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| Chapitre 4 Génomique fonctionnelle d’un facteur sigma essentiel in vivo régulant les protéases extracytoplasmiques de Pseudomonas aeruginosa | ||
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Table des matières
Ce troisième et dernier chapitre est un article de recherche soumis pour évaluation à la revue Molecular Microbiology . Il regroupe essentiellement tous les résultats expérimentaux obtenus suite à la caractérisation biochimique et moléculaire de PA2895, un gène essentiel in vivo . Il a été démontré que le mutant STM2895 est incapable de dégrader la caséine. Cependant, nous nous attardons ici à démontrer que le défaut se situe plutôt au niveau du repliement d’au moins deux des quatre protéases extracellulaires de P. aeruginosa , soit les élastases LasA et LasB. Par des analyses génomiques et bioinformatiques, il est suggéré que PA2895 agirait comme senseur périplasmique régulant de façon négative l’activité transcriptionnelle de PA2896, un facteur sigma-70 de la sous-famille des ECF. Des analyses en transcriptome utilisant la technologie des puces à ADN d’Affymetrix ont permis de démontrer un lien étroit entre la régulation transcriptionnelle dépendante de PA2895 et PA2896 et le métabolisme du fer. Tous les travaux ainsi que la rédaction de ce manuscrit ont été réalisés par moi-même, avec l’aide technique et scientifique de nos collaborateurs du VCU et de l’UO.
Functional genomics of an essential in vivo sigma factor regulating extracytoplasmic proteases from Pseudomonas aeruginosa
Potvin E 1, Richard KL 1, Sanschagrin F 1, Grande K 2, Ohman DE 2, Petersen A 3, Whiteley M 3, Levesque RC 1*
1 Centre de Recherche sur la Fonction, Structure et Ingénierie des Protéines, Faculté de Médecine, Pavillon Charles-Eugène Marchand, Université Laval, Ste-Foy, Québec, Canada, G1K 7P4 2 Medical College of Virginia Commonwealth University, Richmond, VA, USA 3University of Oklahoma, Health Sciences Center, Oklahoma City, OK, USA
Key words: Pseudomonas aeruginosa, signature-tagged mutagenesis, sigma factors, extracytoplasmic functions, elastase, in vivo.
Running title: Functional genomics of an essential in vivo sigma factor regulating extracytoplasmic proteases from Pseudomonas aeruginosa
* Corresponding author. Mailing address: Centre de recherche sur la fonction, structure et ingénierie des protéines, Faculté de médecine, Pavillon Charles-Eugène Marchand, Université Laval, Ste-Foy, Québec, Canada, G1K 7P4. Phone : (1) (418) 656-3070. Fax : (1) (418) 656-7176. E-mail : rclevesq@rsvs.ulaval.ca.
By secreting a large arsenal of virulence factors, P. aeruginosa causes lethal respiratory failure in CF patients. To understand host/bacterium interactions, we constructed a library of P. aeruginosa (PAO1) mutants using PCR-Based Signature-Tagged Mutagenesis. The mutant collection was screened in the rat model of chronic lung infection and identified 148 essential in vivo genes. A mutant of the PA2895 gene (STM2895), frequently recovered by the STM screening, was primarily identified by its exoproteases defect. Biochemical investigations using elastin congo-red, staphylolytic assay and poly-L-lysine degradation assays revealed that STM2895 was affected in LasA, LasB and lysine proteases. Immunoblots of STM2895 showed that LasB was present and correctly refolded and inactive whereas LasA accumulates in a processed form. A deletion GmR mutant of PA2896 was constructed and used with STM2895 in microarray experiments. Compared to wild-type PAO1, 128 genes (64 up and 64 down) for STM2895 and 138 genes (59 up and 79 down) for ΔPA2896::Gm were differentially regulated at ≥ 5 fold. Results from both STM2895 and ΔPA2896::Gm, were comparable in 76% of cases for upregulation and 59% for downregulation. Of significance is the number of operons or gene clusters involved in extracellular functions and genes associated to virulence (type III secretion pathway, exoenzymes, secondary metabolites and siderophores production). For repressed genes, 4 sigma-70 and PvdS (-24 fold) were highlighted as well as several genes identified with microarray data monitoring iron metabolism. In the STM2895 background, PA2896 was upregulated more than 9 fold confirming the negative regulation hypothesis and suggesting a feedback loop regulation. Competitive index (CI) confirmed the attenuation in vivo of STM2895 and a deleted mutant ΔPA2895::Gm had CI values of 0.074 and 0.116, respectively. We conclude that PA2895 is a negative regulator of PA2896, a putative ECF sigma-70 factor, and plays a major role in virulence.
Pseudomonas aeruginosa is an opportunistic pathogen commonly found in association with immunocompromised hosts such as those with AIDS or burns and it also colonizes cystic fibrosis airways. P. aeruginosa is one of the leading causes of hospital-acquired nosocomial infections and demonstrates a high level of resistance to most classes of antibiotics (Banerjee and Stableforth, 2000). The production of exoproteases, lipases, phospholipases, exotoxines, motility apparatus, hydrogen cyanide, siderophores and the ability to produce exopolysaccharides such as alginate involved in biofilm formation represents an impressive arsenal used by the bacterium to bypass host defences (Lyczak et al., 2000). Genomic analysis and annotation of the entire 6.3 Mb genome sequence of PAO1 revealed a repertoire of more than 550 transcriptional regulators (Stover et al., 2000). Hence, more than 9 % of the large genome was predicted to encode transcriptional regulators and two-component environmental sensors. When compared to other microbial sequenced genomes, P. aeruginosa encodes 61 AraC, 115 LysR and 19 ECF (extracytoplasmic function) sigma-70 regulator-type families (Stover et al., 2000).
Sigma factors are essential components of the RNA polymerase complex and determine promoter transcription specificity. Alternative sigma factors of the ECF-type respond to environmental changes and stimuli (Helmann, 2002; Lonetto et al., 1994). In most cases, the activity of ECF sigma factors is modulated via one or more negative regulators including a cognate inner-membrane anti-sigma. These inner-membrane proteins are presumed to act as sensors or signalling molecules responding to environnemental changes (Missiakas and Raina, 1998). ECF sigma factors were shown to control responses to a variety of stresses associated with cell wall biosynthesis and protein folding. P. aeruginosa contains a locus homologous to the E. coli stress response regulator rpoE rseABC gene cluster, called the algU mucABC locus. AlgU controls the expression of the alginate biosynthesis pathway which confers the mucoid phenotype to P. aeruginosa cells (Rouviere et al., 1995; Yu et al., 1995). Recently, ECF sigma factors have been shown to be involved in bacterial pathogenesis and specifically in siderophore synthesis and uptake in P. aeruginosa (Bashyam and Hasnain, 2004). The importance of iron uptake and metabolism in maintenance of chronic lung infections was shown to be mediated by PvdS and a number of homologues, all members of ECF subfamily (Visca et al., 2002).
A PCR-Based Signature-tagged mutagenesis (PCR-STM) was used to generate a large P. aeruginosa strain PAO1 library of 7968 mutants to identify essential genes in vivo (Lehoux et al., 2004). Using a PCR-based approach (Lehoux and Levesque, 2002), the complete library was systematically screened in the rat model of chronic lung infection and 148 genes were shown to be defective for in vivo maintenance. STM2895 which contains an insertional mutation in the functional gene PA2895 was previously identified as attenuated in vivo. Phenotypic analysis highlighted a defect in exoprotease production (Potvin et al., 2003). A query search of the Pseudomonas database (http://www.pseudomonas.com) and annotation data predicted no putative function for PA2895. However, genomic analysis suggested that PA2895 is co-transcribed with a putative ECF sigma factor PA2896, sharing 31% identity with the FecI protein from E. coli (Braun et al., 2003). The transcription of FecIR itself was shown to be dependant on the Fur protein and transcription is initiated by iron-limiting conditions (Braun, 1997). The aim of this paper is to define the role of the PA2895-PA2896 operon in the in vivo maintenance of P. aeruginosa. We focused our efforts on understanding the link between the exoprotease defect of STM2895 by western blot analysis and biochemical evaluation of LasA and LasB activity and its global impact on the transcriptional profiling patterns by the GeneChip technology. The competitive index was used to evaluate maintenance of STM2895 and PA2896 deletion mutants and complementation in trans in the rat chronic lung infection model.
Bacterial strains and media
Bacterial strains used in this study are describedin Table 1. P. aeruginosa and E. coli strains were routinely cultured in Luria-Bertani (LB) broth/agar at 37°C with antibiotics as needed. For some experiments, special culturemedia and conditions were used, and these are described furtherbelow. Antibiotics (Sigma) were used at the following concentrations:ampicillin, 100 µg ml-1 for E. coli; carbenicillin 500 µg ml-1 for P. aeruginosa; gentamicin 15 µg ml-1 for E. coli and50 µg ml-1 for P. aeruginosa; tetracycline 10 µg ml-1 for E. coli and 35µg ml-1 for P. aeruginosa. All solid and liquid media were purchased from Difco laboratories.
Identification of the mini-Tn 5 -Tc insertion points
Chromosomal DNA from STM2895 was extracted and purified using DNeasy tissue kit (Qiagen). PstI digested genomic DNA (300 ng) was ligated into the PstI site of pTZ18R (80 ng) (Amersham Biosciences). The ligation mixture was purified using a Microcon-PCR filtration device (Millipore) and electroporated (Gene-Pulser, Bio-Rad) into E. coli DH10B ElectroMAX competent cells (Invitrogen). Transformants were selected by plating on LB agar supplemented with ampicillin 100 µg ml-1. Ampicillin resistant clones were collected and plated on tetracycline 10 µg ml-1. Sequencing of the mini-Tn5/chromosomal DNA junction was performed using an oligonucleotide complementary to the I-end region of Tn5 (5’-AGA TCT GAT CAA GAG ACA G-3’). The results generated by sequence analysis were compared to the Pseudomonas databases (http://www.pseudomonas.com).
Bioinformatics analysis
Bioinformatics analyses were done using software available online from the National Centre for Biotechnology Information(NCBI) (http://www.ncbi.nlm.nih.gov) and the GCG (Genetics Computer Group, Wisconsin Package Version 10.3, Accelrys) software package. The genomic DNA sequence of the PA2895-PA2896 operon and the probable gene function of P. aeruginosa were obtained by using the Pseudomonas Genome Project and the PseudoCAP web database (http://pseudomonas.com). The PA2895-PA2896 ORFs of P. aeruginosa were analysed for similarities to other proteins by comparing entries in the GenBanknon-redundant database using the blast network service of NCBI.
Chromosomal DNA extraction (Southern hybridization)
Chromosomal DNA was extracted from Pseudomonas STM mutant strains using a modified version of this protocol (Meade et al., 1982). Briefly, cells were grew overnight at 37°C in LB broth and subcultured at a 1/20 dilution into 10 ml fresh LB broth and incubated for 3 to 4 h at 37°C with aeration. Cells were harvested by centrifugation for 15 min at 3500 g and 4°C. The pellet was then washed twice in 10 ml cold saline and centrifuged as above. Cells were suspended in 10 ml cold TE 10 (10 mM Tris-HCl pH 7.6, 10 mM EDTA). One ml of lysozyme (Sigma) solution 2 mg ml-1 in TE 10 (freshly prepared) was added to the cell suspension and incubated 15 min at 37°C. Proteins were removed by adding 2 ml of Pronase (Roche Diagnostics) 5 mg ml-1 in TE 10 containing 10% N-lauryl sarcosine, and incubated at 37°C for 1 h. The suspension was extracted at least twice with 15 ml phenol/chloroform (1:1) and mixed thoroughly by inversion. Phases were separated by centrifugation at 5000 g for 10 min. The aqueous phase was recovered and extracted twice with 15 ml chloroform and phases were separated as previously described. For every 10 ml of aqueous layer, 1.2 ml of 3M sodium acetate pH 5.2 was added. DNA was precipitated by adding 2 volumes of isopropanol at room temperature, mixed by inversion and stored at -80°C overnight. DNA was pellet by centrifugation at 5000 g for 10 min. The DNA pellet was then washed twice with cold ethanol 70% and air dried. DNA was dissolved in 1 ml of 10 mM tris-HCl pH 7.6, 1 mM EDTA. DNA concentrations were determined by UV absorption at 260 nm.
Southern Blot hybridization
All reagents for Southern hybridization and labelling were purchased from Roche Diagnostics and used as recommended by the manufacturer. The probe selected corresponds to the GFP region of mini-Tn5 GFPused in the screening of the STM library (Lehoux et al., 2004). The 820 bp DNA fragment was labelled with the dioxygenin-11-dUTP using the PCR DIG Probe Synthesis kit with primers tag24 (5’-CTT GCG GCG TAT TCT AGT AGG-3’) and gfpR2 (5’-ATC CAT GCC ATG TGT AAT CCC-3’). Amplification conditions were: hot start at 95°C for 15 min followed by a touchdown step 22 cycles (95°C, 1 min; 70-60°C, 1 min decreased by 1°C every 2 cycles; 72°C, 1 min), 10 cycles (95°C, 1 min; 60°C, 1 min; 72°C, 1 min) and a final extension step at 72°C for 7 min. DNA fragments obtained from 2 μg of PstI (New England Biolabs) totally digested genomic DNA from PAO1 and STM2895 were separated on 1 % (w/v) agarose gel, and blotted onto a positively charged nylon membrane (Roche Diagnostics). Before hybridization, the agarose gel was treated as follows: depurination for 20 min (250 mM HCl), denaturation 2 X 15 min (0.5 M NaOH, 1.5 M NaCl), 2 X 15 min neutralization (0.5 M Tris-HCl pH 7.5, 1.5 M NaCl), equilibration 10 min (20X SSC: 300 mM sodium citrate, pH 7.0, 3 M NaCl) and fixing (UV cross-linking, 3 min). The membrane was prehybridized with the DIG easy Hyb for 30 min at 37ºC, incubated with the GFP probe (100 ng) for 18 h at 42ºC and washed with 2X SSC and then with 10X SSC. The membrane was blocked with the DIG wash and Block buffer set and revealed using the Anti-Dioxigenin-AP Fab fragments (dilution 1:5000). CSPDchemiluminescent substratewas diluted in the detection buffer (1:100) and used to reveal the hybridization.
Protease assays
For determination of exoprotease activity, P. aeruginosa strains were cultured in 2 ml of LB broth in 13 mm test tubes without agitation at 37°C for 48 h (Laux et al., 2002). Cell-free supernatant samples were collected by centrifugation for 15 min at 7000 g. and filtered on a 0.45 μm filtration device (Millipore). Aliquots of 100 μl were used to inoculate a brain-heart infusion agar plate containing 1% (w/v) skim milk (Difco Laboratories). Aliquots were dropped into wells and proteolysis zones were visualized after an overnight incubation at 37°C. Elastolysis activity (LasB) was determined with the elastin congo-red assay according to a modification of a previously reported procedure (Ohman et al., 1980). To 15 ml polypropylene screw-cap tubes (Falcon), 10 mg of elastin congo-red (Sigma) and 2 ml of reaction buffer (100 mM Tris-maleate buffer, pH 7.0, 1 mM CaCl2) was added. Supernatant samples were prepared as described above and 1 ml was added to the reaction mixture. Tubes were incubated horizontally overnight at 37°C on a rotary plate at 250 r.p.m. The remaining substrate was removed by centrifugation at 1500 g for 5 min and absorbance was read at 495 nm. Elastase activity was expressed as ΔOD at 495 nm and normalized with bacterial densities present in primary static cultures (OD 600 nm). Elastase A (LasA) activity was measured using a staphylolytic assay (Park and Galloway, 1995). Staphylococcus aureus strain ATCC 25923 was cultured into 25 ml tryptic soy broth overnight at 37°C, cells were centrifuged and resuspended into 12 ml of DE buffer (25 mM diethanolamine buffer, pH 9.5). Cells were heat-killed by boiling at 100°C and diluted in DE buffer to a final OD595 of 1.5. In 13 X 100 mm glass tubes, the heat-killed Staphylococcus suspension (1 ml) was incubated in the presence of aliquots of 300 μl of Pseudomonas supernatants and 700 μl of DE buffer at 37°C for 60 min. Optical densities were measured at 595 nm. Elastase A activity was observed by the decreasing of absorbance values compared to a negative control replacing supernatant by LB broth (ΔOD595). Optical values were normalized to the starting amount of bacteria in primary static culture (OD 600 nm).
Plasmid constructions
All plasmids in this study are listed in Table 1 and plasmid DNAs were purified by using the Qiaprep spin miniprep kit or the Qiafilter plasmid midi kit (Qiagen). Standard recombinant DNA manipulations techniques were used (Sambrook and Russell, 2001). Restriction and modification enzymes were purchased from New England Biolabs and used as recommended by the manufacturer. PCR reactions were performed using a Hotstart Taq Kit (Qiagen). Oligonuleotides synthesized by Invitrogen were used for sequencing and PCR. The starting PAO1 chromosomal DNA template used for PCR amplifications was purified by the procedure described above. Plasmid pMON3401, used for complementation in trans, was constructed by cloning a 1037 bp PCR fragment encoding the PA2895 gene using the primers CO2895sense (5’-CAG TTC TCT AGA CCG AGA TCG CCA CGC TCA CCC AGT C- 3’) and CO2895Asense (5’-CAG TAC GCG CTC TTG CGG CAA TGC TGA CGG CAG ACT G-3’) and was ligated into the pUCP19 Pseudomonas shuttle vector (West et al., 1994). PCR conditions used were hot start at 95°C for 15 min followed by a touchdown step 22 cycles (95°C, 1 min; 65-55°C, 1 min decreased by 1°C every 2 cycles; 72°C, 1 min), 10 cycles (95°C, 1 min; 55°C, 1 min; 72°C, 1 min) and a final extension step at 72°C for 7 min. PCR final mix was supplemented with 5% (v/v) DMSO. The plasmid was introduced into E. coli DH10B ElectroMAX competent cells (Invitrogen) and subsequently into P. aeruginosa STM2895 by electroporation (Enderle and Farwell, 1998). Plasmid pMON3429, used as starting material for allelic exchange, was constructed by cloning the 728 bp fragment encoding the gene PA2896 and generated by PCR with primers 2896Sense (5’-CCT AGA GAG CTC ATG CGT GCC GCG AAG GAT G-3’) and 2896ASense (5’-GGA ACC TCT AGA CGG AAA TGC GCC AGC ATC-3’) using SacI/XbaI restriction sites of pUCP19. PCR conditions were a hot start at 95°C for 15 min followed by a touchdown step 22 cycles (95°C, 1 min; 55-45°C, 1 min decreased by 1°C every 2 cycles; 72°C, 1 min), 10 cycles (95°C, 1 min; 45°C, 1 min; 72°C, 1 min) and a final extension step at 72°C for 7 min. The PCR final mix was supplemented with 2.5% (v/v) DMSO. P. aeruginosa strains with deletions of PA2895 and PA2896 were generated using allelic replacement and the levan-sucrase lethality phenotype (5% sucrose) with the counter-selectable Bacillus subtilis SacB marker (Hoang et al., 1998). Plasmid pMON3401 containing PA2895 was deleted of a 604 bp AvaI/BsmI DNA internal gene fragment, the remaining vector portion was gel purified using Perfectprep Gel Cleanup Kit (Eppendorf), blunt-ended and ligated with the 1078 bp Gm-FRT cassette. The 1698 bp SfiI/HindIII DNA fragment was excised and cloned blunt-end into the SmaI site of pEX18Tc (AF047519) to generate pMON3440. For PA2896, an internal BsmI/ClaI 324 bp DNA fragment was deleted and a blunt-end ligation of the Gm-FRT cassette was inserted giving pMON3429. The 1669 bp SfiI/HindIII DNA fragment was cloned blunt-end into SmaI site of pEX18Tc to generate pMON3441. Plasmids were introduced into the E. coli donor strain SM10 and transfered by conjugation on 0.45 μm filter on BHI agar plates into the recipient strain P. aeruginosa PAO1. Transconjugants were selected on Pseudomonas Isolation Agar (PIA) supplemented with tetracycline 35µg ml-1. Tetracycline resistant clones were collected and plated on PIA + gentamicin 150 µg ml-1 supplemented with 5% (w/v) sucrose to select for double events of homologous recombination. The gentamicin and sucrose resistant clones were purified by streaking on PIA + gentamicin 150 µg ml-1 supplemented with 5% (w/v) sucrose and screened for tetracycline sensitivity. Chromosomal deletions mutant strains were confirmed by PCR using complementation primers (see above) and DNA obtained from boiled bacteria.
Western Blot analysis
Immunoblots were done as described by (Olson and Ohman, 1992). Briefly, supernatants from 48 h culture were concentrated 10 fold with the Microcon YM-10 (Millipore) and an aliquot of 20 μl was mixed with 10 μl of concentrated sample buffer (65 mM Tris-HCl pH 6.8, 25% (v/v) glycerol, 2% (w/v) SDS, 0.01% (w/v) bromophenol blue, 0.5% (v/v) β-mercaptoethanol). Samples were boiled 3-5 min and loaded adjacent to a pre-stained protein standard (New England Biolabs) and resolved on 15% (w/v) SDS-PAGE (Laemmli, 1970) or 4-20 % (w/v) gradient Pre Cast acrylamide gels (Gradipore). Proteins were blotted onto a PVDF membrane (Bio-Rad). Polyclonal rabbit anti-LasB 1:300 000 and polyclonal rabbit anti-LasA 1:2000 were used as primary antibodies; and donkey anti-rabbit HRP-coupled used as secondary. Membranes were washed using 1X PBS + 0.5% (v/v) Tween 20 three times for 5 min at room temperature between antibody incubations and blocked with ECL Donkey Anti-rabbit IgG Peroxidase-linked Kit (Amersham Biosciences). The detection was performed with the ECL Advance Western Blotting Detection Kit (Amersham Biosciences) as recommended by the manufacturer.
Sampling, RNA extraction and transcriptional profiling
For GeneChip analysis, P. aeruginosa strains were grown in LB broth. One hundred microlitre from an overnight test tube static culture was used to inoculate 100 ml LB broth incubated in a 0.5 L Erlenmeyer flask at 37° C with aeration. Cells from late-logarithmic phase cultures (OD600 = 0.8) were collected and RNA degradation was minimized by adding 12.5 ml of ice-cold 5% (v/v) phenol in absolute ethanol (pH < 7.0). RNA was extracted and purified using a previously reported procedure (Bowtell and Sambrook, 2003). DNA contamination of purified RNA was monitored using PCR for amplification of the rplU gene with the primers rplU-for (5’-CGC AGT GAT TGT TAC CGG TG-3’) and rplU-rev (5’-AGG CCT GAA TGC CGG TGA TC-3’). RNA integrity was monitored by agarose gel electrophoresis of glyoxylated samples. Preparation of labeled cDNA and processing of the P. aeruginosa GeneChip arrays was performed as described previously (Schuster et al., 2003). Washing, staining, and scanning of the GeneChips were performed by the University of Iowa DNA core facility using an Affymetrix fluidics station. GeneChip data were analyzed using GeneChip Operating Software (Affymetrix).
In vivo competitive index (CI)
The chronic rat lung model of infection previously described (Cash et al., 1979), was used to confirm the in vivo competitive index (CI) and the previously observed defect in in vivo maintenance for STM2895 (Potvin et al., 2003). Three male Sprague-Dawley rats (300-350 g) (Charles River, Canada) were utilized for determination of the CI for each mutant: STM2895, PAO1 ΔPA2895::Gm and PAO1 ΔPA2896::Gm mixed with the P. aeruginosa wild-type strain PAO1 harbouring pUCP19 (that conferred carbenicillin resistance) (see Table 1). All rats were inoculated intratracheally with 5 X 103 CFUs for the wild-type and mutant and mutant strains giving a total dose of 1 X 104 P. aeruginosa CFU embedded in agarose beads prepared as described (van Heeckeren and Schluchter, 2002). Inoculums were injected into rat lungs using a 1 ml tuberculin syringe containing 100 μl of bead slurry. Animals were sacrificed and lungs were collected at 7 days post-infection. Lungs were homogenized with Polytron and total CFUs were determined by plating serial dilutions on PIA. Mutant and wild-type CFUs were determined by plating the dilutions on selective media MHA + gentamicin 15 μg ml-1 for PAO1 ΔPA2895::Gm, PAO1 ΔPA2896::Gm, MHA + tetracycline 15 μg ml-1 for STM2895 and MHA + carbenicillin 500 μg ml-1 for PAO1 + pUCP19. CI was expressed as the number of CFUs from the mutant strain divided by the number of CFUs from the wild-type strain (Lehoux et al., 2000).
Genomic organization and features of PA2895-PA2896
The genomic organization of the PA2895 operon is presented in Fig. 1A and would encompass at least 2 genes. The transcription of PA2895 and PA2896 is polycistronic and there is a clear evidence for overlapping of the initiation start and stop translation codons for both PA2895 and PA2896 ORFs. Moreover, a Shine-Dalgarno sequence was found upstream of the translation start site (Fig. 1B). Bioinformatics analysis of the deduced polypeptides from PA2895 and PA2896 showed interesting features. The blastp program that uses update databases from the NCBI (http://www.ncbi.nlm.nih.gov) found only one close homologue in the recently released unannotated genome of P. aeruginosa strain UCBPP-PA14 but no predicted function. According to Pseudomonas.com databases, PA2895 encodes a predicted 23 amino acid length transmembrane domain between amino acids 67 and 89. PA2896 is predicted to encode a putative transcriptional regulator of the sigma-70 protein with an ECF subfamily signature (PS01063) (Fig. 1A). The PA2896 putative peptide has homology with a high number of uncharacterized ECF-type sigma factor homologues, including two conserved regions r2 (pfam04542) and r4 (pfam04545). These two regions are involved in the recognition of the -10 and -35 promoter sequences. PA2896 also has a 31% identity with the FecI protein from E. coli K-12 (Braun et al., 2003). Significant identities (48%) to AlgU and SigX of P. aeruginosa were also noted. The genomic organization of PA2895-PA2896 is in agreement with the basic characteristics normally attributed to ECF sigma factors-based regulation (Helmann, 2002). Briefly, PA2896 is co-transcribed with PA2895, a putative transmembrane sensor which could possess an anti-sigma function, and a demonstrated influence on extracytoplasmic functions. We hypothesized that PA2895 could be the anti-sigma factor involved in the transducing of a specific, as yet to be identified, signal from the periplasm activating PA2896-dependent transcription, and causing the exoprotease defects described here. STM2895 is defective in P. aeruginosa major virulence factors
PA2895 was shown to be one of 214 essential in vivo genes identified previously by the screening of a large 7968 STM mutants in the rat lung model of chronic infection (Potvin et al., 2003). The mini-Tn5-Tc GFP insertion of STM2895 was determined by nucleotide sequencing of the junction between the chromosomal and the transposon DNAs of a PstI DNA fragment cloned in pTZ18R which confers tetracycline resistance using as a primer an oligonucleotide encoding the I-end (see material and methods section). Sequencing revealed that the transposon was inserted at the first nucleotide of codon 218. The disrupted 254 amino acids length PA2895 protein is not translated in STM2895. (Fig. 2A). Analysis of insertions in the 214 STM clones confirmed the PA2895 had 11 different insertions and was the most frequently isolated. As shown in Fig. 2B, Southern blot hybridization was performed and indicated the presence of one copy of the transposon in a 3.7 kb PstI chromosomal DNA fragment of STM2895 (Fig. 2B).
Using a qualitative skim milk plate degradation assay, P. aeruginosa STM2895 was shown to be defective in production of exoproteases. As shown in Fig. 3, the wild-type P. aeruginosa strain PAO1 generated a halo of casein degradation that can be compared to those produced by the complemented strain in trans with a functional copy of PA2895 STM2895 + pMON3401 and the PDO801 mutant. β-casein degradation is known to be the preferential substrate of the alkaline protease and mainly the LasB elastase (Caballero et al., 2001). The absence of casein degradation by STM2895 confirmed that the LasB elastase is inactive; similar results were obtained with the PDO240-1 strain. Thus, one of the major secreted proteases of P. aeruginosa, LasB elastase, is inactive in the STM2895 mutant. To confirm these results and to analyse exoprotease defect specificity, a combination of assays including the elastin congo-red (ECR), staphylolytic (SA) and poly-L-lysine degradation assays (PLD) were performed with the same strain collection. Preliminary experiments using culture supernatants in anaerobic growth conditions (static 2-days culture) were tested in ECR and SA assays and confirmed results obtained with plate assay. As shown in Table 2, the level of staphylolytic elastase A activity detected for STM2895, for the PAO1 ΔPA2895::Gm and PAO1 ΔPA2896::Gm is virtually zero and comparable to the PDO801 control strain. The activity of LasA elastase is fully recovered in the STM2895 strain harbouring the pMON3401 plasmid in trans expressing the PA2895 protein. The PDO240-1 strain which is deficient in the production of LasB elastase has reduced LasA activity since LasB was shown to be important in the processing of LasA (Kessler et al., 1998). LasB elastase is known to be the most abundant protease secreted by P. aeruginosa (McIver et al., 2004) and is one of the major virulence factors (Woods et al., 1982). An Elastin congo-red substrate assay specific for LasB was used to determine the influence on the activity of LasB elastase in a PA2895-PA2896 mutant background. As shown in Table 3, STM2895, PAO1 ΔPA2895::Gm and PAO1 ΔPA2896::Gm strains produced low amounts of elastinolytic activity due to LasB, when compared to wild-type PAO1. As LasA and LasB enzymes are known to be induced in the late log phase of growth via the induction of quorum-sensing, we wanted to verify if the protease defect associated to LasA and LasB was dependant on a specific growth point when compared to wild-type expression. Fig. 4 shows growth curves realized in aerobic conditions with a collection of the four most relevant strains. His clearly confirmed that none of these strains were altered in their ability to grow when compared to wild-type PAO1. Using supernatant samples of these cultures, ECR and SA assays were performed to verify the exact point of elastases induction via the PAO1 strain and the associate protease defect of the STM2895 mutant strain. As depicted by Fig. 5 and 6, LasA and LasB activities are detectable around 1-1.2 OD 600 as predicted by the QS induction machinery in late log - early stationary phase. On the other hand, STM2895 produce virtually no more exoprotease activity over detectable elastinolytic activity throughout the growth spectrum and confirmed that the LasA and LasB defect is constitutive. Surprisingly, the complemented strain in trans, with the functional copy of PA2895, seems to accumulate expression delay of the proteolytic enzymes and recovered only a small amount of the lost activity. This phenomenon is likely due to the aerobic growth conditions as static culture showed the fully recovered phenotype.These observations proposed a noticible link between the expression of PA2895 and oxygen availability.
P. aeruginosa secretes two other exoproteases in its supernatant; the lysine-protease protease IV encoded by the prpL gene (Wilderman et al., 2001) and an alkaline protease (Morihara et al., 1965). The PLD assay is specific for lysine proteases activity and was previously demonstrated to be able to degrade poly-L-lysine substrate (Caballero et al., 2001). Fig. 8 clearly demonstrates that lysine proteases are affected in STM2895 strains. However, the PLD assay cannot discriminate between protease IV and alkaline protease activities. The presence of a lower spot in lanes 2 and 3 in Fig. 8 confirmed that the poly-L-lysine substrate is partly degraded, in contrast to the PAO1 wild-type strain (lane1, in Fig. 8). This defect can also complemented in trans in STM2895 using a functional copy of PA2895 expressed in the pMON3401 plasmid. These results suggest that LasA, LasB and one or perhaps both lysine exoproteases are inactive in the mutant strains of PA2895 and PA2896.
Synthesis and secretion of elastases is not altered in the STM2895 mutant
To determine the level of production of LasA and LasB elastases and confirm if they are inactive in culture supernatants of STM2895, PAO1 ΔPA2895::Gm and PAO1 ΔPA2896::Gm, we performed immunoblots to detect the presence and the integrity of these two exoprotease proteins. SDS-PAGE analysis demonstrated that the major exoprotease, LasB elastase, is present in a mature form of 33 kDa for the PAO1 wild-type strain, and is shown in lane 1 of Fig. 7A-B (Kessler et al., 1998). When compared to other strains, LasB is also detected in a correctly processed form in mutant strains STM2895, PAO1 ΔPA2895::Gm and PAO1 ΔPA2896::Gm; and is totally absent in the PDO240-1 mutant strain used as a control. Moreover, separation of proteins secreted into the supernatant by SDS-PAGE revealed the presence of a protein of 20 kDa corresponding to LasA elastase that accumulated in the supernatants of STM2895, STM2895 + pUCP19, and both mutants ΔPA2895 and ΔPA2896. Immunoblots performed with anti-LasA and anti-LasB polyclonal antibodies (Fig. 7B and Fig. 7C) confirmed these observations. Fig. 7B shows that the PAO1 wild-type strain and the complemented strain STM2895 + pMON3401 produced a lower amount of LasA elastase when compared to STM2895, PAO1 ΔPA2895::Gm and PAO1 ΔPA2896::Gm strains. LasA is present in the supernatant of STM2895 and in strains with deletions in PA2895 or PA2896, but it accumulates in a processed form of 20 kDa and remains inactive. Western blot analysis using an anti-LasB antibody showed that LasB elastase is also present in culture supernatants of all strains tested, except for PDO240-1 ΔlasB strain (Fig. 7C). When compared to wild-type PAO1 and the complemented strain, STM2895 and the ΔPA2895 and ΔPA2896 mutants produced significantly less detectable LasB elastase. We conclude that LasA and LasB elastases are produced by the STM2895 mutant strain in a correctly processed form, but both remain inactive. It is known that these enzymes have complex periplasmic refolding steps, involving disulfide bonds formation (Braun et al., 2001).
Transcription profiling of genes affected by PA2895-PA2896
To identify genes regulated by PA2895-PA2896 in P. aeruginosa, we used the complete genome transcriptional analysis GeneChip from Affymetrix and compared transcript profiles between the STM2895 mutant and an isogenic mutant of PA2896 with the transcription profile of the parent strain PAO1. For STM2895 and for PAO1 ΔPA2896::Gm, we noted changes in 128 genes (64 up and 64 down) and 138 genes (59 up and 79 down), 4 intergenic regions and 12 tRNA coding regions which were shown to be differentially regulated by ≥ 5 fold. When compared together, transcriptional data from both chips were similar. In fact, the mutation created in PA2896 presumably gave a polar effect for PA2895, aborting its transcription because of the insertion of the gentamicin resistance cassette upstream. Results for upregulational genes are summarized in Table 2. Results for downregulated genes are presented in Table 3. Indeed, there are 76% (53 on 70 different genes listed) of upregulated expressed genes common for both mutants and 59% (53 on 90 different genes listed) for downregulated genes. These percentages take into account only values above a 5 fold threshold, as presented in Tables 2 and 3. Genes regulated by PA2895 and PA2896 in both chips have comparable values in terms of fold change. In 26% of cases upregulation difference between fold change values is greater than 3. For downregulation, 42% of coregulated genes have a significant difference greater than 3 fold in both experiments.
The most interesting and significant data concerning induced genes by PA2895 and PA2896 is the number of operons, or gene clusters, involved in extracellular functions and genes associated with virulence. For instance, several regulated genes are members of the type III secretion system, phenazine, hydrogen cyanide and pyochelin biosynthesis, exoenzymes Y and S synthesis and elastase B; all of which are involved in virulence of P. aeruginosa. Another gene cluster upregulated in STM2895 and PAO1 ΔPA2896::Gm mutants is PA3327-PA3336 including cytochrome P-450 which is probably involved in enzymatic detoxification of antibiotics (Desomer et al., 1992; Munro and Lindsay, 1996). Also upregulated are two operons involved in transport of the small molecules PA2110-PA2114 which include a probable porin PA2113, a probable MFS transporter PA2114 and a group of probable ATP dependent ABC transporters PA3187-3190. Finally, an operon of uncharacterized function PA0492 to PA0496 was upregulated in both mutants tested.
The induced genes identified here also re-group genes that are not yet characterized as part of an operon and include three transcriptional regulators. The denitrification regulator Dnr protein and a probable transcriptional regulator PA1196 in both strains tested and PA2896 was found only in the STM2895 mutant. This would indicate a feedback loop regulation of the PA2895-PA2896 operon. This interesting result supports the hypothesis that PA2895 could act as an anti-sigma negative regulator on sigma-70 ECF PA2896. The high positive values of +55.7 for upregulation of PA1494 and +13.0 for PA4495 found only in STM2895 transcription profiling and not in PAO1 ΔPA2896::Gm suggested that these genes are regulated by PA2896.
The repressed genes for transcription profiling of STM2895 and ΔPA2896::Gm are listed in Table 3. The analysis of repressed genes controlled by the PA2895-PA2896 regulon revealed that the hierarchy of regulation involved at least 5 other sigma-70 factors of the ECF subfamily, such as PA0472, PA1300, PA3899 and PA4896 of uncharacterized function and the iron-regulator PvdS (PA2426). Moreover, PA0149, PA1912, PA2468 and PA3410 are also sigma-70 factors (ECF subfamily) that are downregulated, but less than 5 fold (data not shown). Other transcriptional regulators are significantly repressed such as rsmA, which is involved in post-transcriptional repression of secondary metabolites including exoproteases, hydrogen cyanide and phenazine compounds (Pessi et al., 2001). Downregulation of the RsmA regulator would thus be linked to the induced response of secondary metabolites described above. Another transcriptional regulator identified as repressed is pchR (PA4227), an AraC-like regulatory protein, which is involved in the production of the pyochelin siderophore (Heinrichs and Poole, 1996). We also noted a strong repressive effect on nir, nor and nar genes by the PA2895-PA2896 mutations. These genes are involved in the denitrification pathway of P. aeruginosa and have been shown to be regulated by Anr/Dnr proteins (Arai et al., 1995) (Vollack et al., 1998). The cytochrome o complex (cyo genes), the predominant terminal oxidase in the aerobic respiratory chain of E. coli and related bacteria such as P. aeruginosa grown under conditions of high aeration, are also repressed in the absence of PA2896 (Chepuri et al., 1990). Other genes associated with oxygen transfer and detoxification are significantly repressed and include probable oxidoreductases (PA1137, PA4615), hmgA dioxygenase, superoxyde dismutase and the strongly repressed fhp gene flavohemoprotein which has been recently shown to protect against oxidative shock in E. coli (Justino et al., 2005). Probably the most relevant finding revealed by the study of the PA2895-PA2896 regulon is the high number of genes that are shared with previously reported microarray experiments in relation to iron metabolism. Effectively, Palma et al., 2003 and Ochsner et al., 2002 published two exhaustive lists of genes that are differentially regulated in response to iron and iron starvation identified in bold in Tables 2 and 3. These results suggest that PA2895-PA2896 could control the response to iron at another level by downregulation of PvdS, one of the major iron regulators. This would have a major impact for in vivo maintenance. P. aeruginosa possesses an obligatory oxidative metabolism, the iron-based oxygen transport is highly used during growth in full aeration; this kind of metabolism is the most energetic but also the most toxic (Vasil and Ochsner, 1999). In fact, during oxygen-based respiration processes free-radicals are created and lead to protein damage (Hassett et al., 1999). The absence of this kind of detoxification system is likely leading to changes in the redox potential of the periplasm, resulting in the elastases misfolding.
PA2895 is essential for in vivo maintenance
Competitive Index (CI) aimed to analyse the ability of a specific P. aeruginosa mutant strain to compete for maintenance in the rat lung. As noted in Table 6, STM2895 and PAO1 ΔPA2895::Gm strains are significantly attenuated when challenged with PAO1 parent strain. In fact, CI values (0,074 and 0,116) confirmed that mutations in the PA2895 gene cause a fatal reduction of approximately 1 log CFUs compared to the wild type PAO1 in the rat lung. We also tested the PAO1 ΔPA2896::Gm strain in the same experiments and surprisingly, CI value was greater than 1 (>1) indicating that this strain is able to maintain chronic infection as PAO1 wild-type strain. We hypothesized above that the insertion of the gentamicin resistance cassette (1 kb) in the ΔPA2896::Gm strain could act as a polar effect on the transcription of PA2895 causing the same observable exoprotease defect and the comparable microarray results for the strain STM2895. But it is noticeable to remember that the PA2896 gene was not identified by our STM screening and by any other in vivo studies. This result suggests certainly an important implication of growth phase and culture conditions in the observation of the different phenotypes described on here the PA2895 knock-out strain and also likely suggest a multipartite regulation network.
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Fig. 1. Genetic organization of the P. aeruginosa PAO1PA2896-PA2895 region under study. (A) Schematic depiction of the two ORFs represented by arrows and in-box summarizes of relevant genomic features from both genes. Gene and protein lengths, protein topology and putative function information were imported from pseudomonas.com databases. Homologues were found using BLASTP software from NCBI. (B) Genomic sequence analysis and proper translation showing the overlap of both ORFs on one codon (bold) and a putative Shine-Dalgarno sequence (underline) upstream of the translation start site of PA2895.
Fig. 2. Unique transposon insertion of STM2895. (A) Schematic representation of mini-Tn5 gfp tag 24 inserted in gene PA2895. The mini-Tn5 (light green) is represented by DNA insertion that disrupted PA2895 (blue). The horizontal bar represents the 3.7 kb pst I fragment visualized on Southern blot analysis (B) using the dig-labelled 820 pb gfp probe (in red). STM2895 DNA track 1, PAO1 DNA track 2, control unlabelled PCR product probe track 3.
Fig. 3. Exoproteases analysis of STM2895. Proteolytic activity was measured qualitatively from supernatant from different strains of Pseudomonas aeruginosa using BHI agar plates containing 1% skim milk. Supernatant samples were collected from 48 hour static cultures and 100μl was dropped onto previously removed agar plugs and proteolytic activity was visualized after incubation overnight at 37°C.
Fig. 4 Growth curves. Growing ability in aeration conditions of four relevant Pseudomonas aeruginosa strains was measured using optical densities (OD) at 600 nm following a time course. An aliquot from overnight cultures was used to inoculate tryptic soy broth (supplemented with appropriate antibiotics) to a starting OD of 0.01 from an overnight culture of these strains. Green curve: PAO1 wild-type, red curve: STM2895, black curve: STM2895+pUCP19, blue curve: STM2895+pMON3401.
Fig. 5. Elastase LasB activity. Elastin-Congo red degradation assays were performed on selected triplicate samples from growth curve experiments. LasB activity was quantitated by a single overnight optical density value at 495 nm using the cell-free supernatant from the collected samples and resulting from the degradation of elastin covalently linked to congo-red. Green bar: PAO1 wild-type, red bar: STM2895, black bar: STM2895+pUCP19, blue bar: STM2895+pMON3401.
Fig. 6. Elastase LasA activity. Staphylolytic assays were performed on selected triplicate samples from growth curve experiments. LasA activity was evaluated by a single 60 minute elapsed optical density value at 595 nm using the cell-free supernatant from the collected samples and resulting from the degradation of a heat-killed Staphylococcus aureus suspension. Green curve: PAO1 wild-type, red curve: STM2895, black curve: STM2895+pUCP19, blue curve: STM2895+pMON3401.
Fig. 7. Exoproteins analysis. (A) Culture supernatants were concentrated 10-fold using Amicon ym-10 column and samples were resolved on a 15% SDS-PAGE. (B) Proteins were blotted on a PVDF membrane and polyclonal rabbit anti-LasB 1:300 000 or anti-LasA 1:2000 (C) was used as a primary antibody; donkey anti-rabbit HRP-coupled as secondary. L = ladder, PAO1 = PAO1 wild-type strain, STM = STM2895, 3401 = STM2895+pMON3401, LasA = lasA::gm, LAsB = ΔlasB::sm, Δ2895 = ΔPA2895::gm, Δ2896 = ΔPA2896::gm.
Fig. 8. Lysine proteases analysis. Poly-L-lysine degradation was measured using 10 μl of overnight culture supernatant incubated in the presence of 40 μg of poly-L-lysine (mol. wt. 500-2000) in 10 mM Tris (pH 8.0), final volume 20 μl. Mixtures were incubated at 50°C for 1 h and then spotted on silica-gel TLC. The plate was chromatographed for 1.5 h in 10:1 ammonium hydroxide - n-propanol. Spots were revealed using ninhydrin spray 0,2% in ethanol. PAO1 - Track 1, STM2895 - Track 2, STM2895+pUCP19 - Track 3, STM2895+pMON3401 - Track 4, , Poly-L-lysine negative control - Track 5.
a Elastolytic activity was evaluated by a single staphylolytic assay using the cell-free supernatant from a 48 h static culture and a heat-killed Staphylococcus aureus suspension. Hydrolytic activity was measured by the decrease of the optical density at 595 nm for 60 min at 37°C.
b ΔOD is a comparative value from the negative control LB broth and the supernatant LasA activities. The value given is directly proportional with LasA elastase activity.
c The supernatant sample was substituted by LB broth.
a LasB activity was quantitated by a single elastin congo-red assay using the cell-free supernatant from a 48 hours static culture. The degradation of elastin covalently linked to congo-red was measured by increasing of optical density at 495 nm.
b ΔOD is a comparative value from the negative control LB broth and the supernatant Las B activity. The value is directly in proportion with LasB elastase activity.
c The supernatant sample was substituted by LB broth.
Table 4. Comparative list of genes upregulated in PAO1 ΔPA2896::Gm and STM2895 as determined by one chip hybridization analysis with wild-type strain PAO1.
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a b PA numbers in bold indicate that these genes were previously demonstrated to be regulated by iron. a Genes predicted responding to iron by Palma M. et al., 2003 b Genes involved in response to iron starvation by Ochsner U.A. et al. 2002
c Fold change in upregulation of mRNA level of STM2895 and PAO1 Δ2896::Gm strains compared to the wild-type PAO1. Values in red indicate that the mRNA level is below 5 fold.
Table 5. Comparative list of genes downregulated in PAO1 ΔPA2896::Gm and STM2895 as determined by one chip hybridization analysis with wild-type strain PAO1.
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a b PA numbers in bold indicate that these genes were previously demonstrated to be regulated by iron. a Genes predicted responding to iron by Palma M. et al., 2003 b Genes involved in response to iron starvation by Ochsner U.A. et al. 2002
c Fold change in downregulation of mRNA level of STM2895 and PAO1 Δ2896::Gm strains compared to the wild-type PAO1. Values in red indicate that the mRNA level is below 5 fold.
Table 6. Competitive index of the STM2895, PAO1 ΔPA2895::Gm and PAO1 ΔPA2896::Gm strains compare to parent strain PAO1.
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a Values were calculated in comparison to the wild-type strain PAO1. CI is defined as the output ratio of mutants (CFUs) divided by the output ratio of wild-type PAO1 (CFUs).
© Éric Potvin, 2007
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| Chapitre 3 Génomique fonctionnelle in vivo de Pseudomonas aeruginosa et criblage à haut débit de nouveaux facteurs de virulence et de cibles antibactériennes |  | Discussion |
