Anti-bacterial activity of Achatina CRP and its mechanism of action
Sandip Mukherjee1, Soma Barman2, Shuvasree Sarkar1, Narayan Chandra Mandal2 & Shelley Bhattacharya1*
1Environmental Toxicology Laboratory, Department of Zoology (Centre for Advanced Studies),
2Microbiology, Mycology & Plant Pathology Laboratory,
Department of Botany, Visva-Bharati University, Santiniketan 731 235, India Received 28 June 2013; revised 4 April 2014
The physiological role of C-reactive protein (CRP), the classical acute-phase protein, is not well documented, despite many reports on biological effects of CRP in vitro and in model systems in vivo. It has been suggested that CRP protects mice against lethal toxicity of bacterial infections by implementing immunological responses. In Achatina fulica CRP is a constitutive multifunctional protein in haemolymph and considered responsible for their survival in the environment for millions of years. The efficacy of Achatina CRP (ACRP) was tested against both Salmonella typhimurium and Bacillus subtilis infections in mice where endogenous CRP level is negligible even after inflammatory stimulus. Further, growth curves of the bacteria revealed that ACRP (50 µg/mL) is bacteriostatic against gram negative salmonellae and bactericidal against gram positive bacilli. ACRP induced energy crises in bacterial cells, inhibited key carbohydrate metabolic enzymes such as phosphofructokinase in glycolysis, isocitrate dehydrogenase in TCA cycle, isocitrate lyase in glyoxylate cycle and fructose-1,6-bisphosphatase in gluconeogenesis. ACRP disturbed the homeostasis of cellular redox potential as well as reduced glutathione status, which is accompanied by an enhanced rate of lipid peroxidation. Annexin V-Cy3/CFDA dual staining clearly showed ACRP induced apoptosis-like death in bacterial cell population. Moreover, immunoblot analyses also indicated apoptosis-like death in ACRP treated bacterial cells, where activation of poly (ADP-ribose) polymerase-1 (PARP) and caspase-3 was noteworthy. It is concluded that metabolic impairment by ACRP in bacterial cells is primarily due to generation of reactive oxygen species and ACRP induced anti-bacterial effect is mediated by metabolic impairment leading to apoptosis-like death in bacterial cells.
Keywords: Achatina fulica, Apoptosis-like-death, C-reactive protein, Metabolic enzymes, Oxidative stress
C-reactive protein (CRP), a prototypic acute phase reactant, is a phylogenetically conserved protein expressed in invertebrates like arthropods1 and mollusks2 and induced in all vertebrates3 except mice4. Invertebrates are endowed with a unique innate immune system but do not possess the acquired immunoglobulin-dependent immune system as found in vertebrates. Phylogenetically CRP appeared much before IgG, and plays an important role in innate responses to infection and is capable of protecting these invertebrates having no adaptive immune response. In Limulus, an arthropod, CRP acts as a main front-line innate immune molecule1 which may be the key to a powerful defense mechanism of these animals against microbial infections that are potentially lethal in other organisms. Several authors reported that CRP can protect mice from infections caused by both gram-positive (Streptococcus
pneumoniae)5, and gram negative (Neisseriae lactamica6; Haemophilus influenzae7) bacteria via direct binding with repetitive phosphorylcholine moieties on the lipoteichoic acid or the lipopolysaccharide (LPS) of these pathogens respectively. In addition to protecting mice against gram positive bacterial infections, human CRP has also been shown to protect mice from infection with Salmonella typhimurium, a pathogen that has phosphatidylethanolamine in its lipid bilayers8.
During the period of bacterial invasion, macrophages are deployed to destroy and remove invading micro-organisms or inflammatory debris. The level of CRP also increases dramatically during periods of immunological challenge and boost the bactericidal activities of monocytes and neutrophils by enhancing the release of reactive oxygen intermediates9. CRP also induces oxidative stress in vitro in endothelial cells, smooth muscle cells and monocyte-macrophages10-12. Although there are many reports on properties of CRP in a wide range of in vitro and in vivo model systems, clear understanding of the actual biological functions of this phylogenetically ancient and highly conserved molecule remains elusive. It is also important to note
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*Correspondent author Telephone: +91-3463-261176 Fax: +91-3463-261176 E-mail: shelleyb38@gmail.com
that bacterial cells are strongly dependent on metabolic cycles for their infection and survival13,14, therefore it is postulated that inhibition of metabolic enzymes by CRP may be one reason for CRP mediated anti-bacterial activity. In the present study, attention was drawn to C-reactive protein isolated from a mollusc, Achatina fulica a terrestrial gastropod surviving in the environment for millions of years. It is a unique species where three different life cycle stages occur in the same individual. Despite maintaining their critical life cycle stages, they have survived successfully and gained the disrepute as an agricultural pest in India. Moreover, the presence of high level of endogenous CRP (2-4 mg/mL) in the haemolymph of A. fulica might be the sole reason behind their effective survival in the environment2. A recent report15 clearly demonstrated that Achatina CRP can ameliorate lead induced hepatotoxicity in rodent models. In the present study, effect of Achatina CRP (ACRP) on key metabolic enzymes, such as phosphofructo kinase 1 (PFK1) in glycolysis, isocitrate dehydrogenase (ICDH) in TCA cycle, isocitrate lyase (IL) in glyoxylate cycle and fructose- 1,6-bis phosphatase (FBP1) in gluconeogenesis was assessed in both gram positive and gram negative bacteria. Amongst these pathways, glycolysis is responsible for aerobic and anaerobic respiration and the irreversible glycolytic reaction catalyzed by PFK1, is essential for infections of S. typhimurium16 and B. subtilis17 to occur in macrophages. Similarly, ICDH and IL are necessary to complete the tricarboxylic acid (TCA) cycle and glyoxylate cycle during bacterial infection, respectively18-21. The importance of FBP1 as the key enzyme for gluconeogenesis is also well documented in these bacteria22. It was hypothesized23 that early α-proteobacterial endosymbionts might have been using secreted and membrane proteases such as metacaspases, paracaspases and Htra-like proteases, to kill their host cells. Existence of eukaryote like programmed cell death and involvement of caspase 3 like proteins in bacteria has been reported24,25. Therefore an attempt has been made to delineate the anti-bacterial property of ACRP mediated by inhibition of salient metabolic enzymes to reduce bacterial infection with the administration of an invertebrate CRP. Also it has been demonstrated that treatment of S. typhimurium and B. subtilis with ACRP induces ROS generation and apoptosis-like phenotypes during bacterial cell death.
Materials and Methods
Chemicals and antibodies—Anti-C-reactive protein antibody (PA1-9579) and immobilized р-aminophenyl phosphoryl choline gel were purchased from Pierce Chemical Co. (Rockford, USA). Primary antibodies against poly (ADP-ribose) polymerase-1 (PARP), caspase-3 and β-actin were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).
Fructose-6-phosphate (Cat.No. F3627), aldolase (Cat.
No. A2714), glyceraldehyde-3-phosphate dehydrogenase (Cat. No. G2267), oxalic acid (Cat. No. 75688), phenylhydrazine hydrochloride (Cat. No. 114715), potassium ferricyanide (Cat. No. 702587), fructose 1, 6-bisphosphate (Cat. No. F6803), glucose-6-phosphate dehydrogenase (Cat. No. 7877), mouse anti-rabbit ALP conjugated secondary antibody, annexin V-Cy3/
CFDA apoptosis detection kit (APOAC) and BCIP/
NBT were procured from Sigma Chemical Co.
(St. Louis, MO, USA). Polyvinylidene fluoride (PVDF) membrane and Oxytetracyclin hydrochloride (Cat. No. 500105) were procured from Millipore (Billerica, MA, USA). Agar powder was purchased from Hi-Media Laboratories Pvt. Ltd., Mumbai, India.
NBT, isocitrate (Cat. No. 094932), reduced glutathione (GSH), 1-chloro-2, 4-dinitrobenzene (CDNB) and thiobarbituric acid (TBA) were purchased from Sisco Research Laboratory (SRL, Mumbai, India). All other chemicals used were of analytical grade.
Maintenance of animals—The giant African land snail Achatina fulica was maintained in a terrarium according to Bose and Bhattacharya2. Adult, healthy, male albino mice of Wistar strain were maintained according to Inglis26. All animal work was conducted according to the guidelines of Institutional Animal Ethics Committee (IAEC), Visva Bharati University. The committee has duly approved the use of animals described in the work as mentioned in the manuscript entitled “Anti-bacterial activity of Achatina CRP and its mechanism of action”. All surgery was performed under sodium pentobarbital anesthesia and all efforts were made to minimize suffering.
Affinity purification of CRP from Achatina fulica―
Haemolymph was collected according to the method described by Bose and Bhattacharya2. ACRP was affinity purified from haemolymph using р-aminophenyl phosphoryl choline conjugated agarose column27. The native and SDS PAGE analyses were carried out with purified ACRP along with the crude haemolymph. The purified ACRP
was also checked by Western blot analysis with an anti-rat CRP antibody.
Bacterial strains—The bacterial strains used in the present study were gram-positive strains such as Bacillus subtilis (MTCC121), Staphylococcus aureus (MTCC96), Listeria monocytogenes (MTCC637), Staphylococcus epidermis (MTCC2639) and the gram-negative strains such as Escherichia coli (MTCC68), Pseudomonas aeruginosa (MTCC741), Salmonella typhimurium (MTCC98) and Pantoea ananatis (MTCC2307). These bacterial strains were procured from the Microbial Type Culture Collection, CSIR-Institute of Microbial Technology (IMTECH), Chandigarh, India. The bacteria were grown at 37 °C in nutrient agar during regular culturing or in Mueller- Hinton agar for the agar diffusion assay.
Determination of minimum inhibitory concentration (MIC) value of ACRP―Inhibition zone diameters were assessed by the disk diffusion method28 on Mueller-Hinton agar plates against the bacteria.
For the evaluation of the antibacterial activities, the diameter of the inhibition zones appearing around the wells was measured in millimeter (mm). The inhibitory potential of the ACRP against different bacteria was checked using a range of concentration (0, 50, 100, 150 and 200 µg/mL) of ACRP. An aliquot (100 µl) of each concentration of properly diluted ACRP was loaded in the cups of petri plates with pre- seeded lawns of test bacterial strains. Oxytetracyclin (50 µg/mL) was used as a positive control. MIC was calculated after 24 h. The MIC value of ACRP against these bacteria were also confirmed by CFU count method 29 and broth micro dilution method30.The broth micro-dilution method was performed according to Ethical and Laboratory Standards Institute (ELSI) guidelines for aerobically grown bacteria in Mueller- Hington medium (data not shown). The inhibition zones were highest in B. subtilis and S. typhimurium at 50 µg/mL which was also found to be the MIC value of ACRP. Therefore all further experiments were performed at this MIC value taking these two representative bacterial strains. B. subtilis was chosen as a representative because the strain MTCC 121 is known to have pathogenic potential31.
In vivo experiments in mice―During acute phase response, more than 200 µg/mL levels of plasma CRP is attained32 in humans, whereas in mice CRP levels remain <2 µg/mL, even after an acute phase response4. Therefore adult male albino mice weighing 20±2 g were selected for the present in vivo
experiments. They were segregated into 4 groups of 5 animals each. Group I, served as control and received iv 2 × 105 CFU of S.typhimurium strain. Group II mice were co-administered, iv 50 µg of ACRP in 150 µL of PBS and S. typhimurium. Similarly, Group III served as control and received, iv 2 × 105 CFU of B. subtilis strain. Group IV mice were co-administered, iv 50 µg of ACRP in 150 µL of PBS and B. subtilis.
Quantitation of viable S.typhimurium and B. subtilis in the blood and organs of mice―To determine bacteremia, 50 µL blood was collected from the retro orbital sinus of mice at 0, 1, 2, 3 and 4 h for analysis of early blood clearance of bacteria. For determination of bacterial load in organs, mice at 4 h of infection were anaesthetized by sodium pentobarbital, and spleen and liver were removed. Each organ was homogenized in 5 mL of ice-cold phosphate buffer (10 mM, pH 7.2); aliquots of serially diluted spleen and liver homogenates were seeded onto nutrient agar plates and incubated at 37 °C for 24 h before bacterial colonies were counted. Counting of CFU of both B. subtilis and S. typhimurium was done by plating them on nutrient agar plates containing the antibiotics to which they are resistant. This was done to eliminate the chance of growth of other bacteria. The antibiotic sensitivity of the bacterial strains against 22 antibiotics (Hi Media octodiscs) was checked before the experiments were designed.
In vitro experiments
Mode of action on bacterial growth―Antibacterial effect of ACRP was assessed by counting the colony forming units (CFU). The bacterial strains were cultured at 37 °C until their log phase was attained.
When CFU count reached approx 1 × 106 cells/mL, ACRP (50 µg/mL) was added to the broth culture of each bacterial strain. Concomitantly, in another set, same broth without ACRP was added to the bacterial strains as an untreated set. To know the mode of action of ACRP, growth kinetics of the test organisms under treated and untreated conditions were performed and rate of fall of CFU was assessed over a period of time (2-32 h). A clear and gradual decline in the number of CFU upon treatment of actively growing cultures indicated a bactericidal mode of action, while a slow rate of decline indicated a bacteriostatic inhibition33.
Preparation of cell-free extracts from control and treated cells―The cell-free extracts were prepared by sonication of treated and untreated cells after
12 and 32 h following the method of Mandal and Chakrabartty34. Cell debris and whole cells were removed from the crude extract by centrifugation at 15,000 g for 10 min. The supernatant obtained after centrifugation was used as the enzyme source.
Enzyme assays―The specific activities of phospho- fructokinase (PFK1), isocitrate dehydrogenase (ICDH), fructose-1, 6-bisphosphatase (FBP1) and isocitrate lyase (IL) were assayed in cell-free extracts of both untreated and treated S. typhimurium and B. subtilis cells following standard protocols35-37.
Assessment of oxidative stress in bacterial cells―The amount of superoxides in control or treated cells was determined by NBT assay38. Level of GSH, being an endogenous anti-oxidant was also estimated in cell free extracts of bacterial cells by the method described by Sedlak and Lindsay39. Similarly, TBARS assay was performed to determine the amount of ipid peroxidation (MDA level) in treated and untreated bacterial cells following the method of Buege and Aust40.
ACRP induced apoptosis-like death in bacterial cells AnnexinV-Cy3/CFDA staining―Phosphatidylserine (PS) is reported to be present in bacteria41. Dwyer et al.42 also demonstrated markers of apoptosis in Escherichia coli including PS exposure detection by fluorescently labeled Annexin V, a human anticoagulant that binds PS with high specificity43. Therefore Annexin V-Cy3-CFDA (Sigma-Aldrich) dual staining was employed as per the manufacturer’s protocol to detect apoptosis like event in bacteria. In this method, Annexin V-positive/ 6-CFDA positive are considered apoptotic; Annexin V-positive/ 6-CFDA negative are considered necrotic and Annexin V-negative/ 6- CFDA positive are considered as live cells. Briefly, control and treated cells were plated on poly-L-lysine- coated slides and incubated for 10 min at room temperature (RT). Cells were then washed and incubated with a double label staining solution (AnnCy3 and 6-CFDA) for 10 min in the dark. After several washes with binding buffer, cells were examined under a Zeiss Axio Scope A1 fluorescence microscope (Carl Zeiss, Germany) in randomly selected fields.
Western blot analysis―Putative caspase 3-like- protein23,24, poly- (ADP-ribose) polymerase-1 (PARP) 44 and actin are present in several bacteria45. Therefore presence of eukaryotic like proteins such as caspase 3, PARP and β-actin were assessed in bacteria by immunoblot analysis. Protein (60 µg) from the lysates
of control and treated cells were resolved in 10%
SDS-PAGE at a constant voltage (60 V) for 2.5 h, and then blotted onto a polyvinylidene fluoride (PVDF) membrane with the help of semi-dry trans blot apparatus (Bio-Rad Trans Blot® SD Cell, USA).
The membranes were first incubated with primary antibodies at a dilution of 1:1000 over night at 4 °C, followed by 2 h incubation with corresponding ALP-conjugated secondary antibodies at 1:2000 (Sigma, St. Louis, MO, USA) dilutions with continuous rocking. The immunoreactive bands were detected by using 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium (BCIP/NBT). β-actin served as loading control. Immunoblots are representatives of five independent experiments.
Scanning electron microscopy―Scanning electron microscopy (SEM) was employed to study the morphology of bacterial cells after the treatment with ACRP. The cells were harvested by centrifugation at 10,000 g for 10 min. The pellets were washed thrice with normal saline and prefixed with a mixture of 3% glutaraldehyde and 5% DMSO in 0.05 M sodium acetate buffer (pH 5.0) for 30 min, harvested by centrifugation at 10,000 g for 10 min, and washed thrice with 0.1 M sodium acetate buffer (pH 5.0). The pellets were then post fixed with osmium tetraoxide solution for 30 min. The cells were collected by centrifugation at 10,000 g for 10 min and were dehydrated with a series of increasing concentrations of ethanol (30–100%) for 10 min at each ethanol concentration. The cells were then spread on a clear glass slide (1 cm2). The slide was mounted on a stub and coated slowly with a very thin (2–5 nm) layer of gold in a sputtering unit prior to examination under a scanning electron microscope (Philips PSEM-500, Holland).
Statistical analyses―Data presented are from at least three independent experiments. The specific activities of enzymes of carbohydrate metabolism are given as mean ± SE. All data were subjected to an analysis of variance (ANOVA) to evaluate the significance of differences and probability of P<0.05 was considered as significant.
Results
Purification of ACRP from haemolymph of A.fulica―
Affinity purification of ACRP was done through a phosphoryl choline affinity column (Fig. 1a) and purity of the protein was checked using native gel (Fig. 1b) followed by immunoblot with anti-rat CRP antibody that showed cross reactivity with ACRP (Fig. 1c).
Screening of anti-bacterial effect of ACRP (in vitro)― Anti-bacterial effect of ACRP in vitro was evaluated on four gram positive and four gram negative bacterial strains. The data obtained from agar cup diffusion method (Table 1) clearly demonstrated that ACRP at 50 µg/mL can potentially inhibit bacterial growth by producing prominent inhibition zones around the wells. The CFU count in presence of different concentrations of ACRP also indicates that at 50 µg/mL, it can significantly inhibit the growth of the bacteria (Table 2). Micro-broth dilution also confirms this concentration to be the MIC of ACRP with potential antimicrobial action (unpublished data). After initial screening of these bacterial strains, mechanism of anti-bacterial action of ACRP was evaluated in B. subtilis (gram positive) and S. typhimurium (gram negative) bacteria.
Effect of ACRP against salmonellae and bacilli infections in mice (in vivo)―Clearance of B. subtilis from the blood was 3-fold higher in ACRP plus B. subtilis co-treated mice as compared to the control mice treated with B. subtilis alone (Fig. 2a). Similarly, the livers and spleens of ACRP injected mice harbored one- to two fold fewer bacteria than control mice treated with B. subtilis alone (Fig. 2a, inset).
Concomitantly, in ACRP and S. typhimurium co-treated mice, blood clearance of salmonellae was significantly (P<0.05) elevated at 3 and 4 h against control mice treated with S. typhimurium alone (Fig. 2b). On the other hand, the number of viable S. typhimurium in liver and spleens of ACRP treated mice was three- to four fold less than control mice treated with salmonellae alone (Fig. 2b, inset).
Fig. 1—Affinity purification of ACRP (a) haemolymph was collected from A. fulica and affinity purification was carried out through phosphorylcholine agarose column. Eluted fractions were collected (arrows) in tubes and pooled for further experiments, (b) pooled fractions were dialyzed and subjected to native-PAGE. Gel was stained with Coomassie and arrow indicates the affinity purified band of ACRP, (c) affinity purified protein was then loaded on to SDS-PAGE and immunoblotted with anti-rat CRP antibody. Corresponding bands of four distinct ACRP subunits could be detected in the blot [M= marker, 1= heamolymph, 2= affinity purified CRP].
Table 1—Determination of minimum inhibitory concentration (MIC) value of ACRP
[Anti-bacterial activity of ACRP at different concentrations assessed by agar cup diffusion method and determination of minimum inhibitory concentration (MIC) value for ACRP. Values are mean of 5 independent experiments
Diameter of inhibition zone (mm) Bacterial strains
ACRP (µg/mL)
0 25 50 100 150 200 Oxytetracyclin (50 µg/mL)
Bacillus subtilis 0 7 16 18 20 24 27
Staphylococcus aureus 0 7 12 14 15 16 29
Escherichia coli 0 5 11 12 14 15 31
Pseudomonas aeruginosa 0 6 10 11 12 14 28
Pantoea ananatis 0 5 10 12 13 15 29
Listeria monocytogenes 0 7 12 13 15 16 31
Staphyloccus epidermis 0 6 10 12 13 14 30
Salmonella typhimurium 0 5 14 15 18 19 32
Assessment of antibacterial effect of ACRP in vitro―After evaluating the anti-bacterial effect of ACRP in vivo condition in mice, potentiality of ACRP was also evaluated in vitro. Cfu counts clearly show that ACRP directly inhibited bacterial growth (Fig. 3) where after 24 h incubation, the number of viable B. subtilis and S. typhimurium with ACRP treatment reduced by 2-4 fold against ACRP untreated bacterial cells.
Mode of action of ACRP―The results obtained from the in vivo and in vitro experiments confirmed the anti-bacterial property of ACRP. However, to know the mode of action of ACRP as a bacteriostatic or bactericidal agent, the growth of bacterial cells was monitored after addition of ACRP into the actively growing bacterial culture. Growth curve of B. subtilis treated with ACRP significantly declined over time (Fig. 4a) as compared to slow decline in ACRP treated S. typhimurium cells (Fig. 4b). Thus, it is abundantly clear that ACRP is bactericidal against gram positive B. subtilis and bacteriostatic against gram negative S. typhimirium cells.
Morphological changes in bacterial cells due to ACRP―ACRP treated cells of both B. subtilis and S. typhimurium showed morphological deformations in comparison to their controls. S. typhimurium cells displayed slightly swollen structures, aggregations and some chewed cells (Fig. 5a). On the other hand, there was no aggregation of B.subtilis upon ACRP treatment, rather deformed and lysed cells were quite frequent (Fig. 5b).
Metabolic impairment in bacterial cells by ACRP―Activation of PFK1 (key regulatory enzyme for glycolysis) was inhibited by 26.68% at 12 h and
highest peak was attained at 32 h, where inhibition rate was 77.82% in ACRP treated S. typhimurium cells as compared to untreated S. typhimurium cells.
In B. subtilis cells with ACRP treatment, inhibition rate of PFK1 was remarkably high attaining a peak (76.8%) at 32 h, as compared to the untreated B. subtilis cell population (Fig. 6a).
Isocitrate dehydrogenase (ICDH), being the major regulatory enzyme for the initiation of TCA cycle was also potentially inhibited by 65-80% in ACRP treated S. typhimurium and B. subtilis cells against respective control cell populations (Fig. 6b). Activation of isocitrate lyase (IL), an important enzyme for the execution of glyoxylate cycle in bacterial cells during stress condition was not significantly decreased in ACRP treated S. typhimurium cells. However, rate of inhibition in ACRP treated B. subtilis cells was about 75% at 12 and 32 h against untreated bacterial cells (Fig. 6c). In salmonellae ACRP treatment caused no change in fructose bis phosphatase (FBP) activity while in B. subtilis cells, ACRP treatment inhibited FBP by 31.54% at 12 h and 70.08% at 32 h (Fig. 6d).
ACRP induced oxidative stress in bacterial cells― As shown in Fig.7a, production of superoxides (O2
-) in ACRP treated S. typhimurium cells enhanced by 2.5 fold at 12 h, which reduced at 32 h, but it was still significantly (P<0.05) higher than control cells.
Similarly, B. subtilis cells treated with ACRP showed remarkable increase in the production of O2
- at 12 h (1.5-fold) and 32 h (2-fold) of incubation against control as evidenced by NBT assay.
Level of lipid peroxidation in S. typhimurium and B. subtilis cells treated with ACRPdemonstrated a significant time dependent increase in MDA
Table 2—Colony forming units (cfu/mL) of B. subtilis and S. typhimurium upon exposure to increasing concentrations of ACRP and positive control- Oxytetracyclin. Values are mean of 5 independent experiments
Concentration of ACRP
(µg/mL)
ACRP+
B.subtilis (cfu/mL)
Concentration of Oxytetracyclin
(µg/mL)
Oxytetracyclin +B.subtilis
(cfu/mL)
Concentration of ACRP
(µg/mL)
ACRP+
S. typhimuium (cfu/mL)
Concentration of Oxytetracyclin
(µg/mL)
Oxytetracyclin + S. typhimuium
(cfu/mL)
0 4.2 x 108 0 4.2 x 108 0 4.36 x 108 0 4.36 x 108
10 3.3 x 108 10 0.8 x 108 10 4.0 x 108 10 1.1 x 108
20 3.1 x 108 20 0.2 x 108 20 5.6 x 108 20 0.2 x 108
30 5.8 x 108 30 0 30 6.5 x 108 30 0
40 4.6 x 108 40 0 40 2.5 x 108 40 0
50 0.5 x 108 50 0 50 0.3 x 108 50 0
60 0 60 0 60 0 60 0
70 0 70 0 70 0 70 0
80 0 80 0 80 0 80 0
90 0 90 0 90 0 90 0
100 0 100 0 100 0 100 0
formation, (P<0.05) as compared to the untreated cells (Fig. 7b and 7c). A significant depletion in GSH was also noted in S. typhimurium cells at 12 h (56%) and 32 h (25%) of incubation with ACRP (Fig.7b).
On the other hand, ACRP treated B. subtilis cells showed 12.5 and 22.54% depletion in GSH level at 12 and 32 h, respectively as compared to the untreated B. subtilis cells (Fig. 7c).
ACRP induced apoptosis-like cell death in bacterial cells―ACRP induced apoptosis-like cell death in bacterial cells was examined by annexin V/Cy3- CFDA dual staining. Increased numbers of apoptotic cells were visualized in S. typhimurium and B. subtilis cell populations treated with ACRP against the untreated ones, where lesser number of apoptotic cells and higher number of CFDA positive cells (live cells) were observed (Fig. 8).
Fig. 3―Colony forming units (CFU) of bacteria. Salmonellae and bacilli were cultured at 37 °C till their log phase was attained and ACRP (50 µg/mL) was added to the broth culture of each bacterial strain. Counting of CFU (CFU/mL) of these bacterial cells was done after 24 h by plating them on nutrient agar plates. Values are mean ± SE of three individual experiments.*P<0.05 represents comparison between untreated and ACRP treated bacterial cells.
Fig. 4―Mode of action of ACRP. (a) ACRP (50 µg/mL) was applied to the bacilli at their log phase followed by counting of cfu (cfu/mL) at an interval of 2 h for 32 h. (b) Similarly, ACRP (50 µg/L) was applied to the salmonellae at their log phase and counting of cfu (cfu/mL) was performed for 32 h. Values are means of three individual experiments.
Fig. 2―Clearance of bacilli and salmonellae in mice. (a and b) At the indicated times, blood was collected from the retro orbital sinus of all groups of mice and serially diluted with phosphate buffer. After serial dilution, blood was seeded onto nutrient agar plates and incubated at 37 °C for 24 h before bacterial colonies were counted. (Inset) Number of viable bacteria recovered from the livers and spleens of mice 4 h post infection. Value are mean ± SE of three individual experiments. *P<0.05 represents comparison between untreated and ACRP treated bacterial cells.
Fig. 5―Scanning electron microscopy of bacterial cells. Effect of ACRP on the morphology of (a) S.typhimurium and (b) B.subtilis assessed by Scanning Electron Microscopy, where swollen structure of S.typhimurium cells and lysed B.subtilis cells were observed as compared to ACRP untreated bacterial cells.
Fig. 6―Inhibition of key metabolic bacterial enzymes by ACRP. ACRP was applied to the log phase of bacterial culture and specific activity of (a) phosphofructokinase-PFK 1, (b) isocitrate dehydrogenase-ICDH, (c) isocitrate lyase-IL and (d) fructose-1, 6-bisphosphatase- FBP was assessed in the cell-free extracts of untreated and ACRP treated bacterial cells at 12 and 32 h. Values are mean ± SE of three individual experiments.*P<0.05 represents comparison between untreated and ACRP treated bacterial cells.
Programmed cell death and activation of caspase- like-proteins is well documented in bacterial cells46-48. Therefore, in the present study, activation of caspase-3 (the effector caspase) and PARP (substrate of caspase 3) was studied by immunoblot analyses. Processing of PARP to activated PARP occurred in S. typhimurium and B. subtilis cells treated with ACRP, indicating the onset of apoptosis-like cell death in these bacterial cells. Moreover, PARP acts as a substrate for caspase-3, which is an effector caspase causing apoptosis-like cell death42. In the present study, remarkable activation of procaspase-3 to form caspase-3 in ACRP treated bacterial cells against the untreated ones indicates ACRP induced apoptosis- like-cell death in both S. typhimurium and B. subtilis cells populations (Fig. 9).
Discussion
The bacteriostatic and bactericidal action of ACRP is the most significant finding in the present study.
It is noteworthy that exogenous ACRP enhances early clearance of iv injected bacteria from the blood in mice and reduces systemic transfer of bacteria to the liver and spleen. Several authors reported efficacy of human CRP in vivo, where hCRP protects mice from S. pneumoniae5,49 or S. typhimurium infections8 and cause survival of the host animal. The present study evaluated different modes of anti-bacterial action of ACRP in vitro. S. typhimurium growth rate is inhibited by ACRP which clearly indicates bacteriostatic action whereas a sharp decline in the growth curve of B. subtilis treated with ACRP indicates bactericidal action. The present finding is further strengthened by scanning electron microscopy (SEM) where ACRP induced destruction of B. subtilis was more prominent than S. typhimurium. Different modes of anti-bacterial effect of ACRP exists which depends on the availability of phosphorylcholine site in the cell wall of bacteria. Since the binding affinity of ACRP with phosphorylcholine is highly conserved from higher vertebrates to invertebrates2, present findings clearly indicate the availability of phosphorylcholine to be imperative for any antibacterial effect. Although there is no direct evidence of the binding of ACRP with bacterial surfaces, the bacteriostatic or bactericidal properties are reported for the first time in the present study.
These in vitro findings further raise a question regarding the involvement of ACRP in modulation of bacterial proteins essential for survival and host infection, which encouraged us to study the direct
Fig. 7―Measurement of cellular redox status. (a) Percentage of superoxide anion generation was determined by NBT assay. (b and c) GSH content of control or ACRP treated (50 µg/mL) bacterial cells were determined. Results were expressed in terms of µg GSH/ mg of protein. The degree of lipid peroxidation was measured in cells by TBARS assay, in terms of nmoles MDA produced/mg protein. A and C- S. typhimurium (Control), B and D- ACRP+ S.typhimurium, E and G- B. subtilis (control), F and H- ACRP + B subtilis. Values are mean ± SE of three individual experiments.*P<0.05 represents comparison between untreated and ACRP treated bacterial cells.
involvement of ACRP in influencing essential intracellular bacterial signaling cascades.
Bacterial cells have high energetic dependence towards glycolysis and TCA cycle. Therefore, inhibition of PFK1 and ICDH, the regulatory enzymes of glycolysis and TCA cycle respectively, is an
interesting intracellular target and appears to be a new mechanism of ACRP induced bacterial cell death in both gram positive and gram negative types. Further, deficit in intracellular metabolic energy triggers execution of glyoxylate cycle and gluconeogenesis to recover from immediate energy crises50,51. The same phenomenon is reflected by the inhibition of FBP and IL in B. subtilis facilitating bacterial cell death owing to different metabolic impairments. However, in S. typhimurium cells ACRP fails to inhibit IL and ICDH enzymes probably due to absence of phosphorylcholine binding sites in these bacterial cells. Therefore, it can be summarized from the present findings that regulatory enzymes of gram positive bacteria are more sensitive to ACRP induced damage than gram negative bacteria.
The present findings corroborate earlier reports on induction of superoxides by CRP in endothelial cells, smooth muscle cells and monocyte-macrophages10-12, which is considered to be a valid reason to effect metabolic depression in these cells. The depletion
Fig. 8―Determination of apoptotic cells in treated and untreated bacterial cells. AnnexinV-Cy3-CFDA dual staining was performed with untreated (control) and ACRP treated cells (as mentioned in the figure). CFDA positive cells were considered as live, AnnexinV-Cy3 positive cells as necrotic and AnnexinV-Cy3/CFDA dual positive cells as apoptotic. Apoptotic cells are observed in the merged image of ACRP treated cell population. Figures are representatives of three independent experiments.
Fig. 9―Activation of caspase cascade. Bacterial cells were incubated without or with ACRP for indicated time periods.
Lysates were prepared and subjected to immunoblot analyses using the anti-bodies as mentioned. β-actin served as loading control. Figures are representatives of five independent experiments.
Lanes: 1 = Markar, 2= S. typhimurium (Control), 3= S. typhimurium + ACRP, 4= B. subtilis (Control), 5= B. subtilis + ACRP.
in intracellular GSH, being one of the indices of oxidative stress, indicates profound scavenging of O2
- radicals52, however, utilization of GSH beyond a critical level with ACRP treatment drives these bacterial cells towards oxidative damage. Enhanced generation of ROS and depletion of intracellular GSH was concomitant with the enhanced rate of lipid peroxidation, as assessed by the level of MDA production in ACRP treated cells. This finding also lends support to the contention that significant membrane damage is caused by ACRP.
Energy yielding metabolic cycles are inhibited by oxidative damage53. This effect demonstrated in the bacteria appears to be due to ACRP induced apoptosis like programmed cell death (PCD) in bacterial cells.
The present study revealed that stressed bacterial cells exhibit apoptotic PCD after treatment with ACRP.
Further, PCD induced biochemical changes in the membrane of bacterial cells are evidenced by externalization of phosphadidylserine (PS) by the application of ACRP. It is important to note that oxidative damage induced by ROS or environmental insult, immediately recruits DNA repair processes in eukaryotes. If the rate of cellular DNA damage is overwhelming, apoptosis is triggered and caspases are deployed for PCD. The present findings suggest that similar events also occur in prokaryotes exposed to oxidative stress. Clear activation of PARP (substrate for caspase-3) and caspase-3, also confirm apoptosis- like PCD in these bacterial cells. In view of the present data, it appears that PARP and caspase-3 are involved in inducing apoptosis-like death in bacterial cells. The present study demonstrates the need to evaluate the antibacterial property of ACRP in terms of mechanistic signals which induced apoptosis-like cell death in bacteria.
Acknowledgment
SM and SS are grateful to UGC, New Delhi for Research Fellowships in Science for Meritorious Students (RFSMS). SB is grateful to CSIR, New Delhi for Senior Research Fellowship (SRF) and NCM acknowledges Visva-Bharati University. Shelley Bhattacharya acknowledges National Academy of Sciences, India (NASI) for the Senior Scientist Platinum Jubilee Fellowship.
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