IDENTIFICATION OF UNUSUAL 60 KDa OUTER MEMBRANE PROTEINS FROM HAEMOPHILUS INFLUENZAE TYPE B FROM INDIAN ISOLATES [Original Research]
KUMAR A, PhD; SINGH AK, MSc; SINGH AK, MSc
Bharat Immunological and Biological Corporation Limited (BIBCOL), Oral Polio Vaccine Plant, Chola, Bulandshahr, Uttar Pradesh, India-203
[emedpub – International Infectious Diseases Vol 1:3] [Date of Publication: 03.30.2011]
ISSN 2231-6019

March 27, 2011 at 1:36 PM

Abstract:

Objective: Outer membrane proteins (OMPs) of Haemophilus influenzae type b (Hib) are important and crucial antigens. Therefore a study was conducted for purification and identification of Hib-OMPs with Indian isolates.

Methods: A total of eight Indian isolates of Hib, including five from blood and three from CSF were studied after cultural and microscopic characterization. The isolates were examined for their OMP pattern on 12% SDS-PAGE. Some of them Hib-OMPs were eluted by electro elution method and the OMPs were further identified by SDS-PAGE.

Results: OMP analysis of all isolates was done and identified by using standard molecular weight marker of protein on 12% SDS-PAGE. All isolates had similar OMP pattern as previously reported. But Hib isolates from cerebrospinal fluid (CSF) had one additional and prominent 60 kDa OMP.

Conclusion: On the basis of the study observations, we found similar Hib-OMP pattern in all the isolates. In addition, one unusual 60 kDa OMP was identified in Hib isolates from the CSF. The latter OMP may play an important role in their diagnosis and may also be crucial in epidemiological study of Hib in India.

Introduction:

Haemophilus influenzae type b (Hib) is a leading cause of bacterial meningitis, pneumonia, and other infections in children, killing an estimated 386,000 children under the age of five each year [1]. Countries have been slow to implement the life-saving vaccine against Hib, in part because the burden of disease caused by Hib is difficult to detect [2]. Almost all infected with Hib are children under the age of five, with those between 4 and 18 months of age are especially vulnerable. However, in many developing countries where Hib vaccination is not routine, invasive Hib disease is still a significant cause of morbidity and mortality. In India, there are many studies on Hib and its infections and these have reported disease manifestations of Hib in the population. In 2003, Shah reported that Hib vaccine has significantly reduced the disease occurrence in India in the past few years. Despite this, there is emerging problem of multidrug resistance to Hib in India [3]. Overall, mortality due to meningitis is reported at 60% and a significant proportion develop long-term sequelae in India [4, 5, 6].

Clinical specimens of cerebrospinal fluid (CSF) and blood were used as main source for isolation of Hib in these studies and successfully isolated and subtyped the Indian isolates of Hib [7]. The clinical isolates were used to study protein profile of surface components, especially the outer membrane proteins (OMPs) [8]. It is well established that gel electrophoresis is a less-expensive technique for study of OMP pattern. The OMPs are also recognized as a useful tool for epidemiological studies of Hib. In this study, Indian clinical isolates of Hib from blood and CSF were used to determine the OMPs using gel electrophoresis techniques.

Methods:

A total of eight Indian isolates of Haemophilus influenzae type b (Hib) were included in this study; five from blood and three from cerebrospinal fluid (CSF). Culture & microscopic identification of the bacteria was done after growing Hib on chocolate agar medium with Haemophilus Vitamino Growth Supplement & Haemoglobin Powder (Hi-media, India) Growth Supplement, Modified. All isolates of Hib were provided by the Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh. Growth of Hib was harvested in phosphate buffer saline and pellet was taken by centrifugation. Whole cell antigen of Hib was used for lysis to expose their antigens by ultrasonication method.

Preparation, purification and identification of Hib-OMPs for Sodium Dodecyl Sulphate-Polyacrylamyde Gel Electrophoresis (SDS-PAGE). Preparation of OMPs of Hib was done by the method of Carlone et al. (1986) and Kaur et al. (2003) [9, 8]. After ultrasonication, protein profile of OMPs was performed on 12% SDS-PAGE. The sonicated antigen of Hib was mixed with an equal volume of sample buffer (2% sodium dodecyl sulfate, 5% 2-mercaptoethanol, 0.001% bromophenol blue and 10% glycerol in 0.06M Tris-hydrochloride, pH 6.8) at 1000C for 2 minutes and standard protein marker (Banglore Genei, India) ranging between 29 and 205 KDa was used for SDS-PAGE.

The electrophoresis apparatus was assembled according to the manufacturer’s instruction (Minigel system, Bio-Rad Laboratories, California). The apparatus was used to load Hib samples in various concentrations i.e. 2 to 12mg per well and protein molecular weight marker was loaded in separate well. SDS-PAGE was performed at a constant current of 20mA. Coomassie brilliant blue-R-250 (0.25g) dye was used to stain the gel on a slowly rotating platform for 4 hours at room temperature. Further, gel was destained by soaking it in methanol:water:acetic acid solution (30:10:60 V/V/V) without dye on a slowly rocking platform for 4 to 8 hours. After destaining, the gel was examined for resolution of Hib-OMPs in the gel and photographs were taken by gel doc system. Molecular weight of Hib proteins was calculated by running distance of Hib-OMPs as corresponding to the molecular weight of protein marker.

Elution and staining of Hib-OMPs by electro elution. Protein bands were visualized by staining the gel with Coomassie brilliant blue-R-250 dye as described earlier. Protein band of interest was cut, rinsed with distilled water, and protein was electrophoretically eluted into elution buffer (25mM Tris [pH 8.4] containing 192 mM glycine and 0.1% SDS) by using an Electroeluter (Bio-Rad Laboratories, California). Protein was eluted from repeated gel for specific OMP and fractions of the OMP were pooled. Each OMP was further identified by SDS-PAGE, as described above.

Results:

In the study, a total eight Hib isolates, including five from blood and three from CSF were online casinos studied for their cultural and microscopic characteristics in our laboratories (Figure 1).

Figure 1. Growth of Haemophilus influenzae type b on chocolate agar medium with Haemophilus Vitamino Growth Supplement & Haemoglobin Powder after 48 hours.

Ultrasonication antigens of Hib was prepared and protein content of each sample was determined by Bradford method. The protein concentration of Hib antigens was adjusted to 100µg per ml. The purified antigen was resuspended in casino online phosphate buffer saline (PBS, pH 7.2). OMP profile of ultrasonicated Hib antigens was done on 12% SDS-PAGE. A total of 2 to 12µg quantities of Hib OMP were used in individual well of the gel. After electrophoresis and staining with commassie brilliant blue R-250, the best resolution for OMPs was observed at 8μg protein content per well. The OMP profile for each isolate when examined in gel showed 11–21 OMPs with molecular masses of approximately 29 to 97.4 KDa. Hib-OMP profile of one sample of CSF and three samples of blood is shown in figure 2.

Figure 2. OMP analysis of Hib isolates on 12% SDS-PAGE after staining with Coomassie brilliant blue-R-250 dye.

Lane 1 indicates representative OMP profile of Hib from CSF.

Lanes 2 to 4 indicates representative OMP profile of Hib from blood.

Lane 6 indicates molecular weight marker range 205 to 29 KDa

Only five major OMPs were eluted through repeated gels and the fractions of specific OMP were pooled. Further identification of each OMP was done with standard molecular weight marker of protein on 12% SDS-PAGE i.e. 47 kDa, 39kDa, 42 kDa, 37 kDa, 28 kDa and 60 kDa. Out of six OMPs, four OMPs (37, 39, 42, & 47 kDa) were found in all isolates. However, 60 kDa OMP was observed in isolates that were obtained from the CSF (Figure 3).

Figure 3. Identification of OMP of 60 kDa of Hib isolate from CSF on 12% SDS-PAGE is seen in lane 3. Lane 1 shows molecular weight marker with the range 205 to 29 kDa.

Discussion:

The incidence of diseases caused by H. influenzae and their severity, sequelae and complication rates in India are reported by John et al. (1998) [10]. Of six serotypes of H. influenzae (a to f), serotype b is most prevalent in India [5,8]. On the evidence of many studies, it is well understood that the Hib- outer membrane proteins (OMPs) play an important role in the pathogenesis of this infection. The OMPs have been a major focus of research aimed at identifying conserved and antigenic surface components for inclusion in vaccines and for their ability to separate non-typable strains into subtypes corresponding to a particular epidemiological area. The outer membrane of H. influenzae contains about 36 proteins, of which between six and eight make up the major protein content, and the remainder exist as relatively minor proteins [11]. The major OMPs include heat-modifiable 47 kDa P1 [12], 39 kDa P2 porin [13], 28 kDa P4 lipoprotein [14], 37 kDa P5 [11] and the high-molecular-mass proteins HMW1 and HMW2 [15]. The minor OMPs include OMP26 (26 kDa) [16] and 42 kDa protein D [17] and several high-molecular-mass proteins ranging from 120 to 125 kDa.

Of all Hib-OMPs, some are consistently present in all strains of H. influenzae, while others vary among Hib strains. During the study, we confirmed the presence of crucial OMPs in the isolates; major like 47 kDa, 39kDa, 37 kDa & 28 kDa and minor like 42 kDa. The OMP profile of the isolates was found to be similar to previously reported profile except for the presence of 60 kDa in CSF isolates. Authors feel that the unusual 60 kDa OMP in CSF isolates can be used as a tool for epidemiological studies in India.

Acknowledgement:

The corresponding author has made principal contributions in study concept and design. Out of corresponding author, other two authors have substantially contributed to perform experiments and compilation of study data.

References:

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