Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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CHARACTERIZATION AND MPLECLAR DETECTION OF PURIFIED
PROTEUS MIRABILIS PMBS41 ALPHA-HEMOLYSIN
Essam F. A. Al-Jumaily* and Sara Hussein Zgaer
Biotechnology Dept., Genetic Engineering and Biotechnology Institute for postgraduate studies ,
Baghdad University, Baghdad , Iraq.
Key words: Molecular detection, Proteus mirabilis, Alpha-hemolysin..
ABSTRACT
The Alpha- hemolysin, produced by Proteus mirabilis PMBS41 grown in a chemical defined
medium was purified 9.77 fold with a yield (14.90 %) . Alpha-hemolysin was estimate the
molecular weight which was shown to be 88,750KD by using gel filtration chromatography using
Sepharose -6B and 109.64 KD by using SDS-electrophoresis and exhibited an optimum temperature
of 35 and 40°C, and pH optimas at 8.0. Whereas hemolysin reserve a full of its activity at a pH 8
and a temperature at 25° - 30°C.Molecular dectection was done by using specific primer to each
HpmA and HpmB genes that encode for Hemolysin as a virulence factor of proteus mirabilis by
using MPCR and electrophoresis technique.
The PCR assay results identified (53) isolate possessed hpmA and hpmB genes of the proteus
mirabilis bacteria diagnosed , This explains the blood analysis of all pathogenic bacterial isolates but
with different ratio and the importance of PCR in detection of virulence of proteus mirabilis in
clinical urine samples of urinary tract infection (UTI) .
INTODUCTION
Hemolysin is one of the major virulence factors in pathogenic bacteria, and a variety of haemolysins
were identified in a wide range of bacterial species. It is a toxin that inserted to the membranes of
target eukaryotic cell forming a pore causing the efflux of ions and subsequent cell damage [1].
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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P.mirabilis hemolysin is a calcium independent hemolysin system consisting of two proteins
HpmA and HpmB. hemolysin (HpmA) is the protein identified as a cytotoxin in P. mirabilis. This
pore-forming secreted cytotoxin is produced by P. mirabilis during the mid-exponential or late
exponential phase of growth [2]. Hemolysin, which is induced during swarmer cell differentiation of
P. mirabilis and during infection [3], lyses nucleated cells and erythrocytes [4]. P. mirabilis
produces hemolysin HpmA, that used to damage the kidney tissues. This hemolysin is associated to
the cell, calcium-independent, former of pores, Encodes by two genes, hpmA and hpmB, that codify
the HpmA (166 kDa) and HpmB (63 kDa) proteins, respectively [5]. HpmA hemolysin is
responsible for tissue damage and is activated when its N-terminal peptide is cleaved, resulting in
active HpmA (140 kDa), and HpmB is responsible for HpmA activation and transport [6,5].The
HpmA haemolysin toxin of Proteus mirabilis production is upregulated co-ordinately with the
synthesis and assembly of flagella during differentiation into hyperflagellated swarm cells [3]. The
levels of hemolysin in P. mirabilis correlate with its ability to invade cultured kidney cells, and an
isogenic P. mirabilis hpmA mutant is minimally invasive in cultured cells [7].
The aim of the study is to study the biochemical characterization of purify of hemolysin and
detection of the genes encodes of Hemolysin including: DNA extraction from P.mirabilis isolates
and detection of hemolysin genes by using PCR and electrophoresis.
MATERIALS AND METHODS
Alpha-hemolysin was extracted from the selected local producer isolate of P. mirabils PMBS41
by centrifugation, applied on an 24 hrs cultured at 37°C of brain heart infusion broth, at 10000 xg for
20 min at 4°C for the removal of cells from the bacterial culture medium. The cell-free supernatant
fluid was decanted and used as crude extract .
The hemolytic activity assay was determined using Senior and Hughes [8] methods with some
modification as follow. 0.1 ml of an overnight nutrient-broth culture of each strain was added to 10
ml of BHI broth and incubated with shaking at 37°C, small amount of samples (150ϻl) were
removed into micro-centrifuge tubes containing 1 ml of a washed suspension of red blood cells 2%
v/v in saline with and without 150 mM CaCl2. The tubes were incubated with gentle agitation in a
water bath at 37°C for 1hr., after that the tubes were then centrifuged at 13000 rpm for 1 min. to
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
121
pellet the red blood cells and bacteria. The amount of free haemoglobin in the supernate (measured
by its absorbance at 540 nm) indicated the degree of hemolytic activity. Protein concentration was
carried out using the Bradford [9].
Determination of Molecular Weight of Hemolysin by Gel- Filtration Chromatography:
Determination of the Void Volume of the Column:-
The column void volume (Vo) was determined, by the estimation of the total volume of fractions
as characterized with start point movement of the blue dextran to that of climax of absorbency of the
blue dextran.
Determination of Hemolysin Elution Volume (Ve)
Sepharose-6B column (80× 1.5 cm) was prepared, packed and Equilibrated for a second time. 5
ml of purified Hemolysin sample was passed through the column carefully, and equilibrated with
0.02 M Tris-base buffer pH 8.0, with a flow rate of 50 ml/hour. Fractions of 5 ml were collected.
The elution volume(Ve) was estimated for the separated fractions of purified hemolysin, by
following the absorbance at 280 nm.
Measurement of Standard Protein Elution Volumn (Ve)
Different standard proteins (Casein , 31KD; Pepsin 34KD; Alkaline phosphatase 80KD ; DNA
Agyrase 199KD and Urease 409KD) were applied through sepharose-4B column, and then eluted
with 0.02 M Tris-HCl buffer pH 8.0, with a flow rate of 50 ml/hour,
The elution volume was estimated for each standard protein by following the absorbance for the
separated fractions at wave length 280 nm. The (Ve/Vo) ratio was calculated for each standard
protein and for the separated fractions of purified Hemolysin . Then standardization was done by
plotting the elution volume (Ve) of each standard proteins to the void volume (Vo) of the blue
dextran 2000 (Ve/Vo) versus the log value of molecular weight [10]. The hemolysin molecular
weight was accordingly calculated.
SDS - Polyacrylamide gel electrophoresis.
Another enzyme purification procedure were applied by utilizing the technique of SDSpolyacrylamide
gel electrophoresis (SDS-PGE ) according to [11] .
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Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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Extraction of the Proteus mirabilis DNA
The extraction of the DNA was carried out. P. mirabilis Isolates were grown in Brain Heart
broth for 24 hrs at 37oC.From these strain cultures, DNA was extracted from bacterial cells using
Genomic DNA Mini kit supplemented by the manufacturing company (Geneaid, Korea). The
classical method of using Genomic DNA mini kit, (Geneaid, Korea) has a protocol for gram negitive
bacteria
RESULTS AND DISCUSSION
The molecular weight was estimated by gel filtration depending on the size of the separated
molecules with their charge. It was possible that the different methods of estimation may be used
(12).Sepharose 6B for gel filtration was used for estimation the molecular weight of hemolysin
that purified from P.mirabilis PMSB41 in the presence of five standard protein, urease
(409KDaltons), DNA gyrase (199 KDaltons), Alkaine phosphatase (80 KDaltons), Pepsin (
34KDaltons), Casein (31KDaltons). Blue dextran 2000 was applied first to column to detection the
void volume (Vo). -hemolysin and each of standard proteins were applied to column and eluted
separately. The elution volume (Ve)of each of standard protein was recorded, then Ve/Vo was
estimated for each one. The molecular weight of -hemolysin was determined by plotting the
relationship between Ve/Vo and Log of MW of standard proteins as shown in figure (1). By the aid
of the standard curve, the molecular weight of the –hemolysin is estimated and it is nearly (102
KDaltons).
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Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS) was
used for detection the degree of - hemolysin purity, also used as another technique for
determination of molecular weight of purified a-hemolysin. In addition to the five standard proteins
(Aldolase ,154000 Dalton; Alkaline phosphatase ,80000 Dalton; BSA , 67000 Dalton; Ovalbumin,
43000 Dalton; Trypsin, 23000 Dalton) that were migrated in the same gel.
Figure (2) showed that the analysis of protein profile of purified – hemolysin for Proteus mibrils
PMBS41 reveal appearance of one protein band showed high purity of hemolysin with molecular
weight ( 109640 Dalton).
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Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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Figure (2): Sodium dodecyl sulfate-polyacrylamide gel analysis of pure -hemolysine from
Proteus mirabilis PMBS41.
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Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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The optimum pH for Hemolysin activity
Different pH values ranging from (6.0-9.5) were used to determine the optimum pH for
hemolysin activity because the activity of the hemolysin was affected by differences in the pH
buffer. The optimum pH of hemolysin activity purified from P.mirabilis was 8.0 (figure 4) .where as
the optimal activity of E.coli hemolysin was 7.9 that reported by [13]. while the results demonstrated
by [14] that the best hemolysin production from Staphylococcus aureus was in the pH near
neutrality (pH 7.0-7.5).In another study conducted by [15], working with entero invasive E. coli,
they showed that hemolysin expression at optimum pH appeared to be a strain specific feature and
was found maximum inthe pH range 7.5-8.0 coinciding with the results of [16] who reported an
optimum pH of 7.5 for hemolysin production by Aeromonas hydrophila in TSB ,While another
strainspecific study indicated that B. pertussis showed maximum activity at 7.5, whereas B.
parapertussis and B. bronchiseptica showed activity at 7.5-8.0. Both studies revealed results that are
nearly similar to the result of this study and the slight indifference might be attributed to the
involvement of different determinant in host interaction and virulence of various bacterial species
and strains within these species [17].
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Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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Figure(4): Effect of different pH on activity of purified hemolysin of
production from Proteus mirabilis PMBS41.
The optimum pH for Hemolysin stability
Essential to assay of an eproteins or preparation at regulare intervals under standared
conditions is to determine that it remains stable; or if an protein was unstable, It steadily loses
activity with time [18]. In this study used deferent buffer pH (6.0-9.5) to detect the optimal pH for
the hemolysin stability. The results show that the optimum pH for the hemolysin purified form
P.mirabilis stability was 8.0 as well as the hemolysin keeps 100% of its activity. This means that the
protein stays at its optimum state in this pH compared with other. Figure (5) shown the pH stability
decrease gradually from pH (8.5 and 9.0) because of the effect of the alkaline on the nature of
protein, while at pH 6.0 stability was very few, This result refers to a decrease in the protein
stability at a hydrogen number which is far of pH 8.0. This decline is due to the pH effect on the
enzymatic protein structure or irreversible denaturation may occur in a high acidic or basic solution
which leads to change in the active site of the enzyme making it lose. its activity [19].
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Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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Figure ( 5): Effect of pH on purified hemolysin stability from Proteus mirabilis PMBS41 . The
purified enzyme incubated at different pH values 6-10 for 1 hours at 37o C.
The optimum temperature for Hemolysin activity
Different incubation temperature was used to study the effect of temperature on
Hemolysin activity because each proteins and enzyme works within a range of temperature specific
to the kind organism. The effect of temperature on the purified hemolysin activity was studied; the
results show that the activity of purified hemolysin reached its maximum value at (25-30)⁰C see
figure (6), the hemolysin activity then decreased with the continuous increase in temperature and the
activity was lost. These results were nearly agree with those of Li et al.,(2011) who mentioned that
the optimal temperature for enzyme and proteins was most active at 35 ⁰C an increase in the enzyme
activity was observed when the temperature was increased until 35⁰C and then begin to decrease see
Figure (6). An increase in temperature generally leads to an increase in reaction rates, a given
increase because higher temperatures lead to a sharp decrease in reaction rates [18].
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Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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Figure (6): Effect of different temperatures on activity of purified hemolysin of production
from Proteus mirabilis PM BS41.
The Optimum Temperature for Hemolysin Stability
The purified hemolysin was affect by an increase in the temperature because of their
proteinaceous nature. The hemolysin production by P.mirabilis PMBS41 was stable at temperature
range of (35-40) ⁰C and then the activity began to decrease with the increase of temperature Figure
(7). This was due to the denaturing of protein structures resulting from the breakdown of the weak
ionic and hydrogen bonding that stabilized three dimensional structure of the enzyme [18] . Most
proteins were more stable at low temperature, and in order to maintain the activity of the proteins is
preferably saved it in low temperatures [21].The results showed that the hemolysin stability began to
decrease gradually with the increase in the temperature that caused distortion in the active site of
protein due to loss of activity to the breakdown of substrates. There may be several explanations
including the facts that (i) a heat-sensitive step occurs early in the reaction sequence and takes place
before heat denaturation occurs, or (ii) the red cell - hemolysin complex is more stable than is the
hemolysin alone.The studies indicate that the hemolysin-erythrocyte interaction is complex.
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Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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Figure ( 7): Effect of temperatues on purified hemolysin stability
from Proteus mirabilis PMBS41 . The purified enzyme
incubated at values 6-10 for 1 hours at 37o C.
Bacterial DNA Extraction
DNA was extracted from colonies identified as P.mirabilis by using genome DNA purification
kit (Geneaid/ korea) by concentration 50-80 ng/μl. The results were detected by electrophoresis on
1% agarose and exposed to U.V light in which the DNA appears as compact bands, see figure (8).
Figure (8): Genomic DNA extraction electrophoresis on 1% agarose at 70 volt/cm for
30min.
Detection of Hemolysin Genes Using Multiplex PCR (MPCR)
P.mirabilis hemolysin differ from other Proteus spp. that codified by two genes, hpmA and
hpmB, that encodes for the HpmA and HpmB proteins respectively [5, 6].
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Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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Amplification and melting conditions were optimized for the MPCR assay, using specific
primers sequences for HpmA and HpmB genes. in this study all P.mirabilis isolates presented the
hpmA and hpmB genes by MPCR see figure (9) ,these result agree with the result reported by
[22].The presence of the hpmB gene in the isolates that had the hpmA gene was in line with the
description [6)]and [23] who showed the need to cleave the N-terminal peptide of the HpmA by
HpmB to activate and transport the hemolytic HpmA protein to outside the cell, This suggests that
HpmA is a factor in the pathogenesis of P. mirabilis samples isolated from human urine.
Figure (9): Agarose gel electrophoresis (1.5% agarose, 75 V/cm for 1
hour) of hpmA and hpmB PCR products (709bp and 422 amplicon) codify for hemolysin of
P.mirabilis isolates. Lane L (DNA ladder) 100-1100bp molecular marker, lanes
2,3,4,5,6,7,8,9,10,11 isolates are positive results while lane1 and 14 show negative results.
The increase in hemolysin HpmA production is coordinately regulated during the cell
differentiation for the swarmer form and during infection, and is correlated with the invasiveness of
P. mirabilis strains [3].
Study results demonstrate that the detection of hpmA and hpmB gene by PCR was
sensitive enough to be used for the detection of these veriulence factor produced by
P.mirabilis and The PCR technique were shown to be precise, fast, cheap and more
acurate, which were in agreement to many studies that listed before.
1 2 3 4 5 6 7 8 9 10 11 12 1
hpmA 709bp
hpmB 422bp
700 bp
400 bp
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Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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Proteus mirabilis PMBS توصیف والکشف الجینی للفا- الھیمولایسین المنقى من بکتریا 41
عصام فاضل علوان الجمیلی* ساره حسین زغیر
*فرع التقنیة الاحیائیة ، معھد الھندسة الوراثیة والتقنیات الاحیائیة للدراسات العلیا ، جامعة بغداد،بغداد،العراق
الخلاصة
والتی نمیت فی الوسط الغذائی المناسب وکانت Proteus mirabilis PMBS تم انتاج الفا- ھیمولایسین بواسطة العزلة 41
عدد مرات النقاوة 9.77 والانتاجیة بنسبة 14.90 % . وقدر الوزن الجزیئی للفا- الھیمولایسین بواسطة کروماتوکرافیا الترشیح
فکان SDS 88,750 .وکذلک قدر بطریقة الترحیل الکھربائی بوجود KD وکان Sepharose 6B الھلامی بأستخدام ھلام
40° والرقم الھیدروجینی الأمثل ھو C 109.64 کلیو دالتن . أظھرت نتائج توصیف الھیمولایسین ان درجة الحراره المثلى ھی 35 و
25° و رقم ھیدروجینی 8. تم - 30°C. 8.0 ، فی حین یظھر الھیمولایسین المنقى اعلى مستویات من الفعالیة عند درجة حرارة
کعامل ضراوة فی HpmB, HpmA التشخیص الجزیئی بأستخدام بوادئ متخصصة للجینات المشفرة للھیمولایسین والتی ھی
Proteus أظھرت النتائج ان جمیع العزلات البکتیریة الممرضھ . PCR وذلک بأستخدام تقنیة Proteus mirabilis بکتریاا
وبذلک HpmA,HpmB المشخصة والمعزولة من عینات الادرار حاویة على جینات عامل الضراوة الھیمولایسین mirabilis
فأنھا قادرة على تحلیل الدم ولکن بنسب متفاوتھ .
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