Expression of IROMPs and effect of Iron on growth

OMP profiles of A. baumannii grown in CDM-Fe and CDM+Fe were compared on SDS-PAGE and it was seen that in absence of iron, bacteria developed 4 new outer membrane proteins which were absent in iron replete conditions. These new proteins are in the range of 77 kDa to 88 kDa and were the IROMPs (Fig. 1).

Monoclonal antibody production

Five best reactive monoclonal antibodies were selected out of 88 clones after 2 fusions based upon their reactivity to IROMPs by ELISA. These monoclonal antibodies were named as 3D₅ABIR, 1D₁₁ABIR, 2G₉ABIR, 1F₇ABIR, and 5D₆ABIR. All of the five monoclonal antibodies were of IgM isotypes (Table 1). These monoclonal antibodies were tested in Western blots. Fig. 2 shows the results of western immunoblots of OMPs. All the monoclonal antibodies reacted with one or more OMP bands corresponding to the range of IROMPs but none reacted with the OMPs of bacteria grown in CDM+Fe media. Two antibodies (5D₆ABIR and 1F₇ABIR) did not show any reactivity on Western blot this may be due to conformational changes of epitope of proteins. All of the antibodies were tested against E.coli and Pseudomonas aerugionsa and none of them were cross reactive with IROMPs of these two bacteria on ELISA.

Characterization of monoclonal antibodies

Uptake of Iron through siderophore

The siderophore was secreted by Acinetobacter buamnnii ATCC 19606 was catechole type as determined by Arnow's method. The uptake of radiolabeled iron siderophore complex by the bacterial whole cells in CDM-Fe was significantly more than that in case of bacterial cells obtained from CDM+Fe medium (Fig. 3). The control tubes incubated only with ⁵⁵Fe without siderophore showed negligible iron uptake. Total radioactivity bound to the filter in case of iron deficient bacteria with radioactive siderophore complex was about 85% out of the 100% radioactivity taken of the neat ⁵⁵Fe. In control as well as with iron rich bacteria it was not more than 15–25%. This suggests that the iron uptake is mediated through siderophore. The data was analyzed by nonparametric Willcoxan Rank Sum Test for two groups. It was found that iron uptake by iron deficient cells was significantly more as compared to iron rich cells (p value < 0.05). Similarly there was a significant difference in CPM reading of iron deficient cells for uptake through siderophore (p value < 0.05) as compared to the control cells in which no siderophore was added.

Binding of ⁵⁹Fe-Siderophore complex to OMP

The outer membrane proteins prepared from cells grown in the absence of iron bound significantly higher amount of radioactive iron siderophore complex as compared to that of the OMP prepared from cells grown in iron rich conditions (p value < 0.05). Similarly there was no binding of radiolabeled ⁵⁹Fe alone to the filters in absence of siderophore which was put up as control. This strongly indicates that OMP of CDM-Fe grown bacteria contains the IROMPs and that they bind to the iron siderophore complex, hence they may be the receptor for siderophore iron complex. (Fig. 4). That binding receptor, a protein was proved by preincubating the OMPs with proteinase K that abolished all the binding activity of the iron siderophore complex (Fig. 4).

Role of monoclonal antibody in Iron pathway

The iron uptake by A. baumannii ATCC 19606 when grown in CDM-Fe was inhibited more than 90% by monoclonal antibodies directed against IROMPs of A. bauamannii, as compared to bacteria grown in CDM+Fe (Fig. 5, p value < 0.05). Similarly, monoclonal antibodies resulted in almost complete inhibition of binding of ⁵⁵Fe siderophore complex to its receptor IROMPs, which are the component of OMPs (p value < 0.05). But there was no effect on binding and uptake of iron siderophore complex in absence of monoclonal antibodies or with control sera (Fig. 5).

Bacterial strains and growth conditions

The Acinetobacter baumannii ATCC 19606 strain was procured from American Type Cell Culture, USA. It was grown in Iron depleted chemically defined media (CDM-Fe) and in iron rich (CDM+Fe) media. Outer membrane proteins were prepared as described by Goel et.al. [32], and the protein concentration was measured by Bradford assay [33]. The OMPs were resolved on SDSPAGE according to Lugtenberg's protocol [34].

Production of monoclonal antibodies

For immunization of Balb/c mice, the area of the SDS-PAGE gel corresponding to the bands representing the IROMPs were cut and used as an antigen [35]. First dose was given Intraperitonealy mixed with 250 μl of complete Freund's adjuvant followed by three doses in Freund's Incomplete Adjuvant at an interval of one week. Final booster was given in normal saline, intraperitonealy, just four days before fusion. The spleen cells were fused with mouse myeloma cell line Sp2O 14/Ag as described previously by using PEG (polyethylene glycol 1450) [36, 37]. Screening of hybridomas was done by testing culture supernatants in ELISA using two different antigens.

(A) OMPs expressed by bacteria grown in CDM-Fe medium i.e. antigen A

(B) OMPs expressed by bacteria grown in CDM+Fe medium i.e. antigen B

Hybridoma supernatants reacting with antigen A alone without any signal with antigen B were selected and subcloned thrice by limiting dilution. Clones of interest were expanded and cultured in the peritoneal cavities of pristane primed Balb/c mice to obtain ascitic fluid.

ELISA

Antigen A and B were diluted to final concentration of 2.5 μg/ml in PBS (pH 7.2). 100 μl of this antigen was added to 96 well ELISA plate (High binding ELISA plates, Costar, USA) and were allowed to adsorb for 16–18 hrs at 4°C. The plates were washed thrice with PBS containing 0.05% Tween 20 (Sigma Co. USA). The nonspecific binding sites on the plate were blocked by 2% nonfat milk powder at 37°C for 3 hrs. 100 μl of the antibody was added to the wells and the plates were further incubated for 3 hrs at 37°C. The rabbit antimouse (Dakopats Switzerland) peroxidase conjugated immunoglobulin was used at working dilution of 1:3000. O-Phenyalenediamine (OPD, Sigma Inc. USA) and H₂O₂ was used as chromogen and substrate, respectively. The optical density were taken by ELISA reader at 492 nm. Cross reactivity of the monoclonal antibodies was checked against E. coli and Pseudomonas aeruginosa IROMPs in ELISA as described above.

Determination of immunoglobulin isotype

The isotyping of monoclonal antibodies was done using commercially available isotyping kit based on ELISA (Sigma Inc., USA) as described by manufacturer.

Immunoblotting

OMPs from SDS-PAGE were transferred electrophoretically to Nitrocellulose paper (0.45 μm Bio-Rad, USA) using semidry blotter (Bio-Rad, USA) as described by manufacturer by a modification of Towbin's [38] procedure. The NC paper was washed twice with PBS pH 7.2. One part of the NC paper was stained with Ponceau S (Merck.Germany) for staining the proteins to check the transfer efficiency and for marking the position of molecular weights. The other part of NC paper was used for immunoblotting. Nonspecific binding sites were blocked by incubating the NC paper in 2% bovine serum albumin and 3% nonfat milk powder at 37°C for 3 hours. NC paper was washed thrice with PBS containing 0.05% Tween 20. The NC paper was incubated at 4°C for overnight with either monoclonal or polyclonal antibodies. The rabbit antimouse peroxidase conjugated immunoglobulin (Dakopats, Switzerland) was used at a dilution of 1:1000 and incubated at 37°C for 3 h. The blots were developed using DAB (diaminobenzimidine) and H₂O₂ as chromogen and substrate, respectively.

Bactericidal assay

Bactericidal assay of monoclonal antibodies was carried out as described by Poolman et.al. [19]. Briefly, 10⁷ cells of A. baumannii ATCC 19606 (grown in iron deficient medium) in 100 μl volume were taken in an eppendrof, 100 μl of monoclonal antibodies culture supernatant (undiluted) was added to it and incubated at 37°C for 1 hour. The human complement from immunoglobulin deficient patient was added (Courtesy of Medical Oncology Unit, AIIMS New Delhi India) to a final concentration of 10% with 10 mM MgCl₂ and CaCl₂, incubated further for 1 hour at 37°C. The reaction mixture were appropriately diluted, plated on to nutrient agar plate and incubated at 37°C for overnight. Bacterial colonies were counted the next day to look for percentage reduction. The percentage reduction was calculated as follows

Control-Test X100/Control

Opsonophagocytic assay

The assay described by Speert et al. [39] was used to measure opsonophagocytic activity of monoclonal antibodies. The polymorphonuclear leucocytes (PMN) were isolated from pooled normal healthy human blood by Ficoll Hypaque density gradient centrifugation. A. baumannii ATCC 19606 (10⁷ bacterial cells/100 μl) grown in CDM-Fe medium were incubated with 100 μl of monoclonal antibody culture supernatant for 30 min at 37°C. To this 10⁶ PMN in 100 μl volume was added and kept in orbital shaker (100 rpm) at 37°C for 90 min. A duplicate set of assay was incubated at 4°C to measure the nonspecific attachment of bacteria. After incubation the tubes were centrifuged at 300 g and cell pellet was washed gently in Hank's balanced salt solution (HBSS) at least thrice. The pellet was resuspended in 0.1 ml of HBSS and one drop of it was placed on glass slide, air-dried heat fixed and stained with Gram Stain. The slide was seen under oil immersion field of light microscope. The number of bacteria within the, cytoplasm of 50 PMNs were counted. The mean number of bacteria per PMN obtained from 4°C assay was subtracted from the number obtained from 37°C assay to get the final results. Same set of experiments was repeated with bacteria grown in CDM+Fe medium.

Electron microscopy

For electron microscopy modified procedure of Root et. al.[40] was followed. Mixture of 10⁷ bacteria and 10⁶ PMNs were combined and processed for opsonization as mentioned above except the last wash, which was in 0.1 M sodium cacodylate buffer pH 7.2. To the pellet 1 ml of 2.5% glutraldehyde (made in 0.1 M sodium cacodylate buffer) was added and centrifuged at 1150 g for 10 min, supernatant was decanted. The cell pellet was fixed by adding 2.5% glutraldehyde for 4 h and the pellet was washed in sodium cacodylate buffer. The pellet was post fixed in 1% osmium tetraoxide, embedded in 1% agar. The embedded sample was dehydrated in graded series of alcohol and finally embedded in Araldite resin. It was sectioned, poststained with uranyl acetate and lead and samples were viewed with a Philips 400 electronmicroscope to look for phagocytosed bacteria.

Blocking of iron uptake by bacteria

For studying the role of monoclonal antibodies in blocking of iron uptake pathway, we purified the siderophore by a modified method of Smith [17]. Briefly the culture supernatant of bacteria grown in CDM-Fe medium was extracted twice with ethyl acetate. The ethyl acetate was reduced to 0.5 ml by rotary evaporation (Buchi rotary evaporator) at 30°C under reduced pressure. This sample was loaded on Whatman 3 mm chromatography paper and developed using solvent system of water:Acetic Acid:Acetone in ratio of 90:10:1. One portion of the chromatogram was stained with 0.1 M FeCl₃ in 0.1 M HCl. The corresponding band was excised and siderophore was eluted in ethyl acetate for 48 hrs at room temperature. After evaporation of ethyl acetate, purified siderophore was re-suspended in distilled water and stored at -80°C. The type of siderophore was determined by Arnow's [41] and Csaky's method [42]. The concentration of siderophore was determined using 2–3 dihydroxybenzoic acid as a standard by Universal Chemical assay as described by Schwyn's [43]. 2 mM of 50 μl purified siderophore was incubated with either 10 μM ⁵⁵FeCl₃ (specific activity 2.068 Mci/g of iron, Bhabha Atomic Research Center Trombay, India) or ⁵⁹FeCl₃ (specific activity 250.49 GBq/g of iron, Bhabha Atomic Research Center Trombay, India). The mixture was incubated at room temperature for 30 minutes. At the end the unbound radiolabelled iron was removed by chromatography using sephadex G-10 as described by Rosenberg [44]. The uptake of radiolabelled iron siderophore was studied by the method described by Rosenberg [44]. Briefly A. baumannii was grown in CDM-Fe medium also in CDM+Fe medium to a mid exponential phase and was washed twice with uptake medium (30 mM Na₂HPO₄, 10 mM K₂HPO₄, 10 mM (NH₄)₂SO₄,1 mM MgCl₂ and 40 μM CaCl₂ pH 6.9) at 4°C and re-suspended in the same medium at a density of A₄₇₀ = 0.4. The washed cells were incubated at 37°C for 15 minutes. The uptake was initiated by adding equal volume of radiolabelled ⁵⁵Fe siderophore to the bacterial suspension and shaken at 300 rpm. Aliquots of 500 μl were withdrawn after every 5 minutes interval and filtered immediately on glass microfiber filter and washed with 2 ml of uptake medium. The membranes were dried at 37°C and measured as counts per minutes on β-scintillation counter (Pharmacia) on the ³H channel. In parallel, bacterial cells were also incubated with radiolabelled iron alone and unlabelled siderophore as a control.

To look for the role of monoclonal antibodies against IROMPs in blocking the iron uptake the bacterial cells were pre-incubated with 100 μl of undiluted monoclonal antibody culture supernatant for 1 h at 37°C before addition of radiolabelled iron siderophore complex. After incubation with antibodies the bacterial cells were washed twice with uptake buffer to remove unbound antibodies. Similarly as like in uptake, radioactivity retained on the filter was counted on β-scintillation counter on ³H channel.

Binding of radiolabelled iron siderophore complex to OMPs

Binding of radiolabelled ⁵⁵Fe siderophore complex to OMPs was carried out as described by Meyer et al.[11]. 100 μg of OMPs in 0.125 M Tris HCl pH 6.8 was mixed with 0.1 ml of siderophore labeled with ⁵⁵Fe (as described above). The total volume of the mixture was adjusted to 1 ml with uptake medium and the reaction mixture were incubated for 10 minutes at 37°C. This was filtered through 0.22 μm filter and washed twice with 2 ml of uptake medium. The radiolabeled iron retained on the filter was counted as counts per minutes on β-scintillation counter on ³H channel. To determine the nature of the binding receptor, the OMP preparation was subjected to digestion with proteinase K (10 mg/ml) for 1 hr at 37°C prior to the incubation with iron siderophore complex.

To test the blocking of iron siderophore to OMP_s, it was preincubated with monoclonal antibodies for 1 h at 37°C as described in uptake experiment and then binding assay was performed