Indian Journal of Experimental Biology Vol. 37, July 1999, pp. 681-690

Purification and characterization of Klebsiella pneumoniae cytotoxins

B R Singh* , V D Sharma and R Chandra

Department of Microbiology, College of Veterinary Sciences, G. B. Pant University of Agriculture and Technology, Pantnagar 263 145, India

Received 9 October 1998; revised March 1999

Iso lation , purification and characterization of 3 new cytotoxins of a K. pneumoniae strain isolated from ready to eat pork sausage are reported . Purificat ion process involved extraction of cytotoxins with polymyxi n B sulphate. salt precipitation, gel filtration and anion exchange chromatography. Klebsiella cytotoxin (KCT) I. a g lycoprotein of about 65 kDa was verocytotoxic, enterotoxic and dermonecrotic. KCT II was erythemogenic, verocytotoxi c and enterotox ic protein of co 55 kDa, while KE:T '" was about double in MW (110 kDa) hadverocytotoxicity but neither enterotox ic ity nor dermatotoxicity. KCT I and II caused granulation, conglomeration, shrinkage, detachment and lysis of MDBK and Vero cells. while KCT '" induced enlargement, vacuolation, granulation, multinucleolation and syncytia formation in exposed cell s. All the three cytotoxins induced specific neutralizing antibodies and cytotoxins were detectable in nanogram quantiti es with enzyme-linked immunosorbant assay using homologous antibodies. None of the anticytotoxin cross-reacted with ei ther heterologous Klebsiella cytotoxins or with verocytotoxic preparations of Shigella dysenteriae .

Klebsiellosis, an emerging nosocomial infection of animals and human beings I, mostly affects infants, aged and breeding population2

• Although, Klebsiella mostly causes gastroenteritis characterized by acute or persistent diarrhoea and haemorrhagic dysentery, it has also been reported to be associated with septicaemia, pneumonia, mastitis and urinary tract, genital tract and wound infections)' 4.

Cytotonic ehterotoxin, already purified, has been shown to be produced by diarrhogenic Klebsiella strains5

. However, pathogenesis of dysentery like disease and hemorrhagic eneterocolitis caused by Klebsiella has not yet been thoroughly understood . Association of K. oxyloca cytotoxin with haemorrhagic eneterocolitis has been documented6

,7

but K. pneumoniae, the commonest group of Klebsiella associated with haemorrhagic enteritis8 has not been thoroughly investigated for cytotoxigenicity. This paper deals with purification and characterisation of cytotoxins of a pathogenic Klebsiella pneumoniae strain offood origin .

Materials and Methods Bacterial strain-A cytotoxigenic and lethal to

mouse K. pneumoniae sub spp. aerogenes strain isolated from ready-to-eat sausage9 (FK-7) was used

*Present address; National Salmonella Centre (Vet), Division of Bacteriology & Mycology, Indian Veterinary Research Institute, lzatnagar 243 122; India

for isolation, purification and characterisation of its cytotoxins. Shigatoxin producer strain of Shigella dysenteriae (E-91) and nontoxigenic strain of Escherichia coli (E-288) were procured from Nationa l Salmonella Centre (Vet.), Indian Veterinary Research Institute, Izatnagar.

Experimental animals - Adult New Zealand white rabbits and Swiss Webster albino mice (0-3 day-old and adults) were procured from Laboratory Animal Section, College of Veterinary Sciences, Pantnagar and were maintained on pathogen-free diet.

Crude cytotoxin preparation - Cell-free culture filtrates (CFCF), cell lysate (CL) and polymyxin B extract (PBE) of K. pneumoniae (FK-7) and reference strains (E-91 and E-288) were prepared from cultures grown in brain-heart infusion broth (BHIB) for 18 hr at 37°C on rotary shaker (200 rpm)IO. All crude toxin preparations were stored at -20°C until tested after sterilization through 0.20 /-lln membrane filtration .

Cytotoxicity testing- To determine the cytotoxicity units in each of the preparations, Vero and Madin Darby bovine kidney (MDBK) ce ll monolayers were used ll

. Cell lines were procured from Indian Veterinary Research Institute, Izatnagar, India.

Purification of cytotoxin (s)- For purification of cytotoxin, polymyxin B extract of K. pneumoniae (FK-7) was salt-precipitated at .20 to 80% ammonium sulphate saturation level , at a regular interval of 10 %.

682 INDIAN 1. EXP. BIOL. , JULY 1999

Different fraction s were desa lted by ge l filtration though Sephadex G-25 co lumn equilibrated with 0.037 M Tris HCI (PH 7.2) and tested for cytotox ic acti vity 11 . The cytotoxic frac ti ons were pooled together and fi Itered through pre-equi I ibrated Seralose-6B co lumn (70 cmx2 cm) at 0.75 ml/min fl ow rate witb Tris HCI (0.037 M, pH 7.2) and monitored at 280 nm to co llect protein peaks8

. The cytotox ic peaks were poo led separately and ,e luted aga in from the same co lumn and furthe r fractionated on Pharmacia Mono-P FPLC co lumn using Tris-HCI (0.037 M; pH7.2 ) buffer for eq uilibration and elution. Sodium chloride (NaCI) sa lt grad ierit (0 .0 to 2.5 M) at a regular step of 0.5 M (for 20 min each) was app lied to e lute different proteins bound to the co lumn . Cytotoxic peaks were pooled separately and subjected further to ion exchange chomatography as above. Thereafter, cytotoxic proteins were chromatofocused on ethanolamine-HCI (0.25 M, pH 9.6) eq uilibrated

10no-P column and e luted with po lybutfe r 96 and 74 accord ing to the recommendations of MIS Pharmacia (FPLC). Chromatofocussed purifi ed cytotox ins were e lectrophoresed on polyacrylamide ge l and were e luted though Sephadex G I 00 (S igma, USA) co lumn (70 cmx2 cm) al ong wi th molecular weigh t markers to determi ne the molecular we ight (MW) and nature of purified toxic proteins l2

. In different cytotox in preparati ons, prote in was estimated by micro-Lowry method 12.

Enterotoxicity assay - Enterotoxic ity of different purified cytotox ins was determined in rabbit ligated ilea l loop model!:l . /\ preparation yielding dil ation index (01 ) of OA or more was considered enterotox ic.

Enterotox ici ty of purified tox ins was also deter­mined after heat treatment (90°C, 30 min) in suck ling mo use assay (SMA) mode l14 . SMA indices 2 0.07 were taken as pos iti ve .

Derlllatotoxicily assay - Dermatotoxicity was determined by inoculating test preparations ( 10- 100 ~I g protein) in rabbi t skin 15 and mouse food pad (MEP)1 6, intradermally.

Preparation 0/ immune serum - After co llecting pre-immune serum, I 00 ~lg of the des ired cytotoxin was inocu lated intradermally in abdomi nal region of each of 3 rabbits. Three boosters, each of I 00 ~g tox in spaced by 15 days, were given sc . Serum was collected on 10lh day of last booster and stored at -20°C ti ll used. Serum neutra liza tion test was carried out in Vero ce lJ assay model 17

Enzyme-linked imrnunosorban! assay (ELISA) -

To perform the test, 20 ~l g of the test preparati on in I 00 ~tI was serially diluted in 0. 1 M carbonate buffer (PH 9.6) and poured into we ll s of 96 \vell ELI SA plate and incubated for 6 hr at 3rC in a humid chamber for coating the antigen. Then pl ates ,vere washed thrice with PBST (PBS with 0.05 % Tween-20) and unsaturated sites were blocked with 5 % skimmed milk . Suitably diluted (reso lved with checker board method) anticytotox in serum ( J 00 p i) was poured to each well of ELI SA plates and incubated for 4 hr at room tem peratu re fo ll owed by washing as above. Anti-rabbi t IgG horse reddi sh peroxidase conjugate (S igma, USA: I: 10000 in PBS) was then poured into each we ll , in cubated for 4 hr as above, then washed repeated ly. The react ion \\ as visualized by adding I 00 ~tI of orth opheny lene diamine (O PD) HP 2 (prepared fresh by disso lvi ng 10

mg OPD in 10 ml of 0.1 M sod ium citrate buffer. p H 4.5, added wi th 4 ~tI of 30 % H20 2) ; after 20 min, plates were read spectrophotometr icall y at 4<)2 nlll (Am ). Test was performed in trip licate and test preparations y ielding A492 at least 50 % aboye the mean A 5.10 of negative contro l (backgro und leve l) \\as considered as one antigeni c unit.

Characterizat ion of cytotoxin - Puri fled cytoto:-:in preparations were characteri zed phys ico-chemi cal ly. For assay ing the effect of heat, aliquots or test preparations were heated at 600- IOODe at a regu lar interva l of I DoC and at 12 1°C for J mi n as we ll as for 30 min separately. The dfect of proteo lytic enzy mes. viz. , trypsi n, chymotrypsin, papain. bacteria l proteases and lipase on cytotoxi ns was determined by the method of jacks and WU l8

.

For assay ing the effect of p I I (2- i 0) on cytoto:-:i­city, the pH of tox ic preparations \\ as adjusted fr0111 :2 to 10 at a regul ar interva l of 0.5 with I N He l or I 1\ NaOH and then incubated at 37De fo r 4 hr and fina lly p H was readjusted to neutra lity. Effect of different chemica ls, namely ethylene di ami ne tetra acetic acid (EDTA, I %), mercuri c chloride (0.5 %), formald e­hyde (0.5 %), acriflavin (0. J % ). acetone (50 % ). ethanol (90 % ), methanol (90 % ), acetic ac id (J Nl. sodium hydroxide (I Nand 5 N), urea ( 5 M)' sodiu m desoxycholate (0.2 %) and sodi um carbonate (0 .2 tv!) on different cytotox in preparation . was studied following the method of Rahman et a/19

.

The cytotoxicity and antigeni city of above treated preparati ons were determined on Vero ce ll s ll and with ELISA, respectively and res ults were compared with that of untreated control.

SINGH el..aI.PUR lfl CATION & CHARACTERI ZATION OF KLEBSIELLA PNEUMONIAECYTOTOX INS 683

Results Results (Table I) revea led that from I Lt culture of

K. pneumoniae (FK-7) a total of 199840 verocytoxic units could be obtained of which only 15 .13 % were detectable in CFCF. Maximum number of cytotoxic units (CTUs) per mg of protein were detected in PBE and least in CFCF. Polymyx in B treatment extracted about 92 % toxicity vested with bacterial cell s. Similar results were observed in dermatotoxicity assay in rabbit skin and mouse foot pad test. The MFPT indices were 127, 196 and 178 for CFCF, CL and PBE, respectively.

Salt precipitation - Salt precipitated protein (SPP, subscript revealing the salt saturation level) fraction s SPP20, SPP70 and SPPso had no cytotoxicity on Vero or MDBK cells (Table I) , while SPP,o had 32 and 64 cytotoxic units (CTU s)/mg of protein on Vero and MDBK ce lls, respectively. In both of the cell lines exposed, it caused enlargement, granu lation, vacuola­ti on, multinucleation and syncytia formati on followed by death and detachment of cell s after 48 hr of exposure. Giant cells and syncytii were best observed in MDBK cell cultures in MDBK ce ll s. Enlargement of nuclei was evident with vacuo les and multiple nucleoli karyoplasm. In rabbit skin, it induced a narrow zone of erythema which vanished within 24-36 hr leav ihg behind a rough surface at the inocu lated site. SPP40, SPP50 and SPP60 had 128, 256 and 16 Vero cell CTU/mg of protein, respectively. They all induced CPE similar to CL preparations of reference shigatoxigenic strains (E-9 1) characterised by granulation, wrinkl ing, conglomeration, rounding,

vacuo lation, death and detachment of exposed ce ll s. In rabbit skin all the 3 SPPs caused erythematous zones and n.ecrosis. SPPso was the most dermatotox ic. The rabb it inocu lated with these SPPs had sloughing of sk in at inoculation site and depilation at remote sites. Thus on the basis of similarity in their cytotox icity, all the 3 SPPs i.e. SPP40, SPPso, SPP(,o were pooled together.

Gel filtration through Serolose 6B column ­Elution of SPP}O through Sera lose 6B 'co lumn formed two major protein peaks eluted in 23rd to 27th ml and 35th to 44th ml of gel filtrate (Fig. I) . Of these, only first peak had cytotoxicity and dermatotoxicity (Tab le 2) similar to that induced by SPp}o.

Gel filtration of SPP40.60 yielded 4 major and two minor peaks in 23 rd to 271h, 35 th to 44th , 45 th to 62"°, 65 th to 88th , 95 th to 97th and 102"d to I 041h ml of e luent (Fig. I). Of the 6 peaks, only first and 3rd peaks had cytotoxic ity and demermatotox icity (Table 2). The first protein peak from SPP}O as we ll as from SPP40-60 had similar CPE in cell lines and dermatotox icity in rabbit skin (Table I). Furthermore, they were eluted in the same elut ion vo lume so both were poo led and designated as S6B-1.

The 3rd peak protein(s) from elution of SPP40-60 wa named as S6B-1I1. It had 512 Vero cel l and 256 MDBK ce ll CTU/mg of protein . The CPE in exposed ce ll s and dermatotoxicity in rabbit skin was similar to that caused by SPP40-60 .

Ion exchange chromatography of S6BI and /// through Mono-P FPLC Pharmacia column - Ion exchange chromatography of S6B I yielded two maj or

Table 1- Dcr!llatotox icity and cytotoxicity of crude and sal t-precipi tated preparation of K. p nelllll olliae subsp. aer og ellase

Type of I uta I protein Dermatotoxiciti' in rabbit Cytotoxici ty in preparation recovered from I L skin Vero ce ll s MDBK Cell s

cu lture in mg Eiythema Necrosis CPE CTU I mg CPE CTU / mg (zone diamcter in 111m) protein protein

CFCF 18')0.0 2 R,G,Y,D,L 16 En, R,G, V ,M.D,L 16 CL 2650.0 32 10 R.G,V,D,L 64 R,G,V,D,L 32 POE 1220.0 27 12 R.G ,V,D,L 128 R,G,Y,D,L 128 SPPzo 25 .5 SPPJO 84.2 5 G,V,M,D 32 En,G,V,M,Q 64 SPP40 40 .0 10 2 R,G,V,D,L 128 R,G,V,D,L 128 SPP50 200.6 17 12 R,G,V,D.L 256 R,G.V,D,L 256 SPP60 248.0 5 R,G,V,D 16 R.G.V.D 8 SPP70 180.0 SPPgO 70.8 A

CFeF, cell-free culture filtrate : CL, cel l lysate ; PBE, polymyxin B extract; SPP, sa lt prec ipitation at different concentrations indicated in % saturati on; CPE, cytopathic effect; CTU. cytotoxic un its; En, enl argement ; R, rounding; G, granulation; V. vacuation; M, multinucleation; D, detachment; L, lysis; -, negative results; A. inocu lated area became red and swollen within 30 min and reddening vanished within 4-6 hr.

684 INDIAN J. EXP. SIOL., JULY 1999

and one minor protein peaks eluted with 1.5 M and 2.5 M salt gradients, respectively (Fig. 2). Of these 3 peaks, 2nd was cytotoxic and dermatotoxic.

Ion exchange chromatography of S6BIIl proteins yielded many protein peaks at different salt gradients (Fig. 2) with different biological activities (Table 2). First major cytotoxic peak consisting of unbound protein contents eluted without any salt gradient, was flanked on either side by one minor peak. Of the three major peaks eluted at 0.5 M salt gradient, first two had similar CPE and dermatotoxicity but the 2nd was more toxic than the first.

Accordingly, the two cytotoxic peaks yielded on ion exchange of S6BIlI at 0.00 M and 0.5 M NaCI gradient were designated as KCT-I and KCT-II, respectively. Cytotoxic peak eluted at 1.5 M salt gradient on anion exchange chromatography of S6BI was designated as KCT-IJI. .

Biological characteristics of KCTs KCT-I-The purified KCT-I had 512 CTUs/mg

protein and was equally active on MDBK and Vero cells (Table 2). It caused rounding of cells within 3 to 4 hr of exposure followed by wrinkling, conglomeration, detachment and lysis within 30-48 hr. Nuclear or cytoplasmic changes like vacuolation, multinucleation, enlargement and syncytia formation were not evident.

On intradermal inoculation in rabbit, it induced no major change except a narrow zone of erythema within first 18 hr but in next 18-24 hr a large area (2-3 cm diam.) around necrosed centre became red, hot and oedematous, oozing out clear serous fluid which formed a thick scab within next 18-24 hr, necrosed mass of skin sloughed off after 3-5 days leaving behind wound which healed up in 15-20 days. After a week of KCT-I inoculation, depilated patches appeared on skin of wither and shoulder regions with underlying erythema area which got covered with brown scab in i -3 days of appearance.

In mouse foot pads, KCT-J caused oedema with a maximum index up to l42 in 24 hr which subsided within 2-3 days. However, foot pads continued to be hard and painful. After 5-7 days of inoculation, mice had focal areas of necrosis on their hind qual1ers and tail. The lesions were covered with black scab and the whole affected regIon lost hair. Subsequently, between 8th and 10th day post inoculation , most of the mice inoculated with > 50 mg of KCT-J in their foot pads developed paraplagia and died .

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SINGH el. aJ. :PURIFICATION & CHARACTERIZATION OF KLEBSIELLA PNEUMONIAECYTOTOXINS 685

The KCT-I induced accumu lation of haemorrhagic fluid in RLIL segments (Table 3) and stomach of infant mouse . It also caused retention of considerable amount of flui~ in stomach, gall bladder and urinary bladder of inoculated animals.

Thereafter, erythematous skin became wrinkled and cracks appeared in 36-72 hr. Cracked and dried superficial skin desquamated in 3-7 days. In mouse foot pads, oedema indices were as high as 210 within 12-18 hr of inoculation . The oedema subs ided in next 2-3 days. KCT-II - Each mg of purified KCT -II had 1024

CTUs. The MDBK cells were half as sensitive as the Vero cel ls (Table 2), though CPE was simi lar. It caused rounding vacuolation and detachment of exposed cells similar to that caused by preparations of shigatoxigenic reference strain (E-91 ).

KCT-II caused serosanguinous fluid accumulation in RLIL and SMA models with test ind ices 0.82 and 0.142, respectively (Table 3) . The inoculated infant mouse died (within 6-8 hr) had diarrhoea, wrinkled and wet skin and blui6h dry mucous membranes.

It was potent erythemogenic in rabbit skin producing erythematous zones of >2 cm in diameter with moderate oedema within 18 hr of inoculation.

KCT-III -It was not enterotoxic in RLIL or SMA. It had 512 CTUs/mg of purified protein (Table 2). The Vero cells were half as sensitive as MDBK cells

0

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Table 3 - Enterotoxicity of different cytotoxic preparation of K pnellmoniae subsp. aerogenes (FK 7). Eneterotoxicity assay Cytotoxic preparation

Rabbit ligated ileal loop assay (100 Jlg protein/ loop) Dilatation index Fluid character Suckling mouse assay- index (40 Jlg protein/ mice)

CFCF CL PBE KCT-I KCT-II

0 .04 H, Y 0.096

0 .62 H, Y 0 .102

0.78 H, Y 0.122

0 .62 H, Y 0.064

0.82 S

0. 148

KCT-1I1

0 .12 M

0.059

CFC F, cell-free cul ture filtrate; CL, cell lysate; PBE, polymyxin B extract; H. haemorrhagic; Y, viscous; S, sero sanguinous; M, mucus type contents; KCT-I ,cytotoxin eluted unbound from column-P as peak I; KCT-II , cytotoxin eluted as 2nd peak with 0 .5 M NaCI grad ient; KCT-I\I, cytotoxin eluted as 1st peak with 1.5 MNaCI gradient.

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686 INDIAN 1. EX P. BlOL. , JULY 1999

and changes were less pronounced in the former. The cellular changes incl uded, enlargement, mu ltinucleation, multinucleolation, syncytia formation, vacuolation and granulation followed by detachment of cells.

It caused only slight erythema fo llowed by a wide zone of wrinkled, dry and rough parchment . Iike surface at the site of inoculation in rabbit ski n. After 5-7 days of inoculation the surface lesion sloughed off leav ing behind a shi il ing healthy skin.

Immunogenicity and serological tests - All the three KCTs were fo und immunogenic in rabbits. Serum neutralization test (SNT) revealed that only homologous cytotox in was neutralized by the respecti ve anti serum. Hyperimmune sera against KCTs neutralized cytotox icity of crude preparations of Klebsiella only part ly, however, anti-KCT-I and anti-KCT-II in combination neutralized dermatotoxi­city and enterotox icity of crude preparations com­pletely. Cytotoxicity of shigatoxic preparations of reference Shigella strain {E-9 1) were not neutralized even partly by any of the antisera. Serum neutrali­zation titres of ant icytotoxin I, II and III were 256, 80 and 32, respecti vely.

ELI SA detected KCT-I , II and III at 180 I-lg, 80 I-lg

and 6 I-lg/ml concentration, respectively. ELI SA ti tre of anti-KCT-I, IJ and III were 1.6x 104

, 3.2 x 104 and 6.4x 104, respectively.

Recovery of cytotoxins during purijication - On Iy 8.04 % cytotoxicity in total was recovered after fina l purification (Table 4). The max im um loss (- 60%) occurred during salt precip itation, desalting and dia lysis followed by 10.7 % and 18.4 % losses durin g

Table 4 - Recovery o f cytotox in(s) at dilferent purification steps

Process product No. of total Recoveryof Step to verocytotox ie cyto toxin (%) step

unitsl l of culture loss (%)

CFCF 3.02x 104 15. 13 A CL 1.696x I 0' 84 .87 NA PB E 1.562x I O ~ 92 .08 7.92 Salt precip itation, 6 .3 14x 104 37.22 59.56 desalting and dialys is Seralose 6B filtrati on 4.492 x I04-· 26 .49 28 .83 Ion exchange 1.6 10 x 104" 8.04 64.25 chromatography

C FCF,ce ll-free cu lture filtrate; CL, ce ll lysate; PB E, polymyx in B extract; ** cytotoxic units from all peaks were cumu lated to calcu late total vcrocytotoxic ity; NA, not appli cab le.

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SINGH et. ato :PURIFICATION & CHARACTERIZATION OF KLEBSIELLA PNEUMONIAECYTOTOX INS 687

gel filtration and Mono-P-column chomatography, respectively.

CFCF contained least number of antigenic units of KCTs per mg.of protein, followed by CL and PBE (Table 5). The final recovery of cytotoxin antigens after Mono-P-column chomatography was calculated to be 23.33, 49.68 and 29.12% for KCT-I, II and III , respectively. The antigenic units/mg protein of purified KCT-] , II and III preparations were 5.6x I0

4,

3 .6x 104 and 1.6x 105 with an overall concentration of about 218.75, 140.63 and 312.5 times, respectively.

Molecular weight - Elution of purified prepara­tions from Seralose 6B column revealed that KCT-I , II and III had molecular weights approximately 65 , 55 and 110 kDa, respectively.

pIs of cytotoxins-Chomatofocusing studies on Mono-P-FPLC column revealed the pIs of KCT-] , II and III were 9 .0, 7.0 and 5.6, respectively.

Nature of cytotoxic proteins - Native polyacryl­amide gel electrophoresis results revealed that KCT-] was a glycoprotein and stained pink with PAS stain . In addition, the preparation also contained two minor proteins as evidenced by PAGE analysis. KCT-II preparation also produced two bands on PAGE, the minor band appeared to be of contaminating protein. KCT-I11 was identified as pure cytotoxin proteins and formed single band on slab gels.

Heat stability-On heat treatment at 90°C (30 min), there was no loss in cytotoxicity of KCT-] and KCT-III , while KCT-II lost 50 % of its cytotoxicity At 100°C (30 min) KCT-II was completely inactivated while KCT-I and III retained 6.25 and 18.75 % of their cytotoxicity, respectively. None of the KCT resisted heat treatment at 12 1°C even for I min. The antigenicity of all the KCTs remained intact at 90°C (30 min) but at 121 °C (I min) all the three KCTs lost their antigenicity completely.

Effect of enzymes - Lipase and papain treatments had no effect on cytotoxicity of KCTs or PBE of FK 7 strain . Protease did not have any adverse effect on cytotoxicity of purified KCTs but 50% cytotoxicity of PBE was lost with the treatment. On the other hand, trypsin and chymotrypsin treatments enhanced the cytotoxicity of PRE, KCT-] and KCT-II by 100 and 60 %, respectively but reduced the activities of KCT­III to zero.

Effect of pJ-J-KCT-I , II and III retained their complete cytotoxicity in pH ranges of 5.5-8.5, 6.5-8 and 5-7, respectively. Beyond the above pH ranges KCTs lost their cytotoxic ity gradually and most of

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688 INDIAN 1. EXP. BIOL.. JULY 1999

them became completely inactive at pH 4 and 10. Antigenicity was also adversely affected at extreme of acidic and alkaline pH . All the toxins retained their antigenicity int{lct between pH 5.5 to 9 but none could tolerate pH s 3.5 , however, alkaline pH was much better tolerated and toxins could be detected with ELISA even after exposure to pH 10.

Effect of chemicals - EDT A treatment reduced the cytotoxic ity and antigenicity of KCT-I11 to zero. Although the antigenicity of KCT-I remained intact, its 75% cytotox icity was lost. KCT-II was resistant to EDT A treatment. Mercuric chloride and acrif1avin had no effect on antigenicity of KCT-I and KCT-II . KCT-III lost its 43.75% antigenicity due to treatment of mercuric chloride but acrif1av in had no effect. Effect of mercuric chl oride and acrif1av in were not determ ined on cytotoxicity.

Acetone (5 0%) and methanol (90 % ) reduced cytotoxicity of KCT-I by 50 and 70 %, respective ly but its antigenicity remained intact; effect on KCT-I1 and III was not assessed . Methanol (90%) treatment of KCT-J resulted in loss of its 75% cytotoxic ity but had no effect on anti genici ty. Ethanol (90%) treatment caused 50 and 87. 5% loss in toxic ity and 21 .4% and 0 0/.0 loss in anti genici ty of KCT-I and II , respective ly . Effect on KCT-JII was not eva luated .

Aoetic acid ( 1 N) treatment completely inactivated C)1otoxicity as well as antigenicity of KCT-J and II. KCT-II1 also lost its cytotoxicity completely and antigenic ity considerably (99. 7 %).

Sodium carbonate (0.2 M) and formaldehyde (0 .5%) treatment resulted in loss of 100% cytotoxic ity of all the KCTs but their antigenicity was not affected . Sodium hydrox ide (5 N) treatment caused comp lete loss of ant igen ic ity and cytotoxicity of all the KCTs. Also, IN NaOH destroyed cytotoxicity of KCT-II and III completely and 99 .2 % of KCT-J while an ti genici ty was also affected considerably (83.9- 100 %).

Urea (5 M) treatment red uced the cytotoxicity of KCT -I by 75 % but had no effect on cytotox i ~ ity of KCT-Il and III . Ant igenic ity of KCT-I , IJ and III was enhanced by 71.4, 11.1 and 50%, respective ly. Sodium desoxycholate (0 .2%) treatment had no effect on cytotoxicity of KCT-II and III , but 75% cytotoxicity of KCT-I was lost, while antigenicity of KCT-I , II and' III was increased by 60.7, 77.8 and 100%, respectively.

Discussion Extracellular release of only 15.13 % of total

cytotoxic activity of Klebsiella culture in C FCF revealed celi-bound nature of KCTs. Serological studies further revealed that only 26.29, 41.63 and 26 .29% of KCT-I , II and Ill , respectively . (Table 5) were released extracellulariy in CFCF by K. pneumoniae (FK7) strain under study, while rest of the cytotoxin could be released only after cell lys is. Cell-bound nature of different enterobacteria l cytotoxins v iz., Shigella, Salmonella and E. coli is well docum ented20

, 22 , 23, but there appears to be no report on ce ll bound nature of Klebsiella cytotoxins . The present study further revealed that there exi sted vanatl on wi th regard to release of diffe rent cytotoxins. Similar variations in extrace llular re lease of different cytoto. ins of Salmonella22 and E. co/i20

stra ins have a lready been reported .

Difference between per cent re lease of cytotoxins detectable with cytotox icity assays and sero logical assays mi ght be attri buted to presence of other minor cytotoxin s, whi ch may be more tightly bound to bacteria l cells, but failing to react in serological tests because of unavailability of specific antibodies to them, though it needs further confi rmation.

PB E of FK-7 stra in conta ined about 5.5 times more cytotoxic ity than in its CFCF and contained about 92 % of cytotoxic ity present in C L revealing potentia l of polymyxin-B to enhance extracell ular re lease of cell­bound cytotoxins of Klebs iella. Furthermore, cytotoxic units/mg of PBE protei n were about twice than in CL prote in . Therefore, polymyxin-B could be used fo r extraction of cell-bound Klebsiella cytotox in(s) for sca ling the purification. Simi lar observations have been a lso reported with Salmonella I 0 . Al though, there appears to be no earlier report on enhancement of extracell ular re lease of K. pneumoniae cytotoxi n with polymyx in B to compare the effi cacy of protoco l adopted in the present study.

Simjlar protocols of cytotox in purification have been used earl ier for puri fi cat ion of shi gatoxin2o

, E. coli cytotoxi ns20 and Salmonella cytotoxi ns21

, 22.

However, in ail earl ier stud ies, e ither DEAE or Mono­Q co lumns were used fo r anion-exchange chomatography; while in this study Mono-P column was preferred because on fo rmer two co lumns KCT­III bound too tightly to be e luted even with 5.0 M NaC l salt or pH (nol. -detrimental to toxin) gradients. Further use of Mono-P column for chromatofocussing , made it economical and convenient.

The overall cytotox in recovery in terms or cytotoxic units was approx imately 8.0% . The

SINGH et. at. :PURIFICATION & CHARACTERIZATION OF KLEBSIELLA PNEUMONfAE CYTOTOXINS 689

maximum loss (~ 60%) in cytotoxic units. of KCTs occurred during salt precipitation as reported earlier in purification of cytotoxins of other enterobacteria22. Comparatively, the loss of antigenicity of cytotoxins was much less than cytotoxicity as 23.3-49.6% antigenicity of different cytotoxins was retained in finally purified preparations. This suggested that during purification some of the cytotoxic activity get inactivated while it remained antigenically detectable as proposed earlier20, 2' .

Investigations on enterotoxicity revealed that of all the 3 cytotoxins, KCT -fI was the most enterotoxic one with dilatation indices much hi gher than KCT-I and III , while haemorrhages were more evident in RLILs inoculated with KCT-I suggest ing that KCT-I and KCT-II play maj or role in pathogenesis of haemorrhagic enterocolitis often reported to be caused by Kpneumoniae4

. Further studies to quantitate the ro le of different cytotoxins in pathogenesis of klebsiellosis using genetically-manupu lated mutants.

Though all the 3 KCTs induced CPE in both the cells, sensitivi ty of two cells varied for different KCTs. Vero cel ls being more sensitive for KCT-II and MOBK cells for KCT-lII, while KCT-I was equa lly toxic to both the cells suggesting benefits of use of more than one cel l line for assessing cytotoxicity. Of the 3 cytotoxins, KCT-llJ was the least cytotoxic and seems to contribute only litt le to cytotoxicity of the crude preparations. On the other hand , CPE induced by KCT-IJ and KCT-I resembled the changes produced by crude cytotoxin preparations of Klebsiella and shigatoxigenic Shige lla suggesting their important role in disease causation similar to shi gatoxin, E. coli SL Ts and Salmonella cytotoxi ns

. " 1 CPE, 02022 causlllg sImI ar . , .

l)ermonecrotic activity was detected on ly in KCT­k whi le the other two KCTs did not cause necrosis in ;abbit skin. Therefore, it seems to be responsible for dermonecrosis caused by crude extract. Klebsiella is known to often contami nate wound and cause abscesses ' . Production of dermonecrotic cytotoxin may be contributing to abscess formation by Klebsiella, similar to necrotoxigenic E. coli, which have often been iso lated from cutaneous abscesses2o

.

However, there appears to be no earl ier report on prod uction of dermonecrotoxin by K pneumoniae, wh ich may be due to lack of attem pts to identify it. Further, studies are requ ired to eva luate the ro le of KCT-I in pathogenesis of abscess/ wound infection by Klebsiell a before reaching any conc lusion.

Chemically, KCT-I was found to be a glycoprotein while other KCTs were proteinous in nature having different molecular weight and pI va lues. Comparatively, high molecular weight of KCT-Ill explains as to why it was eluted much earlier than rest of the 2 KCTs from Seralose 6B column . There appears to be no earlier report on MW of K pneumoniae cytotoxins to compare the finding of th is study. Although K. oxytoca cytotoxin has been reported to be a low MW (217 Oa), nonproteinous, comparatively heat labile (70°C, 30 min) substance6 7

,

no · such cytotoxin was detected 111 Klebsiella pneumoniae strain under present study.

High pI of KCT-l exp lains its fai lu re to bind to Mono-P co lumn even at pH 8.6. Paltial retention of cytotoxicity even after exposure of crude cytotoxin to a wide pH range (4- 10) could be ascribed to wide variation in p I-va lues of different KCTs .

Bacterial protease reduced 50 % cytotox icity of crude cytotoxin , but surprisingly it had no effect on any of the purified KCTs. How it reduced the toxicity of crude preparation is not clear and need further investiga tion. None of the other proteo lytic or lipolyt ic el1.lymes including lipase, papain, trypsi n and chymotrypsin had adverse effect on cytotox ity of crude cytotoxins (PBE). It was interesting to note that tryps in and chymotrypsin significantly enhanced the cytotox icity of KCT-J and KCT-IJ while the enzymes had adverse effect on the activity of KCT-lI1. This is significant as cytotoxin can exert its toxic effect even in presence of enteric enzymes and thereby contribute to the pathogenicity of the organism. Poss ibly overal l increase in cytotoxicity of crude preparations after treatment with trypsin and chymotrypsin could be attri buted to the presence of KCT-I and II therein . Though it has not been reported earli er in Klebsiella, cytotox ic act ivities of some of the E. coli SL Ts have been shown to be enhanced by proteolyti c enzymes whil e that of others get inactivated or remai ned unaffected 20.

Bes ide enzymes, no other chemica l or physical treatment caused increase in cytotoxicity of any of the KCTs. Though urea and sodium desoxycholate enhanced the anti geni city of KCT-I,II and "' (II to 100%), both the treatments had adve rse effect on cytotox icity of all the three KCTs. This Illay be attributed to the fact that both these substances cause dissociation of sulphide bonds and straighten the native protein 1ll0lcc ule' 2 resulting in loss of cytotox icity but making the cytotoxic moiety

690 INDIAN 1. EXP. SIOL., JULY 1999

antigenically more active due to exposure of hidden epitopes.

Another interesting finding was inactivation of biological activities of KCTs while retaining their antigenicity intact with 0.20 Iv! sodium carbonate and formaldehyde treatment. This finding may be of use in toxoid vaccine preparation for immunization against Klebsiellosis but further studies are essential to reach the conclusion.

Native PAGE analysis revealed only partial purification of KCT I and KCT II, while KCT III appeared to be' a homogenous protein and necess itate further studies for purification of KCT I and II to homogen~ity .

The purified KCTs did not share antigenic determinants with each other and Shigatoxin as evidenced by failure to cross-react serologically or cross-neutralize the cytotox icity to Vero cells proving the multiplicity of KCTs as has been suggested by the earlier workers on the basis of their preliminary investigations on clinical isolates of Klebsiella4

,24.

This study further indicates necessity of work on KCTs for better understanding ·of their role in pathogenesis of Klebsiellosis.

Acknowledgement Financial assistance in the form of Senior Research

Fellowship from Council of Scientific and Industrial Research, New Delhi and study leave granted to BRS by Indian Council of Agricultural Research, New Delhi are thankfully acknowledged. The authors are also thankful t{) Dr. J. c.. Verma OIC National Salmonella Centre (Vet.) for providing reference Shigella and E. coli cultures.

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