INFECTION AND IMMUNITY, Apr. 1990, p. 903-908 0019-9567/90/040903-06$02.00/0 Copyright X 1990, American Society for Microbiology

Vol. 58, No. 4

Cloning and Expression in Escherichia coli of LKP Pilus Genes from a Nontypeable Haemophilus influenzae Strain SIDDHARTHA KAR,* SAM C.-M. TO, AND CHARLES C. BRINTON, JR. Department of Biological Sciences, University of Pittsburgh, and Bactex, Inc., Pittsburgh, Pennsylvania 15260 Received 8 September 1989/Accepted 19 December 1989

Nontypeable Haemophilus influenzae HF0295, isolated by aspiration from the middle ear of a patient with otitis media, expresses long, thick, and hemagglutinating pili of a single serotype (LKP1) on its surface. An intact pilus vaccine consisting of the LKP1 serotype protected chinchillas against experimental otitis media (C. C. Brinton, Jr., M. J. Carter, D. B. Derber, S. Kar, J. A. Kramarik, A. C.-C. To, S. C.-M. To, and S. W. Wood, Pediatr. Infect. Dis. J. 8:554-561, 1989; R. B. Karasic, D. J. Beste, S. C.-M. To, W. J. Doyle, S. W. Wood, M. J. Carter, A. C.-C. To, K. Tanpowpong, C. D. Bluestone, and C. C. Brinton, Jr., Pediatr. Infect. Dis. J. 8:562-565, 1989). The genes encoding LKP1 pili were cloned from a genomic library of the clinical strain as a 12.5-kilobase insert on a plasmid vector and inserted into Escherichia coli K-12. Transposon mutagenesis and deletion constructs mapped the pilus-coding region within a 7-kilobase region of insert DNA. The recombinant bacteria were found by electron microscopy to express pili morphologically similar to LKP1 pili. Purified pilus rods from the recombinant and its parental strain were composed of a single detectable protein with an apparent molecular weight of 27,500. Antibodies raised against LKP1 pili purified from H. influenzae immunologically reacted with pili from the recombinant bacteria. Pili from both strains also adhered to human erythrocytes and buccal cells with the same specificity. Pili are bacterial surface antigens expressed by a wide variety of different pathogens (2-6). They are protein appendages consisting of a helically symmetrical assembly of identical major protein (pilin) subunits (3). Some pili can also carry from two to three minor proteins assembled on their tips, one of which, the adhesin, first detected and isolated in this laboratory (8, 9; C. C. Brinton, Jr.,- and M. S. Hanson, U.S. patent 4,801,690, March 1986), carries the active site for pilus adhesion to specific membrane receptors on human and animal cells (11, 15, 19, 20). Pili can project out through the lipopolysaccharide microcapsules as well as the polysaccharide major capsules, and therefore they are accessible to antibodies, whereas outer membrane proteins may be protected from antibodies by capsules. For this reason, pilus antibodies may be a better vaccine candidate against bacterial pathogens than outer membrane proteins. Pili have a complex set of functions including adhesion to host cell receptors, mediation of resistance to phagocytosis, and surface translocation that help to explain their role as important virulence and immunity factors in bacterial disease (4). Pili have been observed on Haemophilus influenzae isolates from a number of disease sources. Many clinical isolates are either piliated or can be enriched for piliation in vitro (23). Piliated H. influenzae colonize mucosal surfaces more efficiently than nonpiliated bacteria (1, 22, 23; N. G. Guerina, S. Langermann, H. W. Clegg, T. W. Kessler, and D. A. Goldman, Pediatr. Res. 16:242A, 1982). The pili of H. influenzae clinical isolates can be classified into several families on the basis of morphology and adhesion specificity (5). The first discovered H. influenzae pili (Guerina et al., Pediatr. Res., 1982) were the LKP family (long, thick, and positive for hemagglutination), probably because they are most easily visualized by electron microscopy. The length and diameter of these pili were about 2 p.m and 4 nm, respectively. The other morphological families are shorter *

and thinner (5). We have found that LKP pili have many properties typical of other pilus families on pathogens. The pilin rod lateral surface antigenic determinants are variable (5). About 12 serotypes account for about 70% of LKP pili on nonencapsulated (nontypeable) strains, while a single type accounts for 89% of LKP pili on H. influenzae type b encapsulated strains and 5 related serotypes account for 100% of pilus types on type b strains (5). There is little or no serological cross-reactivity among LKP pilus types despite extensive amino acid sequence similarities among them (5). H. influenzae diseases are of several kinds. Invasive diseases such as meningitis are caused principally by encapsulated strains, in particular type b. Noninvasive diseases such as otitis media, bronchitis, sinusitis, and pneumonia are caused by nonencapsulated (nontypeable) strains. Bacteria of both kinds can colonize the throat and nasopharynx of humans without causing overt disease. It is believed that disease-causing H. influenzae originate in each individual from those bacteria carried in the throat or nasopharynx or both (17). It follows from this that if carriage could be prevented, disease could be prevented. We are seeking to develop pilus vaccines for H. influenzae diseases beginning with the LKP pilus family. Purified LKP pilus vaccines prepared from nontypeable strains conferred highly significant active and passive protection against experimental otitis media in the chinchilla (10). Protection was correlated with the serological pilus type of vaccine and challenge bacteria. There are several problems in LKP pilus vaccine development that could profit from genetic analysis of LKP pilus genes. The structure-function relationships of the adhesion site and the pilin molecule could be investigated by mutational, deletional, and sequence analysis. If the sequences of the pilins of a number of different serotypes were known, a comparison of them could locate conserved sequences, and among these one would expect to find segments of the adhesive peptides. If the sequence of a single LKP pilus serotype were known it would also contain adhesin se-

Corresponding author. 903

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KAR ET AL. TABLE 1. E. coli, H. influenzae, and phage strains Strain

Description or genotype

Source

H. influenzae HF0295 E. coli HB101 E. coli MB392

Nontypeable strain from clinical otitis media hsdM hsdR recA Apil hsdRJS supE44 supF58 A(argF-lac)Ul69 galK2 gall22 metBl typRSS pro::Tn5(A-, F' traD36 proAB lacIq lacZAM15) Source of vector pUC19 HB101 transformed with pUC19 HB101(pHF1) A b221 TnS (near clII) c1857

Children's Hospital, Pittsburgh, Pa. R. Younga M. Benedika

E. coli MB392(pUC19) E. coli HB101(pUC19) E. coli EC0295 X420 a

M. Benedik This study This study R. Young

Department of Biology and Biochemistry, Texas A&M University, College Station, TX 77840.

quences, which might be located by synthesizing an overlapping series of partial peptides and testing them for the inhibition of binding of adhesion-blocking monoclonals or the inhibition of adhesion. An adhesin peptide vaccine might induce adhesion-blocking, cross-reacting, broadly protecting antibodies to H. influenzae diseases. We report here the beginnings of a genetic analysis of the H. influenzae LKP pilus gene cluster. The genes encoding the LKP1 pili from an H. influenzae otitis media strain were cloned'into Escherichia coli. LKP1 pili expressed by the E. coli recombinant strain were identical to those on the clinical H. influenzae parent isolate in their structural, functional, and immunological properties. The genes for pilus expression were located within a 7-kilobase (kb) region of H. influenzae DNA. Limited transposon mutagenesis did not locate any separate adhesin genes. (These results were presented in brief as a poster at the 89th Annual Meeting of the American Society of Microbiology, New Orleans, May 14 to 18, 1989, D-204, p. 116.) MATERIALS AND METHODS Bacteria and media. Strains are listed in Table 1. H. influenzae HF0295 was grown on brain heart infusion agar or broth supplemented with NAD and hemin (29). E. coli strains were grown on Luria agar (LB agar) and Luria broth (LB) (7). Media were supplemented with 50 xg of ampicillin per ml and 25 ,ug of kanamycin per ml if required. Hemagglutination and serum agglutination. Hemagglutination and serum agglutination assays were done at room temperature in phosphate-buffered saline (PBS). Bacteria grown on agar or in broth were washed twice in PBS and were suspended in PBS. The number of bacteria per milliliter of the suspension was determined with a Petroff-Hausser counter. For hemagglutination assays, different dilutions of bacteria were incubated with a 1% erythrocyte suspension in PBS for 10 min at room temperature on ring slides. The highest dilution of bacteria giving hemagglutination was defined as the hemagglutination titer. For serum agglutination assays, different dilutions of antiserum raised against pure HF0295 pili were incubated for 20 min with bacteria at 108/ml. The highest dilution of antiserum giving positive agglutination was taken to be the antiserum titer. Isolation of plasmid and chromosomal DNA. Chromosomal and plasmid DNA were extracted and purified as described previously (18, 26). DNA library construction. Chromosomal DNA from HF0295 was partially digested with restriction enzyme Sau3A. Fragments of about 12 kb were purified by sucrose gradient centrifugation and were ligated at the dephosphorylated BamHI site of the vector pUC19 DNA (30). Standard procedures for library construction were used (18). Ligated DNA was transformed into E. coli MB392 and plated on LB agar with ampicillin.

Colony blot for library screening and SDS-PAGE. Colony blots were done with antipilus sera raised against pure pili from HF0295 (Bio-Rad Immun.blot [GAR-HRP] Assay Manual). Sodium dodecyl sulfate-polyacrylamide gel electrophoretic (SDS/PAGE) analysis of pilus proteins was performed as described by Laemmli (13). The gel was silver stained (21). Tn5 mutagenesis. Transposon TnS mutagenesis of the recombinant plasmid was done with bacteriophage A420 (24). The positions of transposon insertion were mapped as shown in Fig. 1 by restriction fragment analysis. Construction of deletion mutants. The deletions were constructed by digestion and subsequent ligation employing restriction sites on the plasmid and transposons. DNA between the HpaI site of transposon site 10 and the SmaI site of the vector was deleted to generate pHF11. pHF12 was constructed by deleting between the HincII site of the vector and the HinclI site of transposon site 2. pHF13 had DNA between the two XhoI sites deleted, and pHF14 had a deletion from the BstEII site to the SmaI site of the vector. In the latter case, the BstEII site was first filled in with Klenow fragment and deoxynucleoside triphosphates before blunt-end ligation to the SmaI site. pHF12 was separately mutagenized by Tn5, and a section of DNA was deleted from the HpaI site of a transposon insertion site to the SmaI site of the vector to generate pHF15. Extraction and purification of pili. Pili were purified from HF0295 and the recombinant E. coli strains by procedures to be published in detail elsewhere (C. C. Brinton, Jr., M. J. Carter, and S. Kar, manuscript in preparation). In summary, bacteria were grown on solid medium and harvested. Pili were sheared off the bacteria by blending in a Omni mixer. Pili were crystallized from the crude extract by dialysis against acetate buffer (pH 5). The crystals were pelleted and suspended in 0.01 M CAPS (3-cyclohexamide-1-propanesulfonic acid) buffer at pH 10.5. This crystallization and suspension cycle was repeated three times, and the crystals were suspended in phosphate buffer (pH 10.5) containing 5 mM EDTA and 0.2% Triton X-100. The pH was lowered to 7.5, and pili were crystallized by adding sodium chloride and polyethylene glycol at final concentrations of 0.5 M and 3%, respectively. The crystals were suspended in phosphate buffer (pH 10.5). In the final cycle, SDS and polyethylene glycol were added at concentrations of 5 and 3%, respectively. The pilus crystals were pelleted and washed three times with acetate buffer (pH 5) and suspended in phosphate buffer (pH 10.5) containing 0.02% sodium azide as a preservative. Generation of pilus antiserum. Pure pilus rods from HF0295 were injected (250 ,ug per injection) into rabbits, and after three injections (the first one with Freund complete adjuvant and the subsequent boosters with Freund incomplete adjuvant) 2 weeks apart, blood was collected. The

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blood was allowed to clot for 1 h at room temperature and overnight at 4°C. The clot was separated from the serum by low-speed centrifugation in a tabletop clinical centrifuge. Bacterial adhesion to buccal cells. Buccal cells were freshly collected by scraping inside the cheek of a healthy human volunteer. The cells were washed three times in PBS and resuspended in PBS at a concentration of 107/ml. Bacterial suspensions in PBS were added to the buccal cells at a multiplicity of 100 bacteria per buccal cell and were incubated at room temperature for 30 min. The buccal cells were centrifuged at low speed to pellet only the cells. They were washed four times to remove loosely adhering bacteria. The buccal cells were then examined by phase-contrast microscopy.

Electron microscopy. Pili on bacteria were visualized by the protein monolayer technique (27). Immune electron microscopy was done by a method described previously (14) with immune sera raised in a rabbit against pure pilus rods of strain HF0295 and colloidal gold conjugated anti-rabbit immunoglobulin G (Janssen Pharmaceutica Inc.; goat antirabbit G10). Adherence of pili to erythrocytes and buccal cells. The adherence of pilus rods was detected by the passive serum agglutination method (8). Nonaggregated purified pilus rods were suspended in PBS and were incubated with human erythrocytes or buccal cells in PBS. Neither erythrocytes nor buccal cells were agglutinated by pilus rods. After 1 h of incubation at room temperature, the cells were centrifuged and washed once with PBS. Antiserum raised against intact pilus rods and containing type-specific antibodies to the laterally exposed epitopes was added at a io-3 dilution. Hemagglutination of the erythrocytes and agglutination of buccal cells by the antiserum was taken as a measure of binding of pili to these cells.

RESULTS Pili of HF0295. HF0295 was isolated by tympanic aspiration from a child admitted to Children's Hospital of Pittsburgh with otitis media. The strain was nontypeable and specifically bound to human erythrocytes and buccal cells in vitro. Electron microscopic examination and serological typing showed LKP1 pili on this strain. The purified pilus

rods from this strain were composed of a single detectable protein subunit with an apparent molecular weight of 27,500 (from SDS-polyacrylamide gels). The purified pilus rods retained their properties of binding to human erythrocytes and buccal cells. To study the genetic organization of pilus genes, we cloned them into E. coli. Isolation of recombinant E. coli expressing HF0295 pili. A genomic DNA library was prepared by partially digesting the HF0295 genome with Sau3A and cloning 12-kb fragments into the vector pUC19 at the BamHI site. The recombinant plasmids were transformed into E. coli MB392. A total of 11,000 recombinant colonies were screened for pilus expression, without induction of the lac promoter in the pUC19 vector, on colony blots with antiserum raised against pure LKP1 pili from the HF0295 strain. Twenty-four colonies reacted positively with the pilus antiserum. Seven were selected for examination of their recombinant plasmids with restriction enzymes. They all exhibited similar restriction enzyme cleavage patterns. One of them, pHF1, was selected for further analysis. pHF1 was also transformed into E. coli HB101, which lacks the entire gene cluster for type 1 pili (12), to produce strain EC0295. The EC0295 strain produced HF0295 strain-specific pili. Physical map of cloned DNA. Plasmid pHF1 was digested with various restriction enzymes to prepare a physical map (Fig. 1). The DNA insert was approximately 12.5 kb. Transposon TnS insertions were used to map the boundaries of the pilus genes. Insertions 1, 2, 8, 10, and 11 did not affect pilus expression, whereas insertions 3, 4, 5, 6, 7, and 9 abolished pilus expression. Five deletions described in the Materials and Methods were also constructed. Pilus expression was unaffected in pHF11, pHF12, and pHF15, whereas pili were not expressed in pHF13 and pHF14. These results map the pilus genes in a 7-kb region of the inset DNA on pHF15. Adhesion properties of HF0295 and EC0295. The recombinant EC0295 and the parent HF0295 strains were tested for their ability to bind to human erythrocytes and buccal cells. Hemagglutination titers for the two strains were determined to be 6.2 x 107/ml and 7.8 x 107/ml, respectively. Both bacteria bound strongly to human buccal cells. As a control, E. coli HB101(pUC19) without H. influenzae DNA inserted in its vector did not bind to buccal cells (Fig. 2) or hemag-

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glutinate human erythrocytes. Adherence was also specific for human erythrocytes. Erythrocytes from rats, mice, cattle, chickens, or guinea pigs did not hemagglutinate with these strains. Characterization of pili from HF0295 and EC0295. Bacteria from the two strains were examined in the electron microscope (Fig. 3). Similar pilus structures were seen on both strains. Both strains could be agglutinated by antiserum raised against pure pilus rods. The lateral surface of pili on bacteria also could be labeled by antiserum as seen by immune electron microscopy (Fig. 4). These results indicate that the pili on both strains are morphologically and antigenically identical. Purified pilus rods from both strains were seen to be composed of a subunit protein with an apparent molecular weight of 27,500 (Fig. 5). No minor pilus-associated proteins were detected. The purified pilus rods from both strains had the ability to bind to human erythrocytes and buccal cells as determined by indirect hemagglutination and indirect buccal cell agglutination.

DISCUSSION The H. influenzae LKP1 pilus gene cluster was cloned into E. ccli. The size of the pilus gene region was determined by

FIG. 3. Electron micrograph of bacterial strains H. influenzae HF0295 and E. coli EC0295. Strains HF0295 and EC0295 were mixed and visualized by electron microscopy. The larger bacterium is EC0295, and the smaller ones are HF0295. Bar represents 1.0 pum.

transposon mutagenesis and deletion analysis to be about 7 kb. LKP pili were expressed by the recombinant E. coli carrying the plasmid with the H. influenzae HF0295 LKP1 genes. The pili on the recombinant bacteria could be purified by the same technique used to purify the pili from the parent

strain H. influenzae HF0295. The SDS-PAGE molecular weight, the antigenicity for LKP1 pilus-typing antiserum, and the specificity of attachment to buccal cells and erythrocytes were the same for both pilus preparations. These results strongly suggest that the recombinant pili are identical to the parental pili. These results also suggest that the single gene cluster cloned into E. coli contains all the genes necessary for the expression of functional LKP1 pili. If other genes regulating the expression of LKP1 pili occur in H. influenzae strains at other chromosomal or plasmid loci, they must exert only negative control on LKP1 pilus expression. The existence of a single LKP pilus gene cluster in H. influenzae HF0295 is also suggested by our finding that a number of different independently generated clones from the same parental genomic library were genetically identical. The cloned pilus genes were probably expressed from their natural promoters, since induction of the lac promoter in the vector was unnecessary for pilus expression. The expression and assembly of LKP1 pilus genes were not dependent on the presence of type 1 pilus genes in the host bacteria, since strain HB101, which is deleted for the type 1 pilus genes,

EXPRESSION OF H. INFLUENZAE PILUS GENES IN E. COLI

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FIG. 4. Immune electron microscopic labeling of pili from E. coli EC0295 and H. influenzae HF0295 bacteria. The larger bacterium is EC0295, and the smaller ones are HF0295. Bar represents 1.0 ,um.

also produced LKP1 pili. The fact that TnS insertion 8 made pili but insertion 9 did not suggests that there is more than one gene and operon in the pilus-expressing region. Biochemical evidence suggests that LKP pili consist only of a polymerized pilin subunit and have no detectable minor proteins as found in other pili. When LKP pili were purified by using strong detergent conditions that can remove minor proteins from other pilus families, only a single protein band was seen on Coomassie blue- or silver-stained SDS-polyacrylamide gels. Such pili were fully competent for adhesion to human erythrocytes and buccal cells. The genetic evidence presented here is consistent with this finding since no mutants were isolated which expressed pilus rods that could not adhere to erythrocytes. The possibility must be reserved, however, that a minor adhesin protein undetectable in our experimental conditions is part of the LKP1 pilus structure and that our limited transposon mutagenesis missed the particular adhesin mutant. The study of LKP pilus-mediated adhesion by H. influenzae is simplified by our demonstration that an LKP1piliated E. coli containing a single LKP pilus gene cluster adheres to buccal cells and erythrocytes with the same specificity as H. influenzae. A number of reports that H. influenzae can adhere with several different specificities to human cells have appeared in the literature (16, 25, 28). Other families of H. influenzae pili (5) that do not adhere to human erythrocytes may be responsible for multiple specificities. The adhesion of LKP-piliated recombinant E. coli or

of purified LKP pili can be used as a probe to determine the distribution of LKP receptors on human cells and tissues. It is likely that the gene cluster for LKP pili is located on the chromosome of H. influenzae since no evidence for plasmid DNA in the DNA extracted for library preparation was found. However, the presence of the pilus genes on a very large plasmid cannot be ruled out since precautions were not taken during DNA extraction to preserve large plasmids. Since the binding properties of the recombinant E. coli were similar to those of the parent H. influenzae strain, it can be concluded that LKP pili are probably a major component of H. influenzae involved in adhesion. The number and location of specific genes in the LKP1 cluster, the sizes and other characteristics of the gene products, and the number, location, and transcription direction of operons in the cluster will be published elsewhere. ACKNOWLEDGMENTS We gratefully acknowledge the technical assistance of Melinda Carter, Marcia Hughes, and Agnes C.-C. To. We thank Jan Stephenson and Charles Bluestone for H. influenzae HF0295. LITERATURE CITED 1. Anderson, P. W., M. E. Pichichero, and E. M. Connor. 1985. Enhanced nasopharyngeal colonization of rats by piliated Haemophilus influenzae type b. Infect. Immun. 48:565-568. 2. Brinton, C. C., Jr. 1959. Non-flagellar appendages of bacteria. Nature (London) 83:782-786. 3. Brinton, C. C., Jr. 1965. The structure, function, synthesis and genetic control of bacterial pili and a molecular model for DNA and RNA transport in gram negative bacteria. Trans. N.Y. Acad. Sci. 27:1003-1054. 4. Brinton, C. C., Jr. 1978. The piliation phase syndrome and the uses of purified pili in disease control, p. 33-60. In C. Miller (ed.), 13 U.S.-Japan Conference on Cholera, Atlanta, Ga. National Institutes of Health, Bethesda, Md. 5. Brinton, C. C., Jr., M. J. Carter, D. B. Derber, S. Kar, J. A. Kramarik, A. C.-C. To, S. C.-M. To, and S. W. Wood. 1989. Design and development of pilus vaccines for Haemophilus

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influenzae diseases. Pediatr. Infect. Dis. J. 8:554-561. 6. Christensen, G. D., W. A. Simpson, and E. H. Beachy. 1985. Microbial adherence in infection, p. 6-23. In G. L. Mandell, R. G. Douglas, Jr., and J. E. Bennett (ed.), Principles and practice of infectious diseases, 2nd ed. John Wiley & Sons, Inc., New York. 7. Davis, R. W., D. Botstein, and J. R. Roth. 1980. Advanced bacterial genetics, p. 201. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 8. Hanson, M. S., and C. C. Brinton, Jr. 1988. Identification and characterization of E. coli type-1 tip adhesion protein. Nature 9.

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