The Role of Neurotransmitters in the Protection of Caenorhabditis Elegans for Salmonella Infection by Lactobacillus

Abstract

Salmonellosis is a common foodborne disease. We previously reported the protection of Caenorhabditis elegans from Salmonella Typhimurium DT104 infection by Lactobacillus zeae LB1. However, the mechanism is not fully understood. C. elegans exhibits behavior plasticity when presented with diverse pathogenic or commensal bacteria. Whether it can exert approach avoidance to S. Typhimurium through altering its neurological activity remains to be determined. In the current study, both the wild type and mutants defective in serotonin or dopamine production of C. elegans were used to investigate olfactory preference of the nematode to L. zeae LB1, DT104, and Escherichia coli OP50 by choice assays, and its resistance to DT104 infection and the protection offered by L. zeae LB1 using a life-span assay. The expression of target genes in C. elegans was also examined by real-time quantitative PCR. Results showed that pre-exposure to L. zeae LB1 did not elicit aversive olfactory behavior of the nematode toward DT104. Both mutants tph-1 and cat-2 succumbed faster than the wild type when infected with DT104. While pre-exposure to L. zeae LB1 significantly increased the survival of both the wild type and mutant tph-1, it provided no protection to mutant cat-2. Supplementation of dopamine resulted in both the resistance of mutant cat-2 to S. Typhimurium infection and the protection from L. zeae LB1 to the same mutant. Gene expression data also supported the observations in the life-span assay. These results suggest that both serotonin and dopamine play a positive role in the host defense of C. elegans to S. Typhimurium infection and that the L. zeae LB1 protection is not dependent on modifying olfactory preference of the nematode but mediated by dopamine that may have involved the regulation of p38-mitogen-activated protein kinase and insulin/insulin-like growth factor signaling pathways.

Keywords: Caenorhabditis elegans; Lactobacillus; Salmonella Typhimurium; neurotransmitters; olfactory behavior.

Figure 1

Two-choice behavior assay of Caenorhabditis elegans. L4 stage worms including the wild type (WT) (N2), mutant tph-1, and mutant cat-2 were treated with Escherichia coli OP50 or Lactobacillus zeae LB1, and positioned in the middle of the assay plate, and exposed to test bacteria with equal distance; then choice index was calculated. (A) Worms were treated with OP50 only (OP50 control). (B) Worms were pretreated with L. zeae LB1. (C) Choice index of L. zeae LB1 against OP50. (D) Choice index of DT104 against L. zeae LB1. (E) Choice index of DT104 against OP50. (F) L4 stage worms were positioned in the middle of the plate and exposed to L. zeae LB1 cells or L. zeae LB1 culture supernatant with equal distance. (G) Choice index of L. zeae LB1 culture supernatant against L. zeae LB1 cells. Choice index of A against B = (number of worms in the lawn of bacterium A – number of worms in the lawn of bacterium B)/total worm number. Results are presented as mean ± S.D. with triplicate measurement.

Figure 2

The potential involvement of serotonin and dopamine in the resistance of Caenorhabditis elegans to Salmonella Typhimurium DT104 infection. The mutants are defective in (A) tph-1 or (B) cat-2. N2: the wild type (WT) of C. elegans. N2/OP50, tph-1/OP50, and cat-2/OP50: the WT or each mutant was treated with OP50 only. N2/OP50-Sal, tph-1/OP50-Sal, and cat-2/OP50-Sal: the WT or each mutant was incubated with OP50 first, followed by DT104 infection in the life-span assay. The number of live worms was recorded daily and calculated as follows: survival rate (%) = (live worms/total worms used) × 100. A worm was considered to be dead when it failed to respond to touch. Results are presented as mean ± S.D. with triplicate measurement.

Figure 3

Effect of Lactobacillus zeae LB1 on the resistance of Caenorhabditis elegans mutants to Salmonella Typhimurium DT104 infection. The mutants are defective in (A) tph-1 or (B) cat-2. N2: the wild type (WT) of C. elegans. N2/OP50, tph-1/OP50, and cat-2/OP50: the WT or each mutant was treated with OP50 only. N2/OP50-Sal, tph-1/OP50-Sal, and cat-2/OP50-Sal: the WT or each mutant was incubated with OP50 first, followed by DT104 infection in the life-span assay; N2/LB1-Sal, tph-1/LB1-Sal, and cat-2/LB1-Sal: the WT or each mutant was incubated with L. zeae LB1 first, followed by DT104 infection in the life-span assay. The number of live worms in each group was recorded daily and calculated as follows: survival rate (%) = (live worms/total worms used) × 100. A worm was considered to be dead when it failed to respond to touch. Results are presented as mean ± S.D. with triplicate measurement.

Figure 4

Dopamine involved in the resistance of mutant cat-2 against Salmonella Typhimurium DT104 infection. Synchronized L1 stage worms of mutant cat-2 were treated with various concentration of dopamine (0, 0.25, 0.5, 0.75, and 1.5 mM) in nematode growth medium (NGM) agar seeded with OP50 until they reached L4 stage. And then the worms were infected with DT104 for 7 days. Then life span was detected. N2: the wild type (WT); cat-2: mutant; Dop0: dopamine at 0 mM; Dop0.25: dopamine at 0.25 mM; Dop0.5: dopamine at 0.5 mM; Dop0.75: dopamine at 0.75 mM; Dop1.5: dopamine at 1.5 mM; OP50Sal: treated with Escherichia coli OP50 followed by DT104 infection; OP50: treated with E. coli OP50 only. Wild-type worms (N2) were used as control. The number of live worms in each group was recorded daily and calculated as follows: survival rate (%) = (live worms/total worms used) × 100. A worm was considered to be dead when it failed to respond to touch. Results are presented as mean ± S.D. with triplicate measurement.

Figure 5

Dopamine resulted in the protection from Lactobacillus zeae LB1 to mutant cat-2 against Salmonella Typhimurium DT104 infection. Synchronized L1 stage worms of mutant cat-2 were treated with various concentration of dopamine (0, 0.25, 0.5, 0.75, and 1.5 mM) in nematode growth medium (NGM) agar seeded with OP50 until they reached L4 stage. And then the worms were treated with L. zeae LB1 for 18 h followed by DT104 infection for 7 days. Then life span was detected. N2: the wild type (WT); cat-2: mutant; Dop0: dopamine at 0 mM; Dop0.25: dopamine at 0.25 mM; Dop0.5: dopamine at 0.5 mM; Dop0.75: dopamine at 0.75 mM; Dop1.5: dopamine at 1.5 mM; LB1Sal: treated with L. zeae LB1 for 18 h followed by DT104 infection for 7 days; OP50: treated with E. coli OP50 only. Wild-type worms (N2) were used as control. The number of live worms in each group was recorded daily and calculated as follows: survival rate (%) = (live worms/total worms used) × 100. A worm was considered to be dead when it failed to respond to touch. Results are presented as mean ± S.D. with triplicate measurement.

Figure 6

Gene expression response in dopamine-involved protection from Lactobacillus zeae LB1 to mutant cat-2 against Salmonella Typhimurium DT104 infection. (A) Gene expression in L4 stage mutant cat-2 treated with or without dopamine. cat-2/L4 stage: L4 stage cat-2 worms; cat-2+Dop/L4 stage: L4 stage cat-2 worms supplemented 1.5 mM of dopamine. (B) Gene expression in mutant cat-2 infected with DT104 treated with or without dopamine. cat-2/OP50+Sal 2 Days: L4 stage cat-2 worms were incubated with OP50 for 18 h and then infected with DT104 for 48 h; cat-2+Dop/OP50+Sal 2 Days: L4 stage cat-2 worms were incubated with OP50 for 18 h and then infected with DT104 for 48 h, and 1.5 mM of dopamine was supplemented in the entire procedure. (C) Gene expression in mutant cat-2 treated with or without dopamine that was also pretreated with L. zeae LB1. cat-2/LB1+Sal 2 Days: L4 stage cat-2 worms were pretreated with L. zeae LB1 for 18 h and then infected with DT104 for 48 h; cat-2+Dop/LB1+Sal 2 Days: L4 stage cat-2 worms were pretreated with L. zeae LB1 for 18 h and then infected with DT104 for 48 h, and 1.5 mM of dopamine was supplemented in the entire procedure. ΔCt represents the difference between the Ct value with the primers to a target gene and the Ct value to housekeeping genes. The ΔΔCt represents the difference between the ΔCt value of mutant cat-2 group either in the presence or absence of dopamine and the ΔCt value of the wild type (WT) control group with the same treatment. The values derived from 2^−ΔΔCt represent fold changes of mutant cat-2 groups in abundance relative to the WT group. The WT control groups had the 2^−ΔΔCt value of 1. Results are presented as mean ± S.D. with triplicate measurement. *P ≤ 0.05 for the same gene among different treatments, and **P ≤ 0.01.