCR, which supported the major stress and cell-wall responses identified by RNA sequencing. FKS2/GSC2 showed the same directional pattern, although differences were not statistically significant by qRT-PCR. Despite this compensatory response, Pgm2-deficient cells were markedly more susceptible to sublethal caspofungin, whereas either Pneumocystis Pgm2 homolog restored tolerance toward wild-type levels. These findings link Pgm2-dependent glucose-phosphate metabolism with {beta}-1,3-glucan levels and cell-wall function. Given the importance of {beta}-1,3-glucan to the Pneumocystis cyst wall and its limited cell-wall biosynthetic pathways, these findings further support Pgm2 as an important component of Pneumocystis cell-wall biology and a potential antifungal target.

microbiology↗

bioRxiv2022

Targeting Pulmonary Fibrosis by SLC1A5 dependent Glutamine Transport Blockade

The neutral amino acid glutamine plays a central role in TGF-{beta}-induced myofibroblast activation and differentiation. Cells take up glutamine mainly through a transporter expressed on the cell surface known as solute carrier SLC1A5. In this current work, we demonstrated that profibrotic actions of TGF-{beta} are mediated, at least in part, through a metabolic maladaptation of SLC1A5 and targeting SLC1A5 abrogates multiple facets of fibroblast activation. This approach could thus represent a novel therapeutic strategy to treat fibroproliferative diseases. We found that SLC1A5 was highly expressed in fibrotic lung fibroblasts and fibroblasts isolated from IPF lungs. The expression of profibrotic targets, cell migration, and anchorage independent growth by TGF-{beta} required the activity of SLC1A5. Loss or inhibition of SLC1A5 function enhanced fibroblast susceptibility to autophagy, suppressed mTOR, HIF, Myc signaling, and impaired mitochondrial function, ATP production and glycolysis. Pharmacological inhibition of SLC1A5 by small molecule inhibitor V-9302 shifted fibroblast transcriptional profiles from profibrotic to fibrosis resolving, and attenuated fibrosis in a bleomycin treated mouse model of lung fibrosis. This is the first study, to our knowledge, to demonstrate the utility of a pharmacological inhibitor of glutamine transport in fibrosis, laying a framework for new paradigm-shifting therapies targeting cellular metabolism for this devastating disease.

molecular biology↗

bioRxiv2021

Gene Expression in Lung Epithelial Cells Following Interaction with Pneumocystis carinii and its Specific Life Forms Yields Insights into Host Gene Responses to Infection

Pneumocystis spp. interacts with epithelial cells in the alveolar spaces of the lung. It is thought that the binding of Pneumocystis to host cell epithelium is needed for life cycle completion and proliferation. The effect of this interaction on lung epithelial cells have previously shown that the trophic form of this organism greatly inhibits p34cdc2 activity, a serine/threonine kinase required for transition from G2 to M phase in the cell cycle. To gain further insight into the host response during Pneumocystis pneumonia (PCP), we used microarray technology to profile epithelial cell (A549) gene expression patterns following Pneumocystis carinii interaction. Furthermore, we isolated separate populations of cyst and trophic forms of P. carinii, which were then applied to the lung epithelial cells. Differential expression of genes involved in various cellular functions dependent on the specific P. carinii life form in contact with the A549 cell were identified. The reliability of our data was further confirmed by Northern blot analysis on a number of selected up or down regulated transcripts. The transcriptional response to P. carinii was dominated by cytokines, apoptotic, and anti-apoptotic related genes. These results reveal several previously unknown effects of P. carinii on the lung epithelial cell and provide insight into the complex interactions of host and pathogen.

microbiology↗

bioRxiv2020

Inhibition of fibrosis with multi-agent therapy in pulmonary fibrosis: Results of a drug library screening

AimsSuccessful management of IPF will likely require multi-drug therapy as its pathogenesis is thought to be both driven by both pro-inflammatory and pro-fibrotic pathways. We hypothesized that the available anti-fibrotic agents, pirfenidone and nintedanib, may exhibit synergy in suppressing lung fibroblast extracellular matrix protein generation when administered in combination with other orally active agents. Materials and MethodsA fibroblastic cell line (AKR-2B) was stimulated with TGF-{beta}1 and used to screen a library of over 1500 FDA approved drugs. Extracellular matrix protein generation was assessed via fibronectin ELISA assay and maintenance of cell viability confirmed with XTT assay. ResultsThe screening revealed sixty-two drugs from the repurposed drug-screening library that were shown to significantly suppress fibronectin expression and not result in cell death. Specifically drugs within the category of NSAIDs, steroids, azole antifungal agents, and antipyrine were associated with significant suppression of fibronectin on ELISA analysis. Surprisingly, we observed anti-fibrotic activity across a number of the azole antifungal compounds. We next assessed whether combination of azoles would exhibit synergy when combined with current anti-fibrotic therapies in the stimulated fibroblasts. As proof of concept, we demonstrated in vitro synergy between oxiconazole and nintedanib in suppressing fibroblast generation of extracellular matrix fibronectin. ConclusionsThese results suggest an approach to identify potential combinations of therapy that may improve patient outcomes by reducing cost and potential toxicities during treatment.

cell biology↗