Type III secretion system expression in oxygen-limited Pseudomonas aeruginosa cultures is stimulated by isocitrate lyase activity

Abstract

Pseudomonas aeruginosa is an opportunistic human pathogen and a common cause of chronic infections in individuals with cystic fibrosis (CF). Oxygen limitation was recently reported to regulate the expression of a major virulence determinant in P. aeruginosa, the type III secretion system (T3SS). Here, we show that expression of the T3SS in oxygen-limited growth conditions is strongly dependent on the glyoxylate shunt enzyme, isocitrate lyase (ICL; encoded by aceA), which was previously shown to be highly expressed in CF isolates. ICL-dependent regulation of the T3SS did not alter the expression level of the master transcriptional regulator, ExsA, but did affect expression of the T3 structural proteins, effectors and regulators (ExsC, ExsD and ExsE). An aceA mutant displayed enhanced biofilm formation during anaerobic growth, which suggested that AceA-dependent modulation of type III secretion might impinge upon the RetS/LadS signalling pathways. Indeed, our data suggest that RetS is able to mediate some of its effects through AceA, as expression of aceA in trans partially restored T3SS expression in a retS mutant. Our findings indicate that AceA is a key player in the metabolic regulation of T3SS expression during oxygen-limited growth of P. aeruginosa. To the best of our knowledge, this is the first demonstration that the T3SS can be regulated by factors that do not affect ExsA expression levels.

Figure 1.

T3SS expression in mutants of the TCA cycle and glyoxylate shunt pathway. (a) Diagram depicting the TCA cycle and glyoxylate shunt pathway. (b) Western blot analysis of PcrV expression in oxygen-limited planktonic cultures of P. aeruginosa PAO1 and the indicated TCA or glyoxylate cycle mutants (sampled at the exponential (PE; 12 h post-inoculation) and stationary (PS; 30 h post-inoculation) phases of growth). Note that as the aceE mutant had a growth defect, PE and PS samples were collected at 24 and 48 h post-inoculation, respectively.

Figure 2.

Effect of ICL activity on T3SS expression. Oxygen-limited planktonic cultures of PAO1 and the indicated TCA or glyoxylate cycle mutants (sampled at PE and PS) were analysed for (a) total isocitrate lyase (ICL) activity, and (b) total pyruvate dehydrogenase (PDH) activity. pr, protein. (c) Transcriptional activities of (i) pS (pSB307) and (ii) pG (pSB308) in PAO1 cultures supplemented with 15 mM itaconic acid (ITA) or 1 mM 3-nitropropionic acid (3NP); pMP220 acted as an empty plasmid control. *p < 0.05.

Figure 3.

T3SS expression in an aceA mutant. T3SS expression in PAO1 and an aceA mutant was investigated by analysing oxygen-limited planktonic cultures (sampled at PE and PS) for the following: (a) T3SS proteins (PcrV, PopN, ExoS and ExsD), as determined by Western blot analysis; (b) transcriptional activities of pG (pJC9), pS (pJC8) and pLP170 (empty plasmid); (c) total ICL activity and corresponding PcrV expression in PAO1 or aceA carrying a plasmid-encoded aceA gene (pJC10) or the empty plasmid (pUCP20). pr, protein. *p < 0.05; **p < 0.02.

Figure 4.

Expression of the master T3 transcriptional regulator (exsA) and other T3S regulators (exsC, exsD and exsE) in an aceA mutant. The origin of diminished T3SS expression in the aceA mutant was investigated: (a) ExsA protein expression in PAO1 and an aceA mutant in oxygen-limited conditions (sampled at PE and PS), as determined by Western blot analysis; (b) RT–PCR analysis of T3SS gene transcripts in PAO1 and aceA: total RNA was isolated from cultures grown under (i) oxygen-limited or (ii) aerobic conditions at PE and PS. 16S rRNA (16S) transcript levels indicate equal loading. Samples lacking RT show that no DNA contamination was present.

Figure 5.

Biofilm formation in TCA and glyoxylate cycle mutants under anaerobic conditions. Anaerobic biofilm formation in (a) PAO1 and the indicated TCA and glyoxylate cycle mutants, and (b) PAO1 and an aceA mutant carrying a plasmid-encoded aceA gene (pJC10) or the empty plasmid (pUCP20). *p < 0.05; ***p < 0.01. (c) Expression of the pelA gene transcript in PAO1 and an aceA mutant, as analysed by RT–PCR analysis of total RNA isolated from oxygen-limited cultures at PE and PS. 16S rRNA (16S) transcript levels indicate equal loading. Samples lacking RT show that no DNA contamination was present.

Figure 6.

Effect of ICL activity on the RetS signalling pathway. T3SS expression and ICL activity were investigated in a retS mutant grown under oxygen-limited conditions. (a) Total ICL activity in PAO1 and mutants in retS, ladS, and aceA (harvested at PS). *p < 0.05. (b) Total ICL activity and corresponding PcrV expression in PAO1 or in a retS mutant carrying a plasmid-encoded aceA gene (pJC10) or empty plasmid control (pUCP20), as indicated, at PE and PS. (c) RT–PCR analysis of the aceA gene transcript in PAO1 and a mutant in retS. Total RNA was isolated from cultures grown under oxygen-limited conditions at PE and PS. 16S rRNA (16S) transcript levels indicate equal loading. Samples lacking RT show that no DNA contamination was present.

Figure 7.

Expression of the RetS signalling pathway in an aceA mutant. (a) RT–PCR analysis of the indicated gene transcripts in PAO1 and an aceA mutant. Total RNA was isolated from cultures grown under oxygen-limited conditions at PE and PS. 16S rRNA (16S) transcript levels indicate equal loading. Samples lacking RT show that no DNA contamination was present. (b) PcrV expression in (i) PAO1 and a ΔrsmA mutant carrying a plasmid-borne aceA gene (pJC10) or empty plasmid control (pUCP20), and (ii) PAO1 and an aceA mutant carrying a plasmid-borne rsmA gene (pP35-rsmA) or empty plasmid control (pP35), as indicated.

Figure 8.

Possible mechanisms for AceA-dependent regulation of T3SS expression in oxygen-limited conditions. AceA-dependent regulation of T3SS expression may involve (i) a direct interaction between AceA and components of the RetS/LadS signalling pathways (e.g. GacS/GacA or RsmY/RsmZ); (ii) modulation of ExsA activity by AceA itself or an AceA-derived metabolite; and/or (iii) direct or indirect modulation of the abundance of one or more metabolites that impinge upon T3SS expression.