Binding and Virulence of Shigella flexneri and Enteroinvasive ...

Vol. 57, No. 11

INFECTION AND IMMUNITY, Nov. 1989, p. 3534-3539

0019-9567/89/113534-06$02.00/0 Copyright C) 1989, American Society for Microbiology

A 101-Kilodalton Heme-Binding Protein Associated with Congo Red Binding and Virulence of Shigella flexneri and Enteroinvasive Escherichia coli Strains CAROL E. STUGARD, PANAGIOTIS A. DASKALEROS,

AND

SHELLEY M. PAYNE*

Department of Microbiology, University of Texas at Austin, Austin, Texas 78712 Received 5 June 1989/Accepted 4 August 1989

The ability of Shigellaflexneri to bind Congo red or hemin is associated with virulence. A 101-kilodalton (kDa) protein responsible for this phenotype (Crb+) in S. flexneri was identified by a tetramethylbenzidine staining procedure which detects heme-protein complexes in polyacrylamide gels. Labeling of cell-surface polypeptides with 125I revealed that the 101-kDa heme-binding protein is expressed on the cell surface. Expression of the protein was regulated by growth temperature and was found to be encoded by the large virulence plasmid of S. flexneri. Deletion mutants and a TnS insertion mutant which were negative for Congo red binding (Crb-) did not express the 101-kDa protein. Enteroinvasive Escherichia coli strains that were Crb+, and whose plasmids shared homology with the S. flexneri virulence plasmid, also expressed the 101-kDa protein. Expression of the protein in S. flexneri and enteroinvasive E. coli correlated with the presence of a 9.2-kilobase EcoRI fragment of these plasmids.

The Shigella species are enteroinvasive pathogens which have the ability to penetrate and multiply within intestinal epithelial cells. Virulence of these bacteria is associated with multiple genes located on the virulence plasmid and on the chromosome. These genes encode functions which include attachment to host cells, induction of endocytosis, intracellular multiplication, and spread to adjacent cells (19). The expression of some of these genes has been shown by Maurelli and co-workers to be regulated by temperature; production of the gene products and virulence are detected at 37°C but not at 30°C (17). One easily identifiable phenotypic marker which has been used to assess virulence of Shigella species is the Congo red binding phenotype (20). Wild-type, virulent isolates bind the dye Congo red from agar media (Crb+), whereas mutants which fail to do so (Crb-) are avirulent (18, 20). The conversion of a given organism from the virulent Crb+ status to Crb- phenotype is usually accompanied by loss or deletions of the 220-kilobase (kb) virulence plasmid (18). In rare cases the conversion from Crb+ to Crb- is not accompanied by detectable molecular alterations of the 220-kb plasmid (4). As is the case with invasiveness, Congo red binding in Shigella flexneri is thermoregulated, and the dye is bound when the cells are grown at 37°C but not at 30°C (17). Although the Congo red phenotype is a convenient marker for the identification of invasive S. flexneri isolates, the role of the crb gene product in the invasion process is not clear. Wild-type S. flexneri which had prebound Congo red exhibited increased infectivity for HeLa cells, pointing to a role for the crb gene product in the attachment of the bacterium onto the epithelial cells or in other early steps in invasion (5). In addition, trypsin treatment of whole S. flexneri cells which had prebound Congo red resulted in the removal of the bound dye (5). This observation is indicative of a cell surface-exposed Congo red gene product and is consistent with a possible role in the attachment of the bacterium. In a liquid binding assay, hemin and protoporphyrin IX could be

*

used interchangeably to differentiate Crb+ from Crb- organisms (5). One problem in elucidating the role of Congo red binding in virulence has been the fact that neither the crb gene nor its product has been unequivocally identified. Several fragments of the virulence plasmid which enable transformed Escherichia coli to bind the dye have been cloned, and these have been found to direct the synthesis of several different proteins (2, 3, 21). Sakai et al. (21, 22) cloned a 1-kb fragment encoding a 30-kilodalton (kDa) protein, VirF, which was sufficient for Congo red binding in E. coli. This was later shown to be a positive regulator for invasion gene expression. Chambers et al. (2) identified a 24-kDa protein encoded by a 2.15-kb PstI fragment which conferred Congo red binding in both E. coli and S. flexneri, and Daskaleros and Payne (3, 4) found that several unrelated fragments of the plasmid would permit Congo red binding. Some of these clones failed to restore the Crb+ phenotype, or produced intermediate phenotypes, when introduced into plasmidless or plasmid deletion mutants of S. flexneri, and others were dependent upon the high copy number of the vector to produce the Crb+ phenotype. The purpose of this study was to identify the Congo red/heme-binding protein on the cell surface of S. flexneri by directly screening proteins for associated hemin. Enteroinvasive E. coli (EIEC) strains were also analyzed in this study. The pathogenesis of EIEC is similar to that of Shigella sp., there is considerable homology among the virulence plasmids (23), and these strains often exhibit Congo red/hemin binding (20). The relationship between the Crb+ phenotype and the presence of a 101-kDa, temperature-regulated heme-binding protein was established for these invasive bacteria. MATERIALS AND METHODS

Bacterial strains and plasmids. Bacterial strains and plasmids are listed in Table 1. The S. flexneri strains SA101 and 102 are spontaneous Crb- mutants of the wild-type 2a strain SA100. The 220-kb virulence plasmid, pTKS220, of strain SF100 (serotype 5a) was used to construct E. coli derivatives

Corresponding author. 3534

HEME-BINDING PROTEIN OF S. FLEXNERI

VOL. 57, 1989 TABLE 1. Bacterial strains and plasmids Crba

Source or reference

220 kb 20-kb deletion Cured of 220 kb pTKS220

+ +

(5) (5) This study

None

+

Plasmid

Strain

S. flexneri SA100

SA101 SA102 SF100 E. coli RM1058 JB807 JB904 JB811 JB801 EIEC strains 1107-81 550-3076 930-78 1885 3189 929 5144 a

b

c

pTKS220::Tn5 pTKS220::mini-Kan pTRS11 pTJD2 Wild type Wild type Wild type Wild type Deletion Deletion Deletion

Congo red binding on TSB agar with TDH, Texas Department of Health. University of Texas, Austin.

+ + + + + +

-

TDHb R. Meyerc This study This study This study This study J. J. J. J. J. J. J.

Crosa Crosa Crosa Crosa Crosa Crosa Crosa

0.01% Congo red at 37°C.

of strain RM1058 (JB807, JB904, JB811, and JB801). The plasmid was tagged in a nonessential region with TnS to provide a kanamycin resistance marker (pTKS220::TnS). Strains carrying this plasmid retain virulence. A second plasmid construct, pTKS221 (E. coli JB904), contained a "'mini-Kan" (28) insertion which was mapped to the 9.2-kb EcoRI fragment of pTKS220. This insertion resulted in the loss of Congo red binding (Crb-) at 37°C and loss of virulence. The plasmid pTRS11 contains a 38-kb fragment of pTKS220 in the cosmid vector pJB8 (16). Strains carrying this cosmid clone show temperature-regulated Congo red binding. pTJD2 contains a 6.1-kb PstI fragment of pTRS11 cloned into the high-copy-number vector pAT153 (16). Strains carrying this plasmid are Crb+. Bacterial stocks were maintained at -80°C in Luria broth (L-broth) (16) supplemented with 20% glycerol. Media and chemicals. Trypticase soy broth (BBL Microbiology Systems, Div. of Becton Dickinson and Co., Cockeysville, Md.) was used for routine growth of all Shigella strains, and L-broth was used for growth of E. coli strains. Congo red was added at 0.01% to Trypticase soy broth solidified with 1.5% agar for determination of Crb phenotype (20). Lithium dodecyl sulfate (LDS), 3,3',5,5'-tetramethylbenzidine (TMBZ), dithiothreitol, and Congo red were obtained from Sigma Chemical Co. (St. Louis, Mo.). All other chemicals were of reagent grade. Heme-binding assay and whole-cell protein preparation. Heme binding was performed essentially as described previously (5). Broth cultures (5 ml) of late-log-phase or earlystationary-phase cells were harvested, washed in 1 ml of 100 mM Tris hydrochloride (pH 8.0), and centrifuged. The cell pellets were then suspended in 1 ml of fresh Trypticase soy broth. Hemin (bovine type I; Sigma Chemical Co.) was added to a final concentration of 15 p.g/ml and was allowed to incubate with the cells at 37°C for 20 to 30 min. The cells were pelleted by centrifugation and solubilized by suspending the cell pellet in 300 p.l of 50 mM Na2CO3-12% sucrose2% LDS as described by Delepelaire and Chua (6), except dithiothreitol was omitted in routine preparations. The cell suspension was lysed at 4°C overnight. DNA was removed by centrifugation at 100,000 x g for 40 min at 5°C.

3535

LDS-PAGE. A modification of the procedure of Delepelaire and Chua (6) for polyacrylamide gel electrophoresis (PAGE) of hemin-protein complexes was used, as follows. The resolving gel was 12% acrylamide in 0.375 M Tris (pH 8.8). The stacking gel contained 4% acrylamide, 1 mM EDTA, and 0.1% LDS in 0.125 M Tris (pH 6.8). Samples were diluted 4:1 with glycerol-bromophenol blue loading buffer. The electrophoresis buffer consisted of 192 mM glycine plus 25 mM Tris hydrochloride and 0.1% LDS. Gels were run on a BioRad (Richmond, Calif.) Mini Protean apparatus. The proteins were electrophoresed at a constant 220 V for 1 h in the dark at 4°C. TMBZ staining. Detection of heme-protein complexes was accomplished by staining gels with TMBZ (10), a chromogen that turns blue in areas of heme-associated peroxidase activity. After electrophoresis, the gels were fixed in the dark for 1 h in a prechilled solution of 6:3:1 sodium acetate (0.25 M, pH 5.0)-methanol-H20. TMBZ was prepared fresh for each usage at a concentration of 6.3 mM in 20 ml of absolute methanol. The TMBZ solution was added to 70 ml of cold sodium acetate (0.25 M, pH 5.0) and 10 ml of H20. The gels were stained in this solution for 35 min in the dark in a shaking ice bath as described by Thomas et al. (26). To lessen gel shrinkage, 2 parts (instead of 3) of TMBZ in methanol was added to a final volume of 100 ml. Hydrogen peroxide was added to the staining solution to a final concentration of 30 mM and incubated for 30 min to allow color development. Gels were washed in acetate-buffered isopropanol at a ratio of 8:2 (26) and dried immediately. Southern hybridization. Plasmid DNA was isolated by the procedure of Kado and Liu (11) and digested with EcoRI (New England Biolabs, Inc., Beverly, Mass.). The DNA was electrophoresed on 0.9% agarose gels and transferred by the Southern method (24) onto Gene Screen membranes (NEN Research Products, Boston, Mass.). The blots were allowed to hybridize at 68°C overnight in 6 x SSC (1 x SSC is 150 mM NaCl plus 15 mM sodium citrate) and were washed at 68°C in lx SSC + 0.5% sodium dodecyl sulfate (SDS). The hybridization probe was the 9.2-kb EcoRI fragment of pTKS220. The fragment was electroeluted from agarose gels and nick translated with [a-32P]dCTP by the protocol of Maniatis et al. (16). Radioiodination of surface proteins. External proteins of S. flexneri were labeled enzymatically with 1251 by a modification of the Keski-Oja et al. lactoperoxidase procedure (13). Five milliliters each of stationary-phase strain SA100 grown at 37 and 30°C were washed in phosphate-buffered saline plus 1 ,uM KI. The cells were suspended in 0.5 ml of the phosphate-buffered saline plus KI solution plus 0.5 ml of 0.01 M glucose plus 1 p.M KI. To each tube was added 0.18 U of glucose oxidase, 0.67 U of lactoperoxidase, and 0.5 mCi of Na1251. Lactoperoxidase and glucose oxidase were obtained from Sigma Chemical Co., and carrier-free Na125I was obtained from ICN Biomedicals (Costa Mesa, Calif.). The reactions were carried out at room temperature for 15 min. The reactions were terminated by washing the cells three times in 750-1.d volumes of phosphate-buffered saline plus KI. Cells were lysed in 300 p.l of the 2% LDS lysis buffer, and the DNA was removed by centrifugation as described above. Autoradiograms of dried gels were obtained by exposure to X-ray film for 1.5 h. RESULTS Identification of heme-binding proteins. To identify proteins which might be responsible for the binding of Congo

STUGARD ET AL.

3536

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B FIG. 1. Detection of heme-binding proteins in S. flexneri. Cells were allowed to bind hemin, and the proteins were solubilized, separated by LDS-PAGE, and stained with either Coomassie blue or TMBZ. (A) Whole-cell proteins stained with Coomassie blue. (B) Duplicate gel, TMBZ stained for heme. Lane 1, SA100 grown at 37°C; lane 2, SA100 grown at 30°C; lane 3, SA101; lane 4, SA102. Coomassie blue-stained sample of SA102 is not shown.

red or hemin to the cell surface, S. flexneri proteins were analyzed for the presence of heme. The cells were allowed to bind hemin, and the proteins were solubilized and separated on polyacrylamide gels under conditions which allowed the heme-protein complexes to remain associated (6). The gels were stained with TMBZ for heme-associated peroxidase activity, and a heme protein with an apparent molecular weight of 101,000 was detected in wild-type 2a strain SA100 by this procedure (Fig. 1). A 101-kDa heme-binding protein was also detected in a serotype Sa strain, SF100 (data not shown). To determine whether the presence of this 101-kDa heme protein correlated with the Crb+ phenotype, the effects of growth temperature and Crb- mutations on its expression were determined. Temperature regulation was assessed by growing SA100 at 30 or 37°C prior to assay. The 101-kDa heme signal was not present in the 30°C sample (Fig. 1B, lane 2), compared with cells grown at 37°C (lane 1). Crbmutants containing a deletion in the virulence plasmid (SA101, lane 3) or cured of the large plasmid (SA102, lane 4) also lacked the 101-kDa signal. The Coomassie staining for total proteins in Fig. 1A shows the loss of some high-molecular-weight bands in the 100-kDa range in cells grown at 30°C (lane 2) as compared with 37°C (lane 1), but no obvious differences were seen when comparing the wild type and the Crb- mutant (lane 3). In all experiments, Coomassie staining of gels for total proteins verified that overall amounts of protein loaded were comparable so that the signals on TMBZ-stained gels could be compared. To retain interactions between heme and protein, the procedures followed involve gentle lysis of cells and omission of reagents which might disrupt heme-protein contacts. Solubilization of proteins with the addition of P-mercaptoethanol at concentrations found in conventional SDS solubilization buffers (15), with or without boiling, or addition of dithiothreitol at concentrations greater than 50 mM eliminated the 101-kDa heme-protein signal on LDS-PAGE gels with TMBZ staining (data not shown). The Coomassiestained gel of the same samples showed loss of some high-molecular-weight bands in the 100-kDa range.

-14.4

FIG. 2. LDS-PAGE of 59Fe-labeled proteins of S. flexneri. SA100 was grown in L-broth with 0.8 ,uCi of 59Fe per ml, and whole-cell proteins were isolated. (A) TMBZ-stained gel for hemeprotein complexes; (B) autoradiogram of panel A. The arrow indicates the position of the 101-kDa protein when assayed for both hemin (A) and 59Fe (B). Sizes of protein standards are shown in kilodaltons.

Additional proteins staining for heme were typically seen but differed in intensity among different gels. These variations appear to be due to differences in the amount of hemin bound or in the length of time the gels were stained or dried. These bands were present in both the wild-type and mutant strains and were present at both temperatures. Thus, they likely represent cytochromes or other heme proteins. Labeling of iron-containing proteins. To ensure that the TMBZ staining was detecting heme, the presence of iron in the protein was determined. Preliminary experiments indicated that while exogenous hemin increased the intensity of the signal, the protein could be detected by TMBZ in the absence of added hemin and, therefore, could be labeled endogenously. Wild-type strain SA100 (Crb+) was grown in L-broth with 0.8 ,uCi of 59Fe per ml to radiolabel proteins which contain iron. Therefore, heme proteins detected on TMBZ should also be labeled with 59Fe. The TMBZ-stained gel (Fig. 2A) showed the presence of the 101-kDa hemebinding protein in addition to other heme-binding proteins which include the cytochromes. Autoradiography of the TMBZ gel indicated that the 101-kDa protein was labeled with 59Fe (Fig. 2B). Polypeptides at 96, 25, and 19 kDa were detected by autoradiography but not by TMBZ staining. These signals presumably represent proteins which contain nonheme iron. The intense band at the top of lane B (Fig. 2) is material which failed to enter the gel. lodination of surface proteins. To verify that the 101-kDa heme-binding protein is a surface protein, iodination of intact S. flexneri was carried out. The gels were stained for heme with TMBZ and autoradiographed to detect 1251 (Fig. 3). The 101-kDa protein was labeled with 1251 in cells grown at 37°C (lane 1) but not at 30°C (lane 2). The intensity of 1251 labeling compared with other surface proteins suggests that this is a relatively minor protein species. Identification of DNA sequences required for expression of the 101-kDa heme-binding protein. Because transfer of the virulence plasmid, pTKS220, into E. coli RM1058 enabled the cells to bind Congo red or hemin, the transconjugates were assayed for the presence of the 101-kDa heme-binding

VOL. 57, 1989

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FIG. 3. LDS-PAGE of radioiodinated surface proteins in S. flexneri. SA100 was grown at 37°C (lane 1) or 30°C (lane 2), and total cell proteins were separated by LDS-PAGE. (A) Autoradiogram showing surface proteins labeled with 1251I. (B) The same gel stained with TMBZ to detect heme proteins. The arrow points to the 101-kDa protein. Sizes of protein standards are shown in kilodaltons.

protein (Fig. 4). Expression of the protein was similar to that observed with the wild-type S. flexneri strain SA100. Strains carrying mutants or subclones of the plasmid were also tested for the presence of the protein. A mini-Kan insertion mutation in a 9.2-kb EcoRI fragment of the plasmid (pTKS221) caused loss of both Congo red binding and the 101-kDa protein (Fig. 4, lane 3). Cloned fragments of the plasmid were tested for their ability to restore Congo red binding and expression of the heme-binding protein to E. coli or Crb- S.flexneri. Although several clones permitted E. coli to produce red colonies on Congo red agar (4), only two clones, pTRS11 and pTJD2, produced detectable amounts of the 101-kDa heme-binding protein on TMBZ staining (Fig. 4, lanes 4 and 5). The insert in pTJD2 is a subcloned PstI fragment of the cosmid clone pTRS11. It is not known whether coding sequences for the heme-binding protein reside on the 6.1-kb PstI fragment of pTJD2 or if they encode a regulatory factor which might influence expression of a chromosomally encoded structural gene(s). If these clones do contain structural genes for the heme-binding protein, additional sequences must be necessary for normal expression of the protein, as only small amounts of the heme-binding protein were detected on gels. 1

_-

2

3

4

5

6

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TABLE 2. Influence of cloned plasmid sequences on infectivity

of S. flexneri for HeLa cells Strain

Crb'

% HeLa cells infected

SA100 SA101 SA101(pTRS11) SA102 SA102(pTRS11)

+

49 4 28 2 3

a

+

+

Congo red binding on TSB agar with 0.01% Congo red at

37°C.

These clones were also tested for their ability to restore invasiveness to Crb- S. flexneri by measuring infectivity for HeLa cells, as described previously (5). These clones were relatively unstable in S.flexneri, giving rise to Crb- colonies at high frequency, but they did enhance invasiveness of HeLa cell monolayers (Table 2). A strain containing a deletion in the virulence plasmid, SA101, showed increased infectivity when transformed with pTRS11, although it was less invasive than the wild-type strain, SA100. The plasmid had no apparent effect on virulence of a strain cured of the plasmid, SA102, indicating that plasmid sequences in addition to those encoding Congo red binding are required for invasion of HeLa cells. The cloned inserts of pTRS11 and pTJD2 were tested for homology with the region in which the mini-Kan insertion mapped (data not shown). No homology was detected between the 9.2-kb EcoRI fragment and the cloned inserts, indicating that these defined two separate regions associated with the Crb+ phenotype. EIEC strains. Due to the similarities in the pathogenesis of EIEC and S. flexneri and the relatedness of their virulence plasmids, EIEC strains were examined for Congo red binding and expression of the 101-kDa protein. Seven isolates were analyzed; four of these were Crb+, while three contained plasmid deletions and were Crb- (Table 1). These deletions all spanned the 9.2-kb EcoRI fragment into which the mini-Kan insertion mapped in pTKS221. Hybridization of EcoRI digests of the plasmids with the 9.2-kb fragment indicated that it was present only in the Crb+ strains (Fig. 5). All of the Crb+ EIEC strains showed the 101-kDa signal on TMBZ-stained gels (Fig. 6, lanes 5, 6, 7, and 8), while the Crb- strains did not (lanes 2, 3, and 4).

DISCUSSION A relationship between Congo red binding and adsorption of hemin and protoporphyrin IX has been established for l

2

3 4 5 6

7 8

67

'20.1

FIG. 4. LDS-PAGE with TMBZ staining of total cell proteins of E. coli containing S. fiexneri plasmid sequences. Lane 1, S. flexneri SA100; lane 2, JB807 [E. coli RM1058(pTKS220) Crb+]; lane 3, JB904 [E. coli RM1058(pTKS220::mini-Kan) Crb-]; lane 4, JB811 [E. coli RM1058(pTRS11) Crb+]; lane 5, JB801 [E. coli RM1058(pTJD2) Crb+]; lane 6, E. coli RM1058. Sizes of protein standards are shown in kilodaltons.

FIG. 5. Southern hybridization of the 32P-labeled 9.2-kb fragment of pTKS220 to EcoRI-digested plasmids of enteroinvasive E. coli strains. Lane 1, 1107-81 Crb+; lane 2, 550-3076 Crb+; lane 3, 930-78 Crb+; lane 4, 1885 Crb+; lane 5, 3189 Crb-; lane 6, 929 Crb-; lane 7, 5144 Crb-; lane 8, S. flexneri SA100.

3538

STUGARD ET AL. It2;,

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FIG. 6. LDS/PAGE of whole-cell proteins of enteroinvasive E. coli strains with TMBZ staining for heme-protein complexes. Lane 1, S. flexneri SA100; lane 2, EIEC strain 5144 Crb-; lane 3, 3189 Crb-; lane 4, 929 Crb-; lane 5, 930-78 Crb+; lane 6, 1107-81 Crb+; lane 7, 550-3076 Crb+; lane 8, 1885 Crb+. The arrow indicates the position of the 101-kDa protein.

Yersinia pestis (25), Aeromonas salmonicida (12), and S. flexneri. The ability to bind these compounds in vitro correlates with virulence, and both Congo red and hemin enhance the ability of shigellae to invade HeLa cell monolayers when the compounds are prebound to either the bacterial or target cell (5). It was, therefore, of interest to identify the protein(s) involved in heme binding in S. flexneri and EIEC and to verify the parallel that exists between binding of Congo red and hemin. Staining of SDS-PAGE-separated proteins for associated heme revealed the presence of a 101-kDa heme protein. The presence of this protein, like other invasive phenotypes associated with S. flexneri (17), was regulated by growth temperature. Wild-type cells grown at 37°C possessed the 101-kDa protein, while cells grown at 30°C did not. The protein appears to be located on the cell surface, as determined by 1251 labeling. It does not appear to be a particularly abundant protein, however, based on Coomassie staining and the intensity of 1251 labeling. Genetic information essential for biosynthesis of the 101kDa heme-binding protein is located on the virulence plasmid of S. flexneri and EIEC strains. Transfer of the plasmid, pTKS220, from S.-flexneri to an E. coli K-12 strain rendered it positive for both Congo red binding and expression of the 101-kDa protein. A mini-Kan insertion into a 9.2-kb EcoRI fragment of the plasmid which resulted in the Crb- phenotype also resulted in loss of the 101-kDa protein. It is not known whether the insertion is associated with loss of a positive regulatory function such as virF (22), is in a structural gene for the protein, or exerts a polar effect on downstream Congo red and 101-kDa coding sequences. A cosmid clone, pTRS11, and its subclone pTJD2, both of which encode Congo red binding, showed reduced levels of the 101-kDa protein in E. coli. The 6.1-kb PstI fragment of pTJD2 may contain coding sequences for the 101-kDa protein but fail to express it at a high level due to lack of a positive regulator or lack of its normal promoter, or it may encode regulatory sequences which enhance production of a chromosomally encoded protein. The 9.2-kb fragment shares little or no homology with pTRS11 sequences, indicating that the mini-Kan insertion is likely to be in a regulatory locus or is creating a polar effect. The 101-kDa heme-binding protein was found to correlate with Congo red binding in EIEC as well as S. flexneri. Hybridization with the 9.2-kb EcoRI fragment indicated that this region of the plasmid was essential for expression of this

protein in E. coli also. All the EIEC strains tested that had plasmid sequences homologous to this fragment possessed the 101-kDa protein, while those that contained deletions encompassing this region were Crb- and lacked the protein. The sensitivity of the 101-kDa heme-binding protein to reduction by dithiothreitol and mercaptoethanol, as well as the absence of a 101-kDa protein on Coomassie-stained SDS-PAGE reducing gels, suggests that the protein is composed of subunits or is a complex of two or more polypeptides. The lysis procedure and electrophoresis conditions used for the LDS-PAGE and TMBZ staining are mild enough so that protein-protein interactions would be preserved. Extraction of the protein from the LDS gel and reanalysis on SDS-polyacrylamide denaturing gels has not provided an unambiguous answer to this question (data not shown). Denaturation results in loss of heme from the heme-protein complex and, therefore, loss of ability to detect the protein. Since there are several proteins which comigrate with the heme-staining band in the LDS system, it is not clear which of the protein species on the SDS gel was originally part of the heme-binding complex. Considering the size of the protein, its link to virulence, the temperature regulation of its expression, and the fact that it is plasmid encoded and surface associated, it is tempting to speculate that this protein is a complex of Ipa proteins. IpaB has been reported to be a 57-kDa (1) or 62-kDa (9) surface protein in S. flexneri strains. A complex of IpaB with IpaC (42 kDa) (27) or IpaD (37 kDa) (27) might create the appropriate size of polypeptide. This hypothesis is the subject of current investigation. The ability to bind Congo red or hemin to the cell surface correlates with virulence of shigellae, and the fact that prebinding of these compounds enhances infectivity suggests a role for the 101-kDa heme-binding protein (or protein complex) in the initial penetration of colonic epithelial cells, an essential step for the development of dysentery (14). The ability to bind hemin to the cell surface may confer a unique advantage in terms of virulence of Shigella spp. in vivo. The binding of heme compounds found within the host to a surface protein may disguise the bacterium as a desirable molecule for the intestinal epithelial cell. The host cells could bind the heme-coated bacteria through heme receptors (7) and then actively endocytose the bacterial cells (8). Since it was found that addition of exogenous heme was not essential for detecting the protein by TMBZ, it is possible that shigellae do not need an exogenous source of heme but may incorporate heme into the protein during synthesis or transport to the cell surface. It has not been determined whether the protein is made as a heme protein within the cell or whether binding of endogenous heme occurred during lysis of the cells. After initial invasion, the 101-kDa hemebinding protein was absent from proteins isolated from S. flexneri multiplying within HeLa cells (V. L. Headley, personal communication). This indicates that once shigellae have gained entrance into the target cells, the 101-kDa heme-binding protein is no longer required for continued virulence functions of the bacterium. ACKNOWLEDGMENTS We thank Rose Evans for technical assistance and Terrence Stull for his advice and helpful discussions throughout the study. This work was supported by Public Health Service grant A116935 from the National Institutes of Health. LITERATURE CITED 1. Buysse, J. M., C. K. Stover, E. V. Oaks, M. Venkatesan, and P. J. Kopecko. 1987. Molecular cloning of invasion plasmid


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