INFECriON AND IMMUNITY, May 1994, p. 1914-1919 Vol. 62, No. 500 1 9-9567/94/$04.00+0Copyright ©) 1994, American Society for Microbiology

Isolation and Characterization of Pseudomonas pseudomalleiFlagellin Proteins

PAUL J. BRETT, DAVID C. W. MAH, AND DONALD E. WOODS*

Department of Microbiology and Infectious Diseases, University of Calgary HealthSciences Centre, Calgaty, Alberta, Canada T2N 4N1

Received 8 September 1993/Returned for modification 2 November 1993/Accepted 22 February 1994

Flagellin proteins from several different strains ofPseudomonas pseudomallei have been isolated and purifiedto homogeneity by mechanical shearing and differential centrifugation techniques. Analysis by sodium dodecylsulfate-polyacrylamide gel electrophoresis yielded flagellin monomer protein bands with an estimated Mr of43,400. No lipopolysaccharide contamination of the purified protein preparations was detectable by silverstaining of flagellin displayed on polyacrylamide gels and by Western immunoblotting with P. pseudomalleiantilipopolysaccharide monoclonal antibody. NH2-terminal amino acid sequence analysis of the flagellinprotein of P. pseudomallei 319a revealed significant homology with flagellins from Proteus mirabilis, Bordetellabronchiseptica, and Pseudomonas aeruginosa PAK. Rabbit polyclonal antiserum raised against the 319a flagellinprotein reacted with 64 of 65 P. pseudomallei strains tested. The polyclonal antiserum proved effective ininhibiting the motility of these organisms in motility agar plates. Passive immunization studies demonstratedthat 319a flagellin-specific antiserum was capable of protecting diabetic rats from challenge with aheterologous P. pseudomallei strain.

Pseudomonaspseudomallei is endemic to Southeast Asia andnorthern Australia (4, 19). Although the clinical manifesta-tions of disease caused by this organism are most commonlyobserved in these regions, the organism is not strictly confinedto this geographical domain. P. pseudomallei isolates are mostfrequently recovered from regions that lie 200 north and southof the equator; however, isolates from the Western Hemi-sphere are not uncommon (9). These organisms are inhabi-tants of environmental niches, such as soils, streams, andstagnant waters (7, 19, 36). It is therefore not surprising to findthat the highest incidences of infection caused by these bacte-rial pathogens occur during the rainy season in these tropicalregions. A significant percentage of persons succumbing tothese infections are known to be rice farmers and their families(4).

P. pseudomallei is the causative agent of the disease melioi-dosis (4), which may manifest itself in acute, subacute, andchronic forms (9). Acute forms characterized by overwhelmingsepticemia are rapidly fatal, even when vigorously treated withantimicrobial agents (4, 30). A number of reports have sug-gested that this organism may also establish chronic infectionswith incubation periods ranging up to 26 years prior to thedevelopment of clinical symptoms; the host-parasite relation-ships in these cases, however, are not well understood (22, 32).P. pseudomallei infections are thought to be acquired byinhalation or aspiration or through breaks in surface tissues (9,36). Recent studies have provided evidence indicating thatdiabetes mellitus may be a predisposing factor in the acquisi-tion of melioidosis (27).The pathogenesis of melioidosis is still poorly understood,

and although several putative virulence determinants havebeen identified, they have been relatively uncharacterized.Putative extracellular virulence determinants include a ther-

* Corresponding author. Mailing address: Department of Microbi-ology and Infectious Diseases, University of Calgary Health SciencesCenter, 3330 Hospital Dr., NW, Calgary, Alberta, Canada T2N 4N1.Phone: (403) 220-6885. Fax: (403) 270-4572. Electronic mail address:woods@acs.ucalgary.ca.

molabile toxin and a protease, and cell-associated virulencedeterminants include lipopolysaccharide (LPS), pili, extracel-lular polysaccharide, and flagella (5, 6, 36). In addition to beingidentified as virulence determinants, cell-associated macromol-ecules have also been identified as possible candidates for useas protective immunogens against P. pseudomallei infections.We have previously described the use of LPS as a protectiveimmunogen in an animal infection model of P. pseudomalleidisease (2). In this study, we have examined the ability ofantibody against purified flagellin protein to protect againstdisease caused by P. pseudomallei.We have isolated and purified flagellin proteins from a

number of different P. pseudomallei strains, the purity of whichhas been confirmed by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) and immunoblotting tech-niques. In this communication, we present experimental datawhich suggest that antibody to purified flagellin protein may beeffective in the prevention of P. pseudomallei infections.

MATERIALS AND METHODS

Bacterial strains. P. pseudomallei strains were generouslydonated by D. A. Dance, Wellcome-Mahidol-Oxford TropicalMedicine Research Programme, Bangkok, Thailand. All stockcultures were maintained in a 10% skim milk suspension andstored at - 70°C.

Flagellin isolation and purification. Luria agar (Difco Lab-oratories, Detroit, Mich.) plates containing 0.2% glucose wereinoculated with P. pseudomallei and incubated for 48 h at 37°C.Ten flasks, each containing 500 ml of Luria broth (DifcoLaboratories) plus 0.2% glucose, were inoculated with thebacterial colonies from the plates incubated for 48 h, and theseflasks were gently agitated at 100 rpm overnight at 37°C. Thecells were removed from the culture medium by centrifugationat 7,000 x g at 4°C for 20 min before being resuspended in 300ml of 50 mM sodium phosphate buffer (pH 7.0). The cellsuspension was then blended at the low setting in a Waringcommercial blender for 1.5 min to remove the flagella fromcells. The homogenate was centrifuged at 12,000 x g at 4°C for

1914

P. PSEUDOMALLEI FLAGELLIN CHARACTERIZATION 1915

20 min to remove the cells from the supernatant (15, 35). Thesupernatant was saturated in 5% increments to a final concen-tration of 20% with ammonium sulfate (16). After eachaddition of ammonium sulfate, the solutions were allowed tosit at room temperature for 4 to 5 h and stirred. The differen-tially saturated supernatants were centrifuged at 12,000 x g at4°C to remove the insoluble materials from solution. Each ofthe insoluble fractions was redissolved with a minimum volumeof 50 mM sodium phosphate buffer (pH 7.0) prior to beingdialyzed against the same buffer at 4°C overnight. The 15 and20% fractions typically contained the greatest amount offlagellin protein with the least amount of contaminants, asshown by SDS-PAGE and a bicinchoninic acid protein quan-titation assay (Pierce Chemical Co., Rockford, Ill.).

Although the above procedures removed most contami-nants, overloaded SDS-polyacrylamide gels displayed someresidual proteins at MrS of 40,000 and 66,000. These contam-inants could be easily removed by modification of the ultra-centrifugation, acid-disassociation, differential centrifugation-pH reassociation method outlined by Logan et al. (20). Afterreducing the pH of the proteinaceous fractions to pH 3.0,which was accomplished by the addition of 2.0 M citric acidand stirring at 4°C for approximately 10 min, the contaminat-ing proteins were removed from the solution by ultracentrifu-gation at 100,000 x g at 4°C for 30 min. The supernatant wasthen carefully removed, brought to pH 7.0 with 5.0 M NaOH,and stirred at 4°C for 20 min before being dialyzed overnightagainst the 50 mM sodium phosphate buffer (pH 7.0). Analysisof this protein fraction by SDS-PAGE yielded a single band ona gel loaded with 50 ,ug of protein. The final yield of purifiedflagellin was approximately 40% of the starting material.

Electrophoresis. SDS-PAGE (18) was performed in a mini-gel apparatus (Bio-Rad Laboratories, Richmond, Calif.) with a5% stacking and 12.5% separating (wt/vol) acrylamide gelsystem. Samples were solubilized under denaturing conditionsand heated to boiling for 2 to 3 min prior to being added to thegel wells. Protein separation was accomplished at 170 mAwhile the minigel apparatus was submerged in an ice bath, andproteins were visualized by staining with Coomassie blueR-250.

Electrophoretic transfer to a nitrocellulose (BRL Life Tech-nologies Inc., Gaithersburg, Md.) or polyvinylidene difluoride(Bio-Rad Laboratories) membrane was performed as de-scribed by Towbin et al. (35) using a minigel apparatus(Bio-Rad Laboratories) at a constant voltage of 80 V for 4 h at40C.LPS assays. Flagellin protein was digested with proteinase K

(Boehringer Mannheim, Laval, Quebec, Canada), electropho-resed by SDS-PAGE, and stained with silver as described byHitchcock and Brown (8), in order to detect the presence ofpossible contaminating LPS molecules. Immunoblots were alsoreacted with monoclonal antibody specific for P. pseudomalleiLPS (2).

N-terminal sequencing. Homologous P. pseudomallei 319aflagellin protein was electroblotted onto a polyvinylidene di-fluoride membrane and stained with Coomassie blue. Theflagellin band was excised, and the N-terminal amino acidsequence was determined by S. Kielland at the Department ofBiochemistry and Microbiology, University of Victoria, Victo-ria, British Columbia, Canada, employing standard Edmandegradation procedures.Amino acid composition. Amino acid composition analysis

was performed on the P. pseudomallei 319a flagellin protein. Avacuum-dried 10-,ul sample containing approximately 6.8 ,ug ofprotein was mixed with 5 nmol of norleucine and hydrolyzedwith 6 N HCl plus 0.1% 3-mercaptoethanol for 1 h in vacuo at

150°C. The samples were dried and redissolved in samplebuffer for analysis. Cysteine content was analyzed by performicacid hydrolysis, and tryptophan residues were assessed bytryptophan HCl hydrolysis. The amino acid composition anal-ysis was performed by D. McKay at the Protein SequencingFacility located at the University of Calgary Health SciencesCentre, Calgary, Alberta, Canada.Antibody production. Antibodies to P. pseudomallei 319a

flagellin protein were prepared by administration of Freund'scomplete adjuvant emulsified with a filter-sterilized prepara-tion of 319a flagellin protein in 10 mM phosphate-bufferedsaline (PBS). The rabbits (New Zealand White, 2 to 2.5 kg)each received an intramuscular injection of 0.50 ml of theemulsion in both their right and left hind thigh muscles. Eachinjection contained approximately 100 p.g of protein per ml.The rabbits were reinjected with the same preparation 14 dayslater; however, in this case, Freund's incomplete adjuvant wasused as the emulsifying agent. Serum antibody titers wereexamined by enzyme-linked immunosorbent assay at 5-dayintervals. The antiserum was found to have a high titer(reactive at >1/1,048,576) 10 days after the second immuniza-tion. At this point, the animals were exsanguinated by cardiacpuncture under anesthesia and serum samples were collectedand stored at - 70°C until required for use.

Isolation of IgG from rabbit antisera. An Affi-Gel Protein AMAPS II kit (Bio-Rad Laboratories) was used to isolate theimmunoglobulin G (IgG) fraction of the crude rabbit poly-clonal antiserum produced against 319a flagellin.

Immunoblotting. Following electrophoretic transfer of pro-tein to nitrocellulose membrane, unbound sites were blockedwith a PBS-Tween solution for 1 h (3). The membranes werereacted with primary antibody, followed by either proteinA-horseradish peroxidase (HRP) conjugate or goat anti-mouseIgM-HPP conjugate. Blots were developed with HRP ColorDevelopment Reagent (Bio-Rad Laboratories) (3).

Dot blot assay. P. pseudomallei cultures were streaked ontoLuria agar plates and incubated at 37°C for 48 h. Individualcolonies were picked from each plate and resuspended in500-,ul aliquots of 10 mM PBS. One hundred microliters ofeach strain suspension was vacuum blotted onto nitrocellulosepaper, and the membrane support was left to air dry. Thenitrocellulose paper was then reacted with 319a flagellin-specific antiserum as described above.

Motility inhibition assay. Two rows (6 wells in each row)from a 24-well microtiter plate were filled with motility agar(1.5 ml per well) and were designated control rows. Two rows(6 wells in each row) were filled with the same volume of agarwhich had been rehydrated with anti-319a polyclonal rabbitserum diluted 0, 1:10, 1:100, 1:500, 1:1,000, 1:5,000 and weredesignated antibody-containing lanes. One control row andone antibody-containing row were inoculated with Pseudomo-nas aeruginosa DG1, and one control row and one antibody-containing row were inoculated with P. pseudomallei 319a. Theplate was incubated at 37°C for 12 h. After incubation, thewells were examined for signs of cell motility demonstrated bya halo originating from the stab center.Animal protection studies. Immunoprotection studies were

performed in an animal model of P. pseudomallei infectionwhich we have recently described (37). Briefly, 40 Sprague-Dawley rats (male, approximately 30 g; Charles River, Quebec,Canada) were made diabetic by streptozotocin administrationon each of two consecutive days. Onset of diabetes wasconfirmed by an elevation in urine glucose levels. One half ofthe animals served as unimmunized controls and received100-p.l amounts of PBS by intraperitoneal (i.p.) injection. Theremaining 20 animals were passively immunized by i.p. injec-

VOL. 62, 1994

1916 BRETr ET AL.

A B C D E F

97.4

66.2

45.0

31.0s

FIG. 1. SDS-PAGE of purified flagellin proteins from variousstrains of P. pseudomallei. Purified protein (10 ,ug) was denatured insample buffer and electrophoresed on a 5% stacking and a 12.5%separating polyacrylamide gel. Lanes: A, standard protein markers(Bio-Rad) (rabbit muscle phosphorylase b [97.4 kDa], bovine serum

albumin [66.2 kDa], hen egg white ovalbumin [45.0 kDa], bovinecarbonic anhydrase [31.0 kDa], soybean trypsin inhibitor [21.5 kDa],and hen egg white lysozyme [14.4 kDa]); B, crude 319a flagellinpreparation; C, purified 304f flagellin; D, purified 307d flagellin; E,purified 316c flagellin; F, purified 319a flagellin.

tion of 100-,ul amounts of PBS containing 1.5 mg of purifiedIgG directed against P. pseudomallei 319a flagellin protein.Five animals in each of the control and immunized groups

were inoculated i.p. with one of four different doses of P.pseudomallei 316c: 103, 104, 105, and 106. Animals were mon-

itored carefully for 6 consecutive days for signs of morbidityand mortality. Fifty-percent lethal doses (LD50s) were calcu-lated by the method of Reed and Muench (28). In separateexperiments, animals which were passively immunized byintravenous (i.v.) injection of antiflagellin IgG were comparedwith unimmunized control animals for the ability to resist P.pseudomallei infections.

RESULTS

Flagellum isolation and purification. The flagellar filamentsof P. pseudomallei 304f, 307d, 316c, and 319a were isolated bymechanical shearing and differential centrifugation and puri-fied by ammonium sulfate precipitation. SDS-PAGE analysisof the purified extracts yielded a flagellin monomer with an

apparent Mr of 43,400 from all strains (Fig. 1). A homogeneouspreparation of strain 319a flagellin was accomplished by a

modified version of the ultracentrifugation acid-disassociationdifferential centrifugation-reassociation method outlined byLogan et al. (20). SDS-PAGE analysis of the flagellin after thisstep produced a single broad band with an Mr of 43,400 whenthe polyacrylamide gel was overloaded with sample protein.Citric acid was substituted for HCI in the ultracentrifugationpurification protocol because of the tendency for HCI to cause

degradation of the flagellin protein. The use of HCI caused themonomer to lose relative mobilities of up to 10,000 Da as

displayed by SDS-PAGE, whereas citric acid was shown toobviate this problem. Protein quantitation of the purifiedflagellin extracts by a bicinchoninic acid-based assay (PierceChemical Co.) determined that 15 to 20 mg of flagellin proteincould be routinely isolated from a 5.0-liter cell culture grown

overnight.Assessment of LPS contamination. Homogeneous 319a

flagellin was electrophoresed under standard conditions in an

SDS-PAGE minigel apparatus. The polyacrylamide gel was

A B C D EI IC v ~,-f

F G97.466.2 se.. i a+

45.0 V* :: XEE XI11_ 6SPg:.A" &

21.5

14.0

FIG. 2. Western blot of purified flagellin proteins from variousstrains of P. pseudomallei. Lanes: A, standard protein markers (de-scribed in the legend to Fig. 1); B, purified 304f flagellin; C, purified307d flagellin; D, purified 316c flagellin; E, purified 319a flagellin; F,purified 319a flagellin; G, purified 304f LPS. Lanes B to E were reactedwith a 1:2,500 dilution of polyclonal antiserum against 319a flagella.Lanes F and G were reacted with a 1:1,000 dilution of P. pseudomallei-specific anti-LPS monoclonal antibody.

then silver stained by the procedure of Hitchcock and Brown(8). Development of the gel displayed the absence of thetypical laddering patterns associated with purified LPS prepa-rations (data not shown). Western blot (immunoblot) analysisof the flagellin preparation with an anti-LPS monoclonalantibody specific for P. pseudomallei LPS was again unable todetect the presence of any contaminating LPS (Fig. 2). How-ever, LPS purified from P. pseudomallei 304f was shown toreact strongly with the monoclonal antibody (Fig. 2).

Biochemical analysis. Amino acid composition analysis ofthe 319a flagellin monomer revealed the presence of Asx, Thr,Ser, Glx, Pro, Gly, Ala, Val, Met, Ile, Leu, Tyr, Phe, His, Lys,and Arg (Table 1). The presence of Cys and Trp were notdetected during the sample analysis. The relative hydrophobic-ity of the flagellin protein was calculated to be approximately40% (Table 2). From the 394 amino acid residues detected, an

Mr of 40,100 was predicted for flagellin protein. This valuediffers slightly from that derived by SDS-PAGE.

TABLE 1. Amino acid composition of flagellin proteins fromfour species

No. of residues in flagellin from organism:Aminoacid P. pseudomallei Proteus Bordetella P. aeruginosa

319a mirabilis bronchiseptica PAK

Asx 47 71 66 57Thr 35 33 31 36Ser 43 30 32 40Glx 53 41 40 33Pro 6 NDa 4 3Gly 36 26 28 37Ala 58 38 63 61Val 24 26 34 26Met 3 2 5 4Ile 24 24 22 25Leu 34 32 29 29Tyr 3 5 6 2Phe 8 10 6 8His 2 ND ND NDLys 9 28 12 17Arg 9 15 15 16Trp ND ND 1 NDCys ND ND ND ND

I ND, not detected.

INFEC-F. IMMUN.

P. PSEUDOMALLEI FLAGELLIN CHARACTERIZATION 1917

TABLE 2. Characteristics of flagellin proteins from four species

Total no. Mr (103) NOrganism of residues/ Hydrophobic

molecule Predicted" Apparent' residues"

P. pseudomallei 319a 394 40.1 43.4 40Proteus mirabilis 381 41.0 41.0 35Bordetella bronchiseptica 394 40.9 40.0 42P. aeruginosa PAK 394 40.0 45.0 40

a The amino acids used in these calculations are Pro, Ala, Val, Met, Ile, Leu,Phe, and Trp.

b Calculations based on amino acid composition data.c Values obtained by SDS-PAGE.

NH2-terminal sequencing of the 319a flagellin protein wascontinued for 17 amino acids. Of these 17 residues, 47% of theresidues are hydrophobic, while the remaining 53% that weresequenced are neutral (Table 3). No charged residues werepresent in this short peptide sequence. The NH2-terminal endof the 319a flagellin monomer displays many of the character-istic features of some well-documented flagellins. Along withsharing a conservative likeness for the chemical nature of theR groups in the NH2 terminus, the P. pseudomallei 319aflagellin exhibits a high degree of sequence homology withProteus mirabilis, Bordetella bronchiseptica, and P. aeruginosaPAK flagellin proteins (Table 3).Immunochemical analysis. Rabbit polyclonal antiserum

raised against the homogeneous 319a flagellum preparationwas shown to react strongly with the same 43,400-Mr 319amonomer on Western blots. A single immunostained band atan Mr of 43,400 was observed. The MAPS II-purified IgGfraction from the polyclonal serum demonstrated similar re-sults (data not shown).Dot blot analysis of 65 P. pseudomallei strains reacted with

either the crude polyclonal anti-319a serum or the MAPSTI-purified IgG fraction demonstrated that 64 of 65 of thestrains reacted with the flagellin-specific antibodies (data notshown). P. pseudomallei 319a served as a positive control, andP. aeruginosa PAO served as a negative control in this assaysystem.

Motility inhibition assay. To determine the functional ac-tivity of the anti-319a flagellin antiserum, we examined theability of this antiserum to inhibit the motility of the homolo-gous strain. Specificity was examined by testing the ability ofthis antiserum to inhibit motility of a P. aeruginosa strain.Anti-319a polyclonal serum at a dilution of 1/1,000 or lessprevented the cell motility of P. pseudomallei 319a but did notprevent the cell motility of P. aeruginosa DG1 (Fig. 3). Thus,the anti-319a flagellin antiserum was found to be functionaland specific. Anti-P. pseudomallei LPS antibody did not inhibitmotility (Fig. 3).

Passive immunization studies. In the initial studies, diabeticrats were inoculated with various amounts of P. pseudomallei316c and given i.p. either 1.5 mg of anti-319a flagellin IgG in a

TABLE 3. NH2-terminal amino acid sequences of flagellin proteinsfrom four species

Organism Sequencea

P. pseudomallei 319a ..... ........... 2LGINSNINSLVAQQNL"7Proteus mirabilis..................4INTNYLSLVTONNL)7Bordetella bronchiseptica................. 5INTNYLSLVAONNL 8P. aeruginosa PAK ..... ........... 3LTVNTNIASLNTQRNL.U

"Residues homologous with those in the P. pseudomallei 319a sequence areunderlined. Conservative amino acid replacements are denoted by bold type.

9 R ~A s9

FIG. 3. Motility inhibition assessment of P. pseudomallei 319a and

P. aeruginosa DGI reacted with various dilutions of polyclonal anti-

serum directed against 319a flagella. The wells in rows A and B were

inoculated with P. pseudornaliei 319a. The wells in rows C and D were

inoculated with P. aeruginosa DGI. Anti-319a flagellin antiserum

(rows A and C) or anti-304f LPS monoclonal antibody (rows B and D)was added to the wells. Wells: 1, PBS control; 2, 1:5,000 dilution; 3,

1:1,000 dilution; 4, 1:500 dilution; 5, 1:100 dilution; 6, 1:10 dilution.

PBS solution or a PBS solution (control). Of the rats in the

control (PBS) groups, only 4 of 20 were still alive after the sixth

day, whereas the immunized groups displayed a 55% survival

rate over the same time frame (Table 4). The most significantresults were found in the t04 inoculum groups. Four of five

immunized rats survived compared with none of the five rats in

the PBS-treated control group. The calculated LD50, for the

control group was<A03, while that for the immunized group

was 1.0 x i05 (Table 4).In a separate experiment, the PBS and the anti-319a anti-

bodies were introduced i.v. into the tail veins of the rats. The

immunized animals had a greater survival rate when the

immunoglobulins were administered i.v. than when the animals

were immunized i.p. In PBS-treated controls, 16 of 20 were

dead by day six. The immunized animals displayed a mortalityrate of 4 of 20 in the same 6-day period (Table 4). The 104, i05,and 10' inoculum groups displayed much lower mortality rates

when protected with the IgG fraction. The calculated LD501S

for this control group was <i103, while that for the immunized

group was 106 (Table 4).

Unimmunized animals were shown to be bacteremic, since

blood samples obtained by sterile cardiac puncture streaked

onto tryptic soy agar plates and incubated overnight at 370C

TABLE 4. Passive immunoprotection by anti-319a flagellin IgG in

diabetic rats challenged with a heterologous P. pseudomallei strain"

Mortality (n = 5)

Inoculum Expt A Expt Bsize

Control Immunized Control Immunized(PBS i.p.) (IgG i.p.) (PBS i.v.) (IgG i.v.)

103 4 3 3 2104 5 1 5 0105 3 1 4 1106 4 4 4 1

aGroups of diabetic rats (five rats in each group) were inoculated with 10-folddilutions of P. pseludomallei 316c ranging from 103 to 106 CFU per rat andadministered IgG or PBS i.p. or i.v. The LD50s, calculated by the method of Reedand Muench (28), were 4.5 x 104 CFU for rats immunized i.p., > 1.0 x 106 CFUfor rats immunized i.v., and <103 CFU for the control groups in both experi-ments.

VOL. 62, 1994

1918 BRETT ET AL.

demonstrated heavy P. pseudomallei growth. Rats that survivedthe 6-day period displayed no signs of bacteremia when theirblood samples were similarly analyzed.

DISCUSSION

In this study, the flagella of four strains of P. pseudomalleiwere isolated and purified. The apparent Mr of the flagellinmonomer proteins from strains 304f, 307d, 316c, and 319awere determined to be approximately 43,400. This finding is inkeeping with the data reported for other characterized flagellinmolecules which have been reported to have Mrs ranging from15,000 to 62,000 (11, 14). Among the Pseudomonas spp.flagellins of P. stutzeri and P. aeruginosa have estimatedmolecular weights of 45,000 to 55,000 whereas P. cepacia andP. maltophilia possess flagellins with Mrs ranging from 31,000to 45,000 (25). The values obtained in the present study for theP. pseudomallei flagellins closely resemble the latter organisms,since P. pseudomallei has more in common with these speciesaccording to recent taxonomic reclassifications (38).The presence of a single subunit type in the flagellar

filaments of P. pseudomallei strains analyzed to date agreeswith evidence suggesting that nonsheathed bacterial flagelladisplay only a single flagellin species when disaggregated (11).An exception to this rule appears to be exhibited by the flagellaof Bacillus pumilis 101, in which two immunologically distinctmonomers are found to make up the filament (26, 33).Examples of multiple-subunit-type flagella are found in thesheathed flagellar filaments of Vibrio cholerae, Pseudomonasrhodos, Helicobacter pylori, and Caulobacter crescentus (17, 23,29, 31).The 319a flagellin monomer was purified to homogeneity by

a modified version of a procedure outlined by Logan et al. (20).The use of concentrated HCl suggested for the initial protocolwas found to be unacceptable for maintaining the structuralintegrity of the monomer because of its apparent ability tocause degradation leading to loss of a substantial part (up to anMr of 10,000) of the flagellin molecule. Changing the acid to2.0 M citric acid was found to alleviate this problem. Thepresence of degradation at the NH2-terminal end of themolecule, as displayed by NH2-terminal sequencing data, mayindicate that this flagellin is more sensitive to pH fluctuationsthan others of its class.Amino acid analysis of the 319a monomer showed the

presence of all biologically relevant residues, except for tryp-tophan and cysteine. The absence of these two amino acids aswell as the low number of basic molecules corresponds to thecollective amino acid composition data from other flagellinpolypeptides (14). An Mr of 40,100 was calculated for the 319amolecule from the amino acid analysis data. The conflictingresults may be explained by migrational aberrations of mole-cules in SDS-polyacrylamide gels compared with their knownmolecular masses or possible posttranslational modifications.The data suggest that further studies will be needed to evaluatethe true native molecular weight of the flagellin monomer.Sequencing of the flagellin structural gene should greatly aid inthis examination.Examples of cases in which Mr values do not correspond

exactly to calculated molecular masses from gene sequenceand amino acid analysis data are found in P. aeruginosa PAKand Salmonella typhimurium. Sequencing of the P. aeruginosaa-type H-antigen flagellin suggested a molecular mass of40,040, while SDS-PAGE revealed an Mr of 45,000. Thisanomaly appears to be resolved, however, by the finding ofphosphorylated tyrosine residues in the flagellin molecule (13).S. typhimurium phase I antigen i flagellin was shown to have an

Mr of 49,000, while gene sequence and amino acid compositiondata predicted that the true molecular mass should be 51,000.The reasons for this, however, are not clear (10).

NH2-terminal sequencing revealed significant amino acidhomology with the NH2-terminal ends of P. pseudomalleiflagellin and several other characterized flagellins. Residuesthat were shown not to be identical, for the most part displayeda tendency towards a conservative substitution at the same site.This terminal region also exhibited an absence of amino acidsother than those that were neutral or hydrophobic, which againis a characteristic of these structural proteins (11). The simi-larity between the NH2-terminal regions of different flagellinmolecules appears to be a conserved evolutionary trait. Evi-dence suggests that amino acids in this terminal domain, alongwith a number in the CO2 --terminal region, are closely linkedto export of these molecules and their subsequent polymeriza-tion during biosynthesis of the flagella (12, 21, 34).Immunoblotting of 65 strains of P. pseudomallei with poly-

clonal antiserum raised against the homogeneous preparationof 319a flagellin demonstrated that 64 of these 65 strainsreacted with the antiserum. This result suggests that theflagellins of each of the 64 positively testing strains containcross-reacting epitopes. Future studies will focus on isolatingthe flagellins from each of these strains to prove that thecross-reactivity is indeed due to the presence of the flagellinmonomers and not from some other cell-associated source. Itwill also be interesting to isolate and characterize the flagellinsubunit from the nonreactive strain to uncover the reasonsbehind this anomaly.The anti-319a flagellum polyclonal antiserum was used to

assess the presence of cross-reacting epitopes on the flagella ofP. aeruginosa DG1. When whole-cell lysates of this organismwere incubated with the P. pseudomallei-specific immuno-globulins, no cross-reactivity was observed. This result suggeststhat although flagellins appear to be structurally conserved inmany regions of their primary structure (11, 12, 21, 34), thedifferences that they do exhibit are sufficient to allow forserological distinctiveness.

Polyclonal antiserum raised against the homogeneous 319aflagellin preparation was shown to inhibit cell motility of P.pseudomallei 319a, but not P. aeruginosa DG1 in vitro. Theseresults are complementary to those found in the Western blotexperiment in which the P. aeruginosa DG1 whole-cell lysatewas shown to lack cross-reactivity with the P. pseudomallei-specific serum. The mechanism of this inhibition of motility hasnot been well characterized, but previous studies demonstrat-ing similar results suggest that immunoglobulin interactions atthe surface of the bacterial flagella cause disruption of thenatural rotational movements of the structure, thus leading toloss of motility (24). Since mobility has been demonstrated tobe an important factor in microbial pathogenesis (1, 24, 34),disruption of such a function by immobilizing antibodies invivo may prove to be an advantageous prophylactic measureagainst bacteria.

Immunoprotective studies demonstrated that passive admin-istration of immunoglobulins raised against the 319a flagellumpreparation could protect rats against infection by a heterolo-gous P. pseudomallei strain. The mechanisms responsible forthis protection are unknown, but as the in vitro experimentalevidence suggests, interaction of immobilizing antibodies withflagellar filaments may disrupt the function of the flagella andcause attenuated pathogenicity in these organisms (1, 24).Further studies, however, are needed to confirm this.

INFECT. IMMUN.

P. PSEUDOMALLEI FLAGELLIN CHARACTERIZATION 1919

ACKNOWLEDGMENTS

This work was supported by the Canadian Bacterial DiseasesNetwork of Centres of Excellence.We are grateful to P. A. Sokol and A. L. Jones for careful review of

the manuscript.

REFERENCES1. Bahrani, F. K., D. E. Johnson, D. Robbins, and H. L. T. Mobley.

1991. Proteus mirabilis flagella and MR/P fimbriae: isolation,purification, N-terminal analysis, and serum antibody responsefollowing experimental urinary tract infection. Infect. Immun.59:3574-3580.

2. Bryan, L. E., S. Wong, D. E. Woods, D. A. B. Dance, and W.Chaowagul. Antisera recognizing a common antigen of P.pseudomallei lipopolysaccharide which is protective in an animalmodel of infection. Can. J. Infect. Dis., in press.

3. Catty, D., and C. Raykundalia. 1988. Antibodies, vol. I, p. 164-167.IRL Press, Oxford.

4. Chaowagul, W., N. J. White, D. A. B. Dance, Y. Wattanagoon, P.Naigowit, T. M. E. Davis, S. Looareesuwan, and N. Pitakwhat-chara. 1989. Melioidosis: a major cause of community-acquiredsepticemia in northeastern Thailand. J. Infect. Dis. 159:890-899.

5. Colling, M., C. Nigg, and R. J. Heckly. 1958. Toxins of Pseudomo-nas pseudomallei. I. Production in vitro. J. Bacteriol. 76:422-426.

6. Coiling, M., C. Nigg, and R. J. Heckly. 1958. Toxins of Pseudomo-nas pseudomallei. II. Characterization. J. Bacteriol. 76:427-436.

7. Ellison, D. W., H. J. Baker, and M. Mariappan. 1969. Melioidosisin Malaysia. I. A method for isolation of Pseudomonas pseudoma-llei from soil and surface water. Am. J. Trop. Med. Hyg. 18:694-697.

8. Hitchcock, P. J., and T. M. Brown. 1983. Morphological hetero-geneity among Salmonella lipopolysaccharide chemotypes in sil-ver-stained polyacrylamide gels. J. Bacteriol. 154:269-277.

9. Howe, C., A. Sampath, and M. Spotnitz. 1971. The pseudomalleigroup, a review. J. Infect. Dis. 124:598-606.

10. Joys, T. M. 1985. The covalent structure of phase-I flagellarfilament protein of Salmonella typhimurium and its comparisonwith other flagellins. J. Biol. Chem. 260:15758-15761.

11. Joys, T. M. 1988. The flagellar filament protein. Can. J. Microbiol.34:452-458.

12. Kanto, S., H. Okino, A. Shin-Ichi, and S. Yamaguchi. 1991. Aminoacids responsible for flagellar shape are distributed in terminalregions of flagella. J. Mol. Biol. 219:471-480.

13. Kelly-Wintenberg, K., T. Anderson, and T. C. Montie. 1990.Phosphorylated tyrosine in the flagellum filament protein ofPseudomonas aeruginosa. J. Bacteriol. 172:5135-5139.

14. Kirschenbaum, D. M. 1975. A compilation of amino acid analysesof proteins. VII. Residues per molecule -5. Anal. Biochem.66:123-150.

15. Kobayashi, T., J. N. Rinker, and H. Koffier. 1959. Purification andchemical properties of flagellin. Arch. Biochem. Biophys. 84:342-362.

16. Koffler, H., and T. Kobayashi. 1957. Purification of flagella andflagellin with ammonium sulfate. Arch. Biochem. Biophys. 67:246-248.

17. Kostrzynska, M., J. D. Betts, J. W. Austin, and T. J. Trust. 1991.Identification, characterization, and spatial location of two flagel-lin species in Helicobacterpylori flagella. J. Bacteriol. 173:937-946.

18. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

19. Leelarasamee, A. 1986. Epidemiology of melioidosis. Infect. Dis.Antimicrob. Agents 3:84-93.

20. Logan, S. M., L. A. Harris, and T. J. Trust. 1987. Isolation andcharacterization of Campylobacter flagellins. J. Bacteriol. 169:5072-5077.

21. Macnab, R. M. 1987. Flagella, p. 70-83. In F. C. Neidhardt, J. L.Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E.Umbarger (ed.), Escherichia coli and Salmonella typhimurium, vol.I. American Society for Microbiology, Washington, D.C.

22. May, E. E., and E. A. Ricketts. 1975. Melioidosis: recrudescenceassociated with bronchogenic carcinoma twenty-six years followinginitial geographic exposure. Chest 68:261-263.

23. Minnich, S. D., and A. Newton. 1987. Promoter mapping and cellcycle regulation of flagellin gene transcription in Campylobactercrescentus. Proc. Natl. Acad. Sci. USA 84:1142-1146.

24. Montie, T. C., R. C. Craven, and I. A. Holder. 1982. Flagellarpreparations from Pseudomonas aeruginosa: isolation and charac-terization. Infect. Immun. 35:281-288.

25. Montie, T. C., and G. B. Stover. 1983. Flagellar preparations fromPseudomonas species: isolation and characterization by molecularweight. J. Clin. Microbiol. 18:452-456.

26. Oiler, L., F. Kocka, R. W. Smith, and H. KoffIler. 1971. Thepresence of flagellins A and B in the flagellum of Bacillus pumilus101. Bacteriol. Proc. 1971:27.

27. Puthucheary, S. D., N. Parasakthi, and M. K. Lee. 1992. Septace-mic melioidosis: a review of 50 cases from Malaysia. Trans. R. Soc.Trop. Med. Hyg. 86:683-685.

28. Reed, L. J., and H. Muench. 1937. A simple method of estimatingfifty percent endpoints. Am. J. Hyg. 27:493-497.

29. Richardson, K., and C. D. Parker. 1985. Identification and occur-rence of Vibrio cholerae flagellar core proteins in isolated outermembrane. Infect. Immun. 47:674-679.

30. Sanford, J. P. 1985. Chapter 197, p. 1250-1254. In G. L. Mandell,R. G. Douglas, and J. E. Bennet (ed.), Practice and principles ofinfectious diseases. Churchill Livingstone, New York.

31. Schmitt, J. M., I. Raska, and F. Mayer. 1974. The flagella ofPseudomonas rhodos. J. Bacteriol. 117:844-852.

32. Sexton, M. M., L. A. Goebel, A. J. Godfrey, W. Choawagul, N. J.White, and D. E. Woods. 1993. Ribotype analysis of Pseudomonaspseudomallei isolates. J. Clin. Microbiol. 31:238-243.

33. Sullivan, A., J. Bui, H. Suzuki, R. W. Smith, and H. Kofiler. 1969.Possible phase variation in Bacillus pumilis. Bacteriol. Proc. 1969:30.

34. Totten, P., and S. Lory. 1990. Characterization of the type aflagellin gene from Pseudomonas aeruginosa PAK. J. Bacteriol.172:7188-7199.

35. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets: procedure and some applications. Proc. Natl. Acad. Sci.USA 74:4350-4354.

36. Weinberg, A. N. 1989. Unusual bacterial pneumonias, p. 411-415.In J. E. Pennington (ed.), Respiratory infections: diagnosis andmanagement, 2nd ed. Raven Press, New York.

37. Woods, D. E., A. L. Jones, and P. J. Hill. 1993. Interaction ofinsulin with Pseudomonas pseudomallei. Infect. Immun. 61:4045-4050.

38. Yabuuchi, E., Y. Kosako, H. Oyaizu, I. Yano, H. Hota, Y. Hashi-moto, T. Ezaki, and M. Arakawa. 1992. Proposal of Burkholderiagen. nov. and transfer of seven species of the genus Pseudomonashomology group II to the new genus, with the type speciesBurkholderia cepacia (Palleroni and Holmes 1981) comb. nov.Microbiol. Immunol. 36:1251-1275.

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