- Email: [email protected]
threonine protein kinase PpkA contributes to the adaptation and virulence in Pseudomonas aeruginosa
Microbial Pathogenesis 113 (2017) 5–10
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Serine/threonine protein kinase PpkA contributes to the adaptation and virulence in Pseudomonas aeruginosa
MARK
Jianyi Pan∗, Zhenzhong Zha1, Pengfei Zhang1, Ran Chen1, Chen Ye, Ting Ye School of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Pseudomonas aeruginosa Serine/threonine (Ser/Thr) protein kinase PpkA Virulence Adaptation
Pseudomonas aeruginosa is a leading cause of nosocomial infections worldwide and has become a serious public health threat, which is attributed to a large extent to its extraordinary environmental adaptability and diverse virulence factors that result in infection and progression of pathogenesis. The eukaryote-type serine/threonine (Ser/Thr) protein kinases, known for playing major regulatory roles in eukaryotes, have been demonstrated to play a central role in regulating various bacterial cellular processes via catalyzing protein phosphorylation. Although PpkA, a Ser/Thr protein kinase first identified in P. aeruginosa, has been implicated in association with bacterial virulence, little is known about the protein. Therefore, in this study, to assess the potential role of PpkA in the regulation of P. aeruginosa environmental adaptation and virulence, variations of biofilm formation, pyocyanin production, tolerance to stress, cell invasion and plant virulence were determined in wild type PAO1, ppkA gene-deleted and complemented mutant strains. Our results indicate that the mutant strain lacking ppkA exhibited a significant decrease of biofilm formation and pyocyanin production, less tolerance to oxidative and osmotic stresses, inefficient invasion of host cells and a reduction of bacterial virulence. These findings provide new insight into the regulation of various cellular processes by PpkA; this is an important mechanism for adaptation and virulence in P. aeruginosa.
1. Introduction Pseudomonas aeruginosa is a Gram-negative bacterium universally found in natural environments, such as soil, water, and vegetation [1]. As a well-known opportunistic pathogen, the bacterium could infect a diversity of organisms, including insects, plants and animals [2]. In humans, P. aeruginosa is the primary cause of fatal lung infections among patients with cystic fibrosis and is also the leading cause of secondary infections in immunocompromised patients, such as those with AIDS, cancer and burn wounds [3,4]. Although it rarely causes disease in healthy people, P. aeruginosa has become a major cause of nosocomial infections worldwide, accounting for 10%–20% of these infections [5,6], and it is a serious threat to public health. In the past few decades, as a model pathogen, P. aeruginosa has been studied extensively and intensively; it has been revealed that the clinical difficulty in the treatment of infections caused by the bacterium is attributable to its extraordinary ability to adapt, survive and develop resistance to various hostile environments, such as oxidative and osmotic stresses, antibiotics and others, and its diverse virulence factors that result in progression of pathogenesis [7]. The main virulence factors of P.
∗
1
aeruginosa include bacterial surface factors (flagella, pili and lipopolysaccharide) and active processes such as the secretion of toxins (for example of pyocyanin) and biofilm formation [8]. The versatility of P. aeruginosa in adaptation to different environments and the production of many virulence factors is metabolically costly and therefore requires regulatory control [7,9]. Protein phosphorylation is a reversible posttranslational modification and a well-known mechanism for the regulation of cellular activities in bacteria and eukaryotes [10]. The signal transduction by histidine/aspartate phosphorylation via two-component regulatory systems has been considered to be an important mechanism by which bacteria detect environmental stimuli and establish an adaptive response [11]. However, this paradigm has now changed with the increasing evidence for serine/threonine/tyrosine (Ser/Thr/Tyr) phosphorylation in numerous bacterial pathways [12]. The phosphoproteins at the Ser/Thr/ Tyr site have been implicated in important cellular activities such as catabolite repression, morphological differentiation, sugar transport, cell growth and viability [12,13], virulence [14], and stress response [15]. Accordingly, such protein kinases, serine/threonine (Ser/Thr) or tyrosine protein kinases, known for the predominant regulatory protein
Corresponding author. E-mail address: [email protected] (J. Pan). These authors contribute equally to this work.
http://dx.doi.org/10.1016/j.micpath.2017.10.017 Received 10 August 2017; Received in revised form 10 October 2017; Accepted 12 October 2017 Available online 13 October 2017 0882-4010/ © 2017 Elsevier Ltd. All rights reserved.
Microbial Pathogenesis 113 (2017) 5–10
J. Pan et al.
Glacial acetic acid (100 μl per well) was added to dissolve the dye bound to the bacterial cells. The quantification of biofilms was performed by determination of the optical density at 590 nm.
kinases exclusively in eukaryotes, have been demonstrated to play central roles in signal transduction by catalyzing protein phosphorylation in prokaryotes [16–18] and to be involved in regulating various bacterial cellular functions, such as growth, metabolism, stress responses, biofilm formation and virulence characteristics [16–20]. The eukaryote-type Ser/Thr protein kinase has been identified in large numbers of bacterial species, and the presence of multiple genes that encode the kinase was found by survey of the genome in some species [21,22]. In P. aeruginosa, there exist at least three Ser/Thr protein kinases, including two characterized kinases Stk1 [19] and PpkA [23,24]. PpkA is the first identified Ser/Thr protein kinase in P. aeruginosa [23], and the kinase has been shown in association with bacterial virulence in neutropenic mice [23] and to be responsible for type VI secretion activation in P. aeruginosa [25,26], suggesting the protein might play an important regulatory role in virulence. Therefore, in this study, we provided more information on the potential role of PpkA in P. aeruginosa virulence. Variations of biofilm formation, pyocyanin production, tolerance to stress, cell invasion and plant virulence were assayed in wild type PAO1, ppkA gene-deleted and complemented mutant strains. Significantly decreased effects on these aspects in ppkA null mutant strain in comparison with PAO1 and complemented strains were observed, suggesting that the regulation of various cellular processes by PpkA is an important regulatory mechanism of P. aeruginosa.
2.4. Hydrogen peroxide sensitivity assay Exponent-phase (OD600 value approximately 0.50) cultures were used for the hydrogen peroxide (H2O2) sensitivity assay. Bacterial cells were harvested and resuspended in PBS to OD600 value of 1.0. The sensitivity of bacterial cells to oxidative stress was determined by the incubation of 0.9 ml suspensions to final concentrations of 0, 10 and 50 mM H2O2 with shaking at 37 °C for 45 min. Viable cells were counted by plating them onto LB agar plates, and the number of CFU was counted after 14 h of incubation at 37 °C. Results are expressed as survival rates. 2.5. Osmotic stress sensitivity assay Bacterial cells were harvested from cultures with an OD600 of 0.8 and resuspended in LB medium containing final concentrations of 0.17 (normal LB, used for control), 0.7, 1.3 and 1.6 M sodium chloride (NaCl). After incubation with shaking at 37 °C for 14 h, serial dilutions of the samples were plated on LB agar plates to determine the CFU. 2.6. Pyocyanin quantitation assay
2. Materials and methods
The quantitation assay of pyocyanin was performed as described previously [29]. In brief, overnight seeding cultures of three strains were diluted 1:100 in 5 ml fresh LB medium and were cultured at 37 °C for 24. The supernatants were collected by centrifugation at 5000 rpm for 3 min. Pyocyanin production was extracted by mixing with 3 ml chloroform and then re-extraction into 1 ml of 0.2 M HCl. The absorbance of the pyocyanin-rich aqueous phase was determined at 520 nm. Pyocyanin concentrations (μg/ml) were determined by multiplying the OD520 by 12.8.
2.1. Bacterial strains, plasmids, host cells and growth conditions P. aeruginosa PAO1 and its mutant strain that lack ppkA (designated as ΔppkA) were kindly provided by Professor Joseph Mougous at the University of Washington and were constructed as described previously [25]. The complemented strain to ppkA in ΔppkA was constructed in this study as described herein. The bacteria were grown in standard Luria-Bertani (LB) medium at 37 °C with vigorous shaking or on solid LB medium (1.5% (w/v) agar in LB) at 37 °C. Human epithelial cells (HeLa) were grown in Dulbecco's modified eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C.
2.7. Invasion of HeLa cells by P. aeruginosa variants The ability of the P. aeruginosa variants to invade HeLa cells was assessed using a modification of an assay described previously [30]. Briefly, the HeLa cells were seeded in a 6-well plate with 5 × 105 cells per well (2 ml medium) and incubated overnight in an incubator at 37 °C, 5% CO2. The bacterial cells were collected from 1 ml cultures (cultured to an OD600 of 0.7), washed three times with PBS and resuspended in 1 ml DMEM medium. Twenty-microliter suspensions (approximately 5 × 106 CFU bacterial cells) were inoculated into each well, and the plates were incubated at 37 °C, 5% CO2. After incubation for 2 h, the wells were rinsed three times with DMEM medium to remove unattached bacteria and then incubated for 4 h, followed by incubation for another hour after replacement of the medium to DMEM medium containing 200 μg/ml of gentamicin. The wells were washed twice with PBS, and then the HeLa cells were lysed with 1% Triton X100 to release invasive bacteria. The supernatants were serially diluted and plated on LB agar plates to determine the CFU.
2.2. Construction of ppkA complementation strain The ppkA complemented strain was constructed as described previously with slight modifications [27]. Briefly, a DNA fragment of ppkA was PCR amplified using the primers ppkA-c-F (5′-CTAGTCTAGAAACAAGACCGCCGACGACAGCGAAC-3′) and ppkA-c-R (5′-TACGAGCTCCAGCAGGTCGAGCAGGGTGCTC-3′), including restriction enzyme Xba I and Sac I recognition sites (underlined sequences), respectively. The PCR products were cloned into a pBBR1MCS2 vector. The resulting recombinant plasmid, pBBR1MCS2-ppkA, was then transformed into the ΔppkA mutant cells. The complementation strain was designated + ppkA and verified by PCR analysis. 2.3. Biofilm formation assay The biofilm assay was carried out in a 96-well microtiter plate using a method described previously [28] with slight modifications. Typically, overnight cultured bacteria were diluted to an OD600 of 1.0 with fresh LB medium, and then 10 μl of bacterial suspension was inoculated into a well containing 150 μl of LB medium and incubated statically at 37 °C for 24 h. After incubation, the wells were aspired gently and washed three times with 200 μl of phosphate-buffered saline (PBS). The remaining attached bacteria were fixed for 15 min by the addition of 100 μl methanol to each well, and then the methanol was removed. Biofilms were stained by the addition of 100 μl of 0.4% w/v crystal violet, followed by incubation at 37 °C for 15 min. Crystal violet solutions were removed from the wells, and the plate was dried at 37 °C.
2.8. Virulence to plant assay The bacterial virulence to plants (Romaine lettuce leaf) was assayed as described previously [20]. Each bacterial strain that cultured into exponential phase (OD600 approximately 0.50) was washed three times and diluted to OD600 0.050 in 10 mM MgSO4. Bacterial dilution aliquots of 10 μl were then injected into the midribs of a lettuce leaf. The inoculated leaf was placed in a dish containing filter paper soaked with 10 mM MgSO4 and was then maintained in a growth chamber at 28 °C for three days. Symptoms were monitored daily. 6
Microbial Pathogenesis 113 (2017) 5–10
J. Pan et al.
Fig. 1. Confirmation of lacking or complementing ppkA for the ΔppkA and + ppkA stains, respectively. To confirm the lack or complement of the ppkA, primers 5′CTAGTCTAGAAACAAGACCGCCGACGACAGCGAAC-3′ and 5′-TACGAGCTCCAGCAGGTC GAGCAGGGTGCTC-3′ were used for PCR in ΔppkA and +ppkA stains, respectively. The PCR products were assayed by agarose gel electrophoresis.
Fig. 2. Mutation of ppkA leads to defects in biofilm formation. Bacterial cells were grown in 96-well microtiter plates at 37 °C for 24 h. The attached bacteria were then fixed for 15 min with methanol. The remaining adherent biofilms were stained with 0.4% w/v crystal violet, followed by solubilization of the crystal violet with glacial acetic acid and quantification of the stained wells at 590 nm. The data represent the mean ± SD of 3 independent biological repeats. Two asterisks (**) indicate a statistically significant difference (P < 0.01) between the ΔppkA and PAO1.
2.9. Statistical analysis
To examine PpkA regulation in response to osmotic stress, exponentially growing cells of the three strains were exposed to 0.7, 1.3 and 1.6 M NaCl, and the bacterial survival was measured by plating on agar plates. Almost the same survival rates were found in the three strains upon exposure to 0.7 M NaCl. When the strains were exposed to higher concentrations of the salts (1.3 and 1.6 M NaCl), the ppkA mutant showed reduced survival comparison with PAO1 and +ppkA (Fig. 3B). The result of decreased resistance to NaCl-induced osmotic stress when the strain lacked ppkA suggests that PpkA expression in P. aeruginosa contributes to the response to osmotic stress.
The Student's t test was used to determine statistical significance between pairs of experimental groups. P values < 0.01 were considered statistically significant. All experiments were repeated at least three independent times. 3. Results 3.1. Mutation of ppkA causes defection in biofilm formation It has been reported that clinical or environmental isolates of P. aeruginosa have a great ability to form biofilm, and this behavior allows the bacteria to adhere to biotic and abiotic surfaces but make the bacteria resistant to antibiotics [31]. To assess the effect of PpkA on biofilm formation, the ppkA gene-deletion mutant (ΔppkA) (kindly provided by Professor Mougous [25]) and a complemented mutant (+ppkA) (constructed in this work) were used for follow-up experiments. After positive confirmation of lacking or completing the gene for the ΔppkA and +ppkA stains, respectively (Fig. 1), the formation of biofilms of wild type P. aeruginosa PAO1, ΔppkA and +ppkA stains were quantified by the crystal violet staining method. As shown in Fig. 2, ΔppkA bacteria displayed a statistically significant reduction of biofilm formation during growth for 24 h in comparison to the wild type PAO1, while this biofilm defect was restored when ppkA was complemented into ΔppkA (i.e., +ppkA). The result provided definitive evidence that PpkA plays an important role in biofilm formation.
3.3. The ppkA mutant decreased production of pyocyanin P. aeruginosa has been reported to produce a large number of exoproducts, such as pyocyanin, proteases, hemolysin, rhamnolipids and phenazine [33]. Given that pyocyanin is a typically virulence factor that has multiple deleterious effects on host cells and is vital for P. aeruginosa pathogenicity [33], the effect of PpkA on the production of the compound was investigated. The supernatants of PAO1, ΔppkA and +ppkA strains grown for 24 h were harvested and used for the quantification of pyocyanin production using the chloroform-hydrochloric acid extraction method. The results indicated that the pyocyanin production was decreased greatly in the ΔppkA strain compared with that of the PAO1 and +ppkA strains (Fig. 4). This results indicates that PpkA promotes P. aeruginosa virulence factor production.
3.2. Deficiency of ppkA severely decreases tolerance to oxidative and osmotic stress
3.4. The mutation of ppkA compromises invasion of HeLa cells and attenuates plant virulence
P. aeruginosa has ability to naturalize ROS mediated damage with the help of antioxidant enzymes [32]. Tolerance to oxidative stress is a characteristic feature of P. aeruginosa during its infection of patients affected by cystic fibrosis. Therefore, to investigate the effect of PpkA on the tolerance of the bacteria to oxidative stress, the viability of the ΔppkA strain upon exposure to H2O2 was determined and compared with that of the PAO1 and +ppkA strains. After treatment of the three strains with H2O2 and followed by plating onto agar plates, the survival rate of the ppkA-deletion mutant was significantly decreased, as shown in Fig. 3A. This result that the ppkA null mutant strain had decreased H2O2 tolerance and the ppkA complemented strain restored H2O2 tolerance to wild-type levels suggests that PpkA contributes tolerance to H2O2-induced oxidative stress.
P. aeruginosa is a leading opportunistic pathogen that causes infections in persons suffering from immune deficiency, severe burns and cystic fibrosis [34]. The ability of pathogen to invade into non-phagocytic host cells is a key factor for infections and virulence. To evaluate the effect of PpkA on the virulence of P. aeruginosa, we compared the invasion efficiency of HeLa cells with the three strains. The invasion assay was performed by incubation of the bacterial strains with HeLa cells for a total of 6 h, followed by treatment with gentamicin to kill extracellular bacteria. Then, invaded bacterial cells were counted by plating on agar plates. The results showed that the amount of intracellular ΔppkA strain were much less than that of PAO1 and +ppkA strains (Fig. 5A). This dramatically reduced invasion of ppkA mutant into the host cells demonstrates that the kinase is an important regulator of virulence. 7
Microbial Pathogenesis 113 (2017) 5–10
J. Pan et al.
Fig. 4. ppkA deficiency decreased production of pyocyanin. Overnight cultures of PAO1, ΔppkA and +ppkA were inoculated into fresh LB medium (1:100) and cultured at 37 °C for 24 h. The supernatants were harvested for the quantification of pyocyanin production using the chloroform-hydrochloric acid extraction method. Data represent the mean ± SD from 3 independent experiments performed in triplicate. ** indicates P < 0.01 compared to the PAO1 strain.
changes. Protein phosphorylation has been suggested to be the most important of the at least 200 different post-translational modifications that have been identified to date [36] and plays a central role in signal transduction in eukaryotes and prokaryotes. In bacteria, the phosphorylation signaling mediated by two-component systems consisting of histidine kinases and response regulators had been thought to play major roles in environmental adaptation over the past decades [37]. However, in recent years, eukaryotic-like Ser/Thr protein kinases have been demonstrated to regulate growth, antibiotic resistance and virulence of pathogenic bacteria, although their roles in bacterial signal transduction have not been well studied [37]. With the awareness of the important regulation roles exerted by the kinases, the genes encoding the kinases were herein analyzed in bacterial genomes, and the enzyme was found to exist in most of the bacterial species [37], including P. aeruginosa. PpkA is one of the Ser/Thr protein kinases that has been identified in P. aeruginosa and was found to be associated with bacterial virulence several years ago [20,23,25,26], although the regulation roles related to the bacterial adaptation, response, and virulence of the kinase remain unclear. Therefore, we conducted comparative assays of biofilm formation, pyocyanin production, tolerance to stress, cell invasion and plant virulence in ppkA mutants and wild type PAO1. P. aeruginosa has been considered to be the most versatile bacterial pathogen that has an extraordinary capability to survive in certain extreme environments. This key feature to environmental sustainability is attributed in part to the strong ability of P. aeruginosa to form a biofilm [38]. Forming biofilm by P. aeruginosa is also suggested to be the hallmark of chronic infections as well as long-term persistence [7]. Therefore, illuminating the regulatory mechanism of biofilm formation is an essential step for understanding the wide range of environmental adaptability in P. aeruginosa. In this study, a significant decrease of the biofilm formation was found in the ppkA mutant strain compared with the wild type PAO1 strain, whereas the biofilm level was restored when the gene was complemented in the mutant strain (Fig. 2). This finding suggests that PpkA plays an important regulatory role in biofilm formation. To further reveal if the environmental adaptation or stress response was affected in the ppkA mutant strain, the variations of tolerance to oxidative and osmotic stresses were investigated using wild type PAO1, ppkA deletion and complementation strains. The quick response and tolerance to oxidative and osmotic stresses has been described to be a characteristic feature of P. aeruginosa infection and persistence [20]. As expected, similar findings that the ppkA deletion mutant bacteria showed a remarkable reduction of survival were observed when the strain was exposed to H2O2-induced oxidative stress and NaCl-induced
Fig. 3. The ppkA mutant causes decreased tolerance to oxidative and osmotic stress. (A) The sensitivity to H2O2-induced oxidative stress was carried out by determination of the viability of PAO1, ΔppkA and +ppkA strains during exposure to 10 and 50 mM H2O2 for 45 min, respectively. The viable bacterial cells of these three strains were monitored by plating on LB agar plates. (B) In the assay of sensitivity to hyperosmotic stress, three strains were grown in medium containing final concentrations of 0.7, 1.3 and 1.6 M NaCl for 14 h, and the viable bacterial cells were counted by plating on agar plates. The data represent the mean ± SD for three biological replicates. ** indicate the values with significant differences by Student's t test (P < 0.01).
P. aeruginosa could cause infection in animals and is a plant pathogenic bacteria, and studies have shown that plants and animals share functionally common bacterial virulence factors [35]. Therefore, a plant infection model (lettuce leaf) was applied to further measure the bacterial pathogenicity mediated by PpkA. After injection of bacterial cells into the midribs of a lettuce leaf and maintenance for 3 days in a growth chamber, as expected, very slight infection symptoms were caused by ΔppkA bacteria, whereas the wild type PAO1 and +ppkA strains caused severe necrotic lesions of the midribs (Fig. 5B). Considering these findings together, the protein kinase PpkA is crucial for bacterial virulence. 4. Discuss It is well known that signal transduction is essential for living organisms to survive, respond and adapt to dynamic environmental 8
Microbial Pathogenesis 113 (2017) 5–10
J. Pan et al.
studies have been conducted in recent decades, mainly focusing on the adaptation and pathogenesis of P. aeruginosa. However, this bacterial pathogen remains a public health risk worldwide due to its strong adaptability (such as the fact that it could survive multiple classes of antibiotics) and multiple virulence factors, which lead to the ineffectiveness of current therapies [39]. After describing the potential function of PpkA in P. aeruginosa adaptation, we studied if the virulence factors or bacterial pathogenesis were affected by the absence of this kinase. First, the production of pyocyanin was measured in three strains, and a dramatically decreased pyocyanin level was found in the ppkA deletion strain compared with the wild type PAO1 and ppkA complement strains, suggesting the kinase is associated with P. aeruginosa virulence factor production (Fig. 4). Pyocyanin is a blue-green pigment as a secondary metabolite that is produced by P. aeruginosa. The pigment has been revealed as a virulence factor and a quorum sensing signaling molecule that plays an important role in P. aeruginosa pathogenesis [40,41]. In addition to the virulence factors, the ability of a pathogen to invade into host cells is also a principal factor for infections and pathogenesis. Therefore, comparative assays of three P. aeruginosa strain invasions of HeLa cells were performed to assess the potential role of PpkA. The ppkA deletion strain showed an apparently reduced invasion efficiency as shown in Fig. 5A. Moreover, the virulence of the ppkA mutant strain in a plant pathogenesis model was monitored to further confirm the role of PpkA in the regulation of P. aeruginosa virulence, and very slight infection symptoms in lettuce leaf were observed during infection by the ΔppkA strain, while severe necrotic lesions of the midribs were caused by wild-type and complemented strains (Fig. 5B). The result is similar to previous finding observed in the pppA-ppkA deletion mutant [20]. Taken together, our results demonstrate that PpkA is also an important regulator contributing to P. aeruginosa virulence. This study demonstrated that the lack of the Ser/Thr protein kinase PpkA results in a significant decrease of biofilm formation and pyocyanin production, less tolerance to oxidative and osmotic stresses, inefficient invasion of host cells and a reduction of bacterial virulence in P. aeruginosa. These findings provide new insight into the mechanism of Ser/Thr protein kinase in regulating bacterial adaptation and virulence. Acknowledgements This work was supported by grants from the Natural Science Foundation of Zhejiang Province (LY15C010004), the National Natural Science Foundation of China (31770141, 31701572), the 521 Talent Program of Zhejiang Sci-Tech University to J. Pan. We would like to thank Professor Joseph D. Mougous from the University of Washington for kindly providing P. aeruginosa and ppkA null mutant strains.
Fig. 5. ppkA mutant compromises P. aeruginosa virulence. (A) The ΔppkA stain exhibited significantly reduced invasion of HeLa cells compared to PAO1 and +ppkA strains. The invasion assays were carried out by incubation of 5 × 105 HeLa cells per well in 6-well plates with 20 μl of bacterial suspensions (approximately 5 × 106 bacterial cells) for a total of 6 h at 37 °C, 5% CO2 and followed by treatment of 200 μg/ml of gentamicin to kill the extracellular bacteria. The invaded bacterial cells were released from HeLa cells and counted by plating on agar plates. The data represent the mean ± SD for three biological replicates. ** indicate the values with significant differences by Student's t test (P < 0.01). (B) The ΔppkA stain caused slight infection syndromes compared to the PAO1 and +ppkA strains. Ten microliters of bacterial suspensions (OD600 of 0.05 in 10 mM MgSO4) were injected into the midribs of a lettuce leaf. The inoculated leaf was placed in a dish containing filter paper soaked with 10 mM MgSO4, and the infection syndromes were observed after maintaining the leaves in a growth chamber for three days at 28 °C.
Conflict of interest None declared. References [1] A.J. Spiers, A. Buckling, P.B. Rainey, The causes of Pseudomonas diversity, Microbiology 146 (2000) 2345–2350. [2] M.L. Ellison, J.M. Farrow 3rd, W. Parrish, et al., The transcriptional regulator Np20 is the zinc uptake regulator in Pseudomonas aeruginosa, PLoS One 8 (2013) e75389. [3] Y. Okkotsu, P. Tieku, L.F. Fitzsimmons, et al., Pseudomonas aeruginosa AlgR phosphorylation modulates rhamnolipid production and motility, J. Bacteriol. 195 (2013) 5499–5515. [4] D. Balasubramanian, H. Kumari, K. Mathee, Pseudomonas aeruginosa AmpR: an acute-chronic switch regulator, Pathog. Dis. 73 (2015) 1–14. [5] G.M. Matar, M.H. Chaar, G.F. Araj, et al., Detection of a highly prevalent and potentially virulent strain of Pseudomonas aeruginosa from nosocomial infections in a medical center, BMC Microbiol. 5 (2005) 29. [6] P. Cornelis, Pseudomonas: Genomics and Molecular Biology, first ed., Caister Academic Press, 2008.
hyperosmotic stress (Fig. 3). In previous studies, the strong ability to adaptation along with biofilm formation of P. aeruginosa is suggested to rely on complex regulatory networks, including coordination of twocomponent signal transduction systems and quorum sensing [7,9]. Herein, we reported that the phosphorylation signaling mediated by Ser/Thr protein kinase might also be a key regulator for P. aeruginosa in facilitating the environmental response and adaptation. Currently, as the most important cause of nosocomial infections, P. aeruginosa has become a model gram-negative pathogen, and extensive 9
Microbial Pathogenesis 113 (2017) 5–10
J. Pan et al.
[24] S.T. Motley, S. Lory, Functional characterization of a serine/threonine protein kinase of Pseudomonas aeruginosa, Infect. Immun. 67 (1999) 5386–5394. [25] J.D. Mougous, C.A. Gifford, T.L. Ramsdell, et al., Threonine phosphorylation posttranslationally regulates protein secretion in Pseudomonas aeruginosa, Nat. Cell Biol. 9 (2007) 797–803. [26] F. Hsu, S. Schwarz, J.D. Mougous, TagR promotes PpkA-catalysed type VI secretion activation in Pseudomonas aeruginosa, Mol. Microbiol. 72 (2009) 1111–1125. [27] W. Li, L. Wen, C. Li, et al., Contribution of the outer membrane protein OmpW in Escherichia coli to complement resistance from binding to factor H, Microb. Pathog. 98 (2016) 57–62. [28] G.A. O'Toole, Microtiter dish biofilm formation assay, J. Vis. Exp. 47 (2011) 2437. [29] D.W. Essar, L. Eberly, A. Hadero, et al., Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications, J. Bacteriol. 172 (1990) 884–900. [30] L. Zhang, A.M. Krachler, C.A. Broberg, et al., Type III effector VopC mediates invasion for Vibrio species, Cell Rep. 1 (2012) 453–460. [31] G.A. O'Toole, R. Kolter, Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development, Mol. Microbiol. 30 (1998) 295–304. [32] B.E. Britigan, R.A. Miller, D.J. Hassett, et al., Antioxidant enzyme expression in clinical isolates of Pseudomonas aeruginosa: identification of an atypical form of manganese superoxide dismutase, Infect. Immun. 69 (2001) 7396–7401. [33] G.W. Lau, H. Ran, F. Kong, et al., Pseudomonas aeruginosa pyocyanin is critical for lung infection in mice, Infect. Immun. 72 (2004) 4275–4278. [34] A.M. Sousa, M.O. Pereira, Pseudomonas aeruginosa diversification during infection development in cystic fibrosis lungs-a review, Pathogens 3 (2014) 680–703. [35] L.G. Rahme, F.M. Ausubel, H. Cao, et al., Plants and animals share functionally common bacterial virulence factors, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 8815–8821. [36] R. Raggiaschi, S. Gotta, G.C. Terstappen, Phosphoproteome analysis, Biosci. Rep. 25 (2005) 33–44. [37] K. Burnside, L. Rajagopal, Aspects of eukaryotic-like signaling in Gram-positive cocci: a focus on virulence, Future Microbiol. 6 (2011) 747–761. [38] Z. Xu, S. Islam, T.K. Wood, et al., An integrated modeling and experimental approach to study the influence of environmental nutrients on biofilm formation of Pseudomonas aeruginosa, Biomed. Res. Int. 2015 (2015) 506782. [39] J.L. Veesenmeyer, A.R. Hauser, T. Lisboa, et al., Pseudomonas aeruginosa virulence and therapy: evolving translational strategies, Crit. Care Med. 37 (2009) 1777–1786. [40] G.W. Lau, D.J. Hassett, H. Ran, et al., The role of pyocyanin in Pseudomonas aeruginosa infection, Trends Mol. Med. 10 (2004) 599–606. [41] S. Jayaseelan, D. Ramaswamy, S. Dharmaraj, Pyocyanin: production, applications, challenges and new insights, World J. Microbiol. Biotechnol. 30 (2014) 1159–1168.
[7] M.F. Moradali, S. Ghods, B.H. Rehm, Pseudomonas aeruginosa lifestyle: a paradigm for adaptation, survival, and persistence, Front. Cell Infect. Microbiol. 7 (2017) 39. [8] E. Kipnis, T. Sawa, J. Wiener-Kronish, Targeting mechanisms of Pseudomonas aeruginosa pathogenesis, Med. Mal. Infect. 36 (2006) 78–91. [9] Y.T. Chen, H.Y. Chang, C.L. Lu, et al., Evolutionary analysis of the two-component systems in Pseudomonas aeruginosa PAO1, J. Mol. Evol. 59 (2004) 725–737. [10] T. Ouidir, F. Jarnier, P. Cosette, et al., Extracellular Ser/Thr/Tyr phosphorylated proteins of Pseudomonas aeruginosa PA14 strain, Proteomics 14 (2014) 2017–2030. [11] S. Klumpp, J. Krieglstein, Phosphorylation and dephosphorylation of histidine residues in proteins, Eur. J. Biochem. 269 (2002) 1067–1071. [12] A. Ravichandran, N. Sugiyama, M. Tomita, et al., Ser/Thr/Tyr phosphoproteome analysis of pathogenic and non-pathogenic Pseudomonas species, Proteomics 9 (2009) 2764–2775. [13] J. Deutscher, M.H. Saier Jr., Ser/Thr/Tyr protein phosphorylation in bacteria - for long time neglected, now well established, J. Mol. Microbiol. Biotechnol. 9 (2005) 125–131. [14] A.J. Cozzone, Role of protein phosphorylation on serine/threonine and tyrosine in the virulence of bacterial pathogens, J. Mol. Microbiol. Biotechnol. 9 (2005) 198–213. [15] G. Klein, C. Dartigalongue, S. Raina, Phosphorylation-mediated regulation of heat shock response in, Escherichia Coli. Mol. Microbiol. 48 (2003) 269–285. [16] C.C. Zhang, Bacterial signalling involving eukaryotic-type protein kinases, Mol. Microbiol. 20 (1996) 9–15. [17] L. Shi, M. Potts, P.J. Kennelly, The serine, threonine, and/or tyrosine-specific protein kinases and protein phosphatases of prokaryotic organisms: a family portrait, FEMS Microbiol. Rev. 22 (1998) 229–253. [18] S.F. Pereira, L. Goss, J. Dworkin, Eukaryote-like serine/threonine kinases and phosphatases in bacteria, Microbiol. Mol. Biol. Rev. 75 (2011) 192–212. [19] S. Mukhopadhyay, V. Kapatral, W. Xu, et al., Characterization of a Hank's type serine/threonine kinase and serine/threonine phosphoprotein phosphatase in Pseudomonas aeruginosa, J. Bacteriol. 181 (1999) 6615–6622. [20] J. Goldová, A. Ulrych, K. Hercík, et al., A eukaryotic-type signalling system of Pseudomonas aeruginosa contributes to oxidative stress resistance, intracellular survival and virulence, BMC Genomics 12 (2011) 437. [21] M.Y. Galperin, R. Higdon, E. Kolker, Interplay of heritage and habitat in the distribution of bacterial signal transduction systems, Mol. Biosyst. 6 (2010) 721–728. [22] J. Pérez, A. Castañeda-García, H. Jenke-Kodama, et al., Eukaryotic-like protein kinases in the prokaryotes and the myxobacterial kinome, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 15950–15955. [23] J. Wang, C. Li, H. Yang, et al., A novel serine/threonine protein kinase homologue of Pseudomonas aeruginosa is specifically inducible within the host infection site and is required for full virulence in neutropenic mice, J. Bacteriol. 180 (1998) 6764–6768.
10
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Serine/threonine protein kinase PpkA contributes to the adaptation and virulence in Pseudomonas aeruginosa
MARK
Jianyi Pan∗, Zhenzhong Zha1, Pengfei Zhang1, Ran Chen1, Chen Ye, Ting Ye School of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Pseudomonas aeruginosa Serine/threonine (Ser/Thr) protein kinase PpkA Virulence Adaptation
Pseudomonas aeruginosa is a leading cause of nosocomial infections worldwide and has become a serious public health threat, which is attributed to a large extent to its extraordinary environmental adaptability and diverse virulence factors that result in infection and progression of pathogenesis. The eukaryote-type serine/threonine (Ser/Thr) protein kinases, known for playing major regulatory roles in eukaryotes, have been demonstrated to play a central role in regulating various bacterial cellular processes via catalyzing protein phosphorylation. Although PpkA, a Ser/Thr protein kinase first identified in P. aeruginosa, has been implicated in association with bacterial virulence, little is known about the protein. Therefore, in this study, to assess the potential role of PpkA in the regulation of P. aeruginosa environmental adaptation and virulence, variations of biofilm formation, pyocyanin production, tolerance to stress, cell invasion and plant virulence were determined in wild type PAO1, ppkA gene-deleted and complemented mutant strains. Our results indicate that the mutant strain lacking ppkA exhibited a significant decrease of biofilm formation and pyocyanin production, less tolerance to oxidative and osmotic stresses, inefficient invasion of host cells and a reduction of bacterial virulence. These findings provide new insight into the regulation of various cellular processes by PpkA; this is an important mechanism for adaptation and virulence in P. aeruginosa.
1. Introduction Pseudomonas aeruginosa is a Gram-negative bacterium universally found in natural environments, such as soil, water, and vegetation [1]. As a well-known opportunistic pathogen, the bacterium could infect a diversity of organisms, including insects, plants and animals [2]. In humans, P. aeruginosa is the primary cause of fatal lung infections among patients with cystic fibrosis and is also the leading cause of secondary infections in immunocompromised patients, such as those with AIDS, cancer and burn wounds [3,4]. Although it rarely causes disease in healthy people, P. aeruginosa has become a major cause of nosocomial infections worldwide, accounting for 10%–20% of these infections [5,6], and it is a serious threat to public health. In the past few decades, as a model pathogen, P. aeruginosa has been studied extensively and intensively; it has been revealed that the clinical difficulty in the treatment of infections caused by the bacterium is attributable to its extraordinary ability to adapt, survive and develop resistance to various hostile environments, such as oxidative and osmotic stresses, antibiotics and others, and its diverse virulence factors that result in progression of pathogenesis [7]. The main virulence factors of P.
∗
1
aeruginosa include bacterial surface factors (flagella, pili and lipopolysaccharide) and active processes such as the secretion of toxins (for example of pyocyanin) and biofilm formation [8]. The versatility of P. aeruginosa in adaptation to different environments and the production of many virulence factors is metabolically costly and therefore requires regulatory control [7,9]. Protein phosphorylation is a reversible posttranslational modification and a well-known mechanism for the regulation of cellular activities in bacteria and eukaryotes [10]. The signal transduction by histidine/aspartate phosphorylation via two-component regulatory systems has been considered to be an important mechanism by which bacteria detect environmental stimuli and establish an adaptive response [11]. However, this paradigm has now changed with the increasing evidence for serine/threonine/tyrosine (Ser/Thr/Tyr) phosphorylation in numerous bacterial pathways [12]. The phosphoproteins at the Ser/Thr/ Tyr site have been implicated in important cellular activities such as catabolite repression, morphological differentiation, sugar transport, cell growth and viability [12,13], virulence [14], and stress response [15]. Accordingly, such protein kinases, serine/threonine (Ser/Thr) or tyrosine protein kinases, known for the predominant regulatory protein
Corresponding author. E-mail address: [email protected] (J. Pan). These authors contribute equally to this work.
http://dx.doi.org/10.1016/j.micpath.2017.10.017 Received 10 August 2017; Received in revised form 10 October 2017; Accepted 12 October 2017 Available online 13 October 2017 0882-4010/ © 2017 Elsevier Ltd. All rights reserved.
Microbial Pathogenesis 113 (2017) 5–10
J. Pan et al.
Glacial acetic acid (100 μl per well) was added to dissolve the dye bound to the bacterial cells. The quantification of biofilms was performed by determination of the optical density at 590 nm.
kinases exclusively in eukaryotes, have been demonstrated to play central roles in signal transduction by catalyzing protein phosphorylation in prokaryotes [16–18] and to be involved in regulating various bacterial cellular functions, such as growth, metabolism, stress responses, biofilm formation and virulence characteristics [16–20]. The eukaryote-type Ser/Thr protein kinase has been identified in large numbers of bacterial species, and the presence of multiple genes that encode the kinase was found by survey of the genome in some species [21,22]. In P. aeruginosa, there exist at least three Ser/Thr protein kinases, including two characterized kinases Stk1 [19] and PpkA [23,24]. PpkA is the first identified Ser/Thr protein kinase in P. aeruginosa [23], and the kinase has been shown in association with bacterial virulence in neutropenic mice [23] and to be responsible for type VI secretion activation in P. aeruginosa [25,26], suggesting the protein might play an important regulatory role in virulence. Therefore, in this study, we provided more information on the potential role of PpkA in P. aeruginosa virulence. Variations of biofilm formation, pyocyanin production, tolerance to stress, cell invasion and plant virulence were assayed in wild type PAO1, ppkA gene-deleted and complemented mutant strains. Significantly decreased effects on these aspects in ppkA null mutant strain in comparison with PAO1 and complemented strains were observed, suggesting that the regulation of various cellular processes by PpkA is an important regulatory mechanism of P. aeruginosa.
2.4. Hydrogen peroxide sensitivity assay Exponent-phase (OD600 value approximately 0.50) cultures were used for the hydrogen peroxide (H2O2) sensitivity assay. Bacterial cells were harvested and resuspended in PBS to OD600 value of 1.0. The sensitivity of bacterial cells to oxidative stress was determined by the incubation of 0.9 ml suspensions to final concentrations of 0, 10 and 50 mM H2O2 with shaking at 37 °C for 45 min. Viable cells were counted by plating them onto LB agar plates, and the number of CFU was counted after 14 h of incubation at 37 °C. Results are expressed as survival rates. 2.5. Osmotic stress sensitivity assay Bacterial cells were harvested from cultures with an OD600 of 0.8 and resuspended in LB medium containing final concentrations of 0.17 (normal LB, used for control), 0.7, 1.3 and 1.6 M sodium chloride (NaCl). After incubation with shaking at 37 °C for 14 h, serial dilutions of the samples were plated on LB agar plates to determine the CFU. 2.6. Pyocyanin quantitation assay
2. Materials and methods
The quantitation assay of pyocyanin was performed as described previously [29]. In brief, overnight seeding cultures of three strains were diluted 1:100 in 5 ml fresh LB medium and were cultured at 37 °C for 24. The supernatants were collected by centrifugation at 5000 rpm for 3 min. Pyocyanin production was extracted by mixing with 3 ml chloroform and then re-extraction into 1 ml of 0.2 M HCl. The absorbance of the pyocyanin-rich aqueous phase was determined at 520 nm. Pyocyanin concentrations (μg/ml) were determined by multiplying the OD520 by 12.8.
2.1. Bacterial strains, plasmids, host cells and growth conditions P. aeruginosa PAO1 and its mutant strain that lack ppkA (designated as ΔppkA) were kindly provided by Professor Joseph Mougous at the University of Washington and were constructed as described previously [25]. The complemented strain to ppkA in ΔppkA was constructed in this study as described herein. The bacteria were grown in standard Luria-Bertani (LB) medium at 37 °C with vigorous shaking or on solid LB medium (1.5% (w/v) agar in LB) at 37 °C. Human epithelial cells (HeLa) were grown in Dulbecco's modified eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C.
2.7. Invasion of HeLa cells by P. aeruginosa variants The ability of the P. aeruginosa variants to invade HeLa cells was assessed using a modification of an assay described previously [30]. Briefly, the HeLa cells were seeded in a 6-well plate with 5 × 105 cells per well (2 ml medium) and incubated overnight in an incubator at 37 °C, 5% CO2. The bacterial cells were collected from 1 ml cultures (cultured to an OD600 of 0.7), washed three times with PBS and resuspended in 1 ml DMEM medium. Twenty-microliter suspensions (approximately 5 × 106 CFU bacterial cells) were inoculated into each well, and the plates were incubated at 37 °C, 5% CO2. After incubation for 2 h, the wells were rinsed three times with DMEM medium to remove unattached bacteria and then incubated for 4 h, followed by incubation for another hour after replacement of the medium to DMEM medium containing 200 μg/ml of gentamicin. The wells were washed twice with PBS, and then the HeLa cells were lysed with 1% Triton X100 to release invasive bacteria. The supernatants were serially diluted and plated on LB agar plates to determine the CFU.
2.2. Construction of ppkA complementation strain The ppkA complemented strain was constructed as described previously with slight modifications [27]. Briefly, a DNA fragment of ppkA was PCR amplified using the primers ppkA-c-F (5′-CTAGTCTAGAAACAAGACCGCCGACGACAGCGAAC-3′) and ppkA-c-R (5′-TACGAGCTCCAGCAGGTCGAGCAGGGTGCTC-3′), including restriction enzyme Xba I and Sac I recognition sites (underlined sequences), respectively. The PCR products were cloned into a pBBR1MCS2 vector. The resulting recombinant plasmid, pBBR1MCS2-ppkA, was then transformed into the ΔppkA mutant cells. The complementation strain was designated + ppkA and verified by PCR analysis. 2.3. Biofilm formation assay The biofilm assay was carried out in a 96-well microtiter plate using a method described previously [28] with slight modifications. Typically, overnight cultured bacteria were diluted to an OD600 of 1.0 with fresh LB medium, and then 10 μl of bacterial suspension was inoculated into a well containing 150 μl of LB medium and incubated statically at 37 °C for 24 h. After incubation, the wells were aspired gently and washed three times with 200 μl of phosphate-buffered saline (PBS). The remaining attached bacteria were fixed for 15 min by the addition of 100 μl methanol to each well, and then the methanol was removed. Biofilms were stained by the addition of 100 μl of 0.4% w/v crystal violet, followed by incubation at 37 °C for 15 min. Crystal violet solutions were removed from the wells, and the plate was dried at 37 °C.
2.8. Virulence to plant assay The bacterial virulence to plants (Romaine lettuce leaf) was assayed as described previously [20]. Each bacterial strain that cultured into exponential phase (OD600 approximately 0.50) was washed three times and diluted to OD600 0.050 in 10 mM MgSO4. Bacterial dilution aliquots of 10 μl were then injected into the midribs of a lettuce leaf. The inoculated leaf was placed in a dish containing filter paper soaked with 10 mM MgSO4 and was then maintained in a growth chamber at 28 °C for three days. Symptoms were monitored daily. 6
Microbial Pathogenesis 113 (2017) 5–10
J. Pan et al.
Fig. 1. Confirmation of lacking or complementing ppkA for the ΔppkA and + ppkA stains, respectively. To confirm the lack or complement of the ppkA, primers 5′CTAGTCTAGAAACAAGACCGCCGACGACAGCGAAC-3′ and 5′-TACGAGCTCCAGCAGGTC GAGCAGGGTGCTC-3′ were used for PCR in ΔppkA and +ppkA stains, respectively. The PCR products were assayed by agarose gel electrophoresis.
Fig. 2. Mutation of ppkA leads to defects in biofilm formation. Bacterial cells were grown in 96-well microtiter plates at 37 °C for 24 h. The attached bacteria were then fixed for 15 min with methanol. The remaining adherent biofilms were stained with 0.4% w/v crystal violet, followed by solubilization of the crystal violet with glacial acetic acid and quantification of the stained wells at 590 nm. The data represent the mean ± SD of 3 independent biological repeats. Two asterisks (**) indicate a statistically significant difference (P < 0.01) between the ΔppkA and PAO1.
2.9. Statistical analysis
To examine PpkA regulation in response to osmotic stress, exponentially growing cells of the three strains were exposed to 0.7, 1.3 and 1.6 M NaCl, and the bacterial survival was measured by plating on agar plates. Almost the same survival rates were found in the three strains upon exposure to 0.7 M NaCl. When the strains were exposed to higher concentrations of the salts (1.3 and 1.6 M NaCl), the ppkA mutant showed reduced survival comparison with PAO1 and +ppkA (Fig. 3B). The result of decreased resistance to NaCl-induced osmotic stress when the strain lacked ppkA suggests that PpkA expression in P. aeruginosa contributes to the response to osmotic stress.
The Student's t test was used to determine statistical significance between pairs of experimental groups. P values < 0.01 were considered statistically significant. All experiments were repeated at least three independent times. 3. Results 3.1. Mutation of ppkA causes defection in biofilm formation It has been reported that clinical or environmental isolates of P. aeruginosa have a great ability to form biofilm, and this behavior allows the bacteria to adhere to biotic and abiotic surfaces but make the bacteria resistant to antibiotics [31]. To assess the effect of PpkA on biofilm formation, the ppkA gene-deletion mutant (ΔppkA) (kindly provided by Professor Mougous [25]) and a complemented mutant (+ppkA) (constructed in this work) were used for follow-up experiments. After positive confirmation of lacking or completing the gene for the ΔppkA and +ppkA stains, respectively (Fig. 1), the formation of biofilms of wild type P. aeruginosa PAO1, ΔppkA and +ppkA stains were quantified by the crystal violet staining method. As shown in Fig. 2, ΔppkA bacteria displayed a statistically significant reduction of biofilm formation during growth for 24 h in comparison to the wild type PAO1, while this biofilm defect was restored when ppkA was complemented into ΔppkA (i.e., +ppkA). The result provided definitive evidence that PpkA plays an important role in biofilm formation.
3.3. The ppkA mutant decreased production of pyocyanin P. aeruginosa has been reported to produce a large number of exoproducts, such as pyocyanin, proteases, hemolysin, rhamnolipids and phenazine [33]. Given that pyocyanin is a typically virulence factor that has multiple deleterious effects on host cells and is vital for P. aeruginosa pathogenicity [33], the effect of PpkA on the production of the compound was investigated. The supernatants of PAO1, ΔppkA and +ppkA strains grown for 24 h were harvested and used for the quantification of pyocyanin production using the chloroform-hydrochloric acid extraction method. The results indicated that the pyocyanin production was decreased greatly in the ΔppkA strain compared with that of the PAO1 and +ppkA strains (Fig. 4). This results indicates that PpkA promotes P. aeruginosa virulence factor production.
3.2. Deficiency of ppkA severely decreases tolerance to oxidative and osmotic stress
3.4. The mutation of ppkA compromises invasion of HeLa cells and attenuates plant virulence
P. aeruginosa has ability to naturalize ROS mediated damage with the help of antioxidant enzymes [32]. Tolerance to oxidative stress is a characteristic feature of P. aeruginosa during its infection of patients affected by cystic fibrosis. Therefore, to investigate the effect of PpkA on the tolerance of the bacteria to oxidative stress, the viability of the ΔppkA strain upon exposure to H2O2 was determined and compared with that of the PAO1 and +ppkA strains. After treatment of the three strains with H2O2 and followed by plating onto agar plates, the survival rate of the ppkA-deletion mutant was significantly decreased, as shown in Fig. 3A. This result that the ppkA null mutant strain had decreased H2O2 tolerance and the ppkA complemented strain restored H2O2 tolerance to wild-type levels suggests that PpkA contributes tolerance to H2O2-induced oxidative stress.
P. aeruginosa is a leading opportunistic pathogen that causes infections in persons suffering from immune deficiency, severe burns and cystic fibrosis [34]. The ability of pathogen to invade into non-phagocytic host cells is a key factor for infections and virulence. To evaluate the effect of PpkA on the virulence of P. aeruginosa, we compared the invasion efficiency of HeLa cells with the three strains. The invasion assay was performed by incubation of the bacterial strains with HeLa cells for a total of 6 h, followed by treatment with gentamicin to kill extracellular bacteria. Then, invaded bacterial cells were counted by plating on agar plates. The results showed that the amount of intracellular ΔppkA strain were much less than that of PAO1 and +ppkA strains (Fig. 5A). This dramatically reduced invasion of ppkA mutant into the host cells demonstrates that the kinase is an important regulator of virulence. 7
Microbial Pathogenesis 113 (2017) 5–10
J. Pan et al.
Fig. 4. ppkA deficiency decreased production of pyocyanin. Overnight cultures of PAO1, ΔppkA and +ppkA were inoculated into fresh LB medium (1:100) and cultured at 37 °C for 24 h. The supernatants were harvested for the quantification of pyocyanin production using the chloroform-hydrochloric acid extraction method. Data represent the mean ± SD from 3 independent experiments performed in triplicate. ** indicates P < 0.01 compared to the PAO1 strain.
changes. Protein phosphorylation has been suggested to be the most important of the at least 200 different post-translational modifications that have been identified to date [36] and plays a central role in signal transduction in eukaryotes and prokaryotes. In bacteria, the phosphorylation signaling mediated by two-component systems consisting of histidine kinases and response regulators had been thought to play major roles in environmental adaptation over the past decades [37]. However, in recent years, eukaryotic-like Ser/Thr protein kinases have been demonstrated to regulate growth, antibiotic resistance and virulence of pathogenic bacteria, although their roles in bacterial signal transduction have not been well studied [37]. With the awareness of the important regulation roles exerted by the kinases, the genes encoding the kinases were herein analyzed in bacterial genomes, and the enzyme was found to exist in most of the bacterial species [37], including P. aeruginosa. PpkA is one of the Ser/Thr protein kinases that has been identified in P. aeruginosa and was found to be associated with bacterial virulence several years ago [20,23,25,26], although the regulation roles related to the bacterial adaptation, response, and virulence of the kinase remain unclear. Therefore, we conducted comparative assays of biofilm formation, pyocyanin production, tolerance to stress, cell invasion and plant virulence in ppkA mutants and wild type PAO1. P. aeruginosa has been considered to be the most versatile bacterial pathogen that has an extraordinary capability to survive in certain extreme environments. This key feature to environmental sustainability is attributed in part to the strong ability of P. aeruginosa to form a biofilm [38]. Forming biofilm by P. aeruginosa is also suggested to be the hallmark of chronic infections as well as long-term persistence [7]. Therefore, illuminating the regulatory mechanism of biofilm formation is an essential step for understanding the wide range of environmental adaptability in P. aeruginosa. In this study, a significant decrease of the biofilm formation was found in the ppkA mutant strain compared with the wild type PAO1 strain, whereas the biofilm level was restored when the gene was complemented in the mutant strain (Fig. 2). This finding suggests that PpkA plays an important regulatory role in biofilm formation. To further reveal if the environmental adaptation or stress response was affected in the ppkA mutant strain, the variations of tolerance to oxidative and osmotic stresses were investigated using wild type PAO1, ppkA deletion and complementation strains. The quick response and tolerance to oxidative and osmotic stresses has been described to be a characteristic feature of P. aeruginosa infection and persistence [20]. As expected, similar findings that the ppkA deletion mutant bacteria showed a remarkable reduction of survival were observed when the strain was exposed to H2O2-induced oxidative stress and NaCl-induced
Fig. 3. The ppkA mutant causes decreased tolerance to oxidative and osmotic stress. (A) The sensitivity to H2O2-induced oxidative stress was carried out by determination of the viability of PAO1, ΔppkA and +ppkA strains during exposure to 10 and 50 mM H2O2 for 45 min, respectively. The viable bacterial cells of these three strains were monitored by plating on LB agar plates. (B) In the assay of sensitivity to hyperosmotic stress, three strains were grown in medium containing final concentrations of 0.7, 1.3 and 1.6 M NaCl for 14 h, and the viable bacterial cells were counted by plating on agar plates. The data represent the mean ± SD for three biological replicates. ** indicate the values with significant differences by Student's t test (P < 0.01).
P. aeruginosa could cause infection in animals and is a plant pathogenic bacteria, and studies have shown that plants and animals share functionally common bacterial virulence factors [35]. Therefore, a plant infection model (lettuce leaf) was applied to further measure the bacterial pathogenicity mediated by PpkA. After injection of bacterial cells into the midribs of a lettuce leaf and maintenance for 3 days in a growth chamber, as expected, very slight infection symptoms were caused by ΔppkA bacteria, whereas the wild type PAO1 and +ppkA strains caused severe necrotic lesions of the midribs (Fig. 5B). Considering these findings together, the protein kinase PpkA is crucial for bacterial virulence. 4. Discuss It is well known that signal transduction is essential for living organisms to survive, respond and adapt to dynamic environmental 8
Microbial Pathogenesis 113 (2017) 5–10
J. Pan et al.
studies have been conducted in recent decades, mainly focusing on the adaptation and pathogenesis of P. aeruginosa. However, this bacterial pathogen remains a public health risk worldwide due to its strong adaptability (such as the fact that it could survive multiple classes of antibiotics) and multiple virulence factors, which lead to the ineffectiveness of current therapies [39]. After describing the potential function of PpkA in P. aeruginosa adaptation, we studied if the virulence factors or bacterial pathogenesis were affected by the absence of this kinase. First, the production of pyocyanin was measured in three strains, and a dramatically decreased pyocyanin level was found in the ppkA deletion strain compared with the wild type PAO1 and ppkA complement strains, suggesting the kinase is associated with P. aeruginosa virulence factor production (Fig. 4). Pyocyanin is a blue-green pigment as a secondary metabolite that is produced by P. aeruginosa. The pigment has been revealed as a virulence factor and a quorum sensing signaling molecule that plays an important role in P. aeruginosa pathogenesis [40,41]. In addition to the virulence factors, the ability of a pathogen to invade into host cells is also a principal factor for infections and pathogenesis. Therefore, comparative assays of three P. aeruginosa strain invasions of HeLa cells were performed to assess the potential role of PpkA. The ppkA deletion strain showed an apparently reduced invasion efficiency as shown in Fig. 5A. Moreover, the virulence of the ppkA mutant strain in a plant pathogenesis model was monitored to further confirm the role of PpkA in the regulation of P. aeruginosa virulence, and very slight infection symptoms in lettuce leaf were observed during infection by the ΔppkA strain, while severe necrotic lesions of the midribs were caused by wild-type and complemented strains (Fig. 5B). The result is similar to previous finding observed in the pppA-ppkA deletion mutant [20]. Taken together, our results demonstrate that PpkA is also an important regulator contributing to P. aeruginosa virulence. This study demonstrated that the lack of the Ser/Thr protein kinase PpkA results in a significant decrease of biofilm formation and pyocyanin production, less tolerance to oxidative and osmotic stresses, inefficient invasion of host cells and a reduction of bacterial virulence in P. aeruginosa. These findings provide new insight into the mechanism of Ser/Thr protein kinase in regulating bacterial adaptation and virulence. Acknowledgements This work was supported by grants from the Natural Science Foundation of Zhejiang Province (LY15C010004), the National Natural Science Foundation of China (31770141, 31701572), the 521 Talent Program of Zhejiang Sci-Tech University to J. Pan. We would like to thank Professor Joseph D. Mougous from the University of Washington for kindly providing P. aeruginosa and ppkA null mutant strains.
Fig. 5. ppkA mutant compromises P. aeruginosa virulence. (A) The ΔppkA stain exhibited significantly reduced invasion of HeLa cells compared to PAO1 and +ppkA strains. The invasion assays were carried out by incubation of 5 × 105 HeLa cells per well in 6-well plates with 20 μl of bacterial suspensions (approximately 5 × 106 bacterial cells) for a total of 6 h at 37 °C, 5% CO2 and followed by treatment of 200 μg/ml of gentamicin to kill the extracellular bacteria. The invaded bacterial cells were released from HeLa cells and counted by plating on agar plates. The data represent the mean ± SD for three biological replicates. ** indicate the values with significant differences by Student's t test (P < 0.01). (B) The ΔppkA stain caused slight infection syndromes compared to the PAO1 and +ppkA strains. Ten microliters of bacterial suspensions (OD600 of 0.05 in 10 mM MgSO4) were injected into the midribs of a lettuce leaf. The inoculated leaf was placed in a dish containing filter paper soaked with 10 mM MgSO4, and the infection syndromes were observed after maintaining the leaves in a growth chamber for three days at 28 °C.
Conflict of interest None declared. References [1] A.J. Spiers, A. Buckling, P.B. Rainey, The causes of Pseudomonas diversity, Microbiology 146 (2000) 2345–2350. [2] M.L. Ellison, J.M. Farrow 3rd, W. Parrish, et al., The transcriptional regulator Np20 is the zinc uptake regulator in Pseudomonas aeruginosa, PLoS One 8 (2013) e75389. [3] Y. Okkotsu, P. Tieku, L.F. Fitzsimmons, et al., Pseudomonas aeruginosa AlgR phosphorylation modulates rhamnolipid production and motility, J. Bacteriol. 195 (2013) 5499–5515. [4] D. Balasubramanian, H. Kumari, K. Mathee, Pseudomonas aeruginosa AmpR: an acute-chronic switch regulator, Pathog. Dis. 73 (2015) 1–14. [5] G.M. Matar, M.H. Chaar, G.F. Araj, et al., Detection of a highly prevalent and potentially virulent strain of Pseudomonas aeruginosa from nosocomial infections in a medical center, BMC Microbiol. 5 (2005) 29. [6] P. Cornelis, Pseudomonas: Genomics and Molecular Biology, first ed., Caister Academic Press, 2008.
hyperosmotic stress (Fig. 3). In previous studies, the strong ability to adaptation along with biofilm formation of P. aeruginosa is suggested to rely on complex regulatory networks, including coordination of twocomponent signal transduction systems and quorum sensing [7,9]. Herein, we reported that the phosphorylation signaling mediated by Ser/Thr protein kinase might also be a key regulator for P. aeruginosa in facilitating the environmental response and adaptation. Currently, as the most important cause of nosocomial infections, P. aeruginosa has become a model gram-negative pathogen, and extensive 9
Microbial Pathogenesis 113 (2017) 5–10
J. Pan et al.
[24] S.T. Motley, S. Lory, Functional characterization of a serine/threonine protein kinase of Pseudomonas aeruginosa, Infect. Immun. 67 (1999) 5386–5394. [25] J.D. Mougous, C.A. Gifford, T.L. Ramsdell, et al., Threonine phosphorylation posttranslationally regulates protein secretion in Pseudomonas aeruginosa, Nat. Cell Biol. 9 (2007) 797–803. [26] F. Hsu, S. Schwarz, J.D. Mougous, TagR promotes PpkA-catalysed type VI secretion activation in Pseudomonas aeruginosa, Mol. Microbiol. 72 (2009) 1111–1125. [27] W. Li, L. Wen, C. Li, et al., Contribution of the outer membrane protein OmpW in Escherichia coli to complement resistance from binding to factor H, Microb. Pathog. 98 (2016) 57–62. [28] G.A. O'Toole, Microtiter dish biofilm formation assay, J. Vis. Exp. 47 (2011) 2437. [29] D.W. Essar, L. Eberly, A. Hadero, et al., Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications, J. Bacteriol. 172 (1990) 884–900. [30] L. Zhang, A.M. Krachler, C.A. Broberg, et al., Type III effector VopC mediates invasion for Vibrio species, Cell Rep. 1 (2012) 453–460. [31] G.A. O'Toole, R. Kolter, Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development, Mol. Microbiol. 30 (1998) 295–304. [32] B.E. Britigan, R.A. Miller, D.J. Hassett, et al., Antioxidant enzyme expression in clinical isolates of Pseudomonas aeruginosa: identification of an atypical form of manganese superoxide dismutase, Infect. Immun. 69 (2001) 7396–7401. [33] G.W. Lau, H. Ran, F. Kong, et al., Pseudomonas aeruginosa pyocyanin is critical for lung infection in mice, Infect. Immun. 72 (2004) 4275–4278. [34] A.M. Sousa, M.O. Pereira, Pseudomonas aeruginosa diversification during infection development in cystic fibrosis lungs-a review, Pathogens 3 (2014) 680–703. [35] L.G. Rahme, F.M. Ausubel, H. Cao, et al., Plants and animals share functionally common bacterial virulence factors, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 8815–8821. [36] R. Raggiaschi, S. Gotta, G.C. Terstappen, Phosphoproteome analysis, Biosci. Rep. 25 (2005) 33–44. [37] K. Burnside, L. Rajagopal, Aspects of eukaryotic-like signaling in Gram-positive cocci: a focus on virulence, Future Microbiol. 6 (2011) 747–761. [38] Z. Xu, S. Islam, T.K. Wood, et al., An integrated modeling and experimental approach to study the influence of environmental nutrients on biofilm formation of Pseudomonas aeruginosa, Biomed. Res. Int. 2015 (2015) 506782. [39] J.L. Veesenmeyer, A.R. Hauser, T. Lisboa, et al., Pseudomonas aeruginosa virulence and therapy: evolving translational strategies, Crit. Care Med. 37 (2009) 1777–1786. [40] G.W. Lau, D.J. Hassett, H. Ran, et al., The role of pyocyanin in Pseudomonas aeruginosa infection, Trends Mol. Med. 10 (2004) 599–606. [41] S. Jayaseelan, D. Ramaswamy, S. Dharmaraj, Pyocyanin: production, applications, challenges and new insights, World J. Microbiol. Biotechnol. 30 (2014) 1159–1168.
[7] M.F. Moradali, S. Ghods, B.H. Rehm, Pseudomonas aeruginosa lifestyle: a paradigm for adaptation, survival, and persistence, Front. Cell Infect. Microbiol. 7 (2017) 39. [8] E. Kipnis, T. Sawa, J. Wiener-Kronish, Targeting mechanisms of Pseudomonas aeruginosa pathogenesis, Med. Mal. Infect. 36 (2006) 78–91. [9] Y.T. Chen, H.Y. Chang, C.L. Lu, et al., Evolutionary analysis of the two-component systems in Pseudomonas aeruginosa PAO1, J. Mol. Evol. 59 (2004) 725–737. [10] T. Ouidir, F. Jarnier, P. Cosette, et al., Extracellular Ser/Thr/Tyr phosphorylated proteins of Pseudomonas aeruginosa PA14 strain, Proteomics 14 (2014) 2017–2030. [11] S. Klumpp, J. Krieglstein, Phosphorylation and dephosphorylation of histidine residues in proteins, Eur. J. Biochem. 269 (2002) 1067–1071. [12] A. Ravichandran, N. Sugiyama, M. Tomita, et al., Ser/Thr/Tyr phosphoproteome analysis of pathogenic and non-pathogenic Pseudomonas species, Proteomics 9 (2009) 2764–2775. [13] J. Deutscher, M.H. Saier Jr., Ser/Thr/Tyr protein phosphorylation in bacteria - for long time neglected, now well established, J. Mol. Microbiol. Biotechnol. 9 (2005) 125–131. [14] A.J. Cozzone, Role of protein phosphorylation on serine/threonine and tyrosine in the virulence of bacterial pathogens, J. Mol. Microbiol. Biotechnol. 9 (2005) 198–213. [15] G. Klein, C. Dartigalongue, S. Raina, Phosphorylation-mediated regulation of heat shock response in, Escherichia Coli. Mol. Microbiol. 48 (2003) 269–285. [16] C.C. Zhang, Bacterial signalling involving eukaryotic-type protein kinases, Mol. Microbiol. 20 (1996) 9–15. [17] L. Shi, M. Potts, P.J. Kennelly, The serine, threonine, and/or tyrosine-specific protein kinases and protein phosphatases of prokaryotic organisms: a family portrait, FEMS Microbiol. Rev. 22 (1998) 229–253. [18] S.F. Pereira, L. Goss, J. Dworkin, Eukaryote-like serine/threonine kinases and phosphatases in bacteria, Microbiol. Mol. Biol. Rev. 75 (2011) 192–212. [19] S. Mukhopadhyay, V. Kapatral, W. Xu, et al., Characterization of a Hank's type serine/threonine kinase and serine/threonine phosphoprotein phosphatase in Pseudomonas aeruginosa, J. Bacteriol. 181 (1999) 6615–6622. [20] J. Goldová, A. Ulrych, K. Hercík, et al., A eukaryotic-type signalling system of Pseudomonas aeruginosa contributes to oxidative stress resistance, intracellular survival and virulence, BMC Genomics 12 (2011) 437. [21] M.Y. Galperin, R. Higdon, E. Kolker, Interplay of heritage and habitat in the distribution of bacterial signal transduction systems, Mol. Biosyst. 6 (2010) 721–728. [22] J. Pérez, A. Castañeda-García, H. Jenke-Kodama, et al., Eukaryotic-like protein kinases in the prokaryotes and the myxobacterial kinome, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 15950–15955. [23] J. Wang, C. Li, H. Yang, et al., A novel serine/threonine protein kinase homologue of Pseudomonas aeruginosa is specifically inducible within the host infection site and is required for full virulence in neutropenic mice, J. Bacteriol. 180 (1998) 6764–6768.
10