Chapter 4shodhganga.inflibnet.ac.in/bitstream/10603/8777/11/11... · 2015-12-04 · Results and...

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Results and Discussion

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4. Results and Discussion The present work was undertaken to identify novel immunogens of A. fumigatus which may find application in specific early diagnosis and immunotherapy of Aspergillus induced infections which are generally untreatable. For this purpose we used immunoproteomic approach to identify immunogenic molecules from secreted and cytosolic fractions of two geographically distinct strains of A. fumigatus. The results obtained from various experiments are described in subsequent sub-sections. 4.1. Culture of pathogenic strains of A. fumigatus Two geographically distinct strains of A. fumigatus (ITCC 6604 and DAYA) were used in the present study. Both strains of A. fumigatus were cultured on Sabouraud dextrose agar plates to produce large inocula of conidia. The growth of A. fumigatus started appearing after overnight incubation of the culture plates at 37 ºC and pathogen covered entire surface of agar medium in the plate by 5th day. Subsequently there developed greenish black mat in the plate which is the characteristic feature of A. fumigatus (Fig 4.1A). A B

Fig 4.1. Growth of A. fumigatus after 5 days of inoculation on Sabouraud dextrose agar plate (A) and in L-asparagine medium (B).


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The mycelial mat of A. fumigatus developed after 5 day culture in a flask containing L-asparagine broth, is shown in Fig 4.1B. The secreted fraction of proteins of A. fumigatus was obtained from 3rd week stationary culture where as that of germinating conidia (i.e. CHC) was prepared from 16 h culture of A. fumigatus.

Fig 4.2A shows the tiny conidia of A. fumigatus harvested from agar plates for inoculating the culture flasks. The microscopic examination of germinating conidia from 16 h culture showed that the size of the germ tube at this stage was predominantly of 5.0-10.0 µm (Fig 4.2B). The germinating conidia were used to prepare CHC fraction of A. fumigatus. A B

Fig 4.2. Microscopic picture of A. fumigatus conidia. Normal conidia harvested from agar plate (A), and germinated conidia at 16 h in L-asparagine medium (B).

The yield of protein in secreted and CHC fractions of A. fumigatus strains was determined. A volume of 500 ml of 3 week culture filtrate produced 20-25 mg of secreted protein. The protein yield in CHC fraction was 3.5-4.0 mg/gm of cell mass.


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4.2. Clinical observations on the subjects 4.2.1. Asthma In the recruited subjects asthma was diagnosed on the basis of clinical features and pulmonary function test evidence of obstructive airway disease with reversibility demonstrated by >12.0% or 200.0 ml increase in FEV1, 15 min after the use of short acting β2 agonist (salbutamol). Asthma has been considered as the first indication towards the presence of ABPA. Once the diagnosis of asthma was established the patients were subjected to SPT with antigen of A. fumigatus. 4.2.2. Skin prick test Aspergillus hypersensitivity skin test using A. fumigatus antigen, positive (Glycerinated histamine acid phosphate) and negative (Glycerinated buffer saline) control was performed on all subjects. Asthmatics, who were sensitized to A. fumigatus developed erythema and wheal with test antigen of A. fumigatus. The size of wheal in ABPA patients was almost similar to that produced by positive control antigen after 20 min of inoculation (Fig 4.3).

Fig 4.3. A positive skin prick test in one of the representative patient where

reaction of A. fumigatus antigen (As) was similar to that of positive


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control (H) with wheal and erythema. No reaction was seen in case of glycerinated buffer saline (BS).

No hypersensitivity reaction was developed at site of inoculation with glycerinated buffer saline in patients of ABPA. All 3 preparations (negative, positive and test antigen) were used in the same arm. Healthy subjects did not develop erythema and wheal in SPT. The patients of ABPA developed bigger size of wheal than that in asthmatics. All the patients of asthma did not show positive SPT. The prevalence of skin reactivity to Aspergillus was shown to be 28.0% in patients with asthma (Virnig and Bush, 2007) and 29.0% in patients with CF (Becker et al, 1996). A positive skin test alone was considered not to be enough to diagnose ABPA. Therefore, further serological and radiographic evaluations were carried out to establish ABPA. 4.2.3. Radiological observations The patients of bronchial asthma, who showed positive skin prick test against A. fumigatus antigen, were referred for radiological examination, where presence of infiltration and central bronchiectasis in the lungs were seen on X-ray and CT scan. The radiographic examinations demonstrated parenchymal abnormalities which included lobar or segmental consolidation, cavitary mass aggregation, and small nodular opacity (Fig 4.4). A B

Fig 4.4. Chest X-ray of an ABPA patient showing infiltrative opacities in bilateral upper lobes (A) and chest CT scan of an ABPA patient showing bronchiectatic changes in lower lobe of right lung (B).


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There are case reports which have described the CT findings of semi-invasive pulmonary aspergillosis including chronic progressive peripheral consolidation or mass like lesion, upper lobe predilection, with or without thickening, and distortion of adjacent pleura (Aquino et al, 1994, Thompson et al, 1995, Broderick et al, 1996, Logan and Muller, 1996). The CT findings varied from case to case but appearance of the areas having lobar or segmental consolidation and a low-attenuation mass with central bronchiectasis were quite often. The accuracy of high-resolution CT scan in the diagnosis of ABPA in asthma patients was evaluated in 2 groups of subjects consisted of 44 asthma patients with ABPA and 38 asthmatics without ABPA. The CT scans were analyzed for bronchial wall thickening, bronchiectasis, centrilobular nodules, mucoid impaction, mosaic perfusion, atelectasis, and consolidation. The lung abnormalities were seen more commonly in patients with ABPA than in patients with asthma alone. Bronchiectasis, centrilobular nodules and mucoid impaction were present in the 95.0%, 93.0% and 67.0% respectively in the patients of ABPA. In the asthmatic control group, bronchiectasis was detected in 29.0% patients whereas centrilobular nodules and mucoid impaction were observed in the 28.0% and 4.0% asthmatics respectively (Ward et al, 1999). It was demonstrated that high-resolution CT scanning has been suggestive of ABPA in asthmatics but still requires estimation of immunoglobulins (IgG and IgE) in the serum of subjects for establishing the confirmed diagnosis of ABPA. 4.2.4. Serum immunoglobulins Blood samples of all subjects were analysed for the levels of total IgE, and specific IgE and IgG against A. fumigatus secreted and CHC antigens. All positive patients showed elevated levels of total serum IgE (< 400 IU/ml) whereas healthy controls had comparatively low level of total IgE. The levels of total serum IgE has been shown to remain high in ABPA patients, which may decrease during remission due to corticosteroid therapy. This decrease usually occurs within 2 months after initiation of corticosteroid treatment. Total serum IgE levels sometimes return to


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normal range after completion of the treatment course. In the present study high level of total IgE was observed. It was due to the fact that all those subjects who received corticosteroids in previous 6 weeks were excluded from the study. The ELISA ODs for specific IgG and IgE determined by using secreted and CHC antigens of A. fumigatus were significantly high (p < 0.05) than in controls (Table 4.1). The patients were considered to be positive for ABPA if their sera showed ELISA OD for Aspergillus specific IgG and IgE atleast 3 times more than that in negative controls (Sharma and Sarma, 1997). The sera obtained from confirmed cases of ABPA were used as the source of Aspergillus specific antibodies for development of immunoproteomes of A. fumigatus. Pooled sera from healthy subjects were used as negative control.

Table 4.1. Details of ABPA patients and control subjects included in the study.

Details Healthy

controls ABPA patients

Number of subjects 12 13 Age, year (range) 25-65 28-60 Gender (M/F) 8/4 5/8 Total IgE (IU/ml) M±SE 328.8±48 1035.5±170.8 Specific IgE against A. fumigatus secreted antigen (OD at 410 nm) M±SE 0.033±0.006 0.420±0.073

Specific IgE against A. fumigatus CHC antigen (OD at 410 nm) M±SE 0.075±0.012 0.308±0.051

Specific IgG against A. fumigatus secreted antigen (OD at 492 nm) M±SE 0.042±0.01 0.973±0.115

Specific IgG against A. fumigatus CHC antigen (OD at 492 nm) M±SE 0.015±0.003 0.589±0.0291


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The secreted and CHC crude antigens of A. fumigatus were used to detect specific IgE and IgG in the sera of subjects to establish the diagnosis of ABPA. In the absence of ideal preparation, the crude antigenic fractions of A. fumigatus have been commonly used for diagnosis of ABPA and IA. The detection of Aspergillus specific IgG antibodies using crude secreted antigens also has been shown to be useful in diagnosis of ABPA in patients of CF (Barton et al, 2008) and asthma (Sharma and Sarma, 1993). The ELISA kits for detection of Aspergillus antigens like, mannan and galactomannan have been considered to be good in specificity with variable sensitivity for IA (Barnes, 2008). Approximately 40 proteins of A. fumigatus capable of binding with the IgE antibodies have been identified which include 21 recombinant allergens also (Abad et al, 2010). Kurup et al (2006) assessed the ability of recombinant Aspergillus allergens (rAsp f1, rAsp f2, rAsp f3 rAsp f4 and rAsp f6) to discriminate the patients of ABPA and cystic fibrosis by their IgE, IgG and IgA reactivity. Hemmann et al (1997) showed that SPT with rAsp f4 and rAsp f6 provoked immediate skin reactions in patients with ABPA but not in controls and, therefore, allowed discrimination between ABPA and sensitization to A. fumigatus. Banerjee et al (2001) showed that 70.0% of patients with ABPA had high levels of serum IgE antibodies to Asp f16, a 43 kDa protein, whereas subjects with positive SPT with Aspergillus crude antigens did not show any reactivity with Asp f16. Although several allergens of A. fumigatus produced recombinantly, they are mostly available for research purposes only and their clinical use is very limited.

4.3. Antigen profile of A. fumigatus on SDS gel The antigens from different strains of A. fumigatus were separated on 12.5% SDS gels. Analysis of gels demonstrated a complex protein profile of secreted and CHC fractions of A. fumigatus (Fig 4.5). Both strains had large number of major and minor proteins in the molecular weight range from 150 to 10 kDa. There were visible differences in the secreted protein profile of Indian and German strain. The ITCC 6604 strain of A. fumigatus had different protein profile than that of DAYA. In DAYA, the


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proteins of molecular weight 92, 86, 58, 27 and 13 kDa were not present. The protein bands of 90, 88 and 33 kDa could not be seen in the lane where the antigen of ITCC 6604 was loaded (Fig 4.5A). A protein of 18 kDa was expressed in higher concentration in DAYA as compared to ITCC 6604. The CHC protein profiles of both A. fumigatus strains (ITCC 6604 and DAYA) in SDS gels appeared to be very similar to each other. However, their profiles were different from respective secreted antigen. Although many of the proteins that appeared in secreted antigen, were also present in CHC antigenic fraction of A. fumigatus, the difference in the protein profile of both the antigenic preparations were evident (Fig 4.5). There is paucity of information on comparison of secreted and

A. Secreted fraction B. CHC fraction

Fig 4.5. Antigen profile of Aspergillus strains on 12.5% SDS gels stained with commassie brilliant blue. Lane 1 : ITCC 6604 Indian strain Lane 2 : DAYA German strain Lane M : Standard protein markers


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cytosolic antigens of A. fumigatus. The analysis of complex and variable protein contents of the secreted and cytosolic fractions, and their immunological characterization may hold a potential to discover new antigenic/allergenic molecules of A. fumigatus. 4.4. Immunoblotting To study the immunoreactivity pattern, the proteins of secreted and CHC fractions of both A. fumigatus strains were separated on SDS gels and probed with anti-human IgG and anti-human IgE conjugated with HRP. Immunoblots of secretory antigens showed number of proteins which reacted with IgG as well as IgE antibodies. In strain ITCC 6604, the IgG reactivity was more prominent in high molecular weight range as compared to DAYA, however, proteins of 28 and 18 kDa were found to be commonly reactive in DAYA strain in both IgG and IgE blots (Fig 4.6A and 4.6B). The results of immunoblot experiments with CHC fraction appeared to be very interesting. Most of the CHC proteins of both strains (ITCC 6604 and DAYA) of A. fumigatus were found to be IgE reactive and very less number of proteins reacted with IgG antibodies (Fig 4.6C and 4.6D). There were fewer IgG reactive proteins in DAYA. The ITCC 6604 showed a strong signal of 60 kDa protein along with some more faint bands of IgG reactive proteins (Fig 4.6C). On the other hand the IgE blots of CHC fraction showed presence of highly reactive proteins in molecular weight range of 130-10 kDa of DAYA as well as ITCC 6604 (Fig 4.6D). This pattern of reactivity showed that CHC fraction contained predominantly IgE reactive proteins. Results demonstrated that the IgG and IgE immunoreactive proteins of A. fumigatus had very complex profile and it was difficult to separate all antigens on single dimension SDS gels. Therefore, development of 2DE proteome and immunoproteome, was essentially required for further characterization of the immunogenic proteins of A. fumigatus.


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Fig 4.6. Immnuoblots of secreted (A and B) and CHC fractions (C and D) A. fumigatus strains, developed with pooled sera of ABPA patients and probed with anti human IgG and IgE antibodies conjugated with HRP. ITCC 6604 (A and C) and DAYA (B and D).

Lane 1 : ITCC 6604 Indian strain Lane 2 : DAYA German strain Lane M : Standard protein markers


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4.5. Proteome of A. fumigatus The 2DE was performed for both secreted and CHC fractions of A. fumigatus strains (ITCC 6604 and DAYA) to resolve their complex proteomes and also for development of their respective immunoproteomes using sera of ABPA patients to identify new immunogenic molecules. 4.5.1. Secreted proteome of A. fumigatus The secreted fractions of both (ITCC 6604 and DAYA) strains of A. fumigatus were used for development of 2DE proteomes. An amount of 40.0 µg of secreted proteins of each fraction was resolved on pH 3-10 and pH 4-7 linear pH gradient strips followed by silver staining. The 2DE gels showed that A. fumigatus proteome was very complex and large number of proteins were distributed in the molecular weight range from > 150 to around 10 KDa having pI ranging from 4 to 8.5 (Fig 4.7). The separation of proteins in pH 3-10 showed that a large number of spots clustered in pI range from 4.5 to 6.5. The separation of clustered spots on pH 4-7 IPG strips improved the resolution of proteins significantly (Fig 4.7A and 4.7C). Therefore, in order to obtain better resolution of these spots, pH 4-7 IPG strips were employed (Fig 4.7B and 4.7D). The spot patterns from 2DE gels of two geographically distinct Aspergillus strains were significantly different from each other, even after growing them under similar culture conditions (Fig 4.7). Pattern of protein profile of the secretome of Aspergillus has been strongly dependent on the culture conditions as well as nutrient source. Difficulties have been associated with deglycosylation of secreted proteins which has been a common challenge, as polysaccharide side chains cause streaking in the gels and also interfere with mass spectrometric identification of proteins (Kim et al, 2008). Therefore, separation of secreted proteins in high resolution has not been so easy. Probably for these reasons we also observed horizontal streaking in parts of the 2DE gels of secreted fraction in both pI ranges (pH 3-10 and pH 4-7).


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Fig 4.7. The 2DE proteome of secreted proteins of A. fumigatus strains ITCC 6604 (A and B) and DAYA (C and D) developed using 12.5% acrylamide gels in pH ranges 3-10 (A and C) and 4-7 (B and D). Forty microgram of protein was loaded onto linear pH gradient strips and separated in first dimension on the basis of their isoelectric points followed by separation in second dimension according to their molecular weight. After silver staining, protein spots were marked based on their reactivity with immune sera in western blots. The marked spots were excised, in-gel digested and subjected to liquid chromatography coupled with tandem mass spectrometry.


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4.5.2. Cytosolic proteome of A. fumigatus

For the development of 2DE proteomes of cytosolic fraction, an amount of 40.0 µg CHC proteins, of both the strains of A. fumigatus (ITCC 6604 and DAYA) were resolved on pH 3-10 and pH 4-7 linear immobilized pH gradient strips. The silver stained 2DE gels demonstrated well separated protein profile of ITCC 6604 and DAYA. It was found that large number of proteins remained concentrated in the acidic to neutral pH range (Fig. 4.8). In order to get better separation of those clustered low pI proteins, we resolved CHC fraction on narrow pH range strips (pH 4-7). This improved the separation of spots significantly, however, 2DE gels showed several isomeric proteins having pIs from 5.0 to 6.8 (Fig. 4.8). There were apparent differences in the proteomes of ITCC 6604 and DAYA. A few proteins with pIs in the range of 4.8-5.3 appeared in DAYA exclusively. Although the spot pattern of CHC proteins of two geographically diverse strains was found to be different, there were several overlapping proteins in Indian and German strain (Fig. 4.8). First report on Aspergillus proteome which was focused on A. fumigatus surface proteins, was published by Bruneau et al (2001) for identification of glycosylphosphatidylinositol-anchored membrane proteins. Thereafter, Melin et al (2002) reported the effect of the antibiotic concanamycin on Aspergillus intracellular proteome and showed the up- and down-regulation of several A. nidulans intracellular proteins. The proteome of A. nidulans during growth in the presence of increased salt concentration, or osmoadaptation was published by Kim et al (2007). The most comprehensive understanding of the Aspergillus intracellular proteome has been provided by Kniemeyer et al (2006) and Carberry et al (2006). Both these studies showed the presence of 40.0% similar proteins. Kniemeyer et al (2006) also provided an optimized 2DE sample preparation protocol which was followed by several researchers. Vodisch et al (2009) generated proteome map of A. fumigatus from cytosolic and mitochondrial fractions. Teutschbein et al (2010) identified proteins from dormant conidia to provide complete profiling of A. fumigatus conidial fraction. Above mentioned reports demonstrated that the work on the proteome of various cellular fractions of Aspergillus


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has been undertaken but the comparison of the proteomes of secreted and cytosolic fractions of geographically different strains (derived from India and Germany) are not carried out yet. Information on this aspect is provided in the present study and importantly, development and characterization of their 2DE immunoproteomes may identify new immunogens of A. fumigatus.

Fig 4.8. The 2DE proteome of CHC proteins of A. fumigatus strains ITCC 6604

(A and B) and DAYA (C and D) developed using 12.5% acrylamide gels in pH ranges 3-10 (A and C) and 4-7 (B and D). An amount of 40 µg protein was loaded on linear pH gradient strip and separated horizontally on the basis of their isoelectric point followed by vertical separation on 12.5% SDS polyacrylamide gels. Silver stained gels were compared with immnuoblots for marking the spot numbers.


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4.6. Immunoproteome of A. fumigatus 4.6.1. Secreted immunoproteome of A. fumigatus Pooled sera from patients of ABPA and negative sera of healthy controls were

used separately for immunoblotting experiments. The IgG and IgE immunoblots for

secreted proteomes of both (ITCC 6604 and DAYA) strains of A. fumigatus were

developed using IPG strips of pH 3-10 as well as pH 4-7. Most of IgG reactive proteins

of ITCC 6604 were found to be concentrated in the high molecular weight (>50 kDa)

area with pIs ranging from 4.5 to 6.0 (Fig 4.9A and B). The IgE reactive proteins of ITCC 6604 were discretely located in the blots (Fig 4.10). There were some differences between the reactive spots of DAYA and ITCC 6604, but a noticeable numbers of reactive spots appeared to be common in both strains. No reactive spots were visible on the control IgG and IgE immunoblots of the secreted fractions of A. fumigatus strains (ITCC 6604 and DAYA) which were probed by pooled sera of healthy control subjects. Very few immunoproteomics based studies have been carried out on A. fumigatus. Gautam et al (2007) identified a panel of secreted allergens of A. fumigatus by IgE reactivity with sera of ABPA patients. Kumar et al (2011) also identified few more IgG reactive antigenic molecules from secreted fractions of A. fumigatus by immunoreactivity with sera derived from ABPA patients and Aspergillus sensitized rabbit and mice. Reports available till date provided limited information on the antigens of A. fumigatus. The present work may identify novel immunogens of A. fumigatus for their possible application in the better management of Aspergillus induced infections.


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Fig 4.9. The 2DE immnuoblots of secreted proteomes of A. fumigatus strains developed with pooled sera of ABPA patients and probed with anti-human IgG antibodies conjugated with HRP. ITCC 6604(A and B) and DAYA (C and D), in pH ranges 3-10 (A and C) and pH 4-7 (B and D). Matched immuno-reactive spots identified in 2DE gels were further excised for mass spectrometric analysis.


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Fig 4.10. The 2DE immnuoblots of secreted proteomes of A.

fumigatus strains developed with pooled sera of ABPA patients and probed with anti-human IgE antibodies conjugated with HRP. ITCC 6604 (A and B) and DAYA (C and D), in pH ranges 3-10 (A and C) and pH 4-7 (B and D). Matched immuno-reactive spots identified in 2DE gels were further excised for mass spectrometric analysis.


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4.6.2. Cytosolic immmunoproteome of A. fumigatus The cytosolic proteins of the A. fumigatus were hardly explored for their immunogenic properties. Therefore, present study was aimed at the identification of novel cytosolic immunogens of A. fumigatus. Two dimensional IgE and IgG specific immunoblots of the CHC proteome were developed with pooled sera of ABPA patients. Large number of unevenly distributed IgE reactive spots appeared on the blots (Fig 4.11A to 4.11D). The CHC fraction of A. fumigatus had fewer number of IgG specific antigens and strong reactivity appeared in the area of low pI and high molecular weight (Fig 4.12A to 4.12D). We observed a significant number of common IgE reactive spots with variable intensity of signals on the immunoblots of ITCC 6604 and DAYA (Fig 4.11B and 4.11D). The control IgE and IgG blots of CHC fraction of A. fumigatus developed with sera of apparently healthy control subject, did not show signal of any immunoreactive protein. Most of the A. fumigatus allergens were identified from its secreted fraction, however, the report of Asif et al (2010) showed that its cytosolic fraction also contained IgG, IgA and IgM reactive antigenic molecules when examined using sera of rabbit which had developed immunity after experimentally induced aspergillosis. The experiments using cytosolic fraction of A. fumigatus for the identification of immunologically active molecules using serum of ABPA patients was not reported until the present study was undertaken. A panel of IgE reactive molecules from cytosolic fraction of A. fumigatus has been identified for further characterization of immunoreactive molecules.


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Fig 4.11. The 2DE immnuoblots of CHC proteomes of A. fumigatus strains ITCC 6604 (A and B) and DAYA (C and D), in pH ranges 3-10 (A and C) and pH 4-7 (B and D) showing proteins which reacted with IgE antibodies derived from sera of ABPA patients and probed with anti-human IgE antibodies conjugated with HRP. IgE blots were compared with 2DE gels and spots were excised from the gels on the basis of their appearance in the immnuoblots. Excised protein spots were in-gel digested and subjected to liquid chromatography coupled with tandem mass spectrometry for identification of proteins.


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Fig 4.12. The 2DE immnuoblots of CHC proteome of A. fumigatus strains

ITCC 6604 (A and B) and DAYA (C and D), in pH ranges 3-10 (A and C) and pH 4-7 (B and D) showing proteins which reacted with IgG antibodies derived from sera of ABPA patients and probed with anti-human IgG antibodies conjugated with HRP. IgG blots were compared with 2DE gels and matched spots were excised from the gels for further analyses by liquid chromatography coupled with tandem mass spectrometry for identification of proteins.


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4.7. Marking of the immunoreactive protein spots For the characterization of the immunoreactive molecules the blots were matched with their respective silver stained gels and protein spots were marked. Eighty six spots from 2DE silver stained gels of secreted fractions, corresponding to the signals of immnuoblots were excised, in-gel digested and subjected to mass spectrometry based characterization (Fig 4.7). These spots included 44 from ITCC 6604 (12 from 2DE gel of pH 3-10 and 32 from 2DE gel of pH 4-7) and 42 spots (13 from 2DE gel of pH 3-10 and 29 from 2DE gel of pH 4-7) from DAYA. Immnuoblots of CHC fraction were matched with their corresponding silver stained 2DE gels and 111 protein spots were marked and excised (Fig 4.8). These spots included 64 from ITCC 6604 (7 from 2DE gel of pH 3-10 and 57 from 2DE gel of pH 4-7) and 47 spots from DAYA (2 from 2DE gel of pH 3-10 and 45 from 2DE gel of pH 4-7). All spots were subjected to tryptic digestion, peptide extraction and Q-TOF MS/MS analysis of extracted peptides. 4.8. Mass spectrometry based characterization 4.8.1. Identification of immunoreactive secreted proteins Proteins underlying marked spots were identified by liquid chromatography separation coupled with tandem mass spectrometry (Q-TOF MS/MS). SwissProt and NCBInr protein database search identified 35 different A. fumigatus proteins (Table 4.2) from 86 excised spots. Twenty four proteins showed immuno-reactivity both with IgG and IgE, whereas 4 (Spot Nos. 11, 24, 25 and 28) reacted only with IgG (Fig 4.9) and 7 proteins (Spot Nos. 4, 14, 19, 30-32 and 35) were exclusively IgE reactive (Fig 4.10). The immunoreactivity of antigenic proteins with IgG and IgE has been considered to be dependent on the presence of specific antigenic epitopes. Therefore, we analysed amino acid sequences of all 35 proteins by an in silico analysis tool DNAstar. This analysis demonstrated their antigenic indices and position of antigenic epitopes within the amino acid sequence.


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Table 4.2. List of identified secreted immunoreactive proteins of A. fumigatus.

Spot No.

Accession No.

Identification in A. fumigatus

MASCOT score

Molecular mass/pI

Signal peptide

Cellular localization

Biological function

1 A46497 Asp f1-

ribonuceoprotein 164 19.6/9.23 Yes Non-Cytoplasmic

Purine-specific ribonuclease

2 P79017 Asp f2-hypothetical protein

175 32.8/5.34 Yes Non-Cytoplasmic

Not known

3 Q4WV60 Asp f4- hypothetical protein

153 32.5/6.64 No Extracellular Not known

4 CAA11266 Asp f 9-probable glycosidase, crf1

193 32.3/4.63 Yes Extracellular Glycosidase Hydrolase

5 O60022 Asp f 13/15-hypothetical protein

80 15.9/4.61 Yes Extracellular Not known

6 Q4WEM3 Hypothetical protein

41 35.5/6.00 No Unknown Not known

7 Q4WTF6 Aminotransferase-class V, putative

196 41.8/6.46 No Cytoplasmic Amino transferase

8 O13479 Dipeptidyl-peptidase-V precursor, DppV

194 79.7/5.59 Yes Unknown Hydrolase Protease

9 AAD26111 Chitosanase 184 21.5/5.76 No Cytolpasmic Chitinolysis

10 XP_752511 1,3-β-glucanosyltransferase, Bgt1

343 33.0/5.02 Yes Extracellular Glycosidase Hydrolase Transferase

11 Q4WXR8 Nuclear transport factor 2, NTF-2

117 14.2/4.63 No Unknown Transcription factor

12 Q4WW81 Fucose specific lectin, FleA

117 34.3/7.79 No Extracellular Sugar binding

13 XP_750162 FG-GAP repeat protein

291 33.7/5.33 Yes Unknown Ligand binding

14 P41748 Aspf10 aspergillopepsin-F

200 41.5/4.85 Yes Extracellular Aspartyl protease

15 BOXT72 1,3- β-glucanosyltransferase,gel1

311 48.1/4.94 Yes Extracellular Transferase activity

16 Q9P8U4 1,3- β-glucanosyltransferase, gel2

179 51.6/4.62 Yes Unknown Transferase activity

17 XP_001481609

Class V chitinase 525 46.4/5.21 Yes Cytolpasmic Glycosidase Hydrolase


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18 Q92405 Catalase-B, Cat-B

640 79.8/5.50 Yes Cytoplasmic Heme binding

19 XP_750863 Thioredoxin reductase, GliT

103 36.0/5.44 No Non-Cytoplasmic

Reductase

20 Q6U819 Lysophospholipase-3, Plb3

76 67.3/5.39 Yes Non-Cytoplasmic

Lipolysis

21 Q6U820 Lysophospholipase-1, Plb1

73 68.1/4.59 Yes Extracellular Lipolysis

22 XP_752825 Mannosidase, MsdS

475 53.8/5.09 Yes Unknown Glycosidase Hydrolase

23 AAP23218 Chitinase, Chi-B 123 47.6/5.10 No Extracellular Glycosidase

24 CAA12162 IgE binding protein/Asp f3/pmp20

156 19.4/4.43 No Extracellular Cell-redox homeostasis

25 Q00050 Asp-hemolysin, Asp-HS

489 15.1/5.29 No Cytoplasmic Cytolysis Hemolysis

26 XP_748349 GPI-anchored cell wall beta-1,3-endoglucanase EglC

146 44.6/4.90 Yes Extracellular Glycosidase Hydrolase

27 XP_748380 Hypothetical protein AFUA_3G00600

452 68.0/6.32 No Cytoplasmic Not known

28 EDP51083 Conserved hypothetical protein

122 30.4/4.82 No Cytoplasmic Not known

29 XP_749213 Pectate lyase A 421 33.8/6.23 Yes Extracellular Lyase

30 XP_747586 NAD-dependent formate dehydrogenase, AciA/Fdh

213 45.7/8.43 No Cytoplasmic Oxido- reductase activity

31 XP_748936 Malate dehydrogenase, NAD-dependent

269 35.8/9.08 No Cytoplasmic Oxido- reductase

32 XP_747039 Bifunctional catalase-peroxidase, Cat2

361 83.7/6.12 No Cytoplasmic Catalase Peroxidase

33 XP_750327 Beta-glucosidase 249 95.0/5.01 Yes Cytoplasmic Glycosidase Hydrolase

34 XP_747715 FAD/FMN-containing isoamyl alcohol oxidase, MreA

135 61.3/5.55 Yes Cytoplasmic FAD binding Oxido- reductase activity

35 EDP54506 Glucose-6-phosphate isomerase

215 61.3/5.84 No Cytoplasmic Isomerase


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The work on identification of immunogens from crude fractions of the

A. fumigatus for diagnostic and therapeutic purposes has remained the priority of

researchers in past decades (Kurup, 2005, de Oliveira et al, 2007, Delhaes et al, 2010).

Twenty one allergens (Asp f1-f13, Asp f15-f18, Asp f22, Asp f23, Asp f27-f29, and Asp

f34) and number of other antigenic/pathogenic proteins of A. fumigatus have been

identified over the time using different identification/isolation strategies (Abad et al,

2010). We have explored the most extensively used secreted fraction of A. fumigatus for

identification of immunogenic molecule by IgG and IgE reactivity using sera of ABPA

patients. Efforts resulted in identification of 35 immunoreactive molecules from secreted

fraction of A. fumigatus. Of these molecules, 7 proteins were already known as allergens

(Asp f1, Asp f2, Asp f3, Asp f4, Asp f9, Asp f10 and Asp f13/15) of A. fumigatus. The

Asp f3, a peroxiredoxin family-2, protein only appeared to be IgG reactive in our study.

This protein is associated with type 1 hypersensitivity and the recombinant Asp f3 is one

of the two characterized proteins (Asp f3 and Asp f16) that so far have been successfully

used to induce a protective immune response in mice (Bozza et al, 2002, Ito et al, 2006).

The Asp f3 was also detected as a possible vaccine candidate in immunocompromised

rabbits which acquired protective immunity (Asif et al, 2010) and also identified as

immunogen in an A. fumigatus cDNA expression library screening system (Denikus et al,

2005). Three other proteins [1, 3-β -glucanosyltransferase-gel1 (Spot No. 15), NAD-

dependent formate dehydrogenase AciA/Fdh (Spot No. 30) and malate dehydrogenase

NAD-dependent (Spot No. 31)] as well as Asp f3 have also been identified by Asif et al

(2010). These molecules with thorough characterization may serve as possible vaccine

candidates in future. Asp f9 and Asp f10 only reacted with the IgE antibodies derived

from sera of ABPA patients. Remaining four allergens (Asp f1, Asp f2, Asp f4 and

Asp f13/15) were reactive with both IgG and IgE antibodies. The detection of a limited

number of already established secreted allergenic proteins in the present study could be

due to the limitation associated with the gel-based strategy. In fact, it is not possible to

identify all of the immunogenic proteins using a single separation technique, therefore,

we do not claim to have discovered all the immunoreactive proteins from secreted


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fraction of A. fumigatus. Furthermore, the source of antigens and the sensitivity of the

patients to different A. fumigatus antigens expressed during various stages of infection

may cause variability in immunoreactivity pattern with ITCC 6604 and DAYA strains of

A. fumigatus.

Three of the characterized proteins, (dipeptidyl-peptidase-5 precursor DppV,

nuclear transport factor 2 NTF-2 and malate dehydrogenase NAD-dependent) were

identified as predicted allergens of A. fumigatus (Fedorova et al, 2008). DppV was

reactive to both IgG and IgE and was a glycoprotein (hyphal invasion enzyme)

responsible for the protein degradation of the host cell during invasion (Beauvais et al,

1997, Rementeria et al, 2005). The NTF-2 was only recognized by IgG in the present

study which was reported earlier to be a cross-reactive allergen produced by Alternaria

alternata and C. herbarum, as it reacted with sera obtained from patients sensitized to A.

fumigatus (Weichel et al, 2003). The malate dehydrogenase NAD-dependent is involved

in citric acid and glyoxalate cycles.

As a part of another study of our group, it was observed that the expression of 4

identified secreted immunoreactive proteins of A. fumigatus (Asp f2, thioredoxin

reductase, Asp f3 and NAD-dependent formate dehydrogenase) corresponding to Spot

Nos. 2, 19, 24 and 30, was altered on the treatment of a coumarin derivative SCD-1

(Singh et al, 2012). It could, therefore, be inferred that the immunogenic molecules of

A. fumigatus could also be targets for antifungal agents.

4.8.1.1. Functional categorization of secreted proteins All 35 identified immunogenic proteins were subjected to functional annotation in accordance with the Uniprot database (www.uniprot.org) and Kognitor (Tatusov et al, 2003). Based on the functions, these proteins could be divided into 10 categories (Fig 4.13).


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Fig 4.13. Functional classification of identified secreted immunogenic

proteins which reacted with IgE and IgG antibodies derived from sera of ABPA patients. Proteins in bold type specifically reacted with IgE; proteins in bold and underlined only reacted with IgG. The remaining proteins showed reactivity with both IgE and IgG antibodies. Functional annotation to the proteins is assigned in accordance with the Uniprot database (www.uniprot.org) and Kognitor.


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Majority of the 35 proteins identified from the secreted fraction of A. fumigatus in present study belonged to carbohydrate transport and metabolism. Only one protein each belonged to the functional groups of nucletoide transport and metabolism, inorganic ion transport and metabolism and energy production and conservation. It was observed that several proteins not belonging to any particular functional category may be involved in various other important functions required for fungal survival and virulence.

In the host, A. fumigatus secretes a large number of proteases and lipases. The role of proteases in conferring virulence still remains controversial (Bergmann et al, 2009). The proteases identified from secreted immunoproteome of A. fumigatus included DppV (Beauvais et al, 1997), a glycoprotein also called the hyphal invasion enzyme and the (Asp f10), a secreted aspartic protease. Many of the secreted proteins of A. fumigatus induce an allergic response in the host. The candidates for such responses could be the proteins that react specifically with IgE antibodies such as, Asp f2, Asp f15 (a 19 kDa allergenic ceratoplatinin) and Asp f3 (PMP20) described by Banerjee et al (1998) and Hemmann et al (1997). The cell wall glycosylated proteins of A. fumigatus have been divided into cell wall biosynthetic proteins and cell wall degrading molecules. The Asp f9 has been a cell wall biosynthesis associated protein, whereas 1,3-β -glucanosyltransferase was described to be involved in the cell wall morphogenesis as an anchoring molecule by incorporating 1,3-beta linkages in between 1,3-beta-glucan molecule (Mounya et al,

1998, Bruneau et al, 2001, Fontaine et al, 2003). The mannosidase has been an essential fungal cell wall glycoprotein (Li et al, 2008). The second category i.e. cell wall degradation molecules include chitin dissolving molecules such as, chitosanase and chitinases (Jaques et al, 2003, Cheng et al, 2006). These chitin dissolving glycosidases/hydrolases are abundant in the cell wall and are important in maintaining appropriate cell wall integrity. These cell wall linked molecules have been considered to be targets for antifungal drug development. Lectins have also been considered as important cell wall associated molecules due to their specific sugar recognition properties. Presence of a fucose specific lectin on the conidial surface reflects a potential


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role of laminin and fibrinogen in the adherence of A. fumigatus conidia to the host cells (Tronchin et al, 2002). Stress proteins are considered to be of an important category of molecules for

pathogen defense. These molecules play an essential role in disease progression and have

been considered as pathogenic factors (Hohl and Feldmesser, 2007). Many proteins

identified in the present study like, DppV (Beauvais et al, 1997), Asp f10 (Lee and

Kolattukudy, 1995), thioredoxin redutase (Gardnier et al, 2005, Crameri et al, 2006),

rAsp f3 (Ito et al, 2006) and bifunctional catalase peroxidase Cat2 (Calera et al, 1997),

have been extensively studied for their biological properties. The catalases secreted by

A. fumigatus had been considered to be important fungal defense enzymes responsible for

escape from the phagocytolytic activity. Therefore, immunogenic catalases identified in

present study such as catalase-B (Spot No. 18) and Cat2 (Spot No. 32) may be involved in

the protection of the pathogen against host defense by partially inactivating H2O2.

However, there have been reports which showed that the catalases of A. fumigatus

remained ineffective in providing protection against macrophage killing (Missall et al,

2004, Shibuya et al, 2006). A. fumigatus produced diverse pathogenic molecules which

made its survival easier in the host and also have been found to be responsible for

development of different disease conditions including ABPA. Although the biological

functions of several proteins of A. fumigatus have been described, the existence of many

other molecules with unknown biological functions has been just predicted based on the

presence of their DNA sequences in the genome (Nierman et al, 2005). The molecules

with unknown biological functions may have potential to play a role in host pathogen

interaction and development of disease. We indeed identified three new such proteins

(Spot No. 6, 27 and 28) without any known experimental or putative functional

elucidation. The characterization of these 3 immunogenic, so far, hypothetical proteins of

unknown function, may reveal their possible role in development of Aspergillus-induced

infections.


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4.8.1.2. Prediction of signal peptide and cellular localization of secreted proteins The in silico analysis of all 35 identified secreted immunoreactive proteins by SIG-Pred revealed the presence of signal peptides in 19 proteins. The remaining 16 proteins which did not have secretary signal peptides included 4 hypothetical proteins recovered from Spot Nos. 5, 6, 27 and 28 (Table 4.3). It is thus considered that the crude secreted fraction of A. fumigatus used in the present study contained major fraction of immunoreactive protein having signal peptide sequence for their extracellular secretion. In order to determine the cellular localization of all identified proteins, their amino acid sequences were analyzed by PSORTb. The analysis showed that out of 35 proteins, 12 were extracellular, 4 were non-cytoplasmic, 12 localized in cytoplasm and 6 had unknown cellular localization with equal probability of being extracellular, non-cytoplasmic or cytoplasmic (Table 4.2). The cellular localization showed that majority of these proteins had signal peptide and were not located in cytoplasm. But it has now been accepted that the presence of signal peptide may not be essential for the secretion of proteins as certain non-classical secretory pathways may also exist in A. fumigatus (Asif et al, 2006). Schwienbacher et al (2005) analyzed in-vitro the major secreted proteins of A. fumigatus and detected presence of anti-mitogilin and anti-chitosanase antibodies in the sera of patients of invasive aspergillosis and aspergilloma. They proposed Asp f1 (Moser et al, 1992) and chitosanase to be potentially relevant for diagnosis of aspergillosis. They also identified 3 more proteins (aspergillopepsin, chitinase-B and 1, 3-endoglucanase) which were secreted by A. fumigatus in different growth media. The results of present study also revealed the presence of these five proteins, identified from the Spot Nos. 1, 9, 14, 23 and 26. The immunoreactivity of these proteases with sera of ABPA patients provided promising targets for further investigation of these proteins as potential allergens. These proteins have been known for their proteolytic, elastinolytic, hemolytic and cytotoxic activity and collectively may contribute to the degradation of host lung tissue during


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invasion. Along with proteolytic enzymes, the A. fumigatus also secretes lipolytic enzymes like, lysphospholipases-3 and lysphospholipases-1. Of the metabolic processes of A. fumigatus, sugar metabolism has been the major process by which energy is provided to the fungus to grow in a relatively oxygen deprived environment in the body of the host. Certain enzymes of sugar metabolism such as, malate dehydrogenase NAD-dependent, beta glucosidase, FAD/FMN-containing isoamyl alcohol oxidase and glucose-6-phosphatase isomerase, reacted with the sera of ABPA patients. The presence of the enzymes of sugar metabolism and energy production in secretome of A. fumigatus was in abundance. The reactivity of these abundant molecules with IgG and IgE antibodies derived from sera of ABPA patients may indicate their usefulness in specific diagnosis of ABPA.

4.8.2. Identification of CHC immunogenic proteins The corresponding protein spots of immunoreactive signals excised from 2DE gels of CHC fraction of both A. fumigatus strains (ITCC 6604 and DAYA) were tryptic digested to generate peptides and subjected to mass spectrometric analysis. The MASCOT search of the sequenced peptides against NCBInr database led to identification of total 66 proteins from CHC fraction of both strains (ITCC 6604 and DAYA) of A. fumigatus with significant MASCOT scores ranging from 59 to 876 (Table 4.3). Sequence coverage in the identified proteins ranged between 5-57%. Out of 66 proteins, 64 showed homology with A. fumigatus proteins. One of remaining 2 proteins which corresponded to Spot No. 54, showed match with ACT_BOTCI actin of A. nidulans, and other of Spot No. 16 showed match with elongation factor-Tu of Klebsiella pneumoniae. Although the MASCOT scores of Spot No. 16 with K. pneumoniae was significant but due to lack of homology with any protein of Aspergillus, it was not considered for further analysis. The analysis of identified spots showed presence of 63 IgE reactive proteins (Fig 4.11). Six proteins fell in category of A. fumigatus allergens, of which 2 proteins, Spot No. 17 (Asp f22) and Spot No. 38 (Asp 12), were known allergens and 4 were


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predicted allergens. These predicted allergens were recovered in Spot Nos. 37, 39, 62 and 63, and identified as molecular chaperone Hsp70 chaperone-Hsp88, Hsp70, malate dehydrogenase NAD-dependent and Zn containing alcohol dehydrogenase putative respectively. However, the protein identified from Spot No. 43 was found to be PEP2, a vacuolar aspartic endopeptidase previously reported in the literature but not so far as allergen (Table 4.3). Interestingly we were able to characterize only 3 proteins (2 in ITCC 6604 and 1 in DAYA) that reacted with IgG antibodies (Fig 4.12), however, few more IgG reactive spots were visible on blots but their amount in corresponding protein spot had insufficient concentration of proteins for Q-TOF MS/MS analysis. Present study identified 62 proteins which were uncharacterized predicted molecules deduced from recently sequenced genome of A. fumigatus for their biological functions.


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Table 4.3. List of identified CHC immunoreactive proteins of A. fumigatus.

Spot No.

Accession No.

Identification in A. fumigatus MASCOT score

Molecular mass/pI

Function

1 XP_755878 Sorbitol/xylulose reductase Sou1-like 278 28.2/5.96 Oxidoreductase

2 XP_754489 Proteasome component Prs2 207 28.2/6.84 Hydrolase

3 XP_750785 Proteasome component Pre9 171 28.2/5.80 Hydrolase

4 XP_001481696 6-phosphogluconolactonase 309 28.8/5.84 Hydrolase

5 XP_753827 Pyridoxine biosynthesis protein 171 32.7/6.04 Catalytic activity

6 XP_752719 Spermidine synthase 238 33.4/5.33 Transferase

7 XP_754985 Phosphoribosyl-aminoimidazole-succinocarboxamide synthase

131 33.2/5.51 Ligase

8 XP_754776 Inorganic diphosphatase 217 43.6/7.63 Hydrolase

9 XP_750257 60S ribosomal protein P0 299 33.5/5.11 Ribosomal ribonucleoprotein,

10 XP_753994 Adenosine kinase 82 36.8/5.02 Kinase, transferase

11 XP_748095 Thiamine biosynthesis protein (Nmt1) 295 38.3/6.04 Function not known

12 XP_754452 Fructose-bisphosphate aldolase, class II

438 39.7/5.55 Lyase

13 XP_753716 Transaldolase 419 35.4/6.04 Transferase

14 XP_752429 S-adenosylmethionine synthetase 343 42.1/5.67 Transferase,

15 XP_751519 Glutamine synthetase 113 39.8/5.48 Ligase

17 Q96X30 Asp f22/ Enolase 806 47.2/5.39 Lyase

18 CAE17673 Putative flavohaemoglobin 134 45.5/5.72 Electron carrier, Oxidoreductase

19 XP_752401 Phosphoglycerate kinase PgkA 762 44.7/6.31 Transferase

20 XP_752379 Adenosylhomocysteinase 677 48.4/5.82 Hydrolase

21 XP_751658 Phosphatidyl synthase 452 67.0/6.11 Hydrolase

22 EDP54506 Glucose-6-phosphate isomerase 447 61.3/5.85 Isomerase

23 XP_750954 ATP citrate lyase subunit (Acl), putatibe

474 52.8/5.88 Lyase, Ligase

24 Q4WFT3 GMP synthase [glutamine-hydrolyzing]

120 61.4/5.85 Ligase, ATP binding


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25 XP_750696 6-phosphogluconate dehydrogenase Gnd1

876 55.7/5.86 Oxidoreductase, NADP binding

26 XP_755071 Gamma-glutamyl phosphate reductase 112 48.7/5.60 Oxidoreductase, NADP binding

27 XP_755056 Glutamatecarboxypeptidase 59 52.9/5.47 Carboxypeptidase, metallopeptidaseprotein dimerizaion

28 XP_755448 Secretory pathway gdp dissociation inhibitor

349 52.2/5.33 Rab-GDP dissociation

29 XP_747854 Glucokinase GlkA 110 54.4/5.33 Kinase, ATP binding

30 XP_755657 Bifunctional tryptophan synthase TRPB

376 78.7/5.66 Pyridoxal phosphate binding,

31 XP_755459 3-isopropylmalate dehydratase 181 84.0/5.62

Iron sulfur cluster binding

32 XP_752826 Phosphoribosyl-AMP cyclohydrolase 86 92.8/5.46 Dehydrogenase, Hydrolase

33 XP_753831 Aminopeptidase P 15 72.6/5.93 Metallopeptidase, Hydrolase

34 XP_755328 Mitochondrial Hsp70 chaperone (Ssc70)

673 74.4/6.02 ATP/unfolded protein binding

35 XP_746746 Heat shock protein (Sti1) 102 64.9/5.83 Stress response

36 XP_747200 Hsp70 chaperone (HscA) 408 66.9/5.30 ATP binding

37 XP_752631 Hsp70 chaperone Hsp88 293 79.9/5.08 ATP binding

38 XP_747926 Asp f 12/ Heat shock protein P90/ /Hsp90/Hsp1

613 80.5/4.95 ATP/unfolded protein binding

39 XP_750490 Molecular chaperone Hsp70 693 69.6/5.09 ATP binding

40 XP_753589 ATP synthase F1, β subunit 412 55.6/5.30 Hydrolase, ATP/metal ion binding

41 XP_750312 40S ribosomal protein S3 615 29.1/9.18 Ribonucleoprotein

42 XP_751171 Mitochondrial aconitate hydratase 389 85.4/6.26 Tricarboxlic acid cycle,

43 XP_754479 Aspartic endopeptidase PEP2 66 43.3/4.81 Protease

44 XP_749744 Protein disulfide isomerase Pdi1 260 56.1/4.58 Ca ion binding, Isomerase

45 XP_755686 Translation elongation factor EF-2 58 93.1/6.51 GTPase,

46 XP_752090 Cobalamin-independent methionine synthase MetH/D

89 86.8/6.33 Methyltransferase Zn ion binding


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47 XP_752650 Isocitrate dehydrogenase, NAD-dependent

281 41.7/8.72 Oxidoreductase

48 XP_752056 Phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase

63 64.9/6.39 Hydrolase, Transferase

49 XP_754077 Nucleosome assembly protein Nap1 94 48.3/4.43 Nucleosome assembly

50 XP_754843 Ran GTPase activating protein 1 (RNA1 protein)

99 46.2/4.62 Protein binding

51 XP_756019 Curved DNA-binding protein 131 46.5/ 7.61 DNA binding, Hydrolase, Aminopeptidase

52 XP_754690 Phosphoglycerate mutase, 2,3-bisphosphoglycerate-independent

276 57.4/5.44 Isomerase, Mn ion binding

53 XP_748938 Pyruvate dehydrogenase complex, dihydrolipoamide acetyltransferase component

101 52.0/6.25 Acetyl transferase, Protein binding

54 XP_664146 ACT_BOTCI Actin A. nidulans 64 41.6/ 5.54 ATP/protein binding

55 XP_752265 UPF0160 domain protein MYG1 88 40.0/5.85 Function not known

56 XP_751780 Homocitrate synthase 334 50.5/ 5.66 Acyltransferase

57 XP_755754 Dihydroxy acid dehydratase Ilv3 284 64.7/ 7.55 Hydratase

58 XP_749237 Myo-inositol-phosphate synthase 204 58.6/5.82 Isomerase

59 XP_752164 Glutamate/Leucine/Phenylalanine/Valine dehydrogenase

481 49.3/5.79 Oxidoreductse

60 XP_750781 Assimilatory sulfite reductase 283 114.5/5.03 Oxidoreductase

61 XP_754687 Serine hydroxymethyltransferase 131 51.8/7.64 Transferase,


559 35.8/9.08 Oxidoreductase

63 XP_754009 Alcohol dehydrogenase 381 37.1/7.55 Oxidoreductase, Zn ion binding

64 XP_748145 Glyceraldehyde 3-phosphate dehydrogenase GpdA

162 36.2/6.96 Oxidoreductase, NAD binding

65 XP_747920 Citrate synthase (Cit1) 258 52.0/ 8.69 Acyltransferase

66 EDP50202 Zinc-containing alcohol dehydrogenase, putative

286 36.8/7.01 Oxidoreductase, Zn ion binding


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In contrast to the late stage secreted proteins, the germinating conidia contained the cytosolic and cell wall associated proteins which are usually expressed during early stages of cell development. Vodisch et al (2009) developed proteome map of A. fumigatus using its cytosolic and mitochondrial fractions. The use of cytosolic fraction of A. fumigatus for the identification of immunologically active molecules was not reported until the current work was undertaken. Therefore, present study has been an attempt to characterize immunoreactive molecules from less explored cytosolic fraction of A. fumigatus. Analysis of identified spots showed the presence of a total of 66 CHC immunoreactive proteins in A. fumigatus, out of which 63 were found to be IgE reactive. These 63 proteins included 6 A. fumigatus allergens, of which 2 proteins, Spot No. 17/Asp f22 (Lai et al, 2002) and Spot No. 38/Asp f12 (Kumar et al, 1993) were known allergens and 4 were predicted allergens (Federova et al, 2008). The predicted allergens have been Hsp70 chaperone-Hsp88, Hsp70, malate dehydrogenase NAD-dependent and Zn containing alcohol dehydrogenase putative respectively. The comparative analysis of 6 (known and predicted) allergens of A. fumigatus by SDAP (http://fermi.utmb.edu/SDAP/sdap_who.html) showed significant sequence similarity with allergens reported from other source. The Asp f12 did not share significant homology with any other fungal allergens, so it could be taken as specific allergen of A. fumigatus with out any predicted cross reactivity and may have more diagnostic value. But allergen Asp f22 showed the matches with 4 fungal allergens Pen c22.0101 of Penicillium citrinum (94%), Alt a5 of Alternaria alternata (88%), Cla h6 of Cladosporium herbarum (86%) and Cur l2.0101 of Curvularia lunata (81%) which indicated that it might be a cross reactive allergen of A. fumigatus. Among the 4 identified predicted allergens of A. fumigatus, the molecular chaperone Hsp70 was found to share sequence homology with 2 fungal allergens namely Cla h5.0101 of C. herbarum, (84%) and Pen c19 of P. citrinum, (72%) where as molecular chaperone Hsp70 chaperone-Hsp88 contained 51% homologous amino acid sequence with a yeast allergen


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Mala s10 reported from Malassezia sympodialis. It is important to note that the protein malate dehydrogenase NAD-dependent which is involved in citric acid and glyoxalate cycles, also showed 63.4% homology with Mala f4 allergen of M. furfur. Alcohol dehydrogenase was found to share 61.3% sequence homology with C. albicans allergen (Cand a1). The protein of Spot No. 43 was found to be an earlier reported vacuolar aspartic endopeptidase (Reichard et al, 2000) but so far not as an allergen. Present study identified 62 CHC proteins which were uncharacterized predicted molecules deduced from recently sequenced genome of A. fumigatus for their biological functions. Possible reason for getting a large number of unidentified immunoreactive proteins may be that we used a less explored complex CHC fraction of A. fumigatus for immunoproteomics studies. The results of the present work demonstrated the physical presence of proteins that were predicted in A. fumigatus (Nierman et al, 2004) and also assigned atleast one biological property to them being immunogenic and reacted specifically with IgE and IgG antibodies derived from sera of ABPA patients. It was not known till we observed that the distribution of IgE and IgG binding epitopes in antigens of A. fumigatus was so protein specific. The analysis of proteins by using DNAstar, an in silico analysis tool, confirmed the presence of antigenic epitopes amongst all 66 identified immunoreactive proteins. It was observed that out of 66 identified proteins 30 were common in both strains. Twenty six proteins were specific for Indian strain (ITCC 6604) and 10 proteins were found to be specific for German strain (DAYA). These differences in the strain specific immunoreactivity can be attributed to the fact that sera from Indian ABPA patients were used in the present study. Recently published proteome map (Vodisch et al, 2009) of A. fumigatus also showed the presence of 24 proteins, identical to those identified by us and which were found to be IgE reactive in the present study. Some of the IgE reactive proteins (mitochondrial Hsp70 chaperone Ssc70 and Hsp70 chaperone Hsp88) of the present study were earlier reported as potential drug targets (Gautam et al, 2009). The


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proteins (malate dehydrogenase NAD-dependent and mitochondrial aconitate hydratase) were reported as the molecules which may be involved in the interaction between A. fumigatus conidia and host cells (Sugui et al, 2008). In a report of Asif et al (2006), the conidial hyphal surface associated proteome also showed the presence of 3 common proteins (PEP2, protein disulfide isomerase and alcohol dehydrogenase) where they were reflected as potential vaccine candidates. Another recent study from similar group identified a panel of A. fumigatus proteins reactive to IgG, IgM and IgA antibodies present in the sera of rabbits which acquired protective immunity after experimentally induced invasive aspergillosis (Asif et al, 2010), indicating their role in conferring protection. Sixteen proteins of their panel were identified in CHC fraction of A. fumigatus used in the present study and all of them were found to be predominantly IgE reactive. Thus these proteins will also be important for diagnosis of allergic aspergillosis. As part of another study, proteome of A. fumigatus was developed after treatment of a novel coumarin derivative SCD-1. It was found that out of 66 identified antigens, 31 showed altered expression due to the treatment of SCD-1. These 31 proteins were corresponded to Spot Nos. 1, 4, 5, 6, 10-13, 17, 19, 20, 23, 25, 27, 32, 33, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 52, 59, 60 and 65 have been identified as sorbitol/xylulose reductase Sou1-like, 6-phosphogluconolactonase, pyridoxine biosynthesis protein, spermidine synthase, adenosine kinase, thiamine biosynthesis protein Nmt1, fructose-bisphosphate aldolase class II, transaldolase, Asp f22, phosphoglycerate kinase A, adenosylhomocysteinase, ATP citrate lyase subunit, 6-phosphogluconate dehydrogenase Gnd1, glutamatecarboxypeptidase, phosphoribosyl AMP cyclohydrolase, Aminopeptidase P, Aminopeptidase, Hsp70 chaperone Hsp88, Asp f12, ATP synthase F1, beta subunit, 40S ribosomal protein S3, mitochondrial aconitate hydratase, aspartic endopeptidase, translation elongation factor EF-2 subunit, cobalamin-independent methionine synthase, isocitrate dehydrogenase NAD dependent, phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase,


109

phosphoglycerate mutase, 2,3-bisphosphoglycerate, glutamate/leucine/phenylalanine/valine dehydrogenase, assimilatory sulfite reductase and citrate synthase 1) respectively. It was important to note that out of 143 proteins showed altered expression in A. fumigatus after treatment of SCD-1 included 32 immunodominant IgE reactive molecules (Singh et al, 2012) which may have potential application in management of aspergillosis. 4.8.2.1. Functional categorization of CHC proteins The biological function annotation of identified proteins within Uniprot (www.uniprot.org) and Kognitor (Tatusov et al, 2006) databases alienated 66 immuno-reactive proteins into 16 different categories (Fig 4.14). The major divisions of these functional categories were transport and/or metabolism of essential constituents (amino acid, carbohydrate, nucleotides and coenzymes), proteins that are involved in energy conversion and/or production, and the stress proteins. The molecular chaperones and the proteins involved in secondary processing such as, posttranslational modifications which help in protein turnover constitutes the factors that may help the pathogen to survive under adverse conditions such as in the body of host during infection. In the literature, 9 known (Asp f3, Asp f5, Asp f10, Asp f11, Asp f12, Asp f18, Asp f27, Asp f28 and Asp f29) (Abad et al, 2010) and 8 predicted (thioredoxin, glutathione-S-transferase, Hsp70, alkaline serine protease Alp1, protein disulfide isomerase, thioredoxin A, DppV and Hsp70-chaperone Hsp88) allergens (Federova et al, 2008) of A. fumigatus have been reported to occur in the category of adaptive stress proteins. Likewise, 11 IgE reactive proteins were also present in the functional category of adaptive stress proteins, which have been identified from the Spot No. 2, 3, 28, 34-39, 43 and 44, and listed in Table 4.3 as proteasome component-Prs2, proteasome component-Pre9, secretory pathway-gdp dissociation inhibitor, Ssc70, heat shock protein Sti1, Hsp70 chaperone-HscA, Hsp70 chaperone-Hsp88, Asp f12 (Lai et al, 2002), Hsp70, PEP2 and protein disulfide isomerase respectively. It was interesting to observe that these 11 proteins included 1 known (Spot No. 38) and 2 predicted allergens (Spot Nos. 37 and 39) of A. fumigatus.


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Fig 4.14. Pie chart showing functional categorization of 66 identified CHC A. fumigatus cytosolic proteins in accordance with Kognitor; a function annotation tool of NCBI database. Proteins underlined only reacted with IgG and the remaining proteins showed reactivity with IgE antibodies.


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This observation reflected that the stress proteins could be a potential group of allergens in A. fumigatus. The amino acid transport and metabolism is one of major biological processes at basal cellular level. We observed that there were 14 immunoreactive proteins in A. fumigatus that belonged to this group but none of them showed homology with any known or predicted allergens (http://www.allergome.org). It was important to note that 4 of these novel proteins of A. fumigatus (Spot Nos. 26, 27, 47 and 61) reacted strongly with IgE antibodies (Fig 4.15). Another major group of proteins was carbohydrate transport/metabolism which contained 11 proteins and 5 of them showed strong IgE reactivity in both strains. These 5 proteins were fructose-bisphosphate aldolase class-II (Spot No. 12), transaldolase (Spot No. 13), Asp f22 (Spot No. 17), phosphoglycerate kinase (Spot No. 19) and mitochondrial aconitate hydratase (Spot No. 42). Among 11 proteins of carbohydrate transport/metabolism group identified in present study, the Asp f22, was a known allergen. Energy has been the basic requirement of any cell and the energy production/conversion also covers the major category of cellular functions. In this category also there were 11 proteins which included a predicted allergen called malate dehydrogenase NAD- dependent (Spot No. 62), and 2 other proteins (ATP citrate lyase subunit Acl and 60S ribosomal protein P0, identified from Spot No. 9 and 23) which had putative biological functions. All these 3 proteins reacted more strongly in ITCC 6604 strain of A. fumigatus as compared to DAYA.

There were immunoreactive proteins in A. fumigatus which could not be attributed to any of the major functional category, but few of them had strong IgE reactivity. These strongly reactive proteins were nuclear binding protein Nap1, ran-GTPase activating protein, alcohol dehydrogenase and zinc containing alcohol dehydrogenase. In the literature 3 proteins namely Asp f2, Asp f4 and Asp f7 have been reported as known allergens with unknown biological functions and 5 as the predicted allergens (RNA binding protein MSSP2, glucan 1, 4-α glucosidase, aryl alcohol dehydrogenase, histidine acid phosphatase and NADH quinone oxidoreductase Pst2) which had only predicted biological functions (Tatusov et al, 2003, Federova et al, 2008,


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Abad et al, 2010). Six allergenic proteins (sorbitol/xylulose reductase Sou1-like, thiamine biosynthesis protein Nmt1, putative flavohaemoglobin, phosphatidyl synthase, curved DNA-binding protein and UPF0160 domain protein MYG1) of the present study also belonged to the category of predicted and unknown biological functions. In this context proteins which have unknown/predicted function are also important in terms of immunogenicity and as potential allergens of A. fumigatus. Present study described the immune responses of the host to the cytosolic proteins of A. fumigatus. It was bit surprising to observe that majority of CHC proteins reacted with IgE. The antigen antibody reaction is a complex phenomenon and resultant host immune responses to CHC fraction of A. fumigatus have not been studied so far. We consider that less explored cytosolic fraction of A. fumigatus had mainly the molecules that contained predominantly the allergenic epitopes for IgE binding. This might be the reason why only 3 proteins out of 66 exclusively reacted with IgG antibodies of patients sera. These IgG reactive proteins were identified as protein disulfide isomerase Pdi1, translation elongation factor EF-2 subunit and assimilatory sulfite reductase, from the Spot No. 44, 45 and 60 respectively. Amongst them, the Pdi1 has been considered as possible vaccine candidate from conidial hyphal wall associated proteome (Asif et al, 2006). Few other studies have also demonstrated successful attempts of immunoprotection in experimental animal models of IA by using A. fumigatus immunogenic proteins (Bozza et al, 2002, Ito et al, 2006). Therefore, these 3 IgG reactive proteins may not have allergenic potential but could be evaluated for their immunotherapeutic properties. Screening of immunoproteomes of secreted and cytosolic fractions of A. fumigatus identified 101 immunoreactive proteins which may have potential for application in early diagnosis and immunotherapy of Aspergillus induced infections. Hence it is extremely important to select out key proteins from them for detailed characterization after expressing them recombinantly.


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4.9. Selection of molecules for cloning

The 29.4 megabase sequenced genome of a clinical isolate Af293 which consisted of eight chromosomes, showed the presence of 9926 predicted genes encoding for various proteins of A. fumigatus (Nierman et al, 2005). But surprisingly a very few of them have been recombinantly produced and, therefore, efforts are on to develop ideal recombinant molecule for use in diagnostics and vaccine preparation. We have identified 101 immunogenic molecules from secreted (35) and CHC (66) fractions of A. fumigatus. For identification of potential molecules for their detailed characterization, the selection criteria were novelty of the protein and consistent reactivity with sera of individual ABPA patients. Therefore, 2DE immunoblots of secreted and CHC antigenic fraction of A. fumigatus were developed with individual sera of ABPA patients. 4.9.1. The 2DE immunoblotting of secreted fraction with sera of individual ABPA patients The immunoreactivity of pooled sera of ABPA patients identified 35 proteins in secreted fraction and 66 in CHC fraction of two different A. fumigatus strains (ITCC 6604 and DAYA) in broad (pH 3-10) and narrow pI (pH 4-7) ranges. However, it was not clear if all of them would react to same extent in all patients. Therefore, in a next step, we investigated their reactivity pattern with IgG/IgE antibodies from sera of individual ABPA patients. For this purpose, we randomly selected sera of 5 individual patients of ABPA and developed IgE and IgG immunoblots with secreted fraction of ITCC 6604 in pH range 4-7 (Fig 4.15 and 4.16). The immunoblots developed with sera of individual ABPA patients were compared with blots of pooled serum to identify consistently reactive common proteins. The IgE immunoblots with pooled serum (Fig 4.15) showed presence of 18 antigens, whereas 10 immunoreactive proteins were present in the IgG blots (Fig 4.16). The reactivity of individual sera revealed the presence of 8 proteins which reacted with IgG and IgE both. Those proteins were identified as Asp f2, DppV, chitosanase, lysophospholipase-1 Plb1, mannosidase, β-glucosidase and FAD/FMN


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containing isoamyl alcohol oxidase which belonged to Spot No. 2, 6, 8, 9, 21, 22, 33 and 34 respectively. Excepting Plb1, all of these proteins reacted consistently with sera of all 5 individual patients (Table 4.4). There were 10 proteins namely Asp f9, aminotransferase class V, 1,3-β-glucanosyltransferase gel2, CatB, Plb3, pectate lyase A, NAD-dependent formate dehydrogenase AciA/Fdh, malate dehydrogenase NAD-dependent, bifunctional catalase peroxidase and glucose-6-phosphate isomerase of Spot Nos. 4, 7, 16, 18, 20, 29, 30, 31, 32 and 35 respectively, that selectively reacted with IgE (Fig 4.15), and only 1 (Spot No. 24/Asp f3) was IgG reactive. The remaining proteins out of 35 reactive to pooled sera, did not appear in the list due to the fact that we have used only Indian strain (ITCC 6604) and the immunoproteome was developed only in narrow pH range (4-7).


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Fig 4.15. The 2DE immnuoblots of secreted proteome of A. fumigatus strain ITCC 6604 in narrow pH range (4-7) developed with pooled (PS) and individual sera (S1-S5) of ABPA patients and probed with anti-human IgE antibodies conjugated with HRP.


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Fig 4.16. The 2DE immnuoblots of secreted proteome of A. fumigatus strain ITCC 6604 in narrow pH range (4-7) developed with pooled (PS) and individual sera (S1-S5) of ABPA patients and probed with anti-human IgG antibodies conjugated with HRP.


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Table 4.4. List of consistently reactive secreted proteins of A. fumigatus. Spot No.

Accession No.

Identifications in A. fumigatus

Immuno- reactivity

Pooled sera

S1 S2 S3 S4 S5

2 P79017 Asp f2-hypothetical protein

IgE and IgG + + + + + +

4 CAA11266 Asp f 9-probable glycosidase, crf1

IgE + + + + + +

6 Q4WEM3 Hypothetical protein IgE and IgG + + + + + +

8 O13479 Dipeptidyl-peptidase-V DppV

IgE and IgG + + + + + +

9 AAD26111 Chitosanase IgE and IgG + + + + + +

16 Q9P8U4 1,3-β -glucanosyltransferase, gel2

IgE + + + + + +

18 Q92405 Catalase-B, Cat-B IgE + + + + + -

20 Q6U819 Lysophospholipase-3, Plb3 IgE + + + + + +

21 Q6U820 Lysophospholipase-1, Plb1 IgE and IgG + + - + - +

22 XP_752825 Mannosidase, MsdS IgE and IgG + + + + + +

24 CAA12162 Asp f3/putative peroxiredoxin-pmp20

IgG + - + - - -

29 XP_749213 Pectate lyase A IgE + + + - + -

30 XP_747586 NAD-dependent formate dehydrogenase, AciA/Fdh

IgE + + + + + -


IgE + + + - - -

32 XP_747039 Bifunctional catalase-peroxidase,

IgE + + + + + +

33 XP_750327 β-glucosidase

IgE and IgG + + + + + +

34 XP_747715 FAD/FMN-containing isoamyl alcohol oxidase,

IgE and IgG + + + + + +

35 EDP54506 Glucose-6-phosphate isomerase

IgE + + + + + +


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4.9.2. The 2DE immunoblotting of CHC fraction with sera of individual ABPA patients In order to identify consistently reactive proteins, 2DE IgE immunoblots of the CHC proteome were developed with sera of 10 individual ABPA patients. Large number of unevenly distributed IgE reactive spots appeared on the blots (Fig 4.17B to 4.17K). We marked the protein spots which showed intense signals on immunoblot and visible spot of protein on corresponding 2DE gel. No signal was appeared on the control blot developed by using the negative pooled sera from healthy controls (Fig 4.17L). Immunoblots were matched with corresponding silver stained 2DE gel (Fig 4.17A) and PVDF membranes (Fig 4.17B to 4.17K). All protein spots were subjected to tryptic digestion, peptide extraction and Q-TOF MS/MS analysis. The MASCOT search of the sequenced files with NCBInr databases led to identification of total 18 proteins with significant MASCOT scores ranging from 84 to 830. The sequence coverage in the identified proteins ranged between 7.0-40.0%. All identified proteins showed substantial homology with the amino acid sequences of A. fumigatus molecules. These 18 proteins reacted variably with serum IgE antibodies of individual ABPA patients sera with different signal intensity on the immunoblots. Out of 18 proteins seventeen were reacted with sera of 10 ABPA patients whereas protein of Spot No. 42 reacted with sera of 7 patients only. Two of 18 identified proteins were found to be known allergens (Asp f22 and Asp f12) and 2 were predicted allergens (Hsp70 chaperone-Hsp88 and Hsp70) of A. fumigatus. Remaining 14 immunoreactive proteins have only predicted biological functions, deduced from genomic sequences of A. fumigatus (Table 4.5). We have first time reported that these proteins are immunoreactive in nature. These consistently immunoreactive proteins could be considered as the potential cytosolic allergens of A. fumigatus.


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Fig 4.17. The 2DE IgE immunoblots of CHC proteome of A. fumigatus strain

ITCC 6604 in narrow pH range (4-7) developed with pooled (A), individual sera (B-K) of ABPA patients and negative pooled sera of healthy individuals (L).


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Table 4.5. List of consistently IgE reactive CHC proteins of A. fumigatus.

Spot No.

Accession No. Identification in A. fumigatus Reacted with number of patients

1 XP_755878 Sorbitol/xylulose reductase Sou1-like 10

3 XP_750785 Proteasome component Pre9 10

5 XP_753827 Pyridoxine biosynthesis protein 10

6 XP_752719 Spermidine synthase 10

8 XP_754776 Inorganic diphosphatase 10

12 XP_754452 Fructose-bisphosphate aldolase, class II 10

13 XP_753716 Transaldolase 10

17 Q96X30 Asp f22/ Enolase 10

20 XP_752379 Adenosylhomocysteinase 10

22 EDP54506 Glucose-6-phosphate isomerase 10

23 XP_750954 ATP citrate lyase subunit (Acl), putatibe 10

25 Q4WFT3 GMP synthase [glutamine-hydrolyzing] 10

28 XP_750696 6-phosphogluconate dehydrogenase Gnd1 10

37 XP_752631 Hsp70 chaperone Hsp88 10

38 XP_747926 Asp f12/ Heat shock protein P90 10

39 XP_750490 Molecular chaperone Hsp70 10

40 XP_753589 ATP synthase F1, beta subunit 10

42 XP_751171 Mitochondrial aconitate hydratase 7


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4.9.3. Target molecules of A. fumigatus for cloning The protein of secreted fraction named as hypothetical protein (H6) from Spot No. 6 was selected for cloning and recombinant expression on the basis of its consistent reactivity with pooled and individual sera of ABPA patients. This protein was selected for recombinant expression as a representative target molecule because of the fact that its biological functions were unknown and so far it has not been characterized by any other researcher. The sequence of H6 contained a 250 amino acid long TauA domain, periplasmic component involved in ABC-type nitrate/sulfonate/bicarbonate transport systems of inorganic ion transport and metabolism (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The H6 being an uncharacterized protein, its biological functions could not be even predicted until we first demonstrated that this protein, as shown from the genome, existed in A. fumigatus and was immunoreactive. It was, therefore, important to produce H6 recombinantly. Three proteins were selected from CHC fraction of A. fumigatus for cloning. Two of them namely sorbitol/xylulose reductase Sou1 like (SOU1) and fructose-bisphosphate aldolase, class-II (FBA-II) from Spot Nos. 1 and 12, were selected on the basis of their consistent reactivity with pooled and individual ABPA patients sera where as third CHC protein obtained from Spot No. 63 and named as alcohol dehydrogenase (ADH), was selected on the basis of its dominant immuno-reactivity with pooled sera and its existence as a predicted allergen in A. fumigatus. The protein SOU1 is a member of family of NADP-mannitol dehydrogenases which catalyzes the conversion of fructose to mannitol and share Rossmann fold, a NAD-binding motif (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The FBA-II is a member of a family which contains a conserved phosphate binding motif having an 8 beta/alpha closed barrel structure which is specific for maintaining conserved phosphate binding site (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The ADH is a member of group of proteins that participate in glucose metabolism. The ADH of A. fumigatus was found to share 61.3% of sequence homology with Cand a1, a major allergen of C. albicans


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(http://fermi.utmb.edu/SDAP/sdap_who.html). These 4 selected molecules (H6, SOU1, ADH and FBA-II) from CHC and secreted fraction of A. fumigatus were subjected to PCR amplification followed by recombinant expression. 4.10. Cloning and recombinant production of selected immunoreactive proteins 4.10.1. PCR amplification and cloning of target proteins Specific primers for the genes encoding for the regions of high antigenic index of each target protein, were designed by in silico primer designing tool (DNAstar). Designed primers were synthesized and specific pairs of primers were used for the amplification of genes namely AFUA_5G03520, AFUA_2G15430, AFUA_3G11690 and AFUA_5G06240 encoding for the proteins H6, SOU1, FBA-II and ADH respectively. The PCR products were run on agarose gels and the DNA bands were visualized. Lanes 1, 2, 3 and 4 in Fig 4.18 are showing the amplified DNA of AFUA_5G03520,

Fig 4.18. Agarose gel (1.5%) showing amplified PCR products of 4 target genes AFUA_5G03520 of 972 bp (Lane 1), AFUA_2G15430 of 801 bp (Lane 2), AFUA_3G11690 of 512 bp (Lane 3) and AFUA_5G06240 of 507 bp (Lane 4). Standard 1 Kb DNA ladder was loaded in Lane M.


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AFUA_2G15430, AFUA_3G11690 and AFUA_5G06240 respectively. The size of each amplified gene product was determined by comparing with standards DNA marker loaded in Lane M (Fig 4.18). The PCR products of different genes AFUA_5G03520 of 972 bp, AFUA_2G15430 of 801 bp, AFUA_3G11690 of 512 bp and AFUA_5G06240 of 507 bp were found to be of desired sizes. The amplified PCR products of genes specific for different proteins were confirmed by DNA sequencing and further processed for cloning in expression vectors for production of recombinant proteins. The prokaryotic system has remained as first choice for expressing recombinant

proteins. Among the prokaryotes E. coli has been considered as the most efficient and

widely used host for manipulation of DNA and proteins. Well-known genetics, high

transformation efficiency, cultivation simplicity, rapidity and inexpensiveness are the

main factors that make E. coli to be the preferred host (Saïda et al, 2006).

Therefore, we also selected prokaryotic expression system and E. coli compatible

two different expression plasmids pET28a and pQE30a were used as cloning vectors. The

vector and insert molecules were digested with restriction endonucleases and gel eluted

prior to performing ligation reaction. The purified linearized vector and digested PCR

products were mixed and incubated with T4 DNA ligase with appropriate buffer for

overnight ligation at 4 ºC. The ligation was confirmed by restriction digestion of the all 4

recombinant plasmids containing inserts of different genes with respective restriction

enzymes. The agarose gel showed the presence of two bands in each lane. The upper

band represented the digested vector plasmid and lower band corresponded to cloned

target gene (Fig 4.19). In case of plasmid containing gene AFUA_2G15430 encoding for

SOU1 (Lane 2, Fig 4.19), position of the vector band was found to be higher (6.5 Kb) in

comparison to actual size of pET28a i.e. 5.4 Kb. This is because the SOU1 was cloned as

a fusion protein in pET28a, downstream to a previously maintained 1.04 Kb nontoxic

bacterial gene (EmbR).


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Fig 4.19. Agarose gel (1.5%) showing restriction digestion of all 4 recombinant plasmids confirming the ligation of each in respective plasmid vector. pET28a- AFUA_5G03520 (Lane 1), pET28a- AFUA_2G15430 (Lane 2), pQE30a- AFUA_3G11690 (Lane 3) and pQE30a- AFUA_5G06240 (Lane 4). Standard 1 Kb DNA ladder was loaded in Lane M.

The insertion of target genes in the plasmid vectors was also confirmed by PCR

amplification of insert molecule from recombinant plasmids using primers specific for

flanking region of vector plasmids. All 4 recombinant plasmids showed the presence of

amplified PCR product inserted genes. The size of all 4 genes was confirmed by

comparing them with standard DNA marker (Fig 4.20).


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Fig 4.20. Agarose gel (1.5%) showing PCR products amplified by using primers specific for plasmid vector regions to confirm ligation reaction of all 4 target genes. AFUA_5G03520 (Lane 1), AFUA_2G15430 (Lane 2), AFUA_3G11690 (Lane 3), AFUA_5G06240 (Lane 4). Standard 1 Kb DNA ladder was loaded in Lane M.

4.10.2. Recombinant expression of proteins The recombinant plasmids expressed the proteins of appropriate size in E. coli,

when cells containing recombinant plasmid were treated with IPTG. The bands of

induced proteins were visualized on the SDS gel after commassie staining. The SDS gels

showed the presence various bands which were similar to the uninduced cell lysate but a

single band of over expressed recombinant protein was present in induced cell lysate of

each clone (Fig 4.21). Successful expression of the recombinant H6 and SOU1 was

achieved in plasmid pET28a. The H6 was expressed as full length protein of 37 kDa

(Fig 4.21A), where as the SOU1 was expressed as fusion protein of 67 kDa (Fig 4.21B).


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Recombinant ADH and FBA-II were expressed in pQE30a as a partial protein of 21 kDa

(Fig 4.21C) and 20 kDa (Fig 4.21D) respectively.

Fig 4.21. Commassie stained SDS gels showing inducible expression of 4 target recombinants, H6 (A), SOU1 (B), ADH (C) and FBA-II (D). Standard protein marker (Lane M), uninduced E. coli BL21DE3 cell lystae (Lane Un), IPTG induced E. coli BL21DE3 cell lysate (Lane Ind).


127

Purification of the expressed recombinant proteins was achieved by affinity

chromatography using Ni-NTA resin specific for C terminal six histidine tag common for

both expression vectors. The commassie stained SDS gels showed the presence of single

band of purified recombinant protein of appropriate molecular weight (Fig 4.22, Lanes

1-4).

Fig 4.22. Commassie stained SDS gels showing purified recombinant proteins. Purified recombinant proteins : FBA-II (Lane 1), ADH (Lane 2), H6 (Lane 3) and SOU1 (Lane 4). Standard protein marker (Lane M).

Expression of full length functional protein has been remained a challenge for molecular biologist. As we also have encountered problems while expressing 3 targeted proteins (SOU1, ADH and FBA-II). The H6 was expressed as full length protein without


128

employing any modifications in its coding region as well as in expression strategy. However, SOU1 was expressed as a protein of 67 Kda including an additional part of 38 Kda, belonged to its fusion partner which was maintained on its C-terminus. It was suggested that the fusion partner neutralizes the toxic effect of the target protein to some extent and increase the stability of recombinant protein in the expression cells. However, it is important to note that the neutralizing partner of fusion protein may not be enough to eliminate the toxicity of target protein completely. But a strongly interaction of fusion partner with target protein, may lead to a suppression of the toxic effect (Saïda et al, 2006). The expression of the mammalian apoptosis modulator protein Bax in E. coli is an important example illustrating the rationale design of a neutralizing partner. The Bax protein leads to the lysis of E. coli cells at concentrations as low as 0.01% of total proteins (Asoh et al, 1998). Attempts to express Bax protein as full length into the periplasm of E.coli failed to diminish its toxic effects, therefore, several strategies have been employed to express it as a fusion protein (Donnelly et al, 2001). We have also applied another expression strategy to express ADH and FBA-II, when we failed to express them as full length protein. Instead of expressing complete gene, we reduced the length of recombinant construct by removing the incompatible regions of DNA by selecting the partial region of target gene. It was speculated that the expression of partial protein may enhance the solubility of recombinants. Interestingly employing this approach, we had been achieved the expression of FBA-II and ADH. It was evident from the literature that many of the genes severely interfere with the survival of E. coli cells during the process of their expression in bacteria and lead to death of host cell or cause significant defects in bacteria growth that dramatically decrease expression capabilities (Saïda et al, 2006). On the other hand less solubility of the recombinant proteins due to their interaction with inclusion bodies and associated toxicity also remained as major problem which is usually encountered several times (Sørensen and Mortensen, 2005). Further, all expressed purified recombinants are subjected for their immunological characterization.


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4.11. Immunological characterization of the recombinant proteins For immunological characterization of recombinants, IgE immunoblots were developed with sera of ABPA patients and healthy subjects. For this purpose all 4 recombinant molecules (H6, SOU1, FBA-II and ADH), crude secreted and CHC antigenic fractions of A. fumigatus and cell lyaste of E. coli strain BL21DE3 (expression cells), were loaded in separate lanes of SDS gels. The control cell lysate of E. coli which was grown in similar conditions without containing any plasmid, was taken to ensure that any contaminating band of E. coli protein would not give false signal on the blots. The proteins loaded in different wells were resolved by SDS gel electrophoresis. The separated proteins from gels were transferred onto PVDF membranes. The IgE immunoblots were developed separately with pooled sera of ABPA patients and healthy subjects using antihuman IgE antibodies conjugated with HRP. The IgE immunoblots demonstrated that the crude secreted and CHC fractions of A. fumigatus showed very high reactivity with positive sera of ABPA patients (Fig 4.23A, Lane 1 and 2). Few faint signals of IgE reactive proteins appeared on the control blots also which were developed with the negative sera of healthy subjects (Fig 4.23B, Lane 1 and 2). The recombinants H6, SOU1 and ADH showed the reactivity with sera of ABPA patients as well as healthy subjects (Fig 4.23A and B, Lane 3, 4 and 5). However, the intensity of band of recombinant proteins (H6, SOU1 and ADH) was higher in the blot which was developed with sera of ABPA patients (Fig 4.23A) in comparison to the blot developed with sera of healthy subjects (Fig 4.23B). The recombinant protein FBA-II showed specific reactivity with anti-Aspergillus IgE as it developed strong band on the blot generated using sera of ABPA patients (Fig 4.23B, Lane 6). It did not show any band with sera of healthy subjects (Fig 4.23B, Lane 6), indicating its usefulness in specific diagnosis of allergic forms of aspergillosis. The H6 was expressed from a clone containing gene encoding for complete protein. The analysis of amino acid sequence of H6 showed that it did not have


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significant sequence match with any allergen available in SDAP database. The immunoreactivity of H6 with both positive and negative sera might be due to the presence of few cross reactive antigenic epitopes in its amino acid sequence. Another

Fig 4.23. Immunoblots showing IgE reactivity of recombinants and

crude antigenic secreted and CHC fractions of A. fumigatus with sera of ABPA patients (A) and control subjects (B). Crude secreted antigen (Lane 1), crude cytosolic antigens (Lane 2), H6 (Lane 3), SOU1 (Lane 4), ADH (Lane 5), FBA-II (Lane 6) and crude E. coli BL21DE3 cell lysate without any plasmid (Lane 7).


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target protein, the SOU1, which was expressed as full length fusion protein showed 78% and 73% homology with fungal allergens, Cla h8 of C. herbarum and Alt a8 of Alternaria alternata respectively. The cross reactivity of SOU1 with both subject groups as observed in the immunblots, may be attributed to its high sequence similarity with other fungal allergens. Further, in an allergen database allergome (http://www.allergome.org/script/dettaglio.php?id_molecule=9530), the SOU1 has been included in the list of allergens of A. fumigatus (Asp fSXR). Therefore, SOU1 could be considered as cross reactive allergen of A. fumigatus. The cross reactivity of ADH also could be due to the fact that it also has high sequence homology (61.3%) with Cand a1 which has been reported to be a potential allergen of C. albicans (Shen et al, 1991). The recombinant FBA-II showed 21% sequence homology with Asp f23 (Saxena et al, 2003) of A. fumigatus and did not show match with any known or predicted fungal allergen. This observation indicated its usefulness in specific diagnosis of allergic forms of aspergillosis. In view of the above observations, it may be concluded that the recombinant

protein FBA-II independently or in combination with other allergenic proteins may have

potential for use in the development of improved more sensitive assays/kits for specific

diagnosis of ABPA. On the other hand, other three recombinants (H6, SOU1 and ADH)

could provide a basis for the development of new therapeutic strategies for

desensitization of patients of ABPA and other allergic disorders. These recombinants and

other consistently reactive secreted and cytosolic proteins of A. fumigatus characterized in

the present study, have also set a panel of leads for the identification of immunogenic

peptides. Such reagents may find more comprehensive use at global level in early

diagnosis and possible immunotherapy of aspergillosis.

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