Mapping the Pharyngeal and Intestinal pH of Caenorhabditis elegans and Real-Time Luminal pH Oscillations Using Extended Dynamic Range pH-Sensitive Nanosensors

Abstract

Extended dynamic range pH-sensitive ratiometric nanosensors, capable of accurately mapping the full physiological pH range, have been developed and used to characterize the pH of the pharyngeal and intestinal lumen of Caenorhabditis elegans in real-time. Nanosensors, 40 nm in diameter, were prepared by conjugating pH-sensitive fluorophores, carboxyfluorescein (FAM) and Oregon Green (OG) in a 1:1 ratio, and a reference fluorophore, 5-(and-6)-carboxytetramethylrhodamine (TAMRA) to an inert polyacrylamide matrix. Accurate ratiometric pH measurements were calculated through determination of the fluorescence ratio between the pH-sensitive and reference fluorophores. Nanosensors were calibrated with an automated image analysis system and validated to demonstrate a pH measurement resolution of ±0.17 pH units. The motility of C. elegans populations, as an indicator for viability, showed nematodes treated with nanosensors, for concentrations ranging from 50.00 to 3.13 mg/mL, were not statistically different to nematodes not challenged with nanosensors up to a period of 4 days (p < 0.05). The nanosensors were also found to remain in the C. elegans lumen >24 h after nanosensor challenge was removed. The pH of viable C. elegans lumen was found to range from 5.96 ± 0.31 in the anterior pharynx to 3.59 ± 0.09 in the posterior intestine. The pharyngeal pumping rate, which dictates the transfer of ingested material from the pharynx to the intestine, was found to be temperature dependent. Imaging C. elegans at 4 °C reduced the pharyngeal pumping rate to 7 contractions/min and enabled the reconstruction of rhythmic pH oscillations in the intestinal lumen in real-time with fluorescence microscopy.

The Contribution of Candida albicans Vacuolar ATPase Subunit V 1 B, Encoded by VMA2 , to Stress Response, Autophagy, and Virulence Is Independent of Environmental pH

“…Therefore, we tested tetR-VMA2 virulence in a C. elegans model of infection (34). C. elegans is an ideal organism for the study of virulence in C. albicans vma mutants, as the pH of the larynx and intestine of C. elegans nematodes, where C. albicans infection occurs (34,35), ranges from 3.5 to 5.5 (57). For these studies, we analyzed infection at the permissive pH and temperature, under acidic pH conditions (pH 4.0) and at 30°C, because tetR-VMA2 is unable to grow at neutral pH and low temperatures (i.e., 25°C).…”

“…The infection culminates in C. albicans hyphae piercing the C. elegans cuticle and killing the worm (35). Because the C. elegans gastrointestinal tract and pharynx have very low pH, ranging from pH 5.5 in the pharynx to pH 3.5 in the lower intestine (57), C. elegans is an ideal model to study V-ATPasemediated pathogenesis in vivo. We tested the contribution of VMA2 to C. albicans virulence in an acidic environment, i.e., under pH conditions permissible for growth of V-ATPase-deficient cells.…”

In vivo testing of gold nanoparticles using the Caenorhabditis elegans model organism

“…3.5 in the posterior part. [45] In vitro experiments showed that the combination of acidic pH and ionic strength of the citrate buffer pH 4.6 promoted the aggregation and precipitation of the AuNPs ( Figure S5). Therefore, these conditions may compromise AuNP stability in the most posterior part of the alimentary system.…”

Development of gold nanoparticles coated with silica containing the antibiofilm drug cinnamaldehyde and their effects on pathogenic bacteria

“…58,59 In another study, the presence of digestive enzymes and acidic environment of the digestive system of C. elegans were found to dissolve metal nanoparticles and thus have toxic effects on C. elegans. 60,61 However, in the case of CNMA-GNPs, the silica coating must be digested before the gold core is accessible to the intestinal environment, which suggests that the silica coating on CNMA-GNPs protects the bare GNPs and thus decreases potential toxicity issues.…”