Modulation of bacterial Type III secretion system by a spermidine transporter dependent signaling pathway

Abstract

Background: Many gram-negative bacterial pathogens employ Type III secretion systems (T3SS) to inject effector proteins into host cells in infectious processes.

Methodology/principal findings: By screening a transposon mutant library of P. aeruginosa, we found that mutation of spuDEFGH, which encode a major spermidine uptake system, abolished the expression of the exsCEBA operon that codes for key T3SS regulators under inducing conditions (low calcium). Whole genome microarray analysis revealed that inactivation of the spermidine uptake system significantly decreased the transcriptional expression of most, if not all, T3SS genes. Consistently, the spermidine uptake mutants showed decreased expression of the T3SS genes in responding to host cell extract and attenuated cytotoxicity. Furthermore, exogenous addition of spermidine to the wild type strain PAO1 enhanced the expression of exsCEBA and also the effector protein genes.

Conclusion/significance: Cumulatively, these data have depicted a novel spermidine transporter-dependent signaling pathway, which appears to play an essential role in modulation of T3SS expression in P. aeruginosa.

Figure 1. Mutation of the genes encoding the SpuDEFGH transporter decreased the expression of T3SS genes.

(A) The exsCEBA expression patterns of five transposon mutants under normal and low calcium condition. (B) Time course analysis of the exsCEBA expression in strain PAO1 and in the spermidine transporter mutants. (C) Deletion of the transporter abolished the low calcium-induced expression of exsCEBA. (D) Deletion of the transporter abolished the low calcium-induced expression of exoT. Bacteria were grown in LB medium with or without 7.5 mM NTA, indicated by solid and open bar/symbol, respectively. The experiment was repeated twice and the data were the means of three replicates.

Figure 2. Inactivation of the spermidine transporter led to decreased transcription of T3SS genes.

Top panel shows the relative location and size of the genes required for secretion, translocation and T3SS-related regulatory activities, which are located contiguously on the chromosome within five operons (based on the genome sequence data of strain PAO1, http://prodoric.tu-bs.de/gsearch.php). The ExsA-dependent promoter and transcriptional direction are indicated by diamond and arrow, respectively. Bottom panel indicates the transcriptional fold changes of these T3SS genes in the deletion mutant ΔspuE in comparison with the wild type strain PAO1. The transcriptional changes of three effector genes, i.e., exoT, exoY, and exoS, which are located elsewhere on the chromosome, were described in the text.

Figure 3. In trans expression of exsA restored the T3SS gene expression and effector production in the spermidine transporter mutants.

(A) Inactivation of the spermidine transporter inhibited the production of the type III effector protein ExoS but the phenotype was rescued by expression of exsA. Bacterial cultures were grown overnight in LB medium with or without 7.5 mM NTA and the extra-cellular proteins in supernatants were collected by trichloroacetic acid precipitation and separated by 10% SDS-PAGE. The proteins were transferred onto nitrocellulose membrane and blotted with anti-ExoS antibody (upper panel). (B) Overexpression of exsA in the transporter mutants resulted in a high level expression of the T3SS genes exsCEBA. Symbol: “+” and “−” indicate whether the plasmid or expression construct was carried by the bacterial strains. The data were the means of three replicates.

Figure 4. Exogenous spermidine induction of the T3SS system required a functional SpuDEFGH transporter.

(A) The exsCEBA promoter directed β-galactosidase activity. Results were the means of three replicates. (B) Immunoblotting detection of the effector ExoS. Bacterial cultures were grown in LB medium supplemented with NTA and spermidine as indicated. The extra-cellular proteins (ECP) and intra-cellular proteins (ICP) from strains PAO1 (P) and ΔspuE (ΔE) were separated by 10% SDS-PAGE. The proteins were transferred onto nitrocellulose membrane and blotted with anti-ExoS antibody. The experiment was repeated for at least three times with similar results.

Figure 5. Inactivation of the spermidine transporter reduced the host cell extract-dependent expression of exsCEBA and attenuated the T3SS-mediated cytotoxicity.

(A) The exsCEBA promoter directed β-galactosidase activity after culture of the bacterial cells for 4 h in minimum medium with various amounts of mouse liver extract (indicated as equivalent to liver tissue fresh weight per ml culture). The bacteria grown in the same minimum medium was used as a blank control. (B) Cytotoxicity was assayed by monitoring LDH release by the HeLa cells infected with a MOI of 40–50. Experiments were performed with DMEM medium supplemented with 1% FBS and 2 mM glutamine. The data were the means of at least three replicates.