The C. elegans CHP1 homolog, pbo-1, functions in innate immunity by regulating the pH of the intestinal lumen

Abstract

Caenorhabditis elegans are soil-dwelling nematodes and models for understanding innate immunity and infection. Previously, we developed a novel fluorescent dye (KR35) that accumulates in the intestine of C. elegans and reports a dynamic wave in intestinal pH associated with the defecation motor program. Here, we use KR35 to show that mutations in the Ca2+-binding protein, PBO-1, abrogate the pH wave, causing the anterior intestine to be constantly acidic. Surprisingly, pbo-1 mutants were also more susceptible to infection by several bacterial pathogens. We could suppress pathogen susceptibility in pbo-1 mutants by treating the animals with pH-buffering bicarbonate, suggesting the pathogen susceptibility is a function of the acidity of the intestinal pH. Furthermore, we use KR35 to show that upon infection by pathogens, the intestinal pH becomes neutral in a wild type, but less so in pbo-1 mutants. C. elegans is known to increase production of reactive oxygen species (ROS), such as H2O2, in response to pathogens, which is an important component of pathogen defense. We show that pbo-1 mutants exhibited decreased H2O2 in response to pathogens, which could also be partially restored in pbo-1 animals treated with bicarbonate. Ultimately, our results support a model whereby PBO-1 functions during infection to facilitate pH changes in the intestine that are protective to the host.

Fig 1. pbo-1 mutations disrupt the intestinal acid wave.

The chemical structures of KR35 under acidic and basic conditions are shown. The protonated ring-opened form is highly fluorescent, and the deprotonated spirocyclic form is non-fluorescent. The intensity map provides the lookup table values for the pixel intensity in each image. Fluorescence micrographs of C. elegans wild type or pbo mutants after feeding on the pH-sensitive probe KR35 for 30 min (10 μM). Time-dependent images during the DMP extracted from video microscopy are shown. Fluorescence is rendered as a heat map of fluorescence intensity with red representing the most intense fluorescence (high acidity), and black the least intense fluorescence (low acidity). The head of the animal is on the left side of each image (scale bar = 50 μm).

Fig 2. pbo-1 loss-of-function increases susceptibility to bacterial pathogens.

(A-D) Survival or longevity of C. elegans wild type versus pbo-1, or pbo-4 mutants placed on nematode growth medium (NGM) with a lawn of E. faecalis (A), E. coli (B), S. aureus (C), or P. aeruginosa (D). In each experiment, 30–100 worms were placed on the pathogen lawn and subsequently transferred every two days to a new NGM plate with a fresh pathogen lawn and monitored for survival. (E) Average relative mortality on pathogen (E. faecalis, P. aeruginosa or S. aureus). Relative mortality is calculated using the lethal time to kill 50% of organisms (LT₅₀) and is the ratio of (wild-type LT₅₀/mutant LT₅₀ on pathogen) over (wild-type LT₅₀/mutant LT₅₀ on E. coli). LT₅₀ and p values for individual experiments used for relative mortality calculations are provided in S1 Table. Relative mortality is shown as the average and standard error of the mean of 3–4 experiments. For each pathogen, statistical analysis by student’s t test compared with wild type: *, p<0.05.

Fig 3. Bicarbonate increases survival of E. faecalis-fed pbo-1 mutants.

(A) Survival of wild-type, pbo-1 or pbo-4 mutants fed E. faecalis on nematode growth medium with 25 mM bicarbonate (bicarbonate NGM). In each experiment, 30 worms were placed on bicarbonate NGM with an E. faecalis lawn, and the worms were subsequently transferred every two days to new bicarbonate NGM plates with a fresh E. faecalis lawn and monitored for survival. (B) Average calculated lethal time to kill 50% of animals (LT₅₀) of 3 independent experiments. LT₅₀ and p values for individual experiments are provided in S2 Table. The average LT₅₀ of WT was not statistically different from either mutant by student’s t-test (p>0.6).

Fig 4. Pathogens alter C. elegans intestinal pH.

The chemical structures of KR35 and KR54 under acidic and basic conditions are shown. The protonated ring-opened form is highly fluorescent, and the deprotonated spirocyclic form is non-fluorescent. Fluorescence micrographs of C. elegans wild type or pbo-1 mutant fed E. coli or pathogen (E. faecalis or P. aeruginosa), followed by administration of KR35 (10 μM) for 30 min or KR54 (10 μM) for 10 minutes. Time-dependent images during the DMP extracted from video microscopy are shown. Fluorescence is rendered as a heat map of fluorescence intensity with red representing the most intense fluorescence (high acidity), and black the least intense fluorescence (low acidity) and the map of the intensity values is shown for each fluorophore. The head of the animal is on the left side of each image. (scale bar = 50 μm).

Fig 5. Pathogen ingestion results in intestinal neutralization in wild-type animals.

We used laser-scanning confocal microscopy to assess KR35 fluorescence in N2 animals fed E. coli (A), E. faecalis (B) or P. aeruginosa (C). Below the panels for each pathogen is a plot of the pixel intensity of a line drawn from anterior (left) to posterior along the entire intestine. Ingestion of either E. faecalis or P. aeruginosa results in reduced KR35 fluorescence, suggesting the pH is more neutral in the intestines of each single animal observed. Scale bars represent 50 μm.

Fig 6. Pathogen ingestion in pbo-1 animals does not change intestinal pH.

We used laser-scanning confocal microscopy to assess KR35 fluorescence in multiple pbo-1(sa7) animals fed E. coli (A), E. faecalis (B) or P. aeruginosa (C). Below the panels for each pathogen is a plot of the pixel intensity of a line drawn from anterior (left) to posterior along the entire intestine. In these animals we did not observe robust differences in KR35 fluorescence, as a consequence of pathogen ingestion, suggesting intestinal pH change in response to pathogen ingestion partially requires pbo-1 function. Scale bars represent 50 μm. pbo-1 mutants exhibit reduced H₂O₂ in response to E. faecalis.

Fig 7. Amplex Red H₂O₂ measurements in E. faecalis-fed worms.

(A) PBO-1, but not PBO-4, is important for H₂O₂ production in response to E. faecalis. (B) H₂O₂ is restored to a pbo-1 mutant by adding bicarbonate (NaHCO₃) to the nematode growth medium. (C) RNAi downregulation of bli-3 does not further decrease H₂O₂ production in E. faecalis-fed pbo-1 mutants. Worms were exposed to bli-3 RNAi E. coli mixed with the E. coli empty vector strain at a 1/10 dilution for 3 days prior to feeding with E. faecalis. Results are of three independent experiments with 30 worms each. Error bars represent standard deviation. **, statistically different by student’s t-test (p < 0.01). For part B, wild type and pbo-1 were not statistically different on NGM + bicarbonate (p > 0.4). For part C, pbo-1 mutants exposed to bli-3 RNAi were not statistically different from those exposed to the vector control (p > 0.5).