The Gene Expression of Virulence Factor Zinc-Metalloprotease GP63 in Mice Infected with Visceral Leishmaniasis
Zainab Abdalameer Kadhem#1, Altamemy A. K. Aakool2, Safaa A Faraj3
#1Minstry of education, 2 University of wasit, Collage of Science, Department of Biology,3 University of wasit, Collage of Midicine, MD pediatric hemato-oncology
Abstract: Visceral leishmaniasis is a zoonotic protozoal disease caused by Leishmania donovani in which the pathogen disseminates to visceral organs inside the macrophage that survive within phagolysosome & evade host defense mechanism result in potential fetal infection associated with hepatosplenomegaly, lymphadenopathy and progressive anemia.The present study was planned and conducted in order to evaluate the role of virulence factor zinc-metalloprotease GP63 in spleen of mice infected with visceral leishmaniasis by both real time PCR techniques. The result detected that the highest mean gene expression, in spleen tissue, recorded in passage four which is 7.21, while the lowest mean gene expression, in spleen tissue, recorded in passage four which is 4.81. (highly significant at P ≤ 0.01).
KEYWORD: Visceral leishmaniasis, virulence factor, zinc-metalloprotease GP63 INTRODUCTION
Visceral leishmaniasis also called kala-azar or black fever/disease, reference to hyperpigmentation of the skin by stimulation of melanocytes during infection , another local name is given Described VL, such as Dumdum fever, Assam fever, and infantile splenomegaly, is the most acute form of leishmaniasis and generally affects the bone marrow, liver, spleen, or other lymphoid tissues. [1].
The major surface protein (MSP or gp63) is metalloproteinase (MP) which belongs to the metzincin class and is abundantly expressed on the surface of Leishmania spp. and other related trypanosomatide protozoans [2]. Gp63 is required for the resistance of promastigotes to complement-mediated lyse in the mammalian host, as the presence of an enzymatically active form of this proteinase significantly reduced the binding of terminal complement components to parasites [3]. Effect of gp63 occurs by activation of protein tyrosine phosphatases (PTPs) in macrophages, leading to minimize production of nitric oxide (NO) and attenuated innate inflammatory reactions, thus increasing the chances of survival of the parasite. [4].
Several functions have been proposed for GP63 and are likely to vary in the different life- cycle stages of Leishmania; GP63 is an endopeptidase that can hydrolyze a range of peptides, and common protein substrates include casein, gelatin, albumin, hemoglobin, and fibrinogen.
[5]. Early studies in the search for a GP63 function have shown that it binds and breaks down the complement component C3, suggesting a role in the elimination of complementary- mediated lyses.[6].
Materials and Methods Experiment design
The study has included 20 Balb/c mice, males, (6~8) weeks-old divided into 4 majer passages, each mice injected intra-peritoneal with (3x106) promastigotes of L. donovani, were the first passage was after 30 days post infection, the second passage was after 30 days post infection, the third passage was after 30 days post infection, finally the fourth passage was after 30 days post infection.
Molecular study:Total RNA extraction
Total RNA was extracted from frozen tissue using (TRIzol ® reagent kit) and performed as follows following company instructions:
1- The spleen sample of 200 mg was homogenized with the addition of 750 μl TRIzol ® reagent.
2- In each tube 2- 200μl of chloroform was added and shaken vigorously for 15 seconds.
3- Incubate the mixture on ice for 5 minutes. Instead it centrifuged for 15 minutes at 12000 rpm, 4 ° C.
4- Supernatant was moved to a new Eppendorf pipeline, with the addition of 500μl isopropanol. Next, mixture is mixed 4-5 times by inverting the tube and incubated for 10 minutes at 4 ° C. Then centrifuged for 10 minutes at 12,000 rpm, 4C °.
5- Supernatant was discarded and 1ml 80 per cent of ethanol was applied and again combined with vortex. Then, centrifuge for 5 minutes at 12000 rpm, 4 ° C.
6- The supernatant was discarded, and the pellet of RNA was left to dry in air.
7- 100μl Nuclease-free water was applied to each sample to dissolve the RNA pellet, then the RNA sample extracted was held at -20.
Estimation of extracted total RNA yield
The total extracted RNA was evaluated and analyzed by Nanodrop spectrophotometer (THERMO. USA), two quality controls on extracted RNA were conducted. The first is to determine the volume of RNA (ng / μL), the second is the purity of the RNA by reading the spectrophotometer absorbance at 260 nm and 280 nm in the same Nanodrop system as follows:
1- The correct application (Nucleic acid, RNA) was chosen after opening the Nanodrop device.
2- A dry wipe was applied, and the measuring pedestals were washed several times. Then carefully piped 2μl of free nuclease water and placed Nanodrop blanking on the surface of the lower measuring pedestal.
3- The pedestals are then washed, and 1μl of the total RNA sample is piped for calculation.
2. 2. 10. DNase I Treatment
The extracted RNA was treated with DNase I enzyme to remove trace amounts of genomic DNA from the eluted total RNA using samples (DNase I enzyme kit) and done using the method defined by the company Promega, USA instructions as follows:
Table (2.8) DNase I Treatment Components Volume
Mix
10ul Total RNA 100ng/ul
1ul DNase I enzyme
4ul 10X buffer
5ul DEPC water
20ul Total
After that, the mixture was incubated at 37 ° C for 30 minutes. Then, for inactivation of the DNase enzyme action 1μl stop reaction was added and incubated at 65C ° for 10 minutes.
cDNA synthesis
In the step of cDNA synthesis for the Pten and GAPDH genes, DNase-I treated RNA samples were also used using the M-MLV Reverse Transcriptase kit and performed as follows:
Step 1
Table (1) RT master mix of step 1 Volume RT master mix
8ul Total RNA 100ng/ul
1ul Random Hexamer primer
1ul DEPC water
10ul Total
Then, RNA and priming were denatured at 65 ° C for 10 minutes, immediately afterwards cooling on ice.
Step 2
Table (2) RT master mix of step 2 Volume RT master mix
10ul Step 1 RT master mix
1ul M-MLV RTase (200u)
4ul 5X M-MLV RTase reaction buffer
2ul 100mM DTT
2ul dNTP
1ul RNase inhibitor
20ul Total
The tubes were then put in a vortex and spinning down briefly. In thermocycler the RNA translated into cDNA under the following thermocycler conditions:
Table (3) Real-Time PCR step cycle condition Time Temperature
Step
1 hour 42 °C
cDNA synthesis (RT step)
5 minutes 95 °C
Heat inactivation
Quantitative Real-Time PCR (qPCR)
The quantitative real-time PCR used in the quantification of GP63 gene expression analysis, which was normalized by the housekeeping gene (GAPDH) using the technique of Real-Time PCR, and this method involves the following steps:
1- The preparation of qPCR master mixes
By using GoTaq ® qPCR Master Mix Kit based on SYBER green dye target detection and GAPDH gene amplification in the Real-Time PCR system, qPCR master mix was prepared and contains the following:
Table (4) q PCR Master Mix Components volume
qPCR master mix Reaction solution
5µL cDNA template (100ng)
1 µL Forward primer(10pmol)
1 µL Reverse primer (10pmol)
12.5 µL qPCR Master Mix
5.5 µL DEPC water
25 µL Total
The reaction solution component placed in qPCR plate strip tubes and mixed for 3 minutes by Exispin vortex centrifuge, then placed in Miniopticon Real-Time PCR.
2- qPCR The conditions of thermocyclers
After that, the qPCR plate and the following thermocycler protocol were loaded into the table below:
Table (5) q PCR thermocycle condition
Repeat cycle Time
Temperature qPCR step
1 5min
95 °C Initial Denaturation
45 20 sec
95 °C Denaturation
30 sec 60 °C
Annealing\Extention Detection(scan)
Data analysis of qRT-PCR
The results of the q RT-PCR data for the target and housekeeping genes were analyzed using the relative quantification of the gene expression (fold change) (the ΔCT method Using the reference gene) as described by (Livak and Schmittgen, 2001). as following equation:
Ratio (reference/target) = 2CT(reference) – CT(target)
RESULTTS The first passage
At this passage, 5 mice were injected intra-peritoneal with promastigotes of L. donovani. The animals dissected after 30 days post infection. Three repeats of spleen were isolated from each animals to measure the gene expression of virulence factor zinc-metalloprotease GP63, the results recorded as shown in table1, the mean of higher value was 4. 14 while the lowest value was 0.544.
Table 6: Virulence factor zinc-metalloprotease GP63 gene fold change expression in the spleen tissue of the first passage.
The repeats
CT
(GP63) CT (GAPDH) ∆CT Fold change (2^∆CT)
Mean of each animal
Mean of total animal
R1 37.14 32.49 -4.65 0.040
4.14
2.847
R2 30.66 33.29 2.63 6.190
R3 30.66 33.29 2.63 6.190
R4 32.18 33.32 1.14 2.204
1.717
R5 35.28 32.59 -2.69 0.155
R6 32.26 33.74 1.48 2.793
R7 31.66 33.49 1.83 3.552 2.332
R8 33.36 32.90 -0.46 0.725
R9 32.28 33.72 1.44 2.721
R10 31.26 32.63 1.37 2.585
1.235
R11 34.35 33.68 -0.67 0.627
R12 33.74 32.73 -1.01 0.495
R13 33.64 33 -0.64 0.642
0.544
R14 34.34 33.82 -0.52 0.695
R15 34.63 32.87 -1.76 0.295
The second passage
At this passage, also 5 mice were injected intra-peritoneal with promastigotes of L.
donovani isolated from the culture of the first passage. Three repeats of spleen from each one were isolated after 30 days post infection to measure the gene expression of virulence factor zinc-metalloprotease GP63 and the results recorded as shown in table 2, the higher value was 7.162 while the lowest value was 0.832.
Table 7: Virulence factor zinc-metalloprotease GP63 gene fold change expression in the spleen tissue of the second passage.
The repeats CT
(GP63) CT (GAPDH) ∆CT Fold change (2^∆CT)
Mean of each animal
Mean of total animal
R1 34.39 33.43 -0.96 0.514
2.077
3.735
R2 31.87 33.80 1.93 3.811
R3 32.97 33.90 0.93 1.905
R4 34.63 33.98 -0.65 0.637
0.832
R5 32.77 33.19 0.42 1.338
R6 35.20 34.26 -0.94 0.521
R7 30.63 33.58 2.95 7.727
7.162
R8 30.80 34.03 3.23 9.383
R9 31.07 33.20 2.13 4.377
R10 32.09 33.88 1.79 3.458
6.912
R11 30.52 34.17 3.65 12.553
R12 31.37 33.61 2.24 4.724
R13 32.41 33.88 1.47 2.770
1.690
R14 33.16 34.28 1.12 2.173
R15 37.22 34.23 -2.99 0.126
The third passage
At this passage, also 5 mice were injected intra-peritoneal with promastigotes of L.
donovani preparation from the culture of the second passage. After dissected the animals, three repeats of spleen from each one were isolated after 30 days post infection to measure the gene expression of virulence factor zinc-metalloprotease GP63 and the results recorded as shown in table 3, the higher value was 6.909 while the lowest value was 2.895 .
Table 8: Virulence factor zinc-metalloprotease GP63 gene fold change expression in the spleen tissue of the third passage
The
repeats CT (GP63) CT
(GAPDH) ∆CT Fold change (2^∆CT)
Mean of each animal
Mean of total animal
R1 30.44 33.03 2.59 6.021
6.660
5.051
R2 29.95 33.40 3.45 10.928
R3 31.90 33.50 1.60 3.031
R4 30.09 33.58 3.49 11.236
6.909
R5 31.23 32.79 1.56 2.949
R6 31.15 33.86 2.71 6.543
R7 33.24 33.18 -0.06 0.959
4.739
R8 30.20 33.63 3.43 10.778
R9 31.49 32.80 1.31 2.479
R10 32.22 33.48 1.26 2.395
2.895
R11 31.79 33.77 1.98 3.945
R12 31.98 33.21 1.23 2.346
R13 34.23 33.48 -0.75 0.595
4.052
R14 31.54 33.88 2.34 5.063
R15 31.13 33.83 2.70 6.498
The fourth passage
At this passage, also five mice were injected intra - peritoneal with promastigotes of L.
donovani preparation from the culture of the third passage. After dissected the animals.
Three repeats of spleen from each one were isolated after 30 days post infection to measure
the gene expression of virulence factor zinc-metalloprotease GP63 and the results recorded as shown in table 4 the higher value was 14.912 while the lowest value was 6.181.
Table 9: Virulence factor zinc-metalloprotease GP63 gene fold change expression in the spleen tissue of the fourth passage
The repeats
CT
(GP63) CT (GAPDH) ∆CT Fold change (2^∆CT)
Mean of each animal
Mean of total animal
R1 32.44 33.07 0.63 1.548
7.668
9.119
R2 29.95 33.87 3.92 15.137
R3 31.90 34.56 2.66 6.320
R4 29.09 33.90 4.81 28.051
14.912
R5 30.23 33.17 2.94 7.674
R6 31.15 34.32 3.17 9.011
R7 31.24 34.07 2.83 7.103
6.181
R8 30.20 33.48 3.28 9.682
R9 33.49 34.30 0.81 1.758
R10 30.22 33.21 2.99 7.945
6.911
R11 32.79 34.26 1.47 2.762
R12 29.98 33.31 3.33 10.026
R13 32.23 33.58 1.35 2.549
9.924
R14 31.54 34.40 2.86 7.239
R15 29.13 33.45 4.32 19.985
Table 10 : Comparison of mean virulence factor zinc-metalloprotease GP63gene fold change expression among passages in the spleen tissue
passages n Mean fold change SD P
passage 1 15 2.86 4.81
0.006
passage 2 15 3.73 3.61 HS
passage 3 15 5.06 3.57
passage 4 15 9.12 7.21
n: number of cases; SD: standard deviation; HS: highly significant at P ≤ 0.01
Figure 1: Bar chart showing comparison of mean virulence factor zinc-metalloprotease GP63gene fold change expression among passages.
DISCUSSION
The use of virulence factor zinc-metalloprotease GP63 gene fold change expression may provide a good tool to evaluate the virulence of Leishmania parasite, on the other hand, mice infected with L. donovani do not get sick; therefore, the sole dependence of clinical features to assess the virulence of Leishmania is not successful [7].
The glycoprotein (GP63) is a zinc-dependent metalloprotease that is found in high amount on the promastigotes surface, connected via a (Glycosyl-phosphatidylinositol) GPI-anchor [8].
Breakdown of the GPI anchor by phospholipase C leads to constant shedding of GP63 to the extracellular compartment. Added to that, GP63 is also directly secreted by the parasite through the pocket of the flagella. It has been observed that intracellular amount of GP63 found that can be produced upon certain triggers in the extracellular compartment [9]. The encoding genes for GP63 found as a multigene chain in the Leishmania genome. Various GP63 genes possess subtle variations in sequence as well as pattern expression; however the exact variation among these GP63 genes are not fully understood [10].
In the first passage, first generation of parasite leads to creation level of virulence that has been measured by expression of the virulence factor zinc-metalloprotease GP63 fold change, while in the second passage appear more expression of the virulence factor zinc- metalloprotease GP63 which means that the second generation parasites were more virulent, The elevation of virulence factor continuous increasing in the next passages. Therefore it be can concluded that with further generations, visceral Leishmania develop more virulence strains. This agree with the results of the study by Alcolea, and Alonso, [11] where
mentioned that infectivity and virulence decreases with culture passages and infection of laboratory animals is frequently required for increase the virulence.
On the other hand, WHO [12] have provided the following explanation for the perpetuation, that is the continuation in nature, of these parasite populations in that it depends on the existence Two hosts: insects that feed on blood and mammals. They also suggested that the production of tissue parasites, including blood, liver, spleen, bone marrow, skin, relies on their ability to weaken the hosts through mechanisms that bare either specific to the parasites or common to other forms of parasites. It has been shown by Baneth et al.,[13] that the comprehensive Research into the events that occur in mammalian hosts that are linked to persistence and infectivity can provide for valuable information about parasite dissemination mechanisms and pathogenesis, and this may provide ability to discover better therapeutic approaches. It appears that the dependence on assessment of virulence by the factor zinc- metalloprotease GP63 will reduce the time and number of animals needed in comparison, with dependence on histological assessment alone.
Beside that, Aslan et al.,[14] have shown few in spleen tissue models that were proposed to study visceral leishmaniasis, and that dogs and hamsters are the most reliable experimental animals, because these animals shown similar clinicopathological features as visceral leishmaniasis in human. Those authors have shown that the experimental models of visceral leishmaniasis in mice The premature regulation of the liver parasite load and the delayed burden of the spleen parasite were highlighted.Therefore, histological evaluation of histological sections is essential to identify virulence of the parasite.
In line with our previous observation that new generations of Leishmania produced by inoculating new animals by strains taken and cultured from previous animals causes the appearance of more virulent strains, this might be related to that, due to their numerous mechanisms of immune subversion and evasion and modulation of the macrophage, Leishmania parasites are able to successfully infect mammalian macrophages. this suggestions correspond with as shown by previous studies Abu-Dayyeh et al., [15] and Contreras et al., [16] where it have been described that upon infection of the macrophage, numerous signaling proteins for example Interleukin-1 receptor-associated kinase 1 (IRAK- 1), Janus kinase 2 (JAK2) and mitogen-activated protein kinase (MAP Kinases), transcription factors sign transducer and activator of transcription 1 (STAT-1), Activator protein 1 (AP-1) and nuclear factor kappa beta (NF-κB), The mammalian/mechanistic target of rapamycin (mTOR) translational protein is also altered.
Previous and recent studies have shown that Leishmania infection affects protein kinase C PKC sensitivity to its natural substrates, for example di-acyl-glycerol, and has established GP63 as the main virulence factor responsible for the abrogate ROS production of reactive oxygen species [17].
However, a study by Isnard et al,. [18] has shown that Leishmania GP63 is capable of reaching the nucleus, degrading multiple Activator Protein 1 AP-1 subunits. In addition, it has recently been shown that GP63 reaches the perinuclear portion by gluing specific nucleo- pore proteins and further explaining its interaction with various transcription factors.
CONCLUSION
The gene expression of virulence factor zinc-metalloprotease GP63 in spleen tissue of passage four has the highest, followed by passages three, then by passages two and finally by passages one. Therefore it be can concluded that with further generations, visceral Leishmania develop more virulence strains.
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