Impact of Inactivated Extracellular Proteases on the Modified Flagellin Type III Secretion Pathway of Bacillus halodurans

The flagellin type III secretion pathway of Bacillus haloduransBhFC01 ( ${Delta}$ hag) was modified by the inactivation of fliD. An in-frameflagellin gene fusion polypeptide construct was expressed, andthe heterologous peptides were secreted as flagellin fusionmonomers. The stability of the secreted monomers was significantlyenhanced through gene-targeted inactivation of extracellularproteases.

INTRODUCTION

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INTRODUCTION

Gram-negative bacteria use specialized systems to secrete selectedproteins outside the cell. One example is the type III secretionsystem, whose substrates include virulence proteins (8). Theflagellar type III secretion apparatus efficiently secretesthe flagellin subunit protein FliC in both gram-negative andgram-positive bacteria. Most flagellar components are translocatedacross the cytoplasmic membrane by the flagellar type III system,and they self-assemble at the distal end of the flagellin channelwith the help of a cap structure. Flagellin filaments polymerizeoutside the cell. However, Escherichia coli mutants with a nonfunctionalFliD protein (cap structure) fail to assemble flagella, andFliC monomers diffuse into the culture medium (11).

Stahl and La Vallie (M. L. Stahl and E. R. La Vallie, 23 April1987, international patent publication no. WO 87/02385) suggestedthe use of the Bacillus subtilis flagellar type III system toexport heterologous proteins from host cells. This process involvedthe inactivation of the B. subtilis hag gene, a homologue offliC (10). Fusion constructs were generated whereby the heterologousgene was attached to the 3' terminus of the full-length flagellingene. The expected fusion proteins were, however, detected onlyby Western blotting. The FliD cap protein responsible for thepolymerization of the FliC monomers was still present and wouldtherefore substantially hinder secretion (11). A modified flagellartype III secretion system for E. coli was created through gene-targetedinactivation of the fliC and fliD genes, and the ${Delta}$ fliC deletionwas successfully complemented with heterologous polypeptidescloned as in-frame fliC fusion products and secreted (11).

The alkaliphilic B. halodurans Alk36 strain produced FliC atelevated levels compared to those produced by B. subtilis. Aflagellin surface display system was therefore developed bythe inactivation of the hag gene and subsequent complementationon a multicopy vector of in-frame chimeric flagellin gene fusions(3). In this report, we describe the development of a host strainfor heterologous peptide expression utilizing the flagellartype III secretion apparatus of B. halodurans BhFC01.

Bacterial strains and plasmids used in this study are listedin Table 1. Growth conditions and DNA techniques were describedby Crampton et al. (3). Primers are listed in Table 2.

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TABLE 1. Bacterial strains and plasmids used in this work

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TABLE 2. List of primers and their corresponding nucleotide sequences

Construction of FliD- and extracellular protease-deficient strains through gene-targeted inactivation.

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Construction of FliD- and...

Due to the high degree of sequence identity between the genomesof B. halodurans Alk36 and B. halodurans C-125, primers forthe inactivation of the fliD and protease genes were designedaccording to the sequenced genome of B. halodurans C-125 aspublished in the DNA Data Bank of Japan (http://gib.genes.nig.ac.jp).The sequential inactivation of protease genes was achieved bycreating a defective copy of the gene of interest by PCR amplificationof two fragments containing part of the 5' and 3' regions ofthe gene (Fig. 1). These fragments were ligated to the temperature-sensitivevector pSEC194 (3), and the appropriate B. halodurans strainwas transformed with the construct. The gene was inactivatedthrough a double-crossover event in a combination of two methodsby Biswas et al. (1) and Poncet et al. (12). A fresh B. haloduranscolony containing the defective gene construct was grown overnightat 52°C in Luria-Bertani broth (LB; pH 8.5) with 10 µgof chloramphenicol/ml, and serial dilutions were plated ontothe same medium and grown at 52°C. A putative single-crossoverclone was identified by PCR amplification and grown in LB (pH8.5) overnight at 30°C to force a double-crossover event.Dilutions were plated onto the same medium. The colonies obtainedwere transferred onto LB (pH 8.5) plates with and without antibiotic.Chloramphenicol-sensitive clones were screened through PCR amplificationfor the presence of the defective gene on the chromosome.

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FIG. 1. Schematic diagram showing the 5' and 3' regions of the protease genes inactivated in the B. halodurans chromosome. Gray boxes indicate fragments within the open reading frames of the genes, and white boxes indicate regions in the 5' and 3' directions from the open reading frames. ${Delta}$ indicates deleted base pairs within the genes. Positions of different genes on the B. halodurans chromosome and primer names used for amplifications are indicated. All the primer sequences are listed in Table 2.

In a similar approach, the successful inactivation of the fliDgene was achieved, giving rise to B. halodurans strain BhFD01( ${Delta}$ wprA ${Delta}$ hag ${Delta}$ fliD). B. halodurans is alkaliphilic and does notharbor metalloproteases. Therefore, in an attempt to improvethe stability of secreted heterologous peptides, the homologuesof key alkaline protease genes previously inactivated in theB. subtilis genome (14) were identified. However, the combinedinactivation of the wprA, apr (homologue of epr), alp (homologueof aprE), and vpr genes, resulting in strain BhFD04, did notsignificantly improve the stability of recombinant peptidesin the medium. Further proteases involved in the degradationof the secreted peptides were identified through a combinationof zymography and bioinformatics.

Directed proteomics for bioinformatics-based identification of extracellular proteases.

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Directed proteomics for...

In order to identify the protease(s) responsible for proteolyticactivity still present in BhFD04, a zymogram was developed byincorporating purified flagellin monomers as substrates insteadof gelatin in a sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) gel (Fig. 2). These flagellin monomers were purifiedfrom culture supernatants of B. halodurans strain BhFD04(pSECFliC)on an ÄKTA fast protein liquid chromatography system usinga Toyopearl 650 M strong anion exchange resin. Proteins wereeluted by an increasing NaCl gradient from 0 to 500 mM over15 column volumes, and the fractions containing flagellin monomerswere identified on an SDS-PAGE gel (results not shown). Sampleswere precipitated with trichloroacetic acid (10%, wt/vol) for30 min on ice. After centrifugation, the pellets were resuspendedin Tris buffer (pH 9.0) and used as substrates in the resolvinggel (100 µg/ml). The strain (BhFD04) was grown in LB (pH8.5) at 37°C to an optical density at 600 nm (OD₆₀₀) of5 for protease analysis. After centrifugation, the supernatant(30 ml) was concentrated via ultrafiltration (10-kDa cutoff)to 0.5 ml, and 20 µl/lane was loaded onto an SDS-PAGEgel with flagellin monomers as substrates. The gel was stainedaccording to the method of Heussen and Dowdle (5), and the regionbetween 18 and 25 kDa containing the zone of proteolytic activitywas excised from the zymogram and sent to the Fingerprints ProteomicsFacility at the University of Dundee, Dundee, Scotland, formass spectrometry-based protein identification. Sequence informationwas obtained using a local Mascot (Matrix Science) search engineagainst the NCBInr database. The protease candidate selectedbased on database search scores and the presence of homologuesin the B. halodurans C-125 genome database was an extracellularalkaline serine protease (BH0855) encoded by a gene designatedasp (another homologue of the B. subtilis aprE gene). The purifiedflagellin monomers migrated in the resolving gel to $~$ 36 kDa (thesize of flagellin monomers) (Fig. 2), making it impossible tovisualize proteolytic activity above 36 kDa. Therefore, an agaroseoverlay (containing flagellin monomers) was employed. However,no other zones of proteolytic activity were observed (resultsnot shown). The asp protease was subsequently inactivated, givingrise to strain BhFD05.

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FIG. 2. Protease zymogram containing flagellin monomers as substrates. Extracellular samples from cultures at an OD₆₀₀ of 5 were tested for proteolytic activity. Lanes: 1, molecular mass marker (Fermentas); 2, BhFD05; and 3, BhFD04. The arrow indicates the zone of proteolytic activity at approximately 22 kDa.

Evaluation of secretion efficiency among the different protease-deficient strains.

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Evaluation of secretion...

The gene encoding the human immunodeficiency virus (HIV) subtypeC antigenic peptide (27 amino acids) (6) was fused as an in-framesandwich fusion into the central variable region of the FliCprotein gene (hag) to create construct pSECNHIVC6 (3) (Table1), which was used as a model construct for evaluating heterologouspeptide expression by the mutant strains in both log and stationaryphases. All strains were grown at 30°C in LB (pH 8.5, withchloramphenicol at 10 µg/ml) to OD₆₀₀ values of 1.6 and5. One milliliter of culture was centrifuged, and the supernatantwas precipitated as reported for the flagellin monomers. Thepellets were each resuspended in 20 µl of SDS sample bufferand evaluated on a 10% SDS-PAGE gel (9). Gels were stained usingcolloidal Coomassie blue G-250 gel stain (7) (Fig. 3A). Westernblotting was carried out as described by Crampton et al. (3)(Fig. 3B).

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FIG. 3. (A) SDS-PAGE comparison of extracellular proteins isolated from five protease-deficient B. halodurans strains secreting HIV antigenic fusion peptide in cultures at OD₆₀₀ values of 1.6 (lanes 1 to 5) and 5 (lanes 6 to 10). Lanes: 1 and 6, strain BhFD01(pSECNHIVC6); 2 and 7, BhFD02(pSECNHIVC6); 3 and 8, BhFD03(pSECNHIVC6); 4 and 9, BhFD04(pSECNHIVC6); 5 and 10, BhFD05(pSECNHIVC6); and 11, molecular mass marker (Fermentas). The arrow indicates the HIV antigenic fusion peptide. (B) Western blot of gel in panel A, generated using polyclonal rabbit antiflagellin antibodies.

B. subtilis strains deficient in extracellular proteases showedincreased susceptibility to cell lysis (13). Cell lysis wassuccessfully monitored by detecting the release of isocitratedehydrogenase into the supernatant (2, 4). Peptide secretionstudies carried out over a 24-h time period with B. haloduransBhFD05 showed no decrease in optical density, and no isocitratedehydrogenase was detected in the supernatant.

Thus, the inactivation of the asp protease, giving rise to strainBhFD05, resulted in major improvement in the stability of recombinantfusion peptides in the supernatant at an alkaline pH and duringstationary-phase growth. The asp protease therefore appearsto be a key protease affecting the stability of unpolymerizedrecombinant flagellin fusion monomers found in the supernatantof B. halodurans Alk36.

ACKNOWLEDGMENTS

We acknowledge funding support from Mbuyu Biotech (Pty.) Ltd.

We also acknowledge the proteomics facility at the Universityof Dundee, Dundee, Scotland, for protein identification.

FOOTNOTES

* Corresponding author. Mailing address: CSIR Biosciences, Box 395, Pretoria 0001, South Africa. Phone: 27 12 841-2167. Fax: 27 12 841-3651. E-mail: melouw{at}csir.co.za

Published ahead of print on 7 November 2008.

REFERENCES

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