- Email: [email protected]
Oligoribonuclease is required for the type III secretion system and pathogenesis of Pseudomonas aeruginosa
Microbiological Research 188 (2016) 90–96
Contents lists available at ScienceDirect
Microbiological Research journal homepage: www.elsevier.com/locate/micres
Oligoribonuclease is required for the type III secretion system and pathogenesis of Pseudomonas aeruginosa Gukui Chen a , Qiang Zhao b , Feng Zhu a , Ronghao Chen a , Yongxin Jin a , Chang Liu a , Xiaolei Pan a , Shouguang Jin a,c , Weihui Wu a , Zhihui Cheng a,∗ a State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 300071, China b College of Life Sciences, Nankai University, Tianjin 300071, China c Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL 32610, USA
a r t i c l e
i n f o
Article history: Received 29 January 2016 Received in revised form 29 April 2016 Accepted 1 May 2016 Available online 4 May 2016 Keywords: Pseudomonas aeruginosa Oligoribonuclease Type III secretion system Pathogenesis
a b s t r a c t Oligoribonuclease (Orn) is a 3 to 5 exonuclease that degrades nanoRNAs, which can serve as primers for transcription initiation at a significant fraction of promoters. One of Orn’s substrates, pGpG inhibits the enzymatic activity of EAL-domain containing phosphodiesterases (PDEs), thereby increasing intracellular cyclic-di-GMP (c-di-GMP) level. Here, we found that an orn mutant of Pseudomonas aeruginosa displayed reduced cytotoxicity, which was mainly due to deficient type III secretion system (T3SS). Given the importance of T3SS in pathogenicity, we examined the bacterial virulence in a mouse acute pneumonia model and found that the orn mutant was highly attenuated compared to the wild type PA14 strain. Overexpression of an EAL domain-containing PDE reduced the c-di-GMP level as well as biofilm formation in the orn mutant. However, no effect was observed on the expression of T3SS genes, suggesting that increased c-di-GMP level is not the solely cause of defective T3SS in the orn mutant. Overall, our results demonstrated an essential role of Orn in the expression of T3SS as well as pathogenesis of P. aeruginosa. © 2016 Elsevier GmbH. All rights reserved.
1. Introduction Pseudomonas aeruginosa is a versatile gram-negative bacterium which causes a variety of acute and chronic infections, such as urinary tract infections, burn and wound infections, sepsis, and severe pneumonia (Driscoll et al., 2007). One of the virulence factors that strongly correlated with acute infection in both animal models and human patients is the type III secretion system (T3SS) (Roy-Burman et al., 2001; Hauser et al., 2002). The T3SS is a complex protein secretion and delivery machinery harbored by many animal and plant pathogens, through which bacterial effector molecules are directly translocated into host cell cytosols, causing disruption of intracellular signaling or cell death (Francis et al., 2002). Loss of the T3SS attenuates P. aeruginsa virulence in various mouse acute infection models (Holder et al., 2001; Goodman et al., 2004). The expression of T3SS genes is controlled by a master regulator ExsA (Hovey and Frank, 1995). Four T3SS effectors have been identified in P. aeruginosa, namely, ExoU, ExoS, ExoT and ExoY (Shaver and
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (Z. Cheng). http://dx.doi.org/10.1016/j.micres.2016.05.002 0944-5013/© 2016 Elsevier GmbH. All rights reserved.
Hauser, 2004). It has been demonstrated that during early pneumonia, ExoU is rapidly induced and injected into phagocytic cells, inducing rapid and complete cell lysis (Diaz and Hauser, 2010). Delayed expression of ExoU leads to decreased bacterial burden in lungs of infected mice (Howell et al., 2013). Thus, the T3SS plays an important role in the killing of neutrophils and is essential for survival and proliferation of the bacteria within the host (Diaz et al., 2008; Diaz and Hauser, 2010; Howell et al., 2013). Oligoribonuclease (Orn) is a highly conserved 3 to 5 exonuclease in bacteria, which converts 2–5-nt RNAs, termed “nanoRNAs” to mononucleotides (Ghosh and Deutscher, 1999). Orn is essential for the viability in Escherichia coli (Ghosh and Deutscher, 1999), however, P. aeruginosa remains viable in the absence of Orn (Jacobs et al., 2003), making it an excellent model for Orn research. In P. aeruginosa, depletion of the Orn causes accumulation of nanoRNAs, which serves as primers for transcription initiation at many promoters, causing global alterations in gene expression (Goldman et al., 2011). Additionally, Orn plays an essential role in cyclic-di-GMP (c-di-GMP) turnover (Cohen et al., 2015; Orr et al., 2015). cdi-GMP is an important secondary messenger in most bacteria (Romling et al., 2013). High level of intracellular c-di-GMP in P. aeruginosa represses bacterial motility and promotes extracellular
G. Chen et al. / Microbiological Research 188 (2016) 90–96
polysaccharides production and biofilm formation; whereas low level of c-di-GMP promotes motility and expression of virulence factors associated with acute infections (Romling et al., 2013). cdi-GMP is synthesized by diguanylate cyclases (DGC) harboring a GGDEF domain. Degradation of c-di-GMP is catalyzed by phosphodiesterases (PDE) harboring an EAL or HD-GYP domain, which results in 5 -phosphoguanylyl-(3 , 5 )-guanosine (pGpG) or GMP (Schmidt et al., 2005; Ryan et al., 2006). pGpG is further hydrolyzed by Orn into two GMPs (Cohen et al., 2015; Orr et al., 2015). Deletion of orn causes elevated levels of pGpG, which exerts feedback inhibition on EAL domain-containing PDEs, leading to increased intracellular c-di-GMP concentration and a hyperbiofilm phenotype (Cohen et al., 2015; Orr et al., 2015). However, whether Orn plays a role in bacterial pathogenesis remains unknown. In this study, we identified orn as an essential gene for the motility and T3SS in P. aeruginosa. Consistent with the in vitro phenotypes, the orn mutant displays attenuated virulence in a mouse acute pneumonia model. Thus, our results reveal novel roles of Orn in bacterial pathogenesis.
2. Materials and methods 2.1. Bacterial strains and plasmids The bacterial strains and plasmids used in this study are listed in Table 1. Bacteria were cultured in Luria–Bertani (LB) broth (Sambrook and Pollack, 1974) or LB agar (LB broth containing 1.5% agar) under aerobic conditions at 37 ◦ C. When needed, the medium was supplemented with tetracycline (100 g/ml) (BBI life sciences, Shanghai, China), gentamicin (100 g/ml) (BBI life sciences), or carbenicillin (150 g/ml) (BBI life sciences) for P. aeruginosa, and ampicillin (100 g/ml) (BBI life sciences) for E. coli. For DNA manipulation, standard protocols or manufacture instructions of commercial products were followed. The PA14 36990 coding region was amplified and cloned into the EcoR I-BamH I sites of pUCP20 (West et al., 1994), resulting in pUCP20PA2133. orn gene with its 325-bp upstream region was amplified using wild type P. aeruginosa PA14 strain chromosomal DNA as a template and cloned into Kpn I-Hind III sites of pUC18T-Mini-Tn7TTc (Choi and Schweizer, 2006), resulting in pRN00. Chromosomal gene mutations were generated as described previously (Hoang et al., 1998). Primers used in this study are listed in Table S1.
2.2. Biofilm formation and motility assay Biofilm formation was measured as previously described (Li et al., 2013). Briefly, overnight bacterial cultures were diluted to an OD600 of 0.025 in LB broth and incubated in each well of a 96well plate at 37 ◦ C for 24 h. For quantification of biofilm formation, each well was washed three times with 1 × phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2 HPO4 , pH 7.4) and stained with 0.1% crystal violet, followed by three washes with 1 × PBS. Then, 200 l ethanol was added to each well. After a 10min incubation at room temperature, the OD590 of each sample was measured. P. aeruginosa motilities were assessed as described previously with slight modifications (Rashid and Kornberg, 2000). LB broth solidified with 1% agar was used for the twitching motility assay. The media used for swimming motility assay consisted of 10 g/l tryptone, 5 g/l NaCl and 0.3% agar. Fresh bacterial colonies were inoculated onto plate bottom (for twitching) or surface (for swimming) with sterile toothpicks. After inoculation, plates for twitching motility test were incubated at 37 ◦ C for 24 h, while plates for swimming motility test were incubated at 37 ◦ C for 12–14 h.
91
2.3. Cytotoxicity assay HeLa cells (1.2 × 105 ) were seeded into each well of a 24-well plate. The cells were cultured in DMEM medium with 10% fetal bovine serum (FBS) at 37 ◦ C in 5% CO2 and 95% air for 24 h. Overnight bacterial culture was sub-cultured in fresh LB broth to the log growth phase (OD600 of 0.8) before infection. Bacteria were washed once with 1 × PBS and resuspended in 1 × PBS to an OD600 of 1.0. HeLa cells were infected with bacteria at a multiplicity of infection (MOI) of 30. At the end of incubation, lactate dehydrogenase (LDH) present in the supernatant was measured in each well using the LDH cytotoxicity assay kit (Beyotime, Haimen, China). HeLa cells treated with Triton X100 (0.9%) were used as a control of total release (100% LDH release). The background level (0% LDH release) was determined with DMEM medium with 10% FBS. The percentage of cytotoxicity was calculated following the manufacture’s instructions. 2.4. Mouse acute pneumonia model All animal experiments complied with Nankai University and national guidelines regarding the use of animals in research. Bacteria were grown in LB broth overnight and then sub-cultured into fresh LB broth at 37 ◦ C with aeration to the exponential phase. Bacteria were centrifuged and adjusted to 1 × 109 CFU/ml in 1 × PBS. Female BALB/c mice (6–8-week old) were anesthetized with an intraperitoneal injection of 7.5% chloral hydrate (100 l per mouse). Anesthetized mice were intranasally inoculated with 10 l of bacterial suspension in each nostril, giving a total infection volume of 20 l. Bacterial colonization in the lung was determined as described previously (Sun et al., 2014). Briefly, at 12 h post infection (hpi), mice were sacrificed by inhalation of CO2 . Lungs were isolated and homogenized in 1% proteose peptone, and bacterial cell numbers were determined by serial dilution and plating. 2.5. Quantification of c-di-GMP Concentration of c-di-GMP was measured as described previously (Nakayama et al., 2011). Bacteria were grown to an OD600 of 1.5 at 37 ◦ C in LB medium (15 ml) with agitation. Bacteria were collected by centrifugation and resuspended to a final volume of 1 ml of 10 mM Tris-HCl (pH 8.0) containing 100 mM NaCl, followed by lysis by sonication. Protein concentrations of the lysates were determined with a Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, California, USA). Perchloric acid was added to the lysate at a final concentration of 12% to precipitate cellular macromolecules. The mixture was incubated on ice for 10 min and then neutralized with 3 M KOH, 0.4 M Tris and 2 M KCl. The mixture was centrifuged, and the supernatant was filtered through a 0.2 m filter and 3 kD exclusion columns. The collection was incubated at 95 ◦ C for 5 min, and then room temperature for 15 min. Thiazole orange was then added to the mixture and incubated at 4 ◦ C overnight. To measure fluorescence, Varioskan Flash (Thermo Scientific, Netherlands) was used. The instrument settings were chosen as follows: excitation, 508 nm, emission, 533 nm. The measurements were carried out at 10 ◦ C. 2.6. RNA extraction from in vitro samples Total RNA was isolated with RNAprep pure cell/bacteria Kit (Tiangen Biotech, Beijing, China). Overnight bacterial cultures were sub-cultured in fresh LB broth to exponential phase or stationary phase. To determine bacterial response during infection of HeLa cells, bacteria were added to the cells at a MOI of 30. After 2 h, cells and bacteria in each well were collected and subjected to RNA isolation.
92
G. Chen et al. / Microbiological Research 188 (2016) 90–96
Table 1 Strains and plasmids used in this study. Strain or plasmid
Relevant characteristics or function
Reference or origin
P. aeruginosa PA14 PA14 orn:Gm PA14 flic:Gm PA14 orn PA14 orn/orn PA14 orn Ptac -exsA PA14 orn/PA2133
Wild type strain Orn insertion mutant of PA14, Gmr FliC insertion mutant of PA14, Gmr Orn deletion mutant of PA14 PA14 orn mutant complemented by chromosome inserted orn gene PA14 orn mutant with a chromosome inserted exsA gene driven by tac promoter PA14 orn mutant containing plasmid pUCP20-pa2133
(Liberati et al., 2006) (Liberati et al., 2006) This study This study This study This study
Plasmids pUCP20 pEX18Tc pUC18T-miniTn7T-Tc pRN00 pTNS3 pPA2133
Escherichia-Pseudomonas shuttle vector; Ampr Gene replacement vector, Tcr For gene insertion in chromosome; Tcr pUC18T-miniTn7T-Tc containing orn gene with its 325-bp upstream region Helper plasmid, for gene insertion in chromosome; Ampr PA14 36990 gene of PA14 on pUCP20 driven by lac promoter; Ampr
(West et al., 1994) (Hoang et al., 1998) (Choi and Schweizer, 2006) (Choi and Schweizer, 2006) This study
2.7. RNA extraction from in vivo samples Mice were inoculated with 2 × 107 CFU bacteria intranasally. At indicated time points mice were sacrificed by CO2 . Bronchoalveolar lavage fluid (BALF) was obtained by annulation of the trachea followed by two instillations of 1 ml sterile 1 × PBS with 0.5 mM EDTA. 100 l of the BALF was used for bacterial counting, while the remaining BALF was centrifuged and the pellets were immediately resuspended in 200 l TRIzol reagent (Invitrogen). Total RNA was isolated using a Direct-zol RNA Miniprep kit (ZYMO research, CA, USA). 2.8. Quantitative real time PCR (qRT-PCR) cDNA was synthesized from each RNA sample using a PrimeScript Reverse Transcriptase (TaKaRa, Liaoning, China) with random primers, then subjected to qRT-PCR using SYBR Premix Ex Taq II (TaKaRa). The 30s ribosomal protein gene rpsL was used as an internal control. 3. Results
Fig. 1. An orn mutant is defective in cytotoxicity. HeLa cells were infected with the indicated strains at a MOI of 30. At 3 hpi, released LDH activity was measured by LDH assay kit. LDH activity of each sample was normalized against the LDH activity of HeLa cells treated with Triton X100 (0.9%). A representative of three independent experiments with the same results is shown. The data shown are means ± standard errors of the means. **, p < 0.001 compared to wild type PA14 strain by Student’s t test.
3.1. Orn is required for bacterial cytotoxicity Ribonuclease, such as polynucleotide phosphorylase (PNPase), RNase R and RNase E, was reported to participate in modulation of virulence in many pathogens (Lawal et al., 2011). Additionally, we have previously identified that PNPase is involved in the regulation of T3SS in P. aeruginosa (Li et al., 2013). To assess the roles of other ribonucleases in the regulation of bacterial virulence, we picked five ribonuclease gene mutants from the nonredundant library of PA14 transposon mutants (Liberati et al., 2006) and examined their cytotoxicities. As shown in Fig. 1, majority of the HeLa cells were lysed at 3 hpi by wild type PA14 strain. However, one of the five mutants, orn:Tn, displayed reduced cytotoxicity. To confirm the role of Orn, we constructed an orn in frame deletion mutant, which also displayed reduced cytotoxicity (Fig. 2A). Complementation with an orn gene restored the cytotoxicity (Fig. 2A), thus confirming the role of Orn in cytotoxicity. 3.2. Orn is required for bacteria-host cell contact and T3SS In the above assay, the bacterial cytotoxicity depends on close contact with the host cells and injection of bacterial effector proteins (Hauser, 2009). Thus, we examined the expression of genes involved in bacterial motility and adherence, including flagella, type IV pilus and CupA system (Comolli et al., 1999; Vallet et al., 2001). The mRNA levels of flgB (encoding flagellum basal body rod
protein FlgB), fliC (encoding flagellin) (Brimer and Montie, 1998), pilH (encoding twitching motility protein PilH) (Mattick et al., 1996) and cupA3 (encoding usher CupA3) decreased significantly in the orn mutant (Fig. 2B and Fig. S1A). Consistently, the orn mutant displayed reduced swimming (Fig. 2C) and twitching (Fig. S1B) motilities, which were restored to wild type level by complementation with an orn gene. Mutation in cupA3 or pilH had no effect on cytotoxicity (Fig. S1C) in our experiments. In contrast, mutation in fliC, resulted in defective cytotoxicity (Fig. 2A). Spinning of the tissue culture plate after addition of the bacteria, which enforces the bacterial contact with HeLa cells, restored the cytotoxicity of the fliC mutant (Fig. 2A), thus confirming the role of flagella in the cytotoxicity. However, spinning of the culture plate was unable to restore the cytotoxicity of the orn mutant or an exsA:Tn mutant, suggesting a defective T3SS in the orn mutant (Fig. 2A). To verify the activity of T3SS in the orn mutant, we examined the mRNA levels of a positive regulatory gene exsC and two structural genes pcrV and popD (Hauser, 2009). Bacteria were added to HeLa cells in each well. The plate was spun and incubated for 2 h. Then, bacteria were collected and subjected to RNA isolation and qRT-PCR. In the absence of HeLa cells, no difference in mRNA levels was observed between the wild type PA14 strain and orn mutant (Fig. 3A). However, in the presence of HeLa cells, the mRNA levels of exsC, pcrV and popD were significantly lower in the orn mutant than those in wild type PA14 strain (Fig. 3A). To further confirm the
G. Chen et al. / Microbiological Research 188 (2016) 90–96
93
Fig. 3. Orn is required for the full activation of T3SS genes. Total RNA was isolated from bacterial cells before (−) or after (+) incubation with HeLa cells (A) or treated with 5 mM EGTA (B). The mRNA levels of exsC, pcrV and popD were determined by qRT-PCR. The 30S ribosomal protein-encoding gene rpsL was used as an internal control. A representative of three independent experiments with the same results is shown. The data shown are means ± standard errors of the means. **, p < 0.001 compared to wild type PA14 strain by Student’s t test.
Fig. 2. Role of Orn in bacterial cytotoxicity and motility. (A) Bacterial cytotoxicity. HeLa cells were infected with the indicated strains at a MOI of 30 with or without spinning of the culture plates. At 3 hpi, released LDH activity was measured by LDH assay kit. LDH activity of each sample was normalized against the LDH activity of HeLa cells treated with Triton X100 (0.9%). A representative of three independent experiments with the same results is shown. The data shown are means ± standard errors of the means. **, p < 0.001 by Student’s t test. (B) The mRNA levels of flgB and fliC. Total RNA was isolated from bacterial culture at stationary growth phase (OD600 = 2.5) and mRNA levels of these genes were determined by qRT-PCR. The 30S ribosomal protein-encoding gene rpsL was used as an internal control. A representative of three independent experiments with the same results is shown. The data shown are means ± standard errors of the means. **, p < 0.001 by Student’s t test. (C) Swimming motility. Fresh colonies of indicated bacterial strains were inoculated onto the surface of a plate with a sterile toothpick, followed by incubation at 37 ◦ C for 12–14.
activity of T3SS, we used calcium depletion as an inducing signal and found lower mRNA levels of exsC, pcrV and popD in the orn mutant (Fig. 3B). Complementation with an orn gene restored the expression of those genes in both cell infection and calcium depletion experiments (Fig. 3A and B). These results suggest that Orn is required for the full activation of T3SS in response to environmental signals. 3.3. Orn is required for the virulence in a mouse acute pneumonia model So far, our in vitro experiments demonstrated that Orn was required for the expression of T3SS. Next, we analyzed the expression levels of those genes in a murine acute pneumonia model. Six week-old female BALB/c mice were infected intranasally with wild type PA14 strain or the orn mutant. At 3 hpi, bacteria were collected from BALF and gene expression was analyzed with qRT-PCR. Consistent with the in vitro results, expression levels of exsC, pcrV and popD were lower in the orn mutant (Fig. 4).
Fig. 4. In vivo expression of T3SS genes in a mouse acute pneumonia model. Mice were infected intranasally with either the wild type PA14 strain or the orn mutant. BALFs were collected at 3 hpi and total RNA was isolated from the BALFs. The expression levels of T3SS genes were determined by qRT-PCR. The 30S ribosomal protein-encoding gene rpsL was used as an internal control. A representative of three independent experiments with the same results is shown. The data shown are means ± standard errors of the means. **, p < 0.001 by Student’s t test.
The lower expression of T3SS might render attenuation in virulence. Therefore, we infected mice intranasally with wild type PA14 strain or the orn mutant. At 12 hpi, bacterial loads were determined by serial dilution and plate counting. The average number of the orn mutant was significantly lower than that of wild type PA14 strain (Fig. 5A). Furthermore, lungs from mice infected with wild type PA14 strain had clear neutrophil infiltration, edema, and tissue damage, whereas infection with the orn mutant resulted in significantly less inflammation and neutrophil infiltration (Fig. 5B). In combination, these results suggest that Orn is essential for the bacterial virulence in the acute pneumonia.
94
G. Chen et al. / Microbiological Research 188 (2016) 90–96
Fig. 5. Orn is required for the virulence in a mouse acute pneumonia model. (A) Mice were inoculated intranasally with 2 × 107 CFU of indicated strains. At 12 hpi, mice were sacrificed and lungs were isolated and homogenized. Bacterial loads were determined by serial dilution and plating. The data shown are means ± standard errors of the means. **, p < 0.001 by the MannWhitney test. (B) Differential inflammatory cell migration and lung pathology were observed in mice inoculated with wild type PA14 strain, orn mutant or the vehicle control (PBS).
Compared to wild type PA14, the orn mutant grew more slowly (Fig. S2). To evaluate to what extent the defective T3SS affects the virulence of the orn mutant, we overexpressed exsA and examined bacterial colonization of lungs. Having no appreciate effect on bacterial growth (Fig. S2), overexpression of exsA in the orn mutant fully restored the cytotoxicity in vitro (Fig. 6A) and partially restored the bacterial colonization in vivo (Fig. 6B). In combination, these results suggest that the defective T3SS partially accounts for the attenuated virulence of the orn mutant. 3.4. Role of c-di-GMP in the function of Orn A direct consequence of Orn deficiency is elevated intracellular c-di-GMP level (Cohen et al., 2015; Orr et al., 2015), which has been demonstrated to repress T3SS and motility while promoting biofilm formation (Moscoso et al., 2011; Almblad et al., 2015; Cohen et al., 2015; Orr et al., 2015). Consistent with previous reports, we observed elevated c-di-GMP levels, biofilm formation and cdrA expression in the orn mutant, which were reduced by the overexpression of PA2133, an EAL-dependent PDE (Fig. S3) (Kulasakara et al., 2006). However, no effect was observed on the swimming motility, cytotoxicity by the overexpression of PA2133 (Fig. 7), suggesting that these phenotypes were not solely caused by elevated c-di-GMP level. 4. Discussion Orn is known to affect transcription initiation and intracellular c-di-GMP concentration (Cohen et al., 2015; Orr et al., 2015).
The hyperbiofilm formation of a P. aeruginosa orn mutant is mainly due to increased intracellular c-di-GMP concentration, as overexpression of a PDE (PA2133) reduces the biofilm formation (Kulasakara et al., 2006). In this work, we found that Orn is required for the full activation of the T3SS genes under the in vitro calcium depletion condition (Fig. 3B). In the cytotoxicity assay, expression of the T3SS genes was lower in the orn mutant than those in the wild type strain. Considering the orn mutant is defective in the production of major adhesins, including flagellin, pillin and the CupA system, it could be possible that the orn mutant can not adhere to HeLa cells tightly enough to effectively trigger the activation of T3SS even after enforced contact by spinning of the plate. In the mouse acute pneumonia model, the orn mutant displayed attenuated virulence, which was partially restored by overexpression of ExsA. These results suggest that the defective production of adhesins might be also responsible for the attenuated virulence of the orn mutant. In addition, it will be interesting to examine whether mutation of orn can affect the production of other virulence factors, such as siderophores and exotoxin A during infection. It has been previously reported that the RetS/GacS/LadS regulatory pathway acts in coordination with c-di-GMP signaling in a switch between T3SS and type VI secretion system (T6SS) (Moscoso et al., 2011). And the SadC DGC was found to be a central link between the RetS/GacS/LadS system and c-di-GMP signaling in P. aeruginosa (Moscoso et al., 2014). Additionally, intracellular c-diGMP level influences cAMP levels, which subsequently affects the expression of genes under the transcriptional control of a cAMP receptor protein Vfr, including T3SS genes (Almblad et al., 2015). These studies provide evidences that a high level of c-di-GMP
G. Chen et al. / Microbiological Research 188 (2016) 90–96
Fig. 6. Overexpression of exsA restores cytotoxicity and virulence in the mouse acute pneumonia model. (A) HeLa cells were infected with wild type PA14 strain, the orn mutant or the orn mutant containing exsA driven by a tac promoter (orn Ptac exsA) at a MOI of 30. After addition of bacteria to the cells, the plates were spun at 2000 × g for 10 min. At 3 hpi, released LDH activity was measured by LDH assay kit. LDH activity of each sample was normalized against the LDH activity of HeLa cells treated with Triton X100 (0.9%). A representative of three independent experiments with the same results is shown. The data shown are means ± standard errors of the means. **, p < 0.001 by Student’s t test. (B) Bacterial colonization in the mouse acute pneumonia model. A representative of three independent experiments with the same results is shown. The data shown are means ± standard errors of the means. **, p < 0.001 by the MannWhitney test.
represses T3SS. However, here we showed reduction of intracellular c-di-GMP failed to restore the cytotoxicity of the orn mutant, indicating that Orn may not regulate T3SS solely through the c-di-GMP. It is likely that besides intracellular concentration of c-di-GMP, expression of several T3SS regulatory genes might be altered in the orn mutant, possibly due to the nanoRNA-primed transcription initiation (Goldman et al., 2011). The T3SS is known to be tightly controlled by the proteins encoded by the regulatory operon exsCEBA (Diaz et al., 2011). ExsA is an AraC-type DNA binding protein that recognizes a consensus sequence located upstream of the transcriptional start sites of T3SS genes, including the exsCEBA operon itself (Hovey and Frank, 1995). However, a recent transcriptomic analysis revealed a gap between the mRNAs of exsA and exsCEB, suggesting an independent promoter for the exsA (Wurtzel et al., 2012). A RNA helicase DeaD was found to regulate the translation of exsA by relieving an inhibitory structure of the exsA mRNA that normally prevents exsA translation (Intile et al., 2015). It is possible that nanoRNAs alter the transcription initiation of exsA, influencing its secondary structure or stability. In addition, many extrinsic regulatory pathways influence T3SS genes at transcriptional or post-transcriptional level. For example, another regulator of T3SS gene expression, PsrA, binds the
95
Fig. 7. Role of c-di-GMP in motility and cytotoxicity in the orn mutant. (A) Swimming motility of indicated strains. (B) HeLa cells were infected with indicated strains at a MOI of 30 with or without spinning of the plates. At 3 hpi, released LDH activity was measured by LDH assay kit. LDH activity of each sample was normalized against the LDH activity of HeLa cells treated with Triton X100 (0.9%). A representative of three independent experiments with the same results is shown. The data shown are means ± standard errors of the means. **, p < 0.001 by Student’s t test.
exsC promoter to positively control its transcription (Shen et al., 2006). Our qRT-PCR data revealed down-regulation of the psrA in the orn mutant (data not shown). However, overexpression of psrA was unable to restore the cytotoxicity of the orn mutant (data not shown), raising the possibility that Orn might regulate T3SS through multiple regulatory pathways. Whether Orn regulates psrA through the accumulated c-di-GMP or nanoRNAs-primed transcription initiation remains to be elucidated. In conclusion, our work demonstrated Orn might play an important role in the regulation of T3SS, which contributes to the in vitro and in vivo virulence of P. aeruginosa. Results from previous and our current work underscore the importance of nanoRNAs, including pGpG, in bacterial global gene regulation.
Acknowlegements This work was supported in part by National Science Foundation of China (31370168, and 31370167), National 973 Basic Research Program of China (2012CB518700), Program of international S&T cooperation (2015DFG32500) and Science and Technology Committee of Tianjin (13JCYBJC36700, 15JCZDJC33000, and 15JCYBJC53900).
96
G. Chen et al. / Microbiological Research 188 (2016) 90–96
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.micres.2016.05. 002. References Almblad, H., Harrison, J.J., Rybtke, M., Groizeleau, J., Givskov, M., Parsek, M.R., Tolker-Nielsen, T., 2015. The cyclic AMP-Vfr signaling pathway in Pseudomonas aeruginosa is inhibited by cyclic di-GMP. J. Bacteriol. 197 (13), 2190–2200. Brimer, C.D., Montie, T.C., 1998. Cloning and comparison of fliC genes and identification of glycosylation in the flagellin of Pseudomonas aeruginosa a-type strains. J. Bacteriol. 180 (12), 3209–3217. Choi, K.H., Schweizer, H.P., 2006. mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa. Nat. Protoc. 1 (1), 153–161. Cohen, D., Mechold, U., Nevenzal, H., Yarmiyhu, Y., Randall, T.E., Bay, D.C., Rich, J.D., Parsek, M.R., Kaever, V., Harrison, J.J., Banin, E., 2015. Oligoribonuclease is a central feature of cyclic diguanylate signaling in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 112 (36), 11359–11364. Comolli, J.C., Hauser, A.R., Waite, L., Whitchurch, C.B., Mattick, J.S., Engel, J.N., 1999. Pseudomonas aeruginosa gene products PilT and PilU are required for cytotoxicity in vitro and virulence in a mouse model of acute pneumonia. Infect. Immun. 67 (7), 3625–3630. Diaz, M.H., Hauser, A.R., 2010. Pseudomonas aeruginosa cytotoxin ExoU is injected into phagocytic cells during acute pneumonia. Infect. Immun. 78 (4), 1447–1456. Diaz, M.H., Shaver, C.M., King, J.D., Musunuri, S., Kazzaz, J.A., Hauser, A.R., 2008. Pseudomonas aeruginosa induces localized immunosuppression during pneumonia. Infect. Immun. 76 (10), 4414–4421. Diaz, M.R., King, J.M., Yahr, T.L., 2011. Intrinsic and extrinsic regulation of type III secretion gene expression in Pseudomonas aeruginosa. Front. Microbiol. 2, 89. Driscoll, J.A., Brody, S.L., Kollef, M.H., 2007. The epidemiology, pathogenesis and treatment of Pseudomonas aeruginosa infections. Drugs 67 (3), 351–368. Francis, M.S., Wolf-Watz, H., Forsberg, A., 2002. Regulation of type III secretion systems. Curr. Opin. Microbiol. 5 (2), 166–172. Ghosh, S., Deutscher, S., 1999. Oligoribonuclease is an essential component of the mRNA decay pathway. Proc. Natl. Acad. Sci. U. S. A. 96 (8), 4372–4377. Goldman, S.R., Sharp, J.S., Vvedenskaya, I.O., Livny, J., Dove, S.L., Nickels, B.E., 2011. NanoRNAs prime transcription initiation in vivo. Mol. Cell 42 (6), 817–825. Goodman, A.L., Kulasekara, B., Rietsch, A., Boyd, D., Smith, R.S., Lory, S., 2004. A signaling network reciprocally regulates genes associated with acute infection and chronic persistence in Pseudomonas aeruginosa. Dev. Cell 7 (5), 745–754. Hauser, A.R., Cobb, E., Bodi, M., Mariscal, D., Valles, J., Engel, J.N., Rello, J., 2002. Type III protein secretion is associated with poor clinical outcomes in patients with ventilator-associated pneumonia caused by Pseudomonas aeruginosa. Crit. Care Med. 30 (3), 521–528. Hauser, A.R., 2009. The type III secretion system of Pseudomonas aeruginosa: infection by injection. Nat. Rev. Microbiol. 7 (9), 654–665. Hoang, T.T., Karkhoff-Schweizer, R.R., Kutchma, A.J., Schweizer, H.P., 1998. A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212 (1), 77–86. Holder, I.A., Neely, A.N., Frank, D.W., 2001. Type III secretion/intoxication system important in virulence of Pseudomonas aeruginosa infections in burns. Burns 27 (2), 129–130. Hovey, A.K., Frank, A.K., 1995. Analyses of the DNA-binding and transcriptional activation properties of ExsA, the transcriptional activator of the Pseudomonas aeruginosa exoenzyme S regulon. J. Bacteriol. 177 (15), 4427–4436. Howell, H.A., Logan, L.K., Hauser, A.R., 2013. Type III secretion of ExoU is critical during early Pseudomonas aeruginosa pneumonia. mBio 4 (2) (e00032-13). Intile, P.J., Balzer, G.J., Wolfgang, M.C., Yahr, T.L., 2015. The RNA helicase DeaD stimulates ExsA translation to promote expression of the Pseudomonas aeruginosa type III secretion system. J. Bacteriol. 197 (16), 2664–2674. Jacobs, M.A., Alwood, A., Thaipisuttikul, I., Spencer, D., Haugen, E., Ernst, S., Will, O., Kaul, R., Raymond, C., Levy, R., Chun-Rong, L., Guenthner, D., Bovee, D., Olson, M.V., Manoil, C., 2003. Comprehensive transposon mutant library of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 100 (24), 14339–14344.
Kulasakara, H., Lee, V., Brencic, A., Liberati, N., Urbach, J., Miyata, S., Lee, D.G., Neely, A.N., Hyodo, M., Hayakawa, Y., Ausubel, F.M., Lory, S., 2006. Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3’-5’)-cyclic-GMP in virulence. Proc. Natl. Acad. Sci. U. S. A. 103 (8), 2839–2844. Lawal, A., Jejelowo, O., Chopra, A.K., Rosenzweig, J.A., 2011. Ribonucleases and bacterial virulence. Microb. Biotechnol. 4 (5), 558–571. Li, K., Xu, C., Jin, Y., Sun, Z., Liu, C., Shi, J., Chen, G., Chen, R., Jin, S., Wu, W., 2013. SuhB is a regulator of multiple virulence genes and essential for pathogenesis of Pseudomonas aeruginosa. mBio 4 (6) (e00419-13). Liberati, N.T., Urbach, J.M., Miyata, S., Lee, D.G., Drenkard, E., Wu, G., Villanueva, J., Wei, T., Ausubel, F.M., 2006. An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants. Proc. Natl. Acad. Sci. U. S. A. 103 (8), 2833–2838. Mattick, J.S., Whitchurch, C.B., Alm, R.A., 1996. The molecular genetics of type-4 fimbriae in Pseudomonas aeruginosa—a review. Gene 179 (1), 147–155. Moscoso, J.A., Mikkelsen, H., Heeb, S., Williams, P., Filloux, A., 2011. The Pseudomonas aeruginosa sensor RetS switches type III and type VI secretion via c-di-GMP signalling. Environ. Microbiol. 13 (12), 3128–3138. Moscoso, J.A., Jaeger, T., Valentini, M., Hui, K., Jenal, U., Filloux, A., 2014. The diguanylate cyclase SadC is a central player in Gac/Rsm-mediated biofilm formation in Pseudomonas aeruginosa. J. Bacteriol. 196 (23), 4081–4088. Nakayama, S., Kelsey, I., Wang, J., Roelofs, K., Stefane, B., Luo, Y., Lee, V.T., Sintim, H.O., 2011. Thiazole orange-induced c-di-GMP quadruplex formation facilitates a simple fluorescent detection of this ubiquitous biofilm regulating molecule. J. Am. Chem. Soc. 133 (13), 4856–4864. Orr, M.W., Donaldson, G.P., Severin, G.B., Wang, J., Sintim, H.O., Waters, C.M., Lee, V.T., 2015. Oligoribonuclease is the primary degradative enzyme for pGpG in Pseudomonas aeruginosa that is required for cyclic-di-GMP turnover. Proc. Natl. Acad. Sci. U. S. A. 112 (36), E5048–E5057. Rashid, M.H., Kornberg, A., 2000. Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 97 (9), 4885–4890. Romling, U., Galperin, M.Y., Gomelsky, M., 2013. Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol. Mol. Biol. Rev.: MMBR 77 (1), 1–52. Roy-Burman, A., Savel, R.H., Racine, S., Swanson, B.L., Revadigar, N.S., Fujimoto, J., Sawa, T., Frank, D.W., Wiener-Kronish, J.P., 2001. Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections. J. Infect. Dis. 183 (12), 1767–1774. Ryan, R.P., Fouhy, Y., Lucey, J.F., Crossman, L.C., Spiro, S., He, Y.W., Zhang, L.H., Heeb, S., Camara, M., Williams, P., Dow, J.M., 2006. Cell-cell signaling in Xanthomonas campestris involves an HD-GYP domain protein that functions in cyclic di-GMP turnover. Proc. Natl. Acad. Sci. U. S. A. 103 (17), 6712–6717. Sambrook, J., Pollack, R., 1974. Basic methodology for cell culture—cell transformation. Methods Enzymol. 32 (Part B), 583–592. Schmidt, A.J., Ryjenkov, D.A., Gomelsky, M., 2005. The ubiquitous protein domain EAL is a cyclic diguanylate-specific phosphodiesterase: enzymatically active and inactive EAL domains. J. Bacteriol. 187 (14), 4774–4781. Shaver, C.M., Hauser, A.R., 2004. Relative contributions of Pseudomonas aeruginosa ExoU, ExoS, and ExoT to virulence in the lung. Infect. Immun. 72 (12), 6969–6977. Shen, D.K., Filopon, D., Kuhn, L., Polack, B., Toussaint, B., 2006. PsrA is a positive transcriptional regulator of the type III secretion system in Pseudomonas aeruginosa. Infect. Immun. 74 (2), 1121–1129. Sun, Z., Shi, J., Liu, C., Jin, Y., Li, K., Chen, R., Jin, S., Wu, W., 2014. PrtR homeostasis contributes to Pseudomonas aeruginosa pathogenesis and resistance against ciprofloxacin. Infect. Immun. 82 (4), 1638–1647. Vallet, I., Olson, J.W., Lory, S., Lazdunski, A., Filloux, A., 2001. The chaperone/usher pathways of Pseudomonas aeruginosa: identification of fimbrial gene clusters (cup) and their involvement in biofilm formation. Proc. Natl. Acad. Sci. U. S. A. 98 (12), 6911–6916. West, S.E., Schweizer, H.P., Dall, C., Sample, A.K., Runyen-Janecky, L.J., 1994. Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 148 (1), 81–86. Wurtzel, O., Yoder-Himes, D.R., Han, K., Dandekar, A.A., Edelheit, S., Greenberg, E.P., Sorek, R., Lory, S., 2012. The single-nucleotide resolution transcriptome of Pseudomonas aeruginosa grown in body temperature. PLoS Pathog. 8 (9), e1002945.
Contents lists available at ScienceDirect
Microbiological Research journal homepage: www.elsevier.com/locate/micres
Oligoribonuclease is required for the type III secretion system and pathogenesis of Pseudomonas aeruginosa Gukui Chen a , Qiang Zhao b , Feng Zhu a , Ronghao Chen a , Yongxin Jin a , Chang Liu a , Xiaolei Pan a , Shouguang Jin a,c , Weihui Wu a , Zhihui Cheng a,∗ a State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 300071, China b College of Life Sciences, Nankai University, Tianjin 300071, China c Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL 32610, USA
a r t i c l e
i n f o
Article history: Received 29 January 2016 Received in revised form 29 April 2016 Accepted 1 May 2016 Available online 4 May 2016 Keywords: Pseudomonas aeruginosa Oligoribonuclease Type III secretion system Pathogenesis
a b s t r a c t Oligoribonuclease (Orn) is a 3 to 5 exonuclease that degrades nanoRNAs, which can serve as primers for transcription initiation at a significant fraction of promoters. One of Orn’s substrates, pGpG inhibits the enzymatic activity of EAL-domain containing phosphodiesterases (PDEs), thereby increasing intracellular cyclic-di-GMP (c-di-GMP) level. Here, we found that an orn mutant of Pseudomonas aeruginosa displayed reduced cytotoxicity, which was mainly due to deficient type III secretion system (T3SS). Given the importance of T3SS in pathogenicity, we examined the bacterial virulence in a mouse acute pneumonia model and found that the orn mutant was highly attenuated compared to the wild type PA14 strain. Overexpression of an EAL domain-containing PDE reduced the c-di-GMP level as well as biofilm formation in the orn mutant. However, no effect was observed on the expression of T3SS genes, suggesting that increased c-di-GMP level is not the solely cause of defective T3SS in the orn mutant. Overall, our results demonstrated an essential role of Orn in the expression of T3SS as well as pathogenesis of P. aeruginosa. © 2016 Elsevier GmbH. All rights reserved.
1. Introduction Pseudomonas aeruginosa is a versatile gram-negative bacterium which causes a variety of acute and chronic infections, such as urinary tract infections, burn and wound infections, sepsis, and severe pneumonia (Driscoll et al., 2007). One of the virulence factors that strongly correlated with acute infection in both animal models and human patients is the type III secretion system (T3SS) (Roy-Burman et al., 2001; Hauser et al., 2002). The T3SS is a complex protein secretion and delivery machinery harbored by many animal and plant pathogens, through which bacterial effector molecules are directly translocated into host cell cytosols, causing disruption of intracellular signaling or cell death (Francis et al., 2002). Loss of the T3SS attenuates P. aeruginsa virulence in various mouse acute infection models (Holder et al., 2001; Goodman et al., 2004). The expression of T3SS genes is controlled by a master regulator ExsA (Hovey and Frank, 1995). Four T3SS effectors have been identified in P. aeruginosa, namely, ExoU, ExoS, ExoT and ExoY (Shaver and
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (Z. Cheng). http://dx.doi.org/10.1016/j.micres.2016.05.002 0944-5013/© 2016 Elsevier GmbH. All rights reserved.
Hauser, 2004). It has been demonstrated that during early pneumonia, ExoU is rapidly induced and injected into phagocytic cells, inducing rapid and complete cell lysis (Diaz and Hauser, 2010). Delayed expression of ExoU leads to decreased bacterial burden in lungs of infected mice (Howell et al., 2013). Thus, the T3SS plays an important role in the killing of neutrophils and is essential for survival and proliferation of the bacteria within the host (Diaz et al., 2008; Diaz and Hauser, 2010; Howell et al., 2013). Oligoribonuclease (Orn) is a highly conserved 3 to 5 exonuclease in bacteria, which converts 2–5-nt RNAs, termed “nanoRNAs” to mononucleotides (Ghosh and Deutscher, 1999). Orn is essential for the viability in Escherichia coli (Ghosh and Deutscher, 1999), however, P. aeruginosa remains viable in the absence of Orn (Jacobs et al., 2003), making it an excellent model for Orn research. In P. aeruginosa, depletion of the Orn causes accumulation of nanoRNAs, which serves as primers for transcription initiation at many promoters, causing global alterations in gene expression (Goldman et al., 2011). Additionally, Orn plays an essential role in cyclic-di-GMP (c-di-GMP) turnover (Cohen et al., 2015; Orr et al., 2015). cdi-GMP is an important secondary messenger in most bacteria (Romling et al., 2013). High level of intracellular c-di-GMP in P. aeruginosa represses bacterial motility and promotes extracellular
G. Chen et al. / Microbiological Research 188 (2016) 90–96
polysaccharides production and biofilm formation; whereas low level of c-di-GMP promotes motility and expression of virulence factors associated with acute infections (Romling et al., 2013). cdi-GMP is synthesized by diguanylate cyclases (DGC) harboring a GGDEF domain. Degradation of c-di-GMP is catalyzed by phosphodiesterases (PDE) harboring an EAL or HD-GYP domain, which results in 5 -phosphoguanylyl-(3 , 5 )-guanosine (pGpG) or GMP (Schmidt et al., 2005; Ryan et al., 2006). pGpG is further hydrolyzed by Orn into two GMPs (Cohen et al., 2015; Orr et al., 2015). Deletion of orn causes elevated levels of pGpG, which exerts feedback inhibition on EAL domain-containing PDEs, leading to increased intracellular c-di-GMP concentration and a hyperbiofilm phenotype (Cohen et al., 2015; Orr et al., 2015). However, whether Orn plays a role in bacterial pathogenesis remains unknown. In this study, we identified orn as an essential gene for the motility and T3SS in P. aeruginosa. Consistent with the in vitro phenotypes, the orn mutant displays attenuated virulence in a mouse acute pneumonia model. Thus, our results reveal novel roles of Orn in bacterial pathogenesis.
2. Materials and methods 2.1. Bacterial strains and plasmids The bacterial strains and plasmids used in this study are listed in Table 1. Bacteria were cultured in Luria–Bertani (LB) broth (Sambrook and Pollack, 1974) or LB agar (LB broth containing 1.5% agar) under aerobic conditions at 37 ◦ C. When needed, the medium was supplemented with tetracycline (100 g/ml) (BBI life sciences, Shanghai, China), gentamicin (100 g/ml) (BBI life sciences), or carbenicillin (150 g/ml) (BBI life sciences) for P. aeruginosa, and ampicillin (100 g/ml) (BBI life sciences) for E. coli. For DNA manipulation, standard protocols or manufacture instructions of commercial products were followed. The PA14 36990 coding region was amplified and cloned into the EcoR I-BamH I sites of pUCP20 (West et al., 1994), resulting in pUCP20PA2133. orn gene with its 325-bp upstream region was amplified using wild type P. aeruginosa PA14 strain chromosomal DNA as a template and cloned into Kpn I-Hind III sites of pUC18T-Mini-Tn7TTc (Choi and Schweizer, 2006), resulting in pRN00. Chromosomal gene mutations were generated as described previously (Hoang et al., 1998). Primers used in this study are listed in Table S1.
2.2. Biofilm formation and motility assay Biofilm formation was measured as previously described (Li et al., 2013). Briefly, overnight bacterial cultures were diluted to an OD600 of 0.025 in LB broth and incubated in each well of a 96well plate at 37 ◦ C for 24 h. For quantification of biofilm formation, each well was washed three times with 1 × phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2 HPO4 , pH 7.4) and stained with 0.1% crystal violet, followed by three washes with 1 × PBS. Then, 200 l ethanol was added to each well. After a 10min incubation at room temperature, the OD590 of each sample was measured. P. aeruginosa motilities were assessed as described previously with slight modifications (Rashid and Kornberg, 2000). LB broth solidified with 1% agar was used for the twitching motility assay. The media used for swimming motility assay consisted of 10 g/l tryptone, 5 g/l NaCl and 0.3% agar. Fresh bacterial colonies were inoculated onto plate bottom (for twitching) or surface (for swimming) with sterile toothpicks. After inoculation, plates for twitching motility test were incubated at 37 ◦ C for 24 h, while plates for swimming motility test were incubated at 37 ◦ C for 12–14 h.
91
2.3. Cytotoxicity assay HeLa cells (1.2 × 105 ) were seeded into each well of a 24-well plate. The cells were cultured in DMEM medium with 10% fetal bovine serum (FBS) at 37 ◦ C in 5% CO2 and 95% air for 24 h. Overnight bacterial culture was sub-cultured in fresh LB broth to the log growth phase (OD600 of 0.8) before infection. Bacteria were washed once with 1 × PBS and resuspended in 1 × PBS to an OD600 of 1.0. HeLa cells were infected with bacteria at a multiplicity of infection (MOI) of 30. At the end of incubation, lactate dehydrogenase (LDH) present in the supernatant was measured in each well using the LDH cytotoxicity assay kit (Beyotime, Haimen, China). HeLa cells treated with Triton X100 (0.9%) were used as a control of total release (100% LDH release). The background level (0% LDH release) was determined with DMEM medium with 10% FBS. The percentage of cytotoxicity was calculated following the manufacture’s instructions. 2.4. Mouse acute pneumonia model All animal experiments complied with Nankai University and national guidelines regarding the use of animals in research. Bacteria were grown in LB broth overnight and then sub-cultured into fresh LB broth at 37 ◦ C with aeration to the exponential phase. Bacteria were centrifuged and adjusted to 1 × 109 CFU/ml in 1 × PBS. Female BALB/c mice (6–8-week old) were anesthetized with an intraperitoneal injection of 7.5% chloral hydrate (100 l per mouse). Anesthetized mice were intranasally inoculated with 10 l of bacterial suspension in each nostril, giving a total infection volume of 20 l. Bacterial colonization in the lung was determined as described previously (Sun et al., 2014). Briefly, at 12 h post infection (hpi), mice were sacrificed by inhalation of CO2 . Lungs were isolated and homogenized in 1% proteose peptone, and bacterial cell numbers were determined by serial dilution and plating. 2.5. Quantification of c-di-GMP Concentration of c-di-GMP was measured as described previously (Nakayama et al., 2011). Bacteria were grown to an OD600 of 1.5 at 37 ◦ C in LB medium (15 ml) with agitation. Bacteria were collected by centrifugation and resuspended to a final volume of 1 ml of 10 mM Tris-HCl (pH 8.0) containing 100 mM NaCl, followed by lysis by sonication. Protein concentrations of the lysates were determined with a Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, California, USA). Perchloric acid was added to the lysate at a final concentration of 12% to precipitate cellular macromolecules. The mixture was incubated on ice for 10 min and then neutralized with 3 M KOH, 0.4 M Tris and 2 M KCl. The mixture was centrifuged, and the supernatant was filtered through a 0.2 m filter and 3 kD exclusion columns. The collection was incubated at 95 ◦ C for 5 min, and then room temperature for 15 min. Thiazole orange was then added to the mixture and incubated at 4 ◦ C overnight. To measure fluorescence, Varioskan Flash (Thermo Scientific, Netherlands) was used. The instrument settings were chosen as follows: excitation, 508 nm, emission, 533 nm. The measurements were carried out at 10 ◦ C. 2.6. RNA extraction from in vitro samples Total RNA was isolated with RNAprep pure cell/bacteria Kit (Tiangen Biotech, Beijing, China). Overnight bacterial cultures were sub-cultured in fresh LB broth to exponential phase or stationary phase. To determine bacterial response during infection of HeLa cells, bacteria were added to the cells at a MOI of 30. After 2 h, cells and bacteria in each well were collected and subjected to RNA isolation.
92
G. Chen et al. / Microbiological Research 188 (2016) 90–96
Table 1 Strains and plasmids used in this study. Strain or plasmid
Relevant characteristics or function
Reference or origin
P. aeruginosa PA14 PA14 orn:Gm PA14 flic:Gm PA14 orn PA14 orn/orn PA14 orn Ptac -exsA PA14 orn/PA2133
Wild type strain Orn insertion mutant of PA14, Gmr FliC insertion mutant of PA14, Gmr Orn deletion mutant of PA14 PA14 orn mutant complemented by chromosome inserted orn gene PA14 orn mutant with a chromosome inserted exsA gene driven by tac promoter PA14 orn mutant containing plasmid pUCP20-pa2133
(Liberati et al., 2006) (Liberati et al., 2006) This study This study This study This study
Plasmids pUCP20 pEX18Tc pUC18T-miniTn7T-Tc pRN00 pTNS3 pPA2133
Escherichia-Pseudomonas shuttle vector; Ampr Gene replacement vector, Tcr For gene insertion in chromosome; Tcr pUC18T-miniTn7T-Tc containing orn gene with its 325-bp upstream region Helper plasmid, for gene insertion in chromosome; Ampr PA14 36990 gene of PA14 on pUCP20 driven by lac promoter; Ampr
(West et al., 1994) (Hoang et al., 1998) (Choi and Schweizer, 2006) (Choi and Schweizer, 2006) This study
2.7. RNA extraction from in vivo samples Mice were inoculated with 2 × 107 CFU bacteria intranasally. At indicated time points mice were sacrificed by CO2 . Bronchoalveolar lavage fluid (BALF) was obtained by annulation of the trachea followed by two instillations of 1 ml sterile 1 × PBS with 0.5 mM EDTA. 100 l of the BALF was used for bacterial counting, while the remaining BALF was centrifuged and the pellets were immediately resuspended in 200 l TRIzol reagent (Invitrogen). Total RNA was isolated using a Direct-zol RNA Miniprep kit (ZYMO research, CA, USA). 2.8. Quantitative real time PCR (qRT-PCR) cDNA was synthesized from each RNA sample using a PrimeScript Reverse Transcriptase (TaKaRa, Liaoning, China) with random primers, then subjected to qRT-PCR using SYBR Premix Ex Taq II (TaKaRa). The 30s ribosomal protein gene rpsL was used as an internal control. 3. Results
Fig. 1. An orn mutant is defective in cytotoxicity. HeLa cells were infected with the indicated strains at a MOI of 30. At 3 hpi, released LDH activity was measured by LDH assay kit. LDH activity of each sample was normalized against the LDH activity of HeLa cells treated with Triton X100 (0.9%). A representative of three independent experiments with the same results is shown. The data shown are means ± standard errors of the means. **, p < 0.001 compared to wild type PA14 strain by Student’s t test.
3.1. Orn is required for bacterial cytotoxicity Ribonuclease, such as polynucleotide phosphorylase (PNPase), RNase R and RNase E, was reported to participate in modulation of virulence in many pathogens (Lawal et al., 2011). Additionally, we have previously identified that PNPase is involved in the regulation of T3SS in P. aeruginosa (Li et al., 2013). To assess the roles of other ribonucleases in the regulation of bacterial virulence, we picked five ribonuclease gene mutants from the nonredundant library of PA14 transposon mutants (Liberati et al., 2006) and examined their cytotoxicities. As shown in Fig. 1, majority of the HeLa cells were lysed at 3 hpi by wild type PA14 strain. However, one of the five mutants, orn:Tn, displayed reduced cytotoxicity. To confirm the role of Orn, we constructed an orn in frame deletion mutant, which also displayed reduced cytotoxicity (Fig. 2A). Complementation with an orn gene restored the cytotoxicity (Fig. 2A), thus confirming the role of Orn in cytotoxicity. 3.2. Orn is required for bacteria-host cell contact and T3SS In the above assay, the bacterial cytotoxicity depends on close contact with the host cells and injection of bacterial effector proteins (Hauser, 2009). Thus, we examined the expression of genes involved in bacterial motility and adherence, including flagella, type IV pilus and CupA system (Comolli et al., 1999; Vallet et al., 2001). The mRNA levels of flgB (encoding flagellum basal body rod
protein FlgB), fliC (encoding flagellin) (Brimer and Montie, 1998), pilH (encoding twitching motility protein PilH) (Mattick et al., 1996) and cupA3 (encoding usher CupA3) decreased significantly in the orn mutant (Fig. 2B and Fig. S1A). Consistently, the orn mutant displayed reduced swimming (Fig. 2C) and twitching (Fig. S1B) motilities, which were restored to wild type level by complementation with an orn gene. Mutation in cupA3 or pilH had no effect on cytotoxicity (Fig. S1C) in our experiments. In contrast, mutation in fliC, resulted in defective cytotoxicity (Fig. 2A). Spinning of the tissue culture plate after addition of the bacteria, which enforces the bacterial contact with HeLa cells, restored the cytotoxicity of the fliC mutant (Fig. 2A), thus confirming the role of flagella in the cytotoxicity. However, spinning of the culture plate was unable to restore the cytotoxicity of the orn mutant or an exsA:Tn mutant, suggesting a defective T3SS in the orn mutant (Fig. 2A). To verify the activity of T3SS in the orn mutant, we examined the mRNA levels of a positive regulatory gene exsC and two structural genes pcrV and popD (Hauser, 2009). Bacteria were added to HeLa cells in each well. The plate was spun and incubated for 2 h. Then, bacteria were collected and subjected to RNA isolation and qRT-PCR. In the absence of HeLa cells, no difference in mRNA levels was observed between the wild type PA14 strain and orn mutant (Fig. 3A). However, in the presence of HeLa cells, the mRNA levels of exsC, pcrV and popD were significantly lower in the orn mutant than those in wild type PA14 strain (Fig. 3A). To further confirm the
G. Chen et al. / Microbiological Research 188 (2016) 90–96
93
Fig. 3. Orn is required for the full activation of T3SS genes. Total RNA was isolated from bacterial cells before (−) or after (+) incubation with HeLa cells (A) or treated with 5 mM EGTA (B). The mRNA levels of exsC, pcrV and popD were determined by qRT-PCR. The 30S ribosomal protein-encoding gene rpsL was used as an internal control. A representative of three independent experiments with the same results is shown. The data shown are means ± standard errors of the means. **, p < 0.001 compared to wild type PA14 strain by Student’s t test.
Fig. 2. Role of Orn in bacterial cytotoxicity and motility. (A) Bacterial cytotoxicity. HeLa cells were infected with the indicated strains at a MOI of 30 with or without spinning of the culture plates. At 3 hpi, released LDH activity was measured by LDH assay kit. LDH activity of each sample was normalized against the LDH activity of HeLa cells treated with Triton X100 (0.9%). A representative of three independent experiments with the same results is shown. The data shown are means ± standard errors of the means. **, p < 0.001 by Student’s t test. (B) The mRNA levels of flgB and fliC. Total RNA was isolated from bacterial culture at stationary growth phase (OD600 = 2.5) and mRNA levels of these genes were determined by qRT-PCR. The 30S ribosomal protein-encoding gene rpsL was used as an internal control. A representative of three independent experiments with the same results is shown. The data shown are means ± standard errors of the means. **, p < 0.001 by Student’s t test. (C) Swimming motility. Fresh colonies of indicated bacterial strains were inoculated onto the surface of a plate with a sterile toothpick, followed by incubation at 37 ◦ C for 12–14.
activity of T3SS, we used calcium depletion as an inducing signal and found lower mRNA levels of exsC, pcrV and popD in the orn mutant (Fig. 3B). Complementation with an orn gene restored the expression of those genes in both cell infection and calcium depletion experiments (Fig. 3A and B). These results suggest that Orn is required for the full activation of T3SS in response to environmental signals. 3.3. Orn is required for the virulence in a mouse acute pneumonia model So far, our in vitro experiments demonstrated that Orn was required for the expression of T3SS. Next, we analyzed the expression levels of those genes in a murine acute pneumonia model. Six week-old female BALB/c mice were infected intranasally with wild type PA14 strain or the orn mutant. At 3 hpi, bacteria were collected from BALF and gene expression was analyzed with qRT-PCR. Consistent with the in vitro results, expression levels of exsC, pcrV and popD were lower in the orn mutant (Fig. 4).
Fig. 4. In vivo expression of T3SS genes in a mouse acute pneumonia model. Mice were infected intranasally with either the wild type PA14 strain or the orn mutant. BALFs were collected at 3 hpi and total RNA was isolated from the BALFs. The expression levels of T3SS genes were determined by qRT-PCR. The 30S ribosomal protein-encoding gene rpsL was used as an internal control. A representative of three independent experiments with the same results is shown. The data shown are means ± standard errors of the means. **, p < 0.001 by Student’s t test.
The lower expression of T3SS might render attenuation in virulence. Therefore, we infected mice intranasally with wild type PA14 strain or the orn mutant. At 12 hpi, bacterial loads were determined by serial dilution and plate counting. The average number of the orn mutant was significantly lower than that of wild type PA14 strain (Fig. 5A). Furthermore, lungs from mice infected with wild type PA14 strain had clear neutrophil infiltration, edema, and tissue damage, whereas infection with the orn mutant resulted in significantly less inflammation and neutrophil infiltration (Fig. 5B). In combination, these results suggest that Orn is essential for the bacterial virulence in the acute pneumonia.
94
G. Chen et al. / Microbiological Research 188 (2016) 90–96
Fig. 5. Orn is required for the virulence in a mouse acute pneumonia model. (A) Mice were inoculated intranasally with 2 × 107 CFU of indicated strains. At 12 hpi, mice were sacrificed and lungs were isolated and homogenized. Bacterial loads were determined by serial dilution and plating. The data shown are means ± standard errors of the means. **, p < 0.001 by the MannWhitney test. (B) Differential inflammatory cell migration and lung pathology were observed in mice inoculated with wild type PA14 strain, orn mutant or the vehicle control (PBS).
Compared to wild type PA14, the orn mutant grew more slowly (Fig. S2). To evaluate to what extent the defective T3SS affects the virulence of the orn mutant, we overexpressed exsA and examined bacterial colonization of lungs. Having no appreciate effect on bacterial growth (Fig. S2), overexpression of exsA in the orn mutant fully restored the cytotoxicity in vitro (Fig. 6A) and partially restored the bacterial colonization in vivo (Fig. 6B). In combination, these results suggest that the defective T3SS partially accounts for the attenuated virulence of the orn mutant. 3.4. Role of c-di-GMP in the function of Orn A direct consequence of Orn deficiency is elevated intracellular c-di-GMP level (Cohen et al., 2015; Orr et al., 2015), which has been demonstrated to repress T3SS and motility while promoting biofilm formation (Moscoso et al., 2011; Almblad et al., 2015; Cohen et al., 2015; Orr et al., 2015). Consistent with previous reports, we observed elevated c-di-GMP levels, biofilm formation and cdrA expression in the orn mutant, which were reduced by the overexpression of PA2133, an EAL-dependent PDE (Fig. S3) (Kulasakara et al., 2006). However, no effect was observed on the swimming motility, cytotoxicity by the overexpression of PA2133 (Fig. 7), suggesting that these phenotypes were not solely caused by elevated c-di-GMP level. 4. Discussion Orn is known to affect transcription initiation and intracellular c-di-GMP concentration (Cohen et al., 2015; Orr et al., 2015).
The hyperbiofilm formation of a P. aeruginosa orn mutant is mainly due to increased intracellular c-di-GMP concentration, as overexpression of a PDE (PA2133) reduces the biofilm formation (Kulasakara et al., 2006). In this work, we found that Orn is required for the full activation of the T3SS genes under the in vitro calcium depletion condition (Fig. 3B). In the cytotoxicity assay, expression of the T3SS genes was lower in the orn mutant than those in the wild type strain. Considering the orn mutant is defective in the production of major adhesins, including flagellin, pillin and the CupA system, it could be possible that the orn mutant can not adhere to HeLa cells tightly enough to effectively trigger the activation of T3SS even after enforced contact by spinning of the plate. In the mouse acute pneumonia model, the orn mutant displayed attenuated virulence, which was partially restored by overexpression of ExsA. These results suggest that the defective production of adhesins might be also responsible for the attenuated virulence of the orn mutant. In addition, it will be interesting to examine whether mutation of orn can affect the production of other virulence factors, such as siderophores and exotoxin A during infection. It has been previously reported that the RetS/GacS/LadS regulatory pathway acts in coordination with c-di-GMP signaling in a switch between T3SS and type VI secretion system (T6SS) (Moscoso et al., 2011). And the SadC DGC was found to be a central link between the RetS/GacS/LadS system and c-di-GMP signaling in P. aeruginosa (Moscoso et al., 2014). Additionally, intracellular c-diGMP level influences cAMP levels, which subsequently affects the expression of genes under the transcriptional control of a cAMP receptor protein Vfr, including T3SS genes (Almblad et al., 2015). These studies provide evidences that a high level of c-di-GMP
G. Chen et al. / Microbiological Research 188 (2016) 90–96
Fig. 6. Overexpression of exsA restores cytotoxicity and virulence in the mouse acute pneumonia model. (A) HeLa cells were infected with wild type PA14 strain, the orn mutant or the orn mutant containing exsA driven by a tac promoter (orn Ptac exsA) at a MOI of 30. After addition of bacteria to the cells, the plates were spun at 2000 × g for 10 min. At 3 hpi, released LDH activity was measured by LDH assay kit. LDH activity of each sample was normalized against the LDH activity of HeLa cells treated with Triton X100 (0.9%). A representative of three independent experiments with the same results is shown. The data shown are means ± standard errors of the means. **, p < 0.001 by Student’s t test. (B) Bacterial colonization in the mouse acute pneumonia model. A representative of three independent experiments with the same results is shown. The data shown are means ± standard errors of the means. **, p < 0.001 by the MannWhitney test.
represses T3SS. However, here we showed reduction of intracellular c-di-GMP failed to restore the cytotoxicity of the orn mutant, indicating that Orn may not regulate T3SS solely through the c-di-GMP. It is likely that besides intracellular concentration of c-di-GMP, expression of several T3SS regulatory genes might be altered in the orn mutant, possibly due to the nanoRNA-primed transcription initiation (Goldman et al., 2011). The T3SS is known to be tightly controlled by the proteins encoded by the regulatory operon exsCEBA (Diaz et al., 2011). ExsA is an AraC-type DNA binding protein that recognizes a consensus sequence located upstream of the transcriptional start sites of T3SS genes, including the exsCEBA operon itself (Hovey and Frank, 1995). However, a recent transcriptomic analysis revealed a gap between the mRNAs of exsA and exsCEB, suggesting an independent promoter for the exsA (Wurtzel et al., 2012). A RNA helicase DeaD was found to regulate the translation of exsA by relieving an inhibitory structure of the exsA mRNA that normally prevents exsA translation (Intile et al., 2015). It is possible that nanoRNAs alter the transcription initiation of exsA, influencing its secondary structure or stability. In addition, many extrinsic regulatory pathways influence T3SS genes at transcriptional or post-transcriptional level. For example, another regulator of T3SS gene expression, PsrA, binds the
95
Fig. 7. Role of c-di-GMP in motility and cytotoxicity in the orn mutant. (A) Swimming motility of indicated strains. (B) HeLa cells were infected with indicated strains at a MOI of 30 with or without spinning of the plates. At 3 hpi, released LDH activity was measured by LDH assay kit. LDH activity of each sample was normalized against the LDH activity of HeLa cells treated with Triton X100 (0.9%). A representative of three independent experiments with the same results is shown. The data shown are means ± standard errors of the means. **, p < 0.001 by Student’s t test.
exsC promoter to positively control its transcription (Shen et al., 2006). Our qRT-PCR data revealed down-regulation of the psrA in the orn mutant (data not shown). However, overexpression of psrA was unable to restore the cytotoxicity of the orn mutant (data not shown), raising the possibility that Orn might regulate T3SS through multiple regulatory pathways. Whether Orn regulates psrA through the accumulated c-di-GMP or nanoRNAs-primed transcription initiation remains to be elucidated. In conclusion, our work demonstrated Orn might play an important role in the regulation of T3SS, which contributes to the in vitro and in vivo virulence of P. aeruginosa. Results from previous and our current work underscore the importance of nanoRNAs, including pGpG, in bacterial global gene regulation.
Acknowlegements This work was supported in part by National Science Foundation of China (31370168, and 31370167), National 973 Basic Research Program of China (2012CB518700), Program of international S&T cooperation (2015DFG32500) and Science and Technology Committee of Tianjin (13JCYBJC36700, 15JCZDJC33000, and 15JCYBJC53900).
96
G. Chen et al. / Microbiological Research 188 (2016) 90–96
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.micres.2016.05. 002. References Almblad, H., Harrison, J.J., Rybtke, M., Groizeleau, J., Givskov, M., Parsek, M.R., Tolker-Nielsen, T., 2015. The cyclic AMP-Vfr signaling pathway in Pseudomonas aeruginosa is inhibited by cyclic di-GMP. J. Bacteriol. 197 (13), 2190–2200. Brimer, C.D., Montie, T.C., 1998. Cloning and comparison of fliC genes and identification of glycosylation in the flagellin of Pseudomonas aeruginosa a-type strains. J. Bacteriol. 180 (12), 3209–3217. Choi, K.H., Schweizer, H.P., 2006. mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa. Nat. Protoc. 1 (1), 153–161. Cohen, D., Mechold, U., Nevenzal, H., Yarmiyhu, Y., Randall, T.E., Bay, D.C., Rich, J.D., Parsek, M.R., Kaever, V., Harrison, J.J., Banin, E., 2015. Oligoribonuclease is a central feature of cyclic diguanylate signaling in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 112 (36), 11359–11364. Comolli, J.C., Hauser, A.R., Waite, L., Whitchurch, C.B., Mattick, J.S., Engel, J.N., 1999. Pseudomonas aeruginosa gene products PilT and PilU are required for cytotoxicity in vitro and virulence in a mouse model of acute pneumonia. Infect. Immun. 67 (7), 3625–3630. Diaz, M.H., Hauser, A.R., 2010. Pseudomonas aeruginosa cytotoxin ExoU is injected into phagocytic cells during acute pneumonia. Infect. Immun. 78 (4), 1447–1456. Diaz, M.H., Shaver, C.M., King, J.D., Musunuri, S., Kazzaz, J.A., Hauser, A.R., 2008. Pseudomonas aeruginosa induces localized immunosuppression during pneumonia. Infect. Immun. 76 (10), 4414–4421. Diaz, M.R., King, J.M., Yahr, T.L., 2011. Intrinsic and extrinsic regulation of type III secretion gene expression in Pseudomonas aeruginosa. Front. Microbiol. 2, 89. Driscoll, J.A., Brody, S.L., Kollef, M.H., 2007. The epidemiology, pathogenesis and treatment of Pseudomonas aeruginosa infections. Drugs 67 (3), 351–368. Francis, M.S., Wolf-Watz, H., Forsberg, A., 2002. Regulation of type III secretion systems. Curr. Opin. Microbiol. 5 (2), 166–172. Ghosh, S., Deutscher, S., 1999. Oligoribonuclease is an essential component of the mRNA decay pathway. Proc. Natl. Acad. Sci. U. S. A. 96 (8), 4372–4377. Goldman, S.R., Sharp, J.S., Vvedenskaya, I.O., Livny, J., Dove, S.L., Nickels, B.E., 2011. NanoRNAs prime transcription initiation in vivo. Mol. Cell 42 (6), 817–825. Goodman, A.L., Kulasekara, B., Rietsch, A., Boyd, D., Smith, R.S., Lory, S., 2004. A signaling network reciprocally regulates genes associated with acute infection and chronic persistence in Pseudomonas aeruginosa. Dev. Cell 7 (5), 745–754. Hauser, A.R., Cobb, E., Bodi, M., Mariscal, D., Valles, J., Engel, J.N., Rello, J., 2002. Type III protein secretion is associated with poor clinical outcomes in patients with ventilator-associated pneumonia caused by Pseudomonas aeruginosa. Crit. Care Med. 30 (3), 521–528. Hauser, A.R., 2009. The type III secretion system of Pseudomonas aeruginosa: infection by injection. Nat. Rev. Microbiol. 7 (9), 654–665. Hoang, T.T., Karkhoff-Schweizer, R.R., Kutchma, A.J., Schweizer, H.P., 1998. A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212 (1), 77–86. Holder, I.A., Neely, A.N., Frank, D.W., 2001. Type III secretion/intoxication system important in virulence of Pseudomonas aeruginosa infections in burns. Burns 27 (2), 129–130. Hovey, A.K., Frank, A.K., 1995. Analyses of the DNA-binding and transcriptional activation properties of ExsA, the transcriptional activator of the Pseudomonas aeruginosa exoenzyme S regulon. J. Bacteriol. 177 (15), 4427–4436. Howell, H.A., Logan, L.K., Hauser, A.R., 2013. Type III secretion of ExoU is critical during early Pseudomonas aeruginosa pneumonia. mBio 4 (2) (e00032-13). Intile, P.J., Balzer, G.J., Wolfgang, M.C., Yahr, T.L., 2015. The RNA helicase DeaD stimulates ExsA translation to promote expression of the Pseudomonas aeruginosa type III secretion system. J. Bacteriol. 197 (16), 2664–2674. Jacobs, M.A., Alwood, A., Thaipisuttikul, I., Spencer, D., Haugen, E., Ernst, S., Will, O., Kaul, R., Raymond, C., Levy, R., Chun-Rong, L., Guenthner, D., Bovee, D., Olson, M.V., Manoil, C., 2003. Comprehensive transposon mutant library of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 100 (24), 14339–14344.
Kulasakara, H., Lee, V., Brencic, A., Liberati, N., Urbach, J., Miyata, S., Lee, D.G., Neely, A.N., Hyodo, M., Hayakawa, Y., Ausubel, F.M., Lory, S., 2006. Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3’-5’)-cyclic-GMP in virulence. Proc. Natl. Acad. Sci. U. S. A. 103 (8), 2839–2844. Lawal, A., Jejelowo, O., Chopra, A.K., Rosenzweig, J.A., 2011. Ribonucleases and bacterial virulence. Microb. Biotechnol. 4 (5), 558–571. Li, K., Xu, C., Jin, Y., Sun, Z., Liu, C., Shi, J., Chen, G., Chen, R., Jin, S., Wu, W., 2013. SuhB is a regulator of multiple virulence genes and essential for pathogenesis of Pseudomonas aeruginosa. mBio 4 (6) (e00419-13). Liberati, N.T., Urbach, J.M., Miyata, S., Lee, D.G., Drenkard, E., Wu, G., Villanueva, J., Wei, T., Ausubel, F.M., 2006. An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants. Proc. Natl. Acad. Sci. U. S. A. 103 (8), 2833–2838. Mattick, J.S., Whitchurch, C.B., Alm, R.A., 1996. The molecular genetics of type-4 fimbriae in Pseudomonas aeruginosa—a review. Gene 179 (1), 147–155. Moscoso, J.A., Mikkelsen, H., Heeb, S., Williams, P., Filloux, A., 2011. The Pseudomonas aeruginosa sensor RetS switches type III and type VI secretion via c-di-GMP signalling. Environ. Microbiol. 13 (12), 3128–3138. Moscoso, J.A., Jaeger, T., Valentini, M., Hui, K., Jenal, U., Filloux, A., 2014. The diguanylate cyclase SadC is a central player in Gac/Rsm-mediated biofilm formation in Pseudomonas aeruginosa. J. Bacteriol. 196 (23), 4081–4088. Nakayama, S., Kelsey, I., Wang, J., Roelofs, K., Stefane, B., Luo, Y., Lee, V.T., Sintim, H.O., 2011. Thiazole orange-induced c-di-GMP quadruplex formation facilitates a simple fluorescent detection of this ubiquitous biofilm regulating molecule. J. Am. Chem. Soc. 133 (13), 4856–4864. Orr, M.W., Donaldson, G.P., Severin, G.B., Wang, J., Sintim, H.O., Waters, C.M., Lee, V.T., 2015. Oligoribonuclease is the primary degradative enzyme for pGpG in Pseudomonas aeruginosa that is required for cyclic-di-GMP turnover. Proc. Natl. Acad. Sci. U. S. A. 112 (36), E5048–E5057. Rashid, M.H., Kornberg, A., 2000. Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 97 (9), 4885–4890. Romling, U., Galperin, M.Y., Gomelsky, M., 2013. Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol. Mol. Biol. Rev.: MMBR 77 (1), 1–52. Roy-Burman, A., Savel, R.H., Racine, S., Swanson, B.L., Revadigar, N.S., Fujimoto, J., Sawa, T., Frank, D.W., Wiener-Kronish, J.P., 2001. Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections. J. Infect. Dis. 183 (12), 1767–1774. Ryan, R.P., Fouhy, Y., Lucey, J.F., Crossman, L.C., Spiro, S., He, Y.W., Zhang, L.H., Heeb, S., Camara, M., Williams, P., Dow, J.M., 2006. Cell-cell signaling in Xanthomonas campestris involves an HD-GYP domain protein that functions in cyclic di-GMP turnover. Proc. Natl. Acad. Sci. U. S. A. 103 (17), 6712–6717. Sambrook, J., Pollack, R., 1974. Basic methodology for cell culture—cell transformation. Methods Enzymol. 32 (Part B), 583–592. Schmidt, A.J., Ryjenkov, D.A., Gomelsky, M., 2005. The ubiquitous protein domain EAL is a cyclic diguanylate-specific phosphodiesterase: enzymatically active and inactive EAL domains. J. Bacteriol. 187 (14), 4774–4781. Shaver, C.M., Hauser, A.R., 2004. Relative contributions of Pseudomonas aeruginosa ExoU, ExoS, and ExoT to virulence in the lung. Infect. Immun. 72 (12), 6969–6977. Shen, D.K., Filopon, D., Kuhn, L., Polack, B., Toussaint, B., 2006. PsrA is a positive transcriptional regulator of the type III secretion system in Pseudomonas aeruginosa. Infect. Immun. 74 (2), 1121–1129. Sun, Z., Shi, J., Liu, C., Jin, Y., Li, K., Chen, R., Jin, S., Wu, W., 2014. PrtR homeostasis contributes to Pseudomonas aeruginosa pathogenesis and resistance against ciprofloxacin. Infect. Immun. 82 (4), 1638–1647. Vallet, I., Olson, J.W., Lory, S., Lazdunski, A., Filloux, A., 2001. The chaperone/usher pathways of Pseudomonas aeruginosa: identification of fimbrial gene clusters (cup) and their involvement in biofilm formation. Proc. Natl. Acad. Sci. U. S. A. 98 (12), 6911–6916. West, S.E., Schweizer, H.P., Dall, C., Sample, A.K., Runyen-Janecky, L.J., 1994. Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 148 (1), 81–86. Wurtzel, O., Yoder-Himes, D.R., Han, K., Dandekar, A.A., Edelheit, S., Greenberg, E.P., Sorek, R., Lory, S., 2012. The single-nucleotide resolution transcriptome of Pseudomonas aeruginosa grown in body temperature. PLoS Pathog. 8 (9), e1002945.