Effect of Vibrio cholerae non-O1 protease on lysozyme, lactoferrin and secretory immunoglobulin A

ELSEVIER

FEMS Microbiology

Letters I35

(I 996) 143- 147

Effect of Vibrio choZerue non-01 protease on lysozyme, lactoferrin and secretory immunoglobulin A Claudia Toma a>*, Yasuko Honma a, Masaaki Iwanaga a-b h Resetrrch lnsrifure

’ Deparrment of Bacteriology, qf Conrprehensil~e Medicine.

Uniwrsir? Faculr\

of the Ryukyus. Nishihuro,

of Medicine,

Received 23 October

Uniwr.si?y

1995; accepted

ofthe

Okina,co Ryky~s.

10 November

YO3-01, Joptrn Nishihura,

Okinmw

903-01. Jopat!

1995

Abstract The effect of Vibrio cholerue non-01 protease on host defense proteins (lysozyme, secretory immunoglobul~in A and lactoferrin) was studied in relation to its virulence mechanism. The proteins treated with the protease were analysed by SDS-PAGE. There was no influence of the protease on lysozyme. The protease cleaved lactoferrin into two fragmgnts of SO kDa and 34 kDa. N-terminal amino acid sequencing of these fragments revealed region, between serine 420 and serine 421. This cleavage could affect the transition is involved in iron binding and release. The anti-bacterial activity of lactoferrin Secretory immunoglobulin A yielded a 42-kDa protein as the cleavage product. globulin A to V. cholerae non-01 protease suggests a mechanism by which immunoglobulin. Keword.~:

Vibrio

cholerae

non-01;

Protease; Lysozyme;

Lactoferrin;

1. Introduction Vibrio cholerue non-01 protease was first purified and reported by Honda et al. 111, and its comparison with the V. cholerue 01 soluble hemagglutinin/protease revealed that they were immunologically and physicochemically identical. Many investigators have postulated the possible roles of V. cholerue protease. At first, it was thought to be involved in bacterial colonization, because digestion of mucin and/or fibronectin might facilitate vibrio approximation to the eukaryotic cell surface [2].

_ Corresponding author. Tel.: + 81 (98) 895 3331; Fax: + 81 (98) 895 295 I ; E-mail: [email protected]. 0378.1097/96/$12.00 0 1996 Federation SSDI 03781097(95)00463-7

of European

Microbiological

Secretory

that the cleavage site was near the hinge from open to closed configuration which was not affected by protease tireatment. The susceptibility of secretory immunobacteria might evade the effect of this

immunoglobulin

A

Nevertheless, recent experiments using protaase-deficient mutants showed that they still had the ability to attach to cultured human intestinal epithelial cells [3] and the protease was likely to play a role io detachment more than in attachment [3]. V. cholarm protease was also considered to nick and activhte the A subunit of cholera toxin (CT) [2]. However, ;the wide distribution of the protease among non-CT-producing V. cholerue non-01 strains suggested more biological roles. Furthermore, proteins that may participate in host defense against cholera, such as mucin, fibronectin and lactoferrin, were digested ob cleaved by V. cholerue 01 protease [2]. Mucosal secretions have an anti-bacterial armamentarium very different from that of serum. There Societies.

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are high concentrations of secretory immunoglobulin A (sIgA), lysozyme, and lactoferrin in the secretions. The killing of V. cholerae by lactoferrin [4], and a bactericidal synergistic effect between lactoferrin and lysozyme [5] or lactoferrin and sIgA [6] have been reported. During in vivo infection, the protease released from bacteria could induce an irreversible degradation or inactivation of locally produced proteins which are potentially important in host defense. Pseudomonas aeruginosa elastase digested lysozyme [7], cleaved lactoferrin [S] and degraded IgA [9]. Although not clear, it is postulated that P. aeruginosa elastase could be an important virulence factor. V. cholerae protease and P. aeruginosa elastase were immunologically and functionally related [81. Therefore, V. cholerae non-01 protease could also be a factor that decreases the host mucosal defense and thus might enhance the pathogenic potential. In the present study, we investigated the effect of non-01 protease on host defense proteins such as lactoferrin, lysozyme, and sIgA.

2. Materials and methods 2. I. Bacterial

strain

Vibrio cholerae non-0 1, strain 93Ag 13, isolated from a cholera-like patient in 1992 in Argentina (provided by Dr. M. Rivas, Instituto National de Microbiologia “C. Malbran”), was used for protease purification. This strain did not produce cholera-like toxin. 2.2. Protease pur@cation Bacteria were precultured in trypticase soy broth (TSB) (Difco, Detroit, MI) with shaking at 37°C for 4 h. The precultured bacteria were inoculated into 500 ml of TSB in a 3-1 Erlenmeyer flask, and incubated for 20 h with shaking at 30°C. The cell-free culture supernatant was fractionated with ammonium sulfate. The 40-55% ammonium sulfate-insoluble material was suspended in 0.02 M Tris . HCl buffer (pH 7.0) and dialysed against the same buffer. The dialysed material was applied to a Biogel A5m column (Bio-Rad Laboratories, Richmond, CA) and the fractions showing protease activity were applied to a

TSK gel G-3000 SW column (Tosoh, Tokyo, Japan) on HPLC as described by Ichinose et al. [IO]. 2.3. Protease acticit?; Human lactoferrin (LF) (Sigma Chemical Co.. St. Louis, MO), human lysozyme (LYJ (Sigma) and human sIgA (Organon Teknika N.V., Cape1 Products, NC) were reacted with purified protease at 37°C for an appropriate time (1 - 16 h) at an enzymeto-substrate molar ratio of I:800 for LF, I :800 and I:5 for LY, and I:300 and I :4 for sIgA. The reaction mixtures were analysed by Laemmli’s sodiumdodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [ 111 with a gel concentration of 9% for LF and sIgA, and 15% for LY. The proteins were visualized by Coomassie brilliant blue staining. 2.4. Amino acid sequence analysis The peptides of LF that appeared on SDS-PAGE were blotted onto a polyvinylidene difluoride (PVDF) membrane (Applied Biosystems, CA). The N-terminal amino acid sequence of the blotted proteins were analysed by automated Edman degradation on a Shimadzu PSQ-I protein sequencer. The cleaving site was determined by comparing with the whole sequence reported by Anderson et al. [ 121. 2.5. Effect of protease on lactoferrin actki8 An inoculum of 5 X 10’ cfu of bacteria was added to 200 ~1 of Bactopeptone 1% containing 330 PM Zincov (a protease inhibitor; Calbiochem, La Jolla, CA). The medium was supplemented with 6 mg ml-’ of LF, or with LF treated with protease. The mixtures were incubated at 37°C aliquots removed at different times and cfu determined.

3. Results and discussion

SDS-PAGE of LY treated with protease revealed that lysozyme was not affected by the protease (data not shown). Although incubation of P. aeruginosa elastase with human lysozyme resulted in disappear-

Ml

175

2

34

56

ante of the native lysozyme band on SDS-PAGE [7], V. cholerue non-01 protease had no effect in this system.

K

3.2. Luctoferrin

83

Treatment of human LF with protease (I $00 for I h at 37°C) yielded two fragments of SO kDa and 34 kDa (Fig. 1. lane 2). Finkelstein et al. 121 also detected two fragments of 55 kDa and 37 kDa when LF was treated with V. cholrme 01 protease. Nterminal amino acid sequencing of the two fragments showed that the cleaving site was in the C-lobe between serine 420 and serine 421 (Fig. 2). LF is a glycoprotein composed of two lobes (N-lobe and C-lobe). Each of them has an iron-binding site in a deep cleft between two domains. The C-lobe is more stable and releases iron slower than the N-lobe [ 121. In the C-lobe. amino acids 407-443 form a ‘backbone‘ strand running the whole length of the lobe that has one interruption in the form of an

62 47.5 32.5

Fig,

I. Coomassie

brilliant blue-stained SDS-PAGE

97~ of LF and

sIgA. Lane M, molecular mass markers; lanes I and 2. human LF: lanes 3-S. slgA. Lane 6. purified protease. Lanes 1 and 3. treated with control buffer; lanes 2. 3 and 5, treated with protease: lane 2. I :8OO: lane 4.

1:300: lane 5.

incubated at 37°C:

I

h for lanes

1:3. All reaction mixtures were I and 2: 3 h for lanes 3 and 3; and

overnight for lane S. Steined hands in lane 3 (from the top): sIgA. IgA. SC. H chain. L chain.

1

N-lobe C-lobe

10

20

GRRRSVQWCAVSNPEATKCFQWQRNMRKVRGPPVSCLKRDSPIQCIQAIAENR ARRAR""WCA"GEQELRKCNQ~SGLS~----GSVTCSSASTTEDCIALVLKG~ 310

350

360

60

70

ADAVTLDGGFIYEAGLAPYKLRPVAAEVYGTE----------RQPRTHYYAVA

C-lobe

A D A M S L D

N-lobe C-lobe

G 1; Y V Y T A G K C

- - G L "

400

C-lobe

FDEYFSQSCAPGSDPR--SNLCAL_IGDEQGENKCVPNSNERYYG~TGAFRCL 490

S

S__D.P_D_P_N_~._.V____~

420 120

P V

E G I

L A " A

430 130

140

470

480 180

500

190

510

210

520

530 240

230

220

KDGAGDVAFIRESTVFEDLSD--------EAERDEYELLCPDNTRKPVDKFKD AENAGDVAFVKDVTVLQNTDGNNNEA~AKDLKLADFALLCLDGKRKPVTEARS 540 550 250

C-lobe

CHLANAPNRAVVSRM--DKVERLKQVLLHQQAKFGRNGSDCPDKFCLFQS~-600

280

290

630

620

610

300 310 QKDLLFKDSAIGFSR"PPRIDSGLYLGSGYFTAIQ~LRKSEKE"AARRAR TKNLLFNDNTECLARLHGKTTYEKYLGPQYVAGITNLKKCSTSPLLEACEFLRK 640 650 660

580

570 270

CHLARVPS~AVVARSVNGKEDAIWNLLRQAQ~KFGKD---KSPKFQLFGSPSG

590

550

260

N-lobe

Fig. 3.

E N Y K S Q Q

VVRRSDTSLTWNSVKGKKSCHTAVDRTAGWNIPMGLLFNQTGSC-------K 440 450 460 150 160 170 "ARFFSASCVPGADKGQFPNLCRLZAGT--GENKCAFSSQEPYFS~SGAFKCL

N-lobe C-lobe

L A

90

4 10

N-lobe

N-lobe c-lobe

P V

100 110 VVKKGG-SFQLNELQGLKSCHTGLRRTAGWNVPIGTLRPFLNWTGPPEPIEAA

200

380

80

N-lobe

390

370

140

330

320

670

690

680

Amino acid sequence of human lactoferrin. Alignment of the N-terminal and C-terminal halves is based on the Uperposition

three-dimensional

structures as reported by Anderson et al. [I?]. The fragments obtained after protease treatement (I:800

transferred to a PVDF

membrane and the N-terminal

SO-kDa fragment. Dashed underline, N-terminal xhaded Ictters.

amino acid sequence was determined. Single underline. N-terminal

sequence of the 34.kDa

fragment.

for

of their

I h) were

sequence of the

Residues involved in iron-binding are indicated by

C. Toma et al./

146

FEMS Microbiology

inserted loop (amino acids 416-426). This arrangement might give some flexibility in opening and closing the inter-domain cleft [ 121. Iron binding occurs to the open form to give an intermediate in which the ligand is only bound to one domain. After that, closure of the domains occurs to allow the ligand to bind to both domains, giving the closed, ligated structure. Therefore, cleavage at this position could affect the transition from open to closed configuration that is involved in iron binding and release [ 121. Although the two lobes present a similar polypeptide chain conformation, the region that was cleaved by protease has no superposition in the N-lobe (Fig. 2). This could explain why the N-lobe was not cleaved by the protease in a homology region. The anti-bacterial domain of LF was identified near the N-terminus (amino acids 18-47) and it was demonstrated that anti-bacterial activity of lactoferrin is independent of its iron-binding property [ 131. Cleavage at the N-terminus might not induce any change in the molecular mass of the whole LF and therefore might not be detected in the SDS-PAGE. To investigate if the anti-bacterial domain was inactivated, the growth of bacteria in media containing LF or LF treated with protease were compared. The anti-bacterial activity of LF was not affected by protease treatment (Fig. 3). Although non-cleaved LF was left after the protease treatment. the effect of LF on bacterial growth is dose-dependent and some change in the growth curve would be expected if the anti-bacterial domain was affected.

Letters

135 (19%)

143-147

3.3. Secretor?, immunoglobulin

A

There was no change in the secretory component (SC) and heavy and light chains of the sIgA after treatment with protease with an enzyme-to-substrate ratio of 1:300. Nevertheless, sIgA disappeared and a 42-kDa protein was detected (Fig. 1, lane 4). When protease concentration and incubation time were increased (1:4, overnight) both the 42-kDa protein and SC disappeared (Fig. 1, lane 5). Finkelstein et al. [2] reported a decrease in trichloroacetic acid-precipitable radioactive material when sIgA was treated with V. cholerae 01 protease. However, they did not observe these changes in the SDS-PAGE. Our results suggest that protease degraded SC, yielding a 42-kDa fragment at low enzyme concentration. When the enzyme concentration and incubation time were increased, further proteolysis of the SC and 42-kDa fragment occurred. Resistance to proteolytic degradation of sIgA due to the binding of SC has been reported [14]. The resistance of sIgA to intestinal proteases makes antibodies of this isotype uniquely well suited to protect intestinal mucosal surfaces. Therefore, the susceptibility of sIgA to V. cholerae non-01 protease reported here might represent a factor that allows the bacteria to escape from the host immune response and to establish infection.

Acknowledgements We thank Tokushu Meneki (Tokyo, Japan) for generously providing bovine lactoferrin used in the preliminary experiments.

References [I] Honda, T.. Lertpocasombat, K., Hata, A., Miwatani, T. and Finkelstein, R.A. (I 989) Purification and characterization of

0

1

2

6

7

incub%on t&e (h) 5

Fig. 3. Effects of lactoferrin (LF) 6 mg ml-’ and lactoferrin 6 mg ml-’ treated with protease (LF+ P) on the growth of V. cholerae non-01 strain in bactopeptone 1% containing Zincov 330 PM.

a protease produced by Vibrio cholerae non-0 I and comparison with a protease of V. cholerue 01. Infect. Immun. 57. 2799-2803. [2] Finkelstein, R.A., Boesman-Finkelstein, M. and Holt, P. (1983) Vibrio cholerue hemagglutinin/lectin/protease hydrolyzes fibronectin and ovomucin: F.M. Burnet revisited. Proc. Natl. Acad. Sci. USA 80, 1092-1095.

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