A complex hierarchical quorum-sensing circuitry modulates phenazine gene expression in Pseudomonas aeruginosa

Introduction: Pseudomonas aeruginosa (P. aeruginosa) modulates the expression of a myriad of virulence factors via two complicated hierarchical quorum-sensing (QS) cascade. This study shed light on the interrelation between P. aeruginosa QS systems and pyocyanin production. Methodology: Transcription analysis of lasR, rhlR, rhlI and phz genes using quantitative real time-reverse transcriptase PCR (qRT–PCR) assay, followed by sequencing of the autoinducer synthase (lasI gene) were applied for 15 P. aeruginosa strains recovered from diverse animal clinical sources. Results: Expression studies revealed that most P. aeruginosa strains demonstrated statistically significant differences (p < 0.05) with a very wide range of transcript levels of QS and phz genes in comparison to P. aeruginosa ATCC 27853. We have identified significant positive correlations (r ≥ 0.3) between the expressions of QS and phz genes in eleven analyzed strains, whereas pyocyanin production positively correlated with the expression of lasR only in three strains (r ≥ 0.6). We further found that there was a negative correlation between the transcript levels of QS and phz genes in one bacterial strain. Analysis of lasI sequences showed point mutations explaining the alterations in pyocyanin expression. The deficiencies of lasI, lasR and rhlI with rhlR-dependent expression of phz in one strain were also recorded. Conclusions: These results provided new insights to the pivotal role of QS signal molecules on pyocyanin production presenting the las system as the dominant regulator.


Introduction
Pseudomonas aeruginosa (P. aeruginosa) is a ubiquitous Gram-negative bacterium and an important opportunistic pathogen with elevated metabolic and physiological versatility. Generally, P. aeruginosa is capable of expressing an impressive array of virulence determinants including extracellular secreted factors such as pyocyanin and pyoverdine pigments with potential roles in pathogenicity, mainly through a variety of iron acquisition mechanisms. Notably, it has since become clear that the coordinated expression of around 600 genes and many virulence-related characteristics such as pyocyanin synthesisis regulated by the utilization of cell-to cell communication system based on signal molecules known as quorum sensing in cell density dependent fashion, where certain target genes are induced or repressed [1,2]. In P. aeruginosa, at least two well-defined intertwined hierarchical QS systems, namely, las and rhl have been characterized. Each signaling QS system is comprised of two components, the autoinducer synthases (lasI and rhlI) and their cognate transcriptional regulators (lasR and rhlR), respectively [3]. The las system triggers the expression of elastase, exotoxin A and alkaline protease, while rhl system enhances the expression of rhamnolipid biosynthesis, alkaline protease, elastase, cyanide and pyocyanin production [4]. The two systems are intimately connected being hierarchically organized with lasI/R system regulating rhlI-rhlR transcription [5]. P. aeruginosa is distinguished by the generation of highly diffusible pigmented toxic secondary metabolites, known as phenazines. Pyocyanin is the major soluble bluish green phenazine derivative that is unique for P. aeruginosa suppurative infections [6].
Herein, a long-term transcriptional study was carried out with the objective of relative quantification of mRNA transcribed by lasR, rhlR and rhlI to elucidate the correlation between the expressions of QS systems and phz gene using qRT-PCR assay. Afterwards, we gain insight in analyzing the presence of mutations that may affect the QS synthetase gene, lasI, among a subset of clinically relevant P. aeruginosa strains.

Bacterial strains and growth conditions
The present study was performed on 15 P. aeruginosa strains recovered from two animal clinical sources [bovine mastitis (5) and broiler breeders with respiratory problems (10)]. All pseudomonas strains were routinely enriched in brain heart infusion broth (Oxoid, Hampshire, UK) and immediately streaked on the selective medium of pseudomonas agar base with pseudomonas selective supplements (Oxoid, Hampshire, UK) to assess pigment production. The observed colonies were presumptively identified as P. aeruginosa according to their colonial pigmentation and conventional biochemical tests using the standard microbiological methods [7]. Subsequently, molecular characterization was performed using a P. aeruginosaspecific PCR amplification of oprL gene following a published protocol [8]. All the strains were stored frozen at -20°C in individual aliquots in brain heart infusion broth with 25% glycerol until further analysis.

RNA extraction and qRT-PCR
Total RNAs from bacterial cells were extracted according to the manufacturer's protocol of the QIAamp RNeasy mini kit (QIAGEN, Hilden, Germany). Quantitative RT-PCR was applied for examining the expression of lasR, rhlR, rhlI and phz genes using SYBR green RT-PCR platform; the housekeeping gene rpoD was used as the normalizing gene [9]. Gene specific primer pairs were synthesized in the Reference laboratory for Veterinary Quality Control on Poultry Production, Egypt based on the previously published sequences [10,11].
In MX3005P real time PCR machine (Stratagene, La Jolla, USA), reverse transcription was performed at 50°C for 30 min. After a preliminary denaturation step, the reaction mixture was subjected to 40 cycles of 94°C, 50°C and 72°C, 30 seconds each for lasR gene;94°C, 50°C (rhlR) or 53°C (rhlI) and 72°C, 45 seconds each and 94°C, 64°C and 72°C,one minute each for phz gene. Melting curves were then analyzed in one cycle of 94°C, 50°C (lasR and rhlR) or 53°C (rhlI) or 64°C (phz) and 94°C, one minute each. Amplification curves and cycle threshold (CT) values were determined by Stratagene MX3005P software. Normalized expression was calibrated against corresponding mRNA expression by P. aeruginosa ATCC 27853. The CT values for each strain were converted into fold differences according to the relative quantification method described previously [12].

LasI system amplification and sequencing studies
Chromosomal DNA was extracted from P. aeruginosa strains using a commercial available QIAamp DNA Mini kit (Qiagen, Hilden, Germany,) in accordance with the manufacturers' recommendations. PCR amplification of lasI quorum sensing synthetase gene was performed using specific primers and protocol described elsewhere [10]. PCR products were purified using QIAquick PCR purification kit (Qiagen Inc., Valencia,USA) according to the manufacturers' instructions and referred to automated sequencing reactions using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, PerkinElmer, Foster City, USA) in ABI 3130 automated DNA Sequencer (Applied BioSystems,Foster City, USA). The resultant product sequences were compared with P. aeruginosa PAO1 wild type strain (accession number NC_002516.2) using the BLAST program from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/BLAST/). Sequence analysis was then performed by the use of MEGA5 program, product version 5.1 (http://www.megasoftware.net). All sequence data reported here were deposited into the GenBank databases with the following accession numbers: KP998820-KP998824 and KR020718-KR020726.

Statistical analysis
The relative expression levels of mRNA transcripts were analyzed statistically using Statistical Package for Social Sciences (SPSS) version 22.0 (IBM Corp., Armonk, USA). Comparison analysis was applied using independent samples t-test and Bonferroni's multiple comparison test and homogeneity of variances was analyzed by the Levene's test. Correlation analysis was performed using Pearson's correlation test. The data were expressed as SEM (mean ± standard error) and all p values were based on a 2-tailed distribution. The p values of < 0.05 were considered statistically significant.

A las/rhl box functions in pyocyanin transcriptional activation
Since P. aeruginosa strains were selected for their range of pigment activities, different levels of pigmentation on isolation media were observed due to varying amounts of pyocyanin produced. This phenotypic behavior suggests the potential activity of QS machinery in a synergistic manner.
Relative expressions (fold-changes or folddifferences of expression levels) of QS systems and phz gene of tested strains to a reference strain of P. aeruginosa ATCC 27853, which is assigned a value of 1, were detected. Nine isolates showed increased expressions (> one-fold increase) of lasR (up to 15.67fold), rhlR (up to 11.47-fold) and rhlI (up to 9.34-fold), which were accompanied by 1.13 to 6.73-fold increase in the transcription of phz gene. However, three isolates demonstrated increased expression of phz gene (up to 2.35-fold); one of them (No. 14) exhibited increased expressions of both lasR (4.20-fold) and rhlR (1.14fold) and the remaining two isolates (No. 2, 12) had increased expressions of lasR only (up to 3.16-fold). Another isolate (No. 8) without elevated phz expression had increased expressions of lasR (2.77-fold), rhlI (1.57-fold) and rhlR (1.15-fold), suggesting other factors involved.
The contribution of las system in regulating the expression of pyocyanin production was verified. Indeed, all previously described thirteen isolates showed various point mutations in lasI gene; nine of them (69.23%) possessed sense mutations with substitutions at lys 13 position and only one isolate (No. 10) with an additional substitution at Asp 12 position. These mutations probably explained the variations in pyocyanin expression in tested strains comparable to that produced by P. aeruginosa ATCC 27853.
A notable exception was recorded in the isolate No. 9, which possessed five point mutations in lasI gene; two of them were evolved in the substitution of Lys 13 to Arg. These mutations account for the decreased expressions of Qs systems conferring consequently a down regulation of phz gene expression. A striking observation in the present study was the deficiency of lasI, lasR and rhlI in an isolate No. 15; but rhlR, which is the known regulator of phz gene, was expressed in a low level (0.27-fold) producing a negligible expression level of pyocyanin (0.19-fold) in comparison with the reference strain. Detailed information on the relative expressions of QS systems and phenazine gene compared to P. aeruginosa ATCC 27853 as well as mutations in lasI gene of P. aeruginosa strains under study are given in Table 1.

Transcriptional analysis of QS communication circuit and controlled phenazine gene
Results of this study have a significant clinical impact. In particular, the changes in transcript levels of QS components and phz gene were significantly higher than those produced by the reference strain (p < 0.05) in majority of analyzed P. aeruginosa strains (Table 1).
Moreover, to determine if there was any relationship between the transcript levels of pyocyanin and the key QS regulators, a correlation analysis was performed. Indeed, there were significant positive (p < 0.05) correlations of phz transcript levels and QS signal molecules in 73.3% (11/15) of P. aeruginosa strains (r ≥ 0.3). However, in three strains (No. 2,12,14), pyocyanin production positively correlated with the expression of lasR only (r ≥ 0.6), but there were no real correlations in the changes in the transcript levels of phenazine with those of rhlR and rhlI in strains No. 2 and 12 nor with that of rhlI in strain No. 14. Conversely, in P. aeruginosa strain No. 8, the target gene transcript level negatively correlated with the expression of its regulators suggesting that factors other than QS-based regulation of phz transcription appeared to have a more common effect than did the QS system transcript levels. Relationships between transcription levels of phenazine and those of individual QS systems are shown in Table  2.
It is conceivable that lasI mutations associate with the relative expression levels of pyocyanin. Overall, there were reasonably high significant variations between the mean levels of lasR, rhlR, rhlI, phz transcripts and mutation rates among 15 tested P. aeruginosa strains (p < 0.0001) ( Table 3). Interestingly, transcription levels of highly mutating strains are more influenced than those with low mutations.
Actually, there were modestly significant differences noted in the levels of lasR transcripts in P. aeruginosa isolates from bovine mastitis and broiler breeders with respiratory disorders (t = 2.19, p = 0.032). It is unclear if this association between clinical source and lasR transcript level is of significance in regard to virulence; in spite of the bovine mastitis P. aeruginosa strains having higher levels of lasR-regulated transcripts for rhlI and phz genes than those of the  broiler breeders' strains, there were no significant associations (p > 0.05) between levels of the rhlR (p = 0.823), rhlI (p = 0.203) or phz (p = 0.167) genes and source of isolation ( Figure 1).

Discussion
Quorum sensing signaling systems play a vital role in the regulation of cell physiology of P. aeruginosa in a population density-dependent fashion [13]. The present study was undertaken to principally define the QS responses in P. aeruginosa clinical isolates by analyzing the relationship between transcriptional analysis of individual QS inducer or regulator component on the expression of pyocyanin production.
Our results provide additional evidence supporting this hypothesis; an extensive analysis of transcriptional responses using qRT-PCR indicated that most P. aeruginosa strains showed significantly higher expression levels of QS systems and their regulated genes in comparison to the reference strain, while they were parallel to that produced by the wild type in a previous study [14]. There were variations in transcript levels of the key QS regulators (rhlR and lasR), rhlI and even among the QS-regulated transcriptional responses regarding phz expressions in comparable P. aeruginosa strains. In addition, statistical analysis revealed positive moderate-to-high correlations in the transcript levels between QS and phz genes in more than one half of the isolates demonstrating that both las and rhl QS systems contributes equally to pyocyanin production. In the light of the published genomic data, further support for the idea that the las system positively regulates the expression of both rhlR and rhlI have been defined [5]. Other studies reported the regulation of pyocyanin by QS previously [10,15,16]. It was also documented that pseudomonas strains can express increased pyocyanin levels despite decreased expressions in one or both QS systems. Moreover, in strains No. 2, 12, pyocyanin production positively correlated with the expression of lasR only. The existence of such strains would indicate that phz production is not stringently controlled by all QS systems. In addition the QS systems are not absolutely essential for P. aeruginosa to establish infection, and other QS independent factors can substitute for the loss of QS-controlled mechanisms [14].
It is clear that the regulation of QS systems is not limited only to lasI/R and rhlI/R, but many other global regulators are interconnected with the QS circuitry. The activation of QS requires a critical cell density, although a quorum of bacteria is not sufficient by itself to trigger the QS-regulated genes as available elsewhere [17]. Therefore, it is not surprising that one strain (No. 8) displayed an impaired QS-dependent phenotype as phz gene transcript level negatively correlated with the expression of all QS systems. Similar to our findings, another study in USA has also identified a P. aeruginosa isolate with no pyocyanin activity, which was negative for both autoinducers, but contained other QS genes [14].
Interestingly, there were moderately significant differences in the mean levels of lasR transcripts in P. aeruginosa isolates from bovine mastitis and broiler breeders with respiratory disorders, thereby; there was no functional link between QS transcriptional response and source of isolation. On the basis of molecular aspects of QS cascade in present and previous studies, a definitely little impact of QS on the strains` sources has been suggested [18].
In light of these elements, the mean levels of lasR, rhlR, rhlI and phz transcripts were observed to be statistically significant with lasI mutations, which explain the plurality of QS-dependent phenotypes observed in 14 P. aeruginosa strains, in which all four QS genes were positive with PCR. The presence of PCR products does not exclude the possibility that QS genes may have inactivating mutations demonstrating that las mutation does not lead to loss of virulence factors as anecdotally observed [19][20][21][22].
Most intriguingly, the present study showed the deficiency of QS components (lasI, lasR and rhlI) but rhlR was expressed in a low level with inadequate The bars represent the means of transcript levels and the error bars represent the standard errors of the means. The p value represents the significant differences in lasR transcript levels using independent samples t-test. pyocyanin expression in isolate No. 15, which contradicts the prevailing concept that the rhl system is inactive in the absence of a functional lasR [5]. Importantly, P. aeruginosa can circumvent the deficiency of one of its QS systems by allowing the other to take over so, rhlR is able to overcome the las system when the latter is deficient by activating specific lasR-controlled functions [15].

Conclusion
Our results do not contradict the theory that the expression of QS genes in P. aeruginosa is definitely linked with the pathogen communal behavior such as pyocyanin production and emphasize that some P. aeruginosa strains are capable of causing clinical infections despite an impaired QS system. In addition, this work demonstrates that the QS hierarchy is more complex than the model simply presenting the las system above the rhl system and provide for the future identification of new factors involved in QS regulation.
Authors' contributions NKA and MIA contributed equally in the conception and design of the study, acquisition of data, analysis and interpretation of the results, writing the paper, revising it critically for important intellectual contents and final approval of the version to be submitted and EYE helped in analysis and interpretation of data and final approval of the version to be submitted