- Research article
- Open Access
Expression of mucoid induction factor MucE is dependent upon the alternate sigma factor AlgU in Pseudomonas aeruginosa
© Yin et al.; licensee BioMed Central Ltd. 2013
- Received: 29 May 2013
- Accepted: 9 October 2013
- Published: 18 October 2013
Alginate overproduction in P. aeruginosa, also referred to as mucoidy, is a poor prognostic marker for patients with cystic fibrosis (CF). We previously reported the construction of a unique mucoid strain which overexpresses a small envelope protein MucE leading to activation of the protease AlgW. AlgW then degrades the anti-sigma factor MucA thus releasing the alternative sigma factor AlgU/T (σ22) to initiate transcription of the alginate biosynthetic operon.
In the current study, we mapped the mucE transcriptional start site, and determined that P mucE activity was dependent on AlgU. Additionally, the presence of triclosan and sodium dodecyl sulfate was shown to cause an increase in P mucE activity. It was observed that mucE-mediated mucoidy in CF isolates was dependent on both the size of MucA and the genotype of algU. We also performed shotgun proteomic analysis with cell lysates from the strains PAO1, VE2 (PAO1 with constitutive expression of mucE) and VE2ΔalgU (VE2 with in-frame deletion of algU). As a result, we identified nine algU-dependent and two algU-independent proteins that were affected by overexpression of MucE.
Our data indicates there is a positive feedback regulation between MucE and AlgU. Furthermore, it seems likely that MucE may be part of the signal transduction system that senses certain types of cell wall stress to P. aeruginosa.
- Pseudomonas aeruginosa
- Sigma factor
P. aeruginosa, a Gram-negative bacterium, is the leading cause of morbidity and mortality in patients with cystic fibrosis (CF) . In CF, P. aeruginosa is often isolated from sputum samples and exhibits a phenotype called mucoidy, which is due to overproduction of an exopolysaccharide called alginate. It is also an environmental bacterium which normally does not overproduce alginate . The emergence of mucoid P. aeruginosa isolates in CF sputum specimens signifies the onset of chronic respiratory infections. Mucoidy plays an important role in the pathogenesis of P. aeruginosa infections in CF, which includes, but is not limited to: increased resistance to antibiotics , increased resistance to phagocytic killing [3, 4] and assistance in evading the host’s immune response .
A major pathway for the conversion to mucoidy in P. aeruginosa is dependent upon AlgU (AlgT, σ22), an alternative sigma factor that drives transcription of algD encoding the key enzyme for alginate biosynthesis [5, 6]. Previous studies have shown that several genes take part in the regulation of AlgU activation and alginate overproduction. MucA is a trans-membrane protein that negatively regulates mucoidy by acting as an anti-sigma factor via sequestering AlgU to the cytoplasmic membrane ; MucB and intra-membrane proteases AlgW, MucP and ClpXP were reported to affect alginate production by affecting the stability of MucA . A small envelope protein called MucE was found to be a positive regulator for mucoid conversion in P. aeruginosa strains with a wild type MucA . The mechanism for mucE induced mucoidy is due to its C-terminal –WVF signal, which can activate the protease AlgW possibly by interaction with the PDZ domain . Upon activation, AlgW initiates the proteolytic degradation of the periplasmic portion of MucA, causing the release of AlgU to drive expression of the alginate biosynthetic operon . While the function of MucE as an alginate inducer was identified, its physiological role, and its role in the regulation of mucoidy in clinical isolates, remains unknown.
Comparative analysis through Basic Local Alignment Search Tool (BLAST) using the genomes of Pseudomonas species from the public databases reveals that MucE orthologues are found only in the strains of P. aeruginosa. In order to study the role and regulation of MucE in P. aeruginosa, we first mapped the mucE transcriptional start site. We then examined the effect of five different sigma factors on the expression of mucE in vivo. Different cell wall stress agents were tested for the induction of mucE transcription. Expression of MucE was also analyzed in non-mucoid CF isolates to determine its ability to induce alginate overproduction.
Bacteria strains, plasmids, and growth conditions
Bacterial strains and plasmids used in this study are shown in Additional file 1: Table S1. E. coli strains were grown at 37°C in Luria broth (LB, Tryptone 10 g/L, Yeast extract 5 g/L and sodium chloride 5 g/L) or LB agar. P. aeruginosa strains were grown at 37°C in LB or on Pseudomonas isolation agar (PIA) plates (Difco). When required, carbenicillin, tetracycline or gentamicin were added to the growth media. The concentration of carbenicillin, tetracycline or gentamycin was added at the following concentrations: for LB broth or plates 100 μg ml-1, 20 μg ml-1 or 15 μg ml-1, respectively. The concentration of carbenicillin, tetracycline or gentamycin to the PIA plates was 300 μg ml-1, 200 μg ml-1 or 200 μg ml-1, respectively.
The mucEprimer extension assay
Total RNA was isolated from P. aeruginosa PAO1 grown to an OD600 of 0.6 in 100 ml LB at 37°C as previously described . The total RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA) per the manufacturer’s instructions. Primers for mucE (PA4033), seq 1 (5′-CCA TGG CTA CGA CTC CTT GAT AG-3′) and seq 2 (5′-CAA GGG CTG GTC GCG ACC AG-3′), were radio-labeled using T4 polynucleotide kinase (New England Biolabs, Ipswich, MA) and γP32-ATP. Primer extensions were performed using the Thermoscript RT-PCR system (Invitrogen, Carlsbad, CA) with either PA4033 seq 1 or seq 2 with 10–20 μg of total RNA. Extensions were performed at 55°C for an hour. Primer extension products then were electrophoresed through a 6% acrylamide/8M urea gel along with sequencing reactions (Sequenase 2.0 kit, USB, Cleveland, OH) using the same primers used in the extension reactions.
Transformation and conjugation
E. coli One Shot TOP10 cells (Invitrogen) were transformed via standard heat shock method according to the supplier’s instructions. Plasmid transfer from E. coli to Pseudomonas was performed via triparental conjugations using the helper plasmid pRK2013 .
Generating PAO1 miniCTX-P mucE -lacZreporter strain
PAO1 genomic DNA was used as a template to amply 618 bp upstream of the start site (ATG) of mucE using two primers with built-in restriction sites, HindIII-mucE-P-F (5′-AAA GCT TGG TCG TTG AAA GTC TGC ACC TCA-3′) and EcoRI-mucE-P-R: (5′-CGA ATT CGG TTG ATG TCA CGC AAA CGT TGG C-3′). The P mucE amplicon was TOPO cloned and digested with HindIII and EcoRI restriction enzymes before ligating into the promoterless Pseudomonas integration vector miniCTX-lacZ. The promoter fusion construct miniCTX-P mucE -lacZ was integrated onto the P. aeruginosa chromosome of strain PAO1 at the CTX phage att site  following triparental conjugation with E. coli containing the pRK2013 helper plasmid .
Screening for a panel of chemical agents that can promote P mucE transcription
Membrane disrupters and antibiotics were first tested by serial dilution to determine the minimum inhibitory concentration (MIC) for strain PAO1::attB::P mucE -lacZ. An arbitrary sub-MIC concentration for each compound was then tested for the induction effect through the color change of 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal, diluted in dimethylformamide to a concentration of 4% (w/v)). The final concentration of the compounds used in this study are listed as follows: triclosan 25 μg/ml, tween-20 0.20% (v/v), hydrogen peroxide 0.15%, sodium hypochlorite 0.03%, SDS 0.10%, ceftazidimine 2.5 μg/ml, tobramycin 2.5 μg/ml, gentamicin 2.5 μg/ml, colisitin 2.5 μg/ml, and amikacin 2.5 μg/ml. PAO1::attB::P mucE -lacZ was cultured overnight in 2 ml LB broth, 10 μl of overnight culture and 10 μl of 4% X-gal was added to each treatment culture tube (2 ml LB broth + cell wall stress agent). The cultures were grown overnight at 37°C with shaking at 150 rpm and were used to visually observe the change of the color. LB broth lacking X-gal was used as a negative control.
The β-galactosidase activity assay
Pseudomonas strains were cultured at 37°C on three PIA plates. After 24 hours, bacterial cells were harvested and re-suspended in PBS. The OD600 was measured and adjusted to approximately 0.3. Cells were then permeabilized using toluene, and β-galactosidase activity was measured at OD420 and OD550. The results in Miller Units were calculated according to this formula: Miller Units = 1000 × [OD420 - (1.75 × OD550)]/[Reaction time (minutes) × Volume (ml) × OD600] . The reported values represent an average of three independent experiments with standard error.
P. aeruginosa strains were grown at 37°C on PIA plates in triplicate for 24 hrs or 48 hrs. The bacteria were collected and re-suspended in PBS. The OD600 was analyzed for the amount of uronic acid in comparison with a standard curve made with D-mannuronic acid lactone (Sigma-Aldrich), as previously described .
iTRAQ®MALDI TOF/TOF proteome analysis
Strains PAO1, VE2 and VE2ΔalgU were cultured on PIA plates for 24 hrs at 37°C. Protein preparation and iTRAQ mass spectrometry analysis was performed according to previously described methods .
Mapping of the mucEpromoter in PAO1
The alternative sigma factor AlgU activates transcription of mucE in vivo
Cell wall stress promotes expression of mucE from P mucE
Alginate production is reduced in the mucEmutant compared to PAO1
Expression of mucE can cause alginate overproduction . However, we wondered if mucE would affect transcriptional activity at P algU and P algD promoters. In order to determine this, both pLP170-P algU and pLP170-P algD with each promoter fused to a promoterless lacZ gene were conjugated into PAO1 and PAO1VE2, respectively. As seen in Additional file 1: Figure S1, the activity of P algU (PAO1VE2 vs. PAO1: 183,612.04 ± 715.23 vs. 56.34 ± 9.68 Miller units) and P algD (PAO1VE2 vs PAO1: 760,637.8 ± 16.87 vs. 138.18 ± 9.68 Miller units) was significantly increased in the mucE over-expressed strain PAO1VE2. Although, Qiu et al.  have reported that AlgU is required for MucE induced mucoidy, we wanted to know whether MucE is required for AlgU induced mucoidy. As seen in Additional file 1: Figure S2, we did not observe that the over-expression of MucE induced mucoidy in PAO1ΔalgU. This result is consistent with what was previously reported by Qiu et al.. However, the alginate production induced by AlgU was decreased in the mucE knockout strain. The alginate production induced by AlgU in two isogenic strains, PAO1 and PAO1mucE::ISphoA/hah is 224.00 ± 7.35 and 132.81 ± 2.66 μg/ml/OD600, respectively (Additional file 1: Figure S2). These results indicate that alginate overproduction in PAO1 does not require MucE. However, MucE can promote the activity of AlgU resulting in a higher level of alginate production in PAO1 compared to the mucE knockout. Previously, Boucher et al. and Suh et al. have reported that sigma factors RpoN and RpoS were involved in alginate regulation. In order to determine whether mucE induced mucoidy was also dependent on other sigma factors besides AlgU, pHERD20T-mucE was conjugated and over-expressed in PAO1ΔrpoN, PAO1rpoS::ISlacZ/hah and PAO1rpoF::ISphoA/hah. The results showed that the mucE induction caused mucoid conversion in PAO1rpoS::ISlacZ/hah and PAO1rpoF::ISphoA/hah when 0.1% L-arabinose was added to the media. However, 0.5% L-arabinose was required for mucoid conversion in PAO1ΔrpoN. The alginate production induced by MucE in PAO1rpoS::ISlacZ/hah, PAO1rpoF::ISphoA/hah and PAO1ΔrpoN is 150.62 ± 5.27, 85.53 ± 4.10 and 31.84 ± 0.25 μg/ml/OD600, respectively. These results suggested that RpoN, RpoS and RpoF are not required for MucE-induced mucoidy in PAO1. Conversely, over-expression of these sigma factors rpoD, rpoN, rpoS and rpoF did not induce mucoid conversion in PAO1. When the strains of PAO1 with sigma factor overexpression were measured for alginate production, the level is as follows: 5.11 ± 1.25 (+rpoD), 13.07 ± 4.16 (+rpoN), 3.50 ± 0.10 (+rpoS) and 7.68 ± 1.23 (+rpoF) μg/ml/OD600.
MucE-induced mucoidy in clinical CF isolates is based on two factors, size of MucA and genotype of algU
Mutant AlgUs display partial activity resulting in decreased amount of alginate
Characterization of the MucE regulon using iTRAQ analysis
In order to determine the effect of mucE expression on the proteome change, we performed iTRAQ proteome analysis via MALDI TOF/TOF. Total protein lysates of PAO1, VE2 (PAO1 with constitutive expression of mucE) and VE2ΔalgU (VE2 with in-frame deletion of algU) were collected and analyzed. Within the three samples, 166 unique proteins were identified with 1455 peptides assayed at/or above 95% confidence. The data set was then filtered to include only proteins that were significantly different between samples and the number of the detected peptides for each protein more than three (Additional file 1: Table S3). By comparing the proteomes of VE2 to PAO1, the effects of increased MucE levels on PAO1 were examined; while comparing VE2ΔalgU to PAO1 allowed for the determination of AlgU-independent protein production in VE2. As seen in Additional file 1: Table S3, compared to PAO1, 11 proteins were differentially expressed due to mucE over-expression, and two of them (elongation factor Tu and transcriptional regulator MvaT) are AlgU-independent.
Qiu et al. have reported that MucE can induce alginate overproduction when over-expressed in vivo. However, nothing was known about the regulation of mucE. Recently, the genome-wide transcriptional start sites of many genes were mapped by RNA-seq in P. aeruginosa strain PA14 . However, the transcriptional start site of the mucE gene (PA14_11670) was not included. In this study, we reported the mapping of the mucE transcriptional start site. Furthermore, we found the transcription of mucE is dependent on AlgU. Analysis of the upstream region of mucE reveals an AlgU promoter-like sequence (Figure 1). Previously, Firoved et al. identified 35 genes in the AlgU regulon, based on scanning for AlgU promoter consensus sequence (GAACTTN16-17TCtgA) in the PAO1 genome . In this study, we found that AlgU can activate the transcription of mucE. In order to determine whether AlgU can bind to P mucE region, AlgU was purified (Additional file 1: Figure S3) and electrophoretic mobility shift assay (EMSA) was performed. As seen in Additional file 1: Figure S4, our results showed that AlgU affected the mobility of P mucE DNA, especially in the presence of E. coli RNA polymerase core enzyme, suggesting a direct binding of AlgU to P mucE . However, whether small regulatory RNAs or other unknown regulator proteins are also involved in the transcriptional regulation of mucE needs further study. LptF is another example of an AlgU-dependent gene, but doesn’t have the consensus sequence in the promoter region . While MucE, as a small envelope protein is positively regulated through a feedback mechanism, it’s not clear how many AlgU-regulated genes follow the same pattern of regulation as MucE.
The mucA mutation is a major mechanism for the conversion to mucoidy. Mutation can occur throughout the mucA gene (585 bps) . These mutations result in the generation of MucA proteins of different sizes. For example, unlike the wild type MucA with 194 amino acid residues, MucA25, which is produced due to a frameshift mutation, results in a protein containing the N-terminal 59 amino acids of MucA, fused with a stretch of 35 amino acids without homology to any known protein sequence . MucA25 lacks the trans-membrane domain of wild type MucA, predicting a cytoplasmic localization. Therefore, different mucA mutations could possibly result in different cellular compartment localization. Identification of MucE’s function as an inducer of alginate in strains with wild type MucA and AlgU strongly suggests MucE acts through interaction with AlgW in the periplasm. On the other hand, the loss of this predicted MucA-AlgW interaction can be seen in two strains, CF11 and CF28, which lack the major cleavage site of AlgW  (Figure 5). Interestingly, we observed that the missense mutation in algU can reduce, but not completely abolish, the activity of AlgU as an activator for alginate production. This data may explain why mutant algU alleles have reduced P mucE activity (Figure 2). Furthermore, since AlgU is an auto-regulated protein , this may explain why the P mucE activity induced by mutant AlgU is lower than that of wild type AlgU. A slightly higher activity of P mucE noted in CF149(+algU) than in PAO1VE1 (Figure 3A) could be due to a combined effect of dual mutation of algU and mucA in CF149. In strains of FRD2 and CF14, the retention of the AlgW cleavage site is not sufficient to restore mucoidy. This is because of the partial function of AlgU, which can be seen with alginate production and AlgU-dependent P algD promoter activity (Figure 6). Altogether, these results suggest that mucoidy in clinical isolates can be modulated by a combination of two factors, the size of the MucA protein and the genotype of the algU allele in a particular strain. MucA size determines its cellular localization and its ability to sequester AlgU, and the algU allele determines whether AlgU is fully or partially active.
The iTRAQ results showed that the expression of two proteins was significantly increased and the expression of nine proteins was decreased in the mucE over-expressed strain VE2 (Additional file 1: Table S3). Of these 11 proteins, nine of them are AlgU dependent, for including flagellin type B. Garrett et al. previously reported that AlgU can negatively regulate flagellin type B and repress flagella expression . However, no AlgU consensus promoter sequences were found within the upstream of the 11 regulated genes through bioinformatics analysis, indicating that these may be indirect effect. In addition, two proteins (elongation factor Tu and transcriptional regulator MvaT) were significantly decreased when compared to PAO1 proteome, but remained unchanged when comparison was made between VE2 and VE2ΔalgU, suggesting the reduction of these two proteins was independent of AlgU in the MucE over-expressed strain. MvaT is a global regulator of virulence in P. aeruginosa, and elongation factor Tu is important for growth and translation. Elongation factor Tu has also been shown to act as a chaperone in E. coli, consistent with induction of proteins involved in responding to heat or other protein damaging stresses . Recently, elongation factor Tu has been shown to have a unique post-translational modification that has roles in colonization of the respiratory tract [36, 37]. The differential expression of Tu due to mucE overexpression suggests there may be signaling networks dependent upon mucE that we have not yet been identified. Although, previous studies have shown that the growth rate is slower in mucoid strains and the virulence is increased after deleting AlgU [15, 38], the relationship between MucE and growth or virulence need further study. Together, iTRAQ analysis suggests that MucE signaling affected both AlgU-dependent and AlgU-independent protein expression.
The alternative sigma factor AlgU was responsible for mucE transcription. Together, our results suggest there is a positive feedback regulation of MucE by AlgU in P. aeruginosa, and the expression of mucE can be induced by exposure to certain cell wall stress agents, suggesting that mucE may be part of the signal transduction that senses the cell wall stress to P. aeruginosa.
This work was supported by the National Aeronautics and Space Administration West Virginia Space Grant Consortium (NASA WVSGC) and the Cystic Fibrosis Foundation (CFF-YU11G0). F.H.D. was supported by grants from the NASA Graduate Student Researchers Program (NNX06AH20H), NASA West Virginia Space Grant Consortium, and a post-doctoral fellowship from the Cystic Fibrosis Foundation (DAMRON10F0). T.R.W. was supported through the NASA WVSGC Graduate Research Fellowship. H.D.Y. was supported by NIH P20RR016477 and P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence.
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