Indole Inhibits IncP-1 Conjugation System Mainly Through Promoting korA and korB Expression

Abstract

Indole works as an interspecies signal molecule to regulate multiple physiological activities, like antibiotic resistance, acid resistance, and virulence. However, the effect of indole on conjugation is unknown. Here, with Escherichia coli SM10λπ as a donor strain that carries a chromosomally integrated conjugative RP4 plasmid, we explored the effect of indole on conjugation of a mobilizable pUCP24T plasmid imparting gentamycin resistance. The results showed that exogenous indole treatment inhibited conjugative transfer of pUCP24T from SM10λπ to recipient strains, Pseudomonas aeruginosa PAO1 and E. coli EC600. Furthermore, raising endogenous indole production through overexpression of TnaA, a tryptophanase, in SM10λπ significantly inhibited both SM10λπ-PAO1 and SM10λπ-EC600 conjugation, whereas deficiency of tnaA reversed the phenotype. Subsequent mechanistic studies revealed that exogenous indole significantly inhibited the expression of mating pair formation gene (trbB) and the DNA transfer and replication gene (trfA), mainly due to the promotion of regulatory genes (korA and korB), and the result was confirmed in tnaA knockout and overexpression strains. Additionally, we found that both extracellular indole production and tnaA expression of SM10λπ were downregulated by ciprofloxacin (CIP). Intriguingly, one-eighth minimum inhibitory concentration of CIP treatment clearly facilitated both SM10λπ-PAO1 and SM10λπ-EC600 conjugation, and indole inhibited CIP-induced conjugation frequency. These data suggest that indole may play a negative role in the process of CIP-induced conjugation. This is the first study to reveal the biological function of indole-inhibiting conjugation and its role in CIP-induced conjugation, which may be developed into a new way of controlling the spread of antibiotic resistance.

Keywords: E. coli; antibiotic resistance; ciprofloxacin; conjugation; indole.

FIGURE 1

Indole inhibited SM10λπ-PAO1 and SM10λπ-EC600 conjugation. (A) Effect of exogenous indole on SM10λπ-PAO1 and SM10λπ-EC600 conjugation. Donor SM10λπ and recipient PAO1 or EC600 cells (1 × 10⁷ CFU/ml each) were mated in the presence of indicated concentrations of indole at 37°C for 6 h. (B) Effect of endogenous indole on SM10λπ-PAO1 and SM10λπ-EC600 conjugation. SM10λπ wild-type strain (SM10λπ-vector), tnaA-deficient strain (ΔtnaA-vector), TnaA overexpression strain in wild-type (SM10λπ-TnaA), and tnaA mutation (ΔtnaA-TnaA) were mated with PAO1 or EC600 cells (1 × 10⁷ CFU/ml each), respectively, at 37°C for 6 h. Values are means ± SEMs from at least three independent experiments; ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001.

FIGURE 2

The mRNA expression level of conjugation-associated genes. (A) Effect of exogenous indole on the expression levels of conjugation genes (trbB, trfA, traI, and traJ) and global regulatory genes (korA, korB, and trbA). SM10λπ (1 × 10⁷ CFU/ml) was treated with different concentrations of indole at 37°C for 3 h, followed by real-time PCR analysis. (B) Effect of endogenous indole on the expression of the aforementioned conjugation-associated genes. The indicated SM10λπ strains were mated (1 × 10⁷ CFU/ml) at 37°C for 3 h, followed by real-time PCR analysis. The rpoD gene of SM10λπ was used as an internal control. Values are means ± SEMs from at least three independent experiments; ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001; ns, not significant.

FIGURE 3

Indole inhibited CIP-induced conjugation. (A) Effect of CIP on indole production. SM10λπ was treated with different sub-MICs of CIP at 37°C for 5 h, followed by measurement of extracellular indole. (B) Effect of CIP on tnaA expression. SM10λπ was treated with different sub-MICs of CIP at 37°C for 4 h, followed by real-time PCR analysis of tnaA expression. (C) Effect of CIP on SM10λπ-PAO1 and SM10λπ-EC600 conjugation. SM10λπ was treated with different sub-MICs of CIP at 37°C for 8 h and then mated with PAO1 or EC600 cells at 37°C for 6 h. (D) Indole inhibited CIP-induced conjugation frequency. SM10λπ was treated with 1/8 MIC of CIP at 37°C for 8 h and then mated with PAO1 or EC600 cells in the presence of 500 μM of indole at 37°C for 6 h; meanwhile, overexpression of TnaA in wild-type (SM10λπ-TnaA) was treated with 1/8 MIC of CIP at 37°C for 8 h and then mated with PAO1 or EC600 cells at 37°C for 6 h. Values are means ± SEMs from at least three independent experiments; ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001.