RNase 1, 2, 5 & 8 Role in Innate Immunity: Strain Specific Antimicrobial Activity
Janet Cheruiyot Kosgey2,1, Lina Jia1, Rose Magoma Nyamao1,3, Yi Zhao1, Teng Xue1, Jianxun Yang1,4, Yong Fang1 and Fengmin Zhang1*
1 Department of Microbiology, WU Lien-Teh Institute, Harbin Medical University, Harbin, 150086, China
2 School of biological and life sciences, The Technical University of Kenya, 52428-00200 Kenya
3 School of Medicine, Kenyatta University, 43844, 00100 Kenya
4 Department of Dermatology, The 2ndHospital of Harbin Medical University, Harbin, 150086, China
fengminzhang@ems.hrbmu.edu.cn (Fengmin Zhang*)
* Corresponding author
Declarations of interest: none
Abstract
The increase in microbial resistance to common antimicrobial agents is driving research for the discovery of new antibiotics and antifungal agents. The greatest challenge in this endeavor is to find antimicrobial agents with broad antimicrobial activity and low toxicity. One of the promising areas being explored is antimicrobial peptides, for example RNases. The production of RNases is increased during infection, but their role is still being explored. Whereas the enzymatic activity of RNases is well documented, their physiological role is still being investigated. This study aimed at evaluating the antimicrobial activity of RNases 1, 2, 5, and 8 against E. coli strains, S. aureus, Streptococcus thermophilus, P. aeruginosa, Candida albicans and Candida glabrata. The results demonstrated that RNases have a strain specific antimicrobial activity. RNase 1 had the highest antimicrobial activity compared to other RNases. All the microorganisms screened had varying levels of susceptibility to RNases, except P. aeruginosa and E. coli DR115. RNase 1 showed dose dependent activity against C. albicans. The RNase killed Candida albicans by lowering the mitochondrial membrane potential, but did not damage the cell membrane. We concluded that strain specific antimicrobial activity is one of the physiological roles of RNases.
Key words: RNase; antimicrobial peptide; Candida; E. coli; mitochondrial membrane potential; cell membrane damage.
Introduction
The use of antibiotics and antifungals are gradually being faced out due to the rapid emergence of microbial resistance. The increasing rate of resistance versus the slow rate at which new antimicrobial agents are being discovered is of great concern [1-3]. Antimicrobial peptides offer some ray of hope as they are ancient evolutionary weapons that form part of the innate immune response [4, 5]. Innate immunity has the advantage of having a rapid reaction as compared to the slower adaptive immunity, and its components do not elicit proinflammatory reaction [5, 6]. The advantage of antimicrobial peptides over antibiotics is that they have lower chances of antimicrobial resistance [7, 8], and have the ability to kill a broad spectrum of microbes [5, 9].
The complex composition of the human microbiome is shaped by health status [10], though it is also linked to genetic [11], age [12] and environmental factors like nutrition, drug intake [12], prebiotics, probiotics and microbiota transplantation [12, 13]. In recent years, it has been demonstrated that RNases can be produced by a variety of host or microbial cells, and participates in host defense, antimicrobial activity [8], neurotoxicity, and angiogenesis [14-17]. However, the role and mechanism of RNases in antimicrobial activity, and whether it may be a natural immune molecule regulating the microbiota balance and microbial diversity are unclear.
Microorganisms utilize RNases for defense and survival. The Bacillus species that are RNase producers include; B. amyloliquifaciens, B. subtilis, B. cereus, B. megaterium and B. pumilus [18]. The role of these extracellular RNases in some of these bacteria is unknown. Neutrophils produce neutrophil extracellular traps (NETS) mainly made up of chromatin (30-40% DNA, 1-10% RNA and 50-60% protein) to trap and destroy pathogens [19]. However, some pathogenic bacteria example Streptococcus pyogenes, Staphylococcus aureus, and Clostridium perfringens utilize extracellular nucleases (DNases and RNases) as virulence factors to destroy and escape NETS [19]. Therefore, pathogenic bacteria produce RNases to colonize the host and survive, but their role in non-pathogenic microorganisms is still undetermined. As such, researchers have scaled up the research on nucleases especially ribonucleases as potential antimicrobial peptides [4].
RNase A superfamily has 8 canonical members; RNase 1/pancreatic RNase, RNase 2/eosinophil derived neurotoxin (EDN), RNase 3/eosinophil cationic protein (ECP), RNase 4, RNase 5/angiogenin (ANG), RNase 6, RNase 7 and RNase 8, all of them are encoded in chromosome 14 [5, 8]. All of these proteins possess enzymatic activity while, their physiological roles which include inflammation and the host defense against infections are still being explored [5, 8]. RNase 1 or pancreatic-type RNase, is an endoribonuclease that specifically degrades single-stranded RNA at C and U residues [20-22]. RNase 1 expression is not restricted to the exocrine pancreas [23] since endothelial cells of the circulatory system [24] and human umbilical vein endothelial cells selectively produce and secrete RNase 1 [16, 24]. Primarily, RNase A degrades vascular polyRNA [14, 25]. In addition, to its enzymatic role, RNase A possesses Anti-HIV-1 activity [26-28] and induces maturation and activation of dendritic cells [29]. The enzymatic role of RNases is well documented, yet their physiological role has barely been evaluated [9], with some studies pointing towards the antimicrobial role. Therefore, more research is needed to evaluate the antimicrobial activity and mechanism of RNase A superfamily.
In this study, RNase 1 antimicrobial activity was observed accidentally and due to cost implications RNase 2, 5 and 8 were selected randomly. They were tested for their specific antimicrobial activities and the mechanisms involved, so as to establish their potential role in regulating microbiota balance and microbial diversity.
Materials and methods
2.1 Strain, media, culture conditions and chemicals
Bovine pancreas RNase A (RNASE1) was purchased from Tiangen (China), human recombinant ribonuclease A2 (RNASE2), human recombinant ribonuclease A5 (RNASE5) and human recombinant ribonuclease A8 (RNASE 8) were purchased from ImmunoClone (I&C) America (USA) as lyophilized powder and were reconstituted following the manufacturer’s instructions. E. coli 25922, S. aureus 25923, and C. glabrata 2.3983 were purchased from China general microbiological culture collection (CGMCC). Drug resistant E. coli SYY89 and E. coli DR115 were kindly provided by Prof. Fu (Harbin Medical University, Department of Microbiology). Streptococcus thermophilus, P. aeruginosa (clinical isolates), and C. albicans ATCC10231 were also used in the study. E. coli 25922, S. aureus 25923, E. coli SYY89, E. coli DR115 and P. aeruginosa were grown in nutrient broth (NB)/Agar. Streptococcus thermophilus was grown in De Man, Rogosa and Sharpe (MRS) broth and agar. Incubation conditions were optimized at 37°C for these bacteria in an aerobic incubator (180rpm for liquid cultures). Bacteria (108) were determined using optical density (OD) and confirmed by viable counts (CFU/mL) on nutrient agar except for Streptococcus thermophilus (MRS agar). C. albicans ATCC10231 and C. glabrata 2.3983 were cultured in yeast peptone dextrose (YPD) broth, and sub-cultured on Sabouraud dextrose agar (SDA) slants. Incubation conditions for the Candida species were optimized at 30°C for 24 hours in an aerobic incubator (180rpm for liquid cultures). Candida species (105) were counted using hemocytometer, optical density (OD) and confirmed by viable counts (CFU/mL) on SDA. Media were purchased from Hopebio (China), except Muller Hinton (MH) from Biotopped (China). Cefotaxime (30µg), ceftazidime (30µg), cefoperozone (75µg), and ceftriaxone (30µg) antibiotic discs were purchased from Hopebio (China).
2.2 Antimicrobial susceptibility of E. coli strains
Six clinical strains of E. coli strains;(a) DR115, (b) SYY89, (c) SYY78, (d) ER, (e) SYY130 and (f) SYY72 with different degree of antibiotic susceptibilities were provided by Prof. Fu. The bacterial strains were screened using agar diffusion assay for their antibiotic susceptibility against cefotaxime (30µg), ceftazidime (30µg), cefoperozone (75µg), and ceftriaxone (30µg) on MH agar, except Streptococcus thermophilus which was subcultured on MRS agar.
2.3 Anticandidal activity of RNase
The test microorganisms were treated with RNase to a final concentration of 20µg/ml, a concentration that was reached from RNA degradation experiment. Untreated negative control was included in all screened microorganisms. The bacteria which included E. coli 25922, S. aureus 25923, E. coli SYY89, E. coli DR115 and P. aeruginosa were inoculated in nutrient broth, Streptococcus thermophilus in MRS broth and incubated at 37°C for 24h in an aerobic incubator (180rpm). Whereas, the yeasts C. albicans and C. glabrata were inoculated in YPD broth and incubated 30°C for 24h in an aerobic incubator (180rpm). Bacteria were then sub-cultured in nutrient agar, Streptococcus thermophilus in MRS agar and the yeasts in YPD agar and their viable colonies (CFU/ml) were recorded.
2.4 Dose dependent experiments
Different concentrations of RNase1 (20 µg/ml, 10 µg/ml, 5 µg/ml, 2.5 µg/ml, 1 µg/ml, 0.5 µg/ml) were incubated with C. albicans (104), at 30°C for 24h. The initial concentration 20 µg/ml was used to degrade RNA in previous experiment. Half dilutions were then made to examine minimum cidal concentration. Negative controls consisted of non-RNase-treated C. albicans. These cultures treated and non-treated negative controls were sub-cultured on YPD agar and viable CFU/ml were recorded and photographs of the agar plates taken.
2.5 Checking the integrity of cell membrane using propidium iodide
The integrity of the cell membrane was determined using propidium iodide (PI) (invitrogen). The C. albicans treated with RNase and untreated negative controls were incubated at 30°C for 24h. They were then centrifuged at 5000×g for 5 minutes. The cells concentration was adjusted to 108 cells/ml, and 100µl were stained with 20µl of 20mg/ml of propidium iodide (PI). Fluorescent intensity was assessed immediately after staining using an Olympus FluoViewFV500/IX laser scanning confocal microscope and Quantitative analysis of membrane integrity (red fluorescence) from three independent experiments was done using Image J software.
2.6 Mitochondria membrane potential
JC-1 (Molecular Probes) (Sigma) was used to ascertain changes in mitochondrial membrane potential (∆Ψm) following the manufacturer’s instructions. The C. albicans treated with RNase and untreated negative control were incubated at 30°C for 24h. The cultures were stained with 1mg/ml of JC-1 at 30oC for 20 minutes [30, 31]. Sodium azide (1mM) (Tianjin Fuchen) a fungal respiratory inhibitor was used as positive control [30]. Immediately after staining the mean of the fluorescence intensities was captured using an Olympus FluoViewFV500/IX laser scanning confocal microscope and Quantitative analysis of mitochondrial membrane potential (green/red fluorescence) from three independent experiments with three replicates each was done using Image J software and the ratio of aggregated JC-1 (FL2) to monomer (FL1) intensity was calculated.
2.7 Statistical analysis
The data is presented as mean ± standard deviation from three independent experiments using GraphPad Prism 5.01 (GraphPad Software, La Jolla, USA). The statistically significant differences between untreated control and RNase-treated samples of RNase dose, and mitochondrial membrane potential assays were subjected to one-way ANOVA tests, followed by Turkey’s multiple comparison tests. A P-value <0.05 was considered to be significant, * denoted p<0.05, ** denoted p<0.01, *** denoted p<0.001 and **** denoted p<0.0001.
Results
3.1 Antimicrobial susceptibility of E. coli strains
The six E. coli strains had different degrees of antibiotic susceptibilities, screened using agar diffusion assay for their antibiotic susceptibility to cefotaxime, ceftazidime, cefoperozone, and ceftriaxone. E. coli strains DR115, SYY89 which had resistance and E. coli 25922 susceptible to the screened drugs (Fig. 1a, b & c) were chosen for further experiments on antimicrobial activity of RNases. Other bacteria; S. aureus, Pseudomonas aeruginosa and Streptococcus thermophilus screened against the same antibiotics (Fig. 1h, i & j) were chosen and used in this study.
3.2 Antimicrobial activity of RNases
The results demonstrated that the antimicrobial activity of RNase 1, 2, 5 and 8 was strain specific rather than species specific. This was shown by the three E. coli strains; antibiotic susceptible E. coli 25922 and two antibiotic resistant E. coli SYY89 and DR115. E. coli SYY89 was susceptible to all RNases (1, 2, 5 & 8) screened while, antibiotic resistant E. coli DR115 was only susceptible to only one RNase (2) (p<0.05). Antibiotic susceptible E. coli 25922 was susceptible to three RNase (1, 2 & 5) (p<0.05). The RNase strain specificity antimicrobial activity was also seen in RNase 1 in which it had complete inhibition against E. coli strains 25922, SYY89 (p<0.0001) and did not have antimicrobial activity against E. coli DR115 (p>0.05).
The microbial susceptibility to the RNases varied. Streptococcus thermophilus, S. aureus, and E. coli SYY89 were susceptible to all RNases i.e. RNase 1, 2, 5 & 8 making them the most susceptible. P. aeruginosa and E. coli DR115 were each susceptible to only one RNase making them least susceptible. Candida species had similar susceptibility as each was susceptible to three RNases.
RNase 1 is the only RNase that had complete inhibition of microorganism, this phenomenon was not observed in the other RNases (2, 5 & 8). RNase 1 had complete inhibition of E. coli strains 25922, SYY89, Candida albicans and Candida glabrata. RNase 2 had significant antimicrobial activity against all screened microorganisms except P. aeruginosa. Of all the RNases screened, RNase 1 had the highest antimicrobial activity in which most of the screened microorganisms were inhibited by half (p<0.0001), except E. coli DR115 and P. aeruginosa (p>0.05). RNase 8 had the least antimicrobial activity as it significantly inhibited only half of the screened pathogens. Of interest, RNase 2 had enhanced growth on E. coli 25922; its growth was increased compared to the non-treated groups (Fig 2 & 3).
3.2 Anticandidal activity of RNase 1
RNase 1 was chosen for subsequent experiments because it possessed a significant anticandidal activity, a microorganism of our research interest. Therefore, RNase 1 treatment was investigated independently and anticandidal activity was observed, which was dose-dependent. The lowest RNase 1 concentration observed that inhibited C. albicans significantly (p<0.05) was 5µg/ml (Fig 4).
3.2 Mechanism in which RNase induces cell death
3.2.1 Integrity of the cell membrane using propidium iodide (PI)
RNase 1 did not induce significant (p>0.05) damage on the cell membrane as compared to permeabilized C. albicans (positive control). Since, PI stains nucleic acids of cells that have damaged cell membrane, the dead cells of C. albicans treated with RNase retained intact cell membrane and PI could not access and stain the nucleic acids. Therefore, cell membrane damage was ruled out and other organelle (mitochondrial) integrity was examined (fig 5a, b).
3.2.2 Mitochondrial membrane potential (JC-1)
The integrity of mitochondria was assessed using mitochondrial membrane potential in which the ratio of aggregated JC-1 (FL2 red fluorescence) to monomer (FL1 green fluorescence) intensity was calculated (fig 6a). Consequently, a decrease in the ratio meant mitochondrial depolarization. There was a significant statistical difference between the negative control and RNase treated groups. Thus, it was concluded that RNase induced mitochondrial damage (Fig 6a, b).
Discussion
The discovery of antimicrobial activity of RNase 1 was accidental. While digesting extracellular RNA in an experiment involving C. albicans, it was observed that the RNase 1 independently could induce cell death in Candida albicans. Therefore, we were interested to probe how this occurred since the literature search showed a knowledge gap. We also randomly selected and investigated if other RNases 2, 5 and 8 possessed the same antimicrobial activity. The selection of the few other RNases used was due to limited resources.
Our experiments portrayed that RNase had both antibacterial and antifungal activity. The results demonstrated that the RNase antimicrobial activity was strain specific rather than species specific, which so far had not been reported. This was demonstrated by E. coli strains which had both most susceptible (SYY89) and most resistant (DR115) microorganisms. RNase 1 had complete inhibition of E. coli strains, Candida albicans and Candida glabrata. Previous research on antibacterial and antifungal activity of RNase 1 is undocumented [8, 9]. RNase 2 had significant antimicrobial activity against all screened microorganisms except P. aeruginosa. RNase 2 antibacterial and antifungal activity is scanty and largely remains unknown [5, 9]. E. coli SYY89, Streptococcus thermophilus, and S. aureus were susceptible to all RNases i.e. RNase 1, 2, 5 & 8, while E. coli DR115 and P. aeruginosa was least susceptible to the RNases. The antibacterial and antifungal activity of RNases 5 and 8 has been documented [5, 8, 9, 32-34]. However, the same has not been documented for RNase 1 and 2. The observed antimicrobial activity supports observation by Becknell et al [7] who affirmed the evidence that antimicrobial peptides protect the urinary tract from infection. These results suggest that the RNase is used by host cells to regulate the microbiota balance and the microbial diversity, and this sheds light on the interaction between the host and microorganisms.
RNase 1 exerted the anticandidal activity at a concentration as low as 5µg/ml (MFC100 10µg/ml). Similar observations were made in RNase 3 and RNase 7 with MFC100 30-60µg/ml [35]. The antifungal activity of RNase 1 against Candida albicans has not been reported previously, but has been reported against Candida utilis [36, 37] and Saccaromyces cereviseae [36]. However, the anticandidal activity of other RNases’ (5 and 8) has been reported against Candida albicans [4, 8, 38, 39]. Bacillus intermedius RNase has been reported to penetrate and influence yeast multiplication in Candida utilis [40] and Candida tropicalis [41]. Furthermore, Caporale et al. [42] reported an RNase named as wheatwin which was pathogen related (PR) with enzymatic property that could hydrolyze wheat RNA and also had antifungal activity against plant pathogen Fusarium culmorum. The mechanism of action of antimicrobial activity of RNases’ has been minimally explored thus, we endeavored to explore the biochemical changes in C. albicans after treatment with RNase 1. Our results showed that the RNase treated C. albicans cell membrane remained intact as it had no significant difference with untreated control group. Previous studies related RNase antifungal activity [37], and general cell death [43] to membrane damage [9], which was not the case with our observation. Furthermore, the antibacterial activity of RNase 3 is associated with disruption of the bacterial cell membrane [5, 44, 45]. Previous studies have also reported that RNases diffuse through cell wall and cell membrane causing extensive disorganization of cell structure leading to the release of cytoplasmic content, RNA fragments and amino acid pool [46] supporting RNase cell death by autolysis [9, 47].
We further probed the killing mechanisms, especially if it induced cell death through mitochondria damage. We observed that RNase 1 damaged mitochondria. Alper et al. largely attributed RNase cell damage to damage of the cell membrane and minimal damage on the mitochondria [46]. Our findings agreed with the report by [15, 48] who linked the cationic nature of RNases to its antimicrobial activity by acting on cell membranes with large electric potential example mitochondria membrane and bacteria cell membrane. Furthermore, RNase 2 and 3 have been documented to kill cells by inducing morphological changes including chromatin condensation, membrane reversion, production of reactive oxygen species (ROS) and activation of caspase like activity [9].
Conclusion
RNase 1 and 2 antimicrobial activity had not been previously reported, which is not the case with RNase 5 and 8. RNase 1 completely inhibited E. coli strains, C. albicans and C. glabrata, while RNase 2 significantly inhibited all screened microorganisms except P. aeruginosa. This implied that RNase activity vary among the RNase A superfamily. The strain specific antimicrobial activity of RNases had not been previously reported. E. coli SYY89 was susceptible to all RNases while E. coli DR115 was least susceptible, which strongly indicated that the RNase antimicrobial activity is strain specific. The anticandidal activity of RNase 1 was found to be dose dependent. Further, RNase 1 induced damage on the mitochondria but not on cell membrane. Therefore, this study confirmed that RNases mediate antimicrobial activity, and participate in the formation of the host’s microbiota balance and microbial diversity. These indirectly influence health and disease. We recommend (1) further research on the mechanism of penetration of RNase. (2) A study on the detailed mechanism of RNase strain specific antimicrobial activity. (3) A study on the potential for antifungal application of RNase 1.
Conflict of interest
The authors declare no conflict of interest.
Authors contribution
JCK designed experiments, executed the experiments, analyzed data, wrote the manuscript, edited and approved the manuscript; ZY, LJ, JY, RMN and TX executed the experiments, edited and approved the manuscript; YF and ZFM funded the experiment, designed the experiments, edited and approved the manuscript.
Compliance with ethical standards
This research did not include experiments on animal or humans.
Acknowledgment
Prof. Fu is thanked for kind provision of the antibiotic resistant E. coli strains.
Funding: This work was supported by National Natural Science Foundation of China (NSFC) (No. 31370164 and 81301703) ，Ministry of Science and Technology of the People’s Republic of China (MOST) (2017ZX10201301-003-005), the China Sponsorship Council (CSC) (No. 2016BSZ783) and Government of Kenya under The Technical University of Kenya financially supported JC. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
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Figures and legends
Fig. 1: Antibiotic susceptibility of microbial strains. E. coli selected for further screening in this study were a typed strain and two strains resistant to the screened antibiotics; (a) E. coli 25922, (b) E. coli DR115, (c) E. coli SYY89 respectively. Other E. coli strains not selected for further screening (d) E. coli SYY78, (e) E. coli SYY72 and (f) E. coli SYY130, (g) E. coli ER133, and other bacterial species which were included in this study (h) S. aureus 25923, (g) P. aeruginosa (clinical), & (g) Streptococcus thermophilus. The drugs screened were; (1) Cefotaxime, (2) Ceftazidime, (3) Cefoperozone, and (4) Ceftriaxone. Negative control was empty disc placed in the middle of the agar plate
Fig. 2: Antimicrobial activity of four RNases; RNase 1, RNase 2, RNase 5 & RNase 8 against six bacteria; E. coli CGMCC25922, E. coli SYY89, E. coli DR115, S. aureus CGMCC25923, Streptococcus thermophilus, P. aeruginosa (clinical isolate), and two yeasts; C. albicans ATCC10231 and C. glabrata CGMCC2.3983 in comparison to untreated control group (none). (* p<0.05, ** p<0.01, *** denoted p<0.001 and p<0.0001
Fig. 3: Colony forming unit (cfu) on agar plates of test organisms after treatment with RNases. The test organisms were E. coli CGMCC25922, E. coli SYY89, E. coli DR115, P. aeruginosa (clinical isolate), S. aureus CGMCC25923, Streptococcus thermophilus, C. albicans ATCC10231 and C. glabrata CGMCC2.3983 after treatment with RNase 1, 2, 5 and 8. Negative untreated control (NC) was included
Fig. 4: Anticandidal activity of different concentrations of RNase 1. (a) 20 µg/ml, (b) 10 µg/ml, (c) 5µg/ml, (d) 2.5 µg/ml, (e) 1 µg/ml, (f) 0.5 µg/ml, (g) 0 µg/ml (negative control)
Fig. 5: PI staining of C. albicans after various treatments. A: Red fluorescence intensity of PI stained cells Ctl- negative control (untreated cells), Ctl+ positive control (permeabilized cells) and RNase treated. B: bright field and red fluorescent panel and merge of C. albicans of PI stained cells Ctl+ positive control (untreated cells), Ctl- negative control (permeabilized cells) and RNase treated.
Fig. 6: JC-1 ratio and fluorescent intensity of C. albicans after various treatments. A: the ratio of aggregated JC-1 (FL2 red fluorescence) to monomer (FL1 green fluorescence) intensity; Ctl- negative control (untreated cells), Ctl+ positive control (respiratory inhibitor sodium azide treated cells) and RNase treated group. B: bright field, green and red fluorescent and merge panel of C. albicans stained with JC-1; Ctl- negative control (untreated cells), Ctl+ positive control (sodium azide treated cells) and RNase treated group.