Regulation of acetyl-CoA synthetase transcription by the CrbS/R two-component system is conserved in genetically diverse environmental pathogens

Abstract

The CrbS/R two-component signal transduction system is a conserved regulatory mechanism through which specific Gram-negative bacteria control acetate flux into primary metabolic pathways. CrbS/R governs expression of acetyl-CoA synthase (acsA), an enzyme that converts acetate to acetyl-CoA, a metabolite at the nexus of the cell's most important energy-harvesting and biosynthetic reactions. During infection, bacteria can utilize this system to hijack host acetate metabolism and alter the course of colonization and pathogenesis. In toxigenic strains of Vibrio cholerae, CrbS/R-dependent expression of acsA is required for virulence in an arthropod model. Here, we investigate the function of the CrbS/R system in Pseudomonas aeruginosa, Pseudomonas entomophila, and non-toxigenic V. cholerae strains. We demonstrate that its role in acetate metabolism is conserved; this system regulates expression of the acsA gene and is required for growth on acetate as a sole carbon source. As a first step towards describing the mechanism of signaling through this pathway, we identify residues and domains that may be critical for phosphotransfer. We further demonstrate that although CrbS, the putative hybrid sensor kinase, carries both a histidine kinase domain and a receiver domain, the latter is not required for acsA transcription. In order to determine whether our findings are relevant to pathogenesis, we tested our strains in a Drosophila model of oral infection previously employed for the study of acetate-dependent virulence by V. cholerae. We show that non-toxigenic V. cholerae strains lacking CrbS or CrbR are significantly less virulent than are wild-type strains, while P. aeruginosa and P. entomophila lacking CrbS or CrbR are fully pathogenic. Together, the data suggest that the CrbS/R system plays a central role in acetate metabolism in V. cholerae, P. aeruginosa, and P. entomophila. However, each microbe's unique environmental adaptations and pathogenesis strategies may dictate conditions under which CrbS/R-mediated acs expression is most critical.

Fig 1. CrbS/R homologs control expression of acsA in Pseudomonas aeruginosa.

Quantitative real-time PCR was used to measure acsA transcript abundance in an mxtR sensor kinase mutant (A), erdR response regulator mutant (B), and REC domain mutants of either mxtR (C) or erdR (D) in Pseudomonas aeruginosa. acsA transcript levels were measured relative to the clpX housekeeping protease transcript levels. Strains were transformed with either an empty vector plasmid pPSV38, or a pPSV38-erdR (pErdR) expression vector, as indicated. Statistical significance was determined by comparing results of each mutant strain to the wild-type strain. (*) denotes a P-value less than 0.05, (**) denotes a P-value less than 0.01, and (***) denotes a P-value less than 0.001.

Fig 2. CrbS/R homologs control expression of acsA in Pseudomonas entomophila.

Quantitative real-time polymerase chain reaction was used to measure acsA transcript abundance in a crbS sensor kinase mutant (A), crbR response regulator mutant (B), and REC domain mutants of either crbS (C) or crbR (D) in Pseudomonas entomophila. acsA transcript levels were measured relative to the clpX housekeeping protease transcript levels. Strains were transformed with either an empty vector plasmid, pPSV38, or a pPSV38-crbR (pCrbR) expression vector. Statistical significance was determined by comparing results for each mutant strain to those of the wild-type strain. (*) denotes a P-value less than 0.05, (**) denotes a P-value less than 0.01, and (***) denotes a P-value less than 0.001.

Fig 3. CrbS/R controls expression of acsA in a non-O1/non-O139 strain of V. cholerae.

Expression of the luxCDABE operon driven by the V. cholerae SIO acsA promoter in plasmid pPT002 was measured after 4, 8, and 10 hours of growth, and normalized to OD_600nm. The pBBRlux plasmid carries no promoter sequence. Luminescence was measured in V. cholerae strains carrying in-frame deletions of crbS, crbR, or the receiver domain of crbR (A); in V. cholerae strains with mutations in the putative conserved histidine within the HisKA domain (H798A and H798Q) (B); and in V. cholerae strains carrying either an in-frame deletion of the crbS receiver domain or a mutation within the putative conserved aspartate residue in the crbS receiver domain (D1081A) (C). Results from two biological replicates, each performed in duplicate or triplicate, are shown. Statistical significance was determined by comparing results from each mutant strain to those of the wild-type strain at that time point using the Mann-Whitney test. (*) denotes a P-value less than 0.05, (**) denotes a P-value less than 0.01, and (***) denotes a P-value less than 0.001.

Fig 4. CrbS/R homologs control expression of an sssA homolog in Pseudomonas aeruginosa.

Quantitative real-time polymerase chain reaction was used to measure the transcript abundance of an sssA homolog in an mxtR sensor kinase mutant (A), erdR response regulator mutant (B), and receiver domain mutants of either mxtR (C) or erdR (D) in Pseudomonas aeruginosa. sssA transcript levels were measured relative to the clpX housekeeping protease transcript levels. Strains were transformed with either an empty vector plasmid pPSV38 or a pPSV38-erdR (pErdR) expression vector. Statistical significance was determined by comparing results of each mutant strain to the wild-type strain. (*) denotes a P-value less than 0.05, (**) denotes a P-value less than 0.01, and (***) denotes a P-value less than 0.001.

Fig 5. Homologs of the CrbS/R system are important for Pseudomonas aeruginosa growth on media with acetate as the sole carbon source.

P. aeruginosa strains were inoculated to a starting OD_600nm of 0.03 in M63 minimal media supplemented with 5mM acetate as the sole carbon source. Growth was observed over a 28-hour period, during which cell density was recorded at the indicated time points by measuring optical density at 600 nm (A–B). Growth assays compared an erdR deletion mutant and an erdR receiver domain mutant strain to wild-type P. aeruginosa (A), and compared an mxtR deletion mutant and an mxtR receiver domain mutant strain to wild-type P. aeruginosa (B). P. aeruginosa strains were inoculated to a starting OD_600nm of 0.03 in M63 minimal media supplemented with 5mM acetate and 5mM glucose as carbon sources (C–D). Growth was observed over a 28-hour period, and cell density was measured at an optical density of 600 nm at the indicated time points. Growth assays comparing an erdR deletion mutant and an erdR receiver domain mutant strain to wild-type P. aeruginosa (C). Growth assays comparing an amxtR deletion mutant and an mxtR receiver domain mutant strain to wild-type P. aeruginosa (D). Strains were transformed with either an empty vector plasmid pPSV38 or a pErdR expression vector, as indicated.

Fig 6. Homologs of the CrbS/R system are important for Pseudomonas entomophila growth on media with acetate as the sole carbon source.

Pseudomonas entomophila strains were inoculated to a starting OD_600nm of 0.03 in M63 minimal media supplemented with 5mM acetate as the sole carbon source. Growth was observed over a 28-hour period, during which cell density was measured at an optical density of 600 nm at the indicated time points (A, B). Growth assays comparing a crbS deletion mutant, a crbS receiver domain mutant strain, and an acsA mutant strain to wild-type P. entomophila (A). Growth assays comparing a crbR deletion mutant and a crbR receiver domain mutant strain to wild-type P. entomophila (B). P. entomophila strains were inoculated to a starting OD_600nm of 0.03 in M63 minimal media supplemented with 5mM acetate and 5mM glucose as carbon sources (C, D). Growth was observed over a 28-hour period, and cell density was measured at an OD_600nm at the indicated time points. Growth assays compared a crbS deletion mutant strain, a crbS receiver domain mutant strain, and an acsA mutant strain to wild-type P. entomophila (C). Growth assays comparing a crbR deletion mutant and a crbR receiver domain mutant strain to wild-type P. aeruginosa (D). Strains were transformed with either an empty vector plasmid pPSV38 or a pCrbR expression vector, as indicated.

Fig 7. The CrbS/R system is important for Vibrio cholerae growth on media with acetate as the sole carbon source.

Vibrio cholerae strains were inoculated to a starting OD_600nm of 0.005 in M63 minimal media supplemented with 15mM acetate. Growth was observed over a 36-hour period, and cell density was measured at an OD_600nm at the indicated time points.

Fig 8. The CrbS/R homologs of Pseudomonas aeruginosa do not significantly contribute to virulence towards Drosophila.

Survival of flies fed bacterial strains in Luria Bertani (Miller) broth was monitored for 136 hours in triplicate vials containing 10 flies each. Statistical significance of survival differences associated with each individual mutant strain relative to the wild-type strain was assessed using the log-rank test. None of the mutants displayed virulence phenotypes significantly different from that of the wild-type strain (P<0.05).

Fig 9. The CrbS/R homologs of Pseudomonas entomophila do not play a dominant role in determining virulence towards Drosophila.

Survival of flies fed bacterial strains in Luria Bertani (Miller) broth was monitored for 125 hours in triplicate vials containing 10 flies each. Statistical significance of differences in fly survival associated with each mutant strain relative to the wild-type strain was assessed using the log-rank test. Some strains of Pseudomonas entomophila exhibited slight differences in fly survival; in this assay, the crbSΔREC, acsA, and crbR mutants were associated with significantly different survival rates than was the wild-type strain (P = 0.0076, P = 0.0034, and P = 0.047, respectively) (A, B). However, none of the mutant strains differed significantly from the wild-type strain in all three biological replicates of the assay (S2 Table).

Fig 10. Mortality of Drosophila infected with V. cholerae strain SIO is dependent upon CrbS/R homologs.

Survival of flies fed bacterial strains in LB broth was monitored for 110 to 125 hours in triplicate vials containing 10 flies each. Statistical significance of differences in fly survival associated with each mutant strain relative to that associated with the wild-type strain was assessed using the log-rank test. Flies infected with the crbSΔREC mutant succumbed more quickly than did flies infected with the wild-type strain in this assay (P = 0.0028) (A), but this result was not reproduced in three additional assays (S2 Table). Survival of flies infected with the crbS, crbR, and crbRΔREC mutants was significantly greater than that of flies infected with the wild-type strain (P<0.0001) (B). These results were reproduced in three additional independent experiments (S2 Table). Mutation of crbS residues important in the phosphorelay pathway demonstrate that mutation of CrbS His-798 to Ala or Gln reduces lethality of V. cholerae SIO (P<0.0001) in three independent assays (S2 Table), of which one representative example is shown (C). Mutation of CrbS Asp-1081 to Ala does not reduce lethality, but instead trends towards increasing virulence of V. cholerae SIO (P = 0.0354) in three independent assays (S2 Table).