The Pseudomonas aeruginosa magnesium transporter MgtE inhibits transcription of the type III secretion system

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that causes life-long pneumonia in individuals with cystic fibrosis (CF). These long-term infections are maintained by bacterial biofilm formation in the CF lung. We have recently developed a model of P. aeruginosa biofilm formation on cultured CF airway epithelial cells. Using this model, we discovered that mutation of a putative magnesium transporter gene, called mgtE, led to increased cytotoxicity of P. aeruginosa toward epithelial cells. This altered toxicity appeared to be dependent upon expression of the type III secretion system (T3SS). In this study, we found that mutation of mgtE results in increased T3SS gene transcription. Through epistasis analyses, we discovered that MgtE influences the ExsE-ExsC-ExsD-ExsA gene regulatory system of T3SS by either directly or indirectly inhibiting ExsA activity. While variations in calcium levels modulate T3SS gene expression in P. aeruginosa, we found that addition of exogenous magnesium did not inhibit T3SS activity. Furthermore, mgtE variants that were defective for magnesium transport could still complement the cytotoxicity effect. Thus, the magnesium transport function of MgtE does not fully explain the regulatory effects of MgtE on cytotoxicity. Overall, our results indicate that MgtE modulates expression of T3SS genes.

FIG. 1.

Mutation of mgtE leads to increased effector molecule secretion through T3SS. (A) Western immunoblotting of supernatants from planktonic cultures of strain PA14 (WT) and its isogenic mgtE deletion mutant. Supernatants were probed with antibodies to ExoU and PcrV in the presence and absence of the calcium chelator EGTA. (B) Cytotoxicity of wild-type PA14 (WT), with vector control (p70, or pMQ70) plasmid or with a plasmid carrying mgtE (pmgtE, or pSMC291), toward CFBE cells at 6 h postinoculation. The value of 100% cytotoxicity represents a maximum release from the CFBE cells by Triton X-100 lysis. *, P < 0.05. (C) Relative exsA expression by qRT-PCR in 6-h coculture biofilms formed by the wild type (WT) with vector control (p70) or the wild type expressing mgtE from plasmid pmgtE. fbp transcript levels were used as a normalization control. *, P < 0.05.

FIG. 2.

Mutation of mgtE results in small increases in T3SS transcription. (A) Transcription of T3SS was measured as exsA transcript levels by qRT-PCR in wild-type PA14 (WT) and the ΔmgtE strain grown as biofilms in coculture with CFBE cells. The y axis measures relative exsA transcript levels, using fbp transcript levels as a normalization control (see Materials and Methods). Data represent the average of three independent experiments, each containing triplicate or quadruplicate samples. Each of these samples was assayed in triplicate by qRT-PCR. *, P < 0.05. (B) β-Galactosidase activity was measured from the P_exsD-lacZ construct in wild-type PA14 (WT) and the ΔmgtE strain grown as biofilms in coculture with CFBE cells. The y axis represents relative β-galactosidase activity from the P_exsD-lacZ transcriptional fusion construct, normalized to the number of CFU (see Materials and Methods). Data are representative of three independent experiments. *, P < 0.05. (C) Transcription of T3SS was also measured in broth cultures as β-galactosidase activity (Miller units) using the P_exsD-lacZ fusion construct in wild-type PA14 (WT) and the isogenic mgtE deletion mutant. Transcriptional activity was assessed in the presence and absence of 1 mM EGTA, as indicated. Replicate samples were additionally treated with 20 mM MgCl₂, as indicated. *, P < 0.05, compared to EGTA-stimulated WT cells without magnesium; #, P < 0.05, compared to EGTA-stimulated ΔmgtE cells without magnesium; †, P < 0.05 compared to EGTA-stimulated WT cells with magnesium.

FIG. 3.

Cytotoxicity effects of MgtE require ExsA-mediated gene transcription. Wild-type PA14 (WT), the mgtE deletion mutant, the exsA deletion mutant, and the double deletion strain ΔmgtE ΔexsA were tested for cytotoxicity toward CFBE cells at 6 h. The value of 100% cytotoxicity represents a maximum release from the CFBE cells by Triton X-100 lysis. *, P < 0.05, compared to the mgtE mutant.

FIG. 4.

MgtE expression impacts the Exs regulatory system. Cytotoxicity assays (A, C, and E) and expression levels of exsA (B, D, and F) were examined in the exsC mutant and the ΔmgtE ΔexsC double mutant (A and B), the exsE mutant complemented with vector control (p70) or pmgtE (C and D), and the exsED double mutant complemented with vector control or pmgtE (E and F). Results were compared to wild-type PA14 (WT) and the isogenic mgtE deletion mutant. Relative exsA expression was determined by qRT-PCR of coculture biofilms at 6 h postinoculation, as described in Materials and Methods. A value of 100% cytotoxicity represents a maximum release from the CFBE cells by Triton X-100 lysis. *, P < 0.05, compared to WT and mgtE strains (A and B), exsE p70 (C), exsED p70 (E), and WT p70 and mgtE p70 (D and F); #, P < 0.05, compared to exsE p70 (D) or exsED p70 (F).

FIG. 5.

pmgtE inhibits ExoU and PcrV secretion. Western blots of supernatants from wild-type (WT), ΔexsE, and ΔexsED strains grown planktonically and complemented with empty vector (pMQ70) or pmgtE. Cultures were grown in broth and treated with 1 mM EGTA to stimulate T3SS, as described in Materials and Methods.

FIG. 6.

Magnesium transport and cytotoxicity can be separated. (A) Growth of S. Typhimurium MM281 on N minimal agar plates. MM281 was complemented with vector control (p70), pmgtE, and plasmids pCHIS and pNHIS. (B) Cytotoxicity at 6 h of strain PA14 and the isogenic mgtE deletion mutant complemented with the plasmids in panel A. A value of 100% cytotoxicity represents a maximum release from the CFBE cells by Triton X-100 lysis. *, P < 0.05, compared to mgtE p70.

FIG. 7.

Point mutations in the magnesium binding sites of MgtE disrupt magnesium transport but not cytotoxicity. (A) Growth of S. Typhimurium MM281 complemented with vector control (p70), mgtE plasmid (pmgtE), or the mgtE plasmid containing point mutations in magnesium binding sites 2 and 3 (pBS2/3). Strains were grown overnight on N minimal medium agar plates. (B) Cytotoxicity at 6 h of PA14 and the mgtE deletion mutant complemented with the plasmids in panel A. A value of 100% cytotoxicity represents a maximum release from the CFBE cells by Triton X-100 lysis. *, P < 0.05, compared to mgtE p70. (C) Western blot of total membrane fractions (left side) and cytoplasmic fractions (right side) from ΔmgtE transformed with vector control (p70), pNHIS, pBS2/3, and pCHIS. Fractions were incubated with anti-six-histidine (HIS) primary antibodies as well as anti-SadB or anti-SecY primary antibodies to detect cytoplasmic and membrane control proteins, respectively. The presence of the lower, nonspecific bands in SadB lanes (marked by an asterisk) has been noted previously (5). Apparent cytoplasmic presence of SecY has also been previously reported (34). Importantly, there appears to be no cytoplasmic contamination of the membrane fraction.