INFECTION AND IMMUNITY, Mar. 1990, p. 828-832

Vol. 58, No. 3

0019-9567/90/030828-05$02.00/0 Copyright C) 1990, American Society for Microbiology

NOTES

Nonreciprocal Complementation of the hlyC and lktC Genes of the Escherichia coli Hemolysin and Pasteurella haemolytica Leukotoxin Determinants CHRISTIANE FORESTIER AND RODNEY A. WELCH*

Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison, Wisconsin 53706 Received 29 August 1989/Accepted 16 November 1989

The genetic organization of the Pasteurella haemolytica leukotoxin operon (lktCABD) is similar to that of the Escherichia coli hemolysin (hlyCABD). Their gene products share a sequence similarity of 66, 62, 90.5, and 75.6%, respectively. We investigated the role of the C proteins (LktC and HlyC) by performing reciprocal transcomplementation analyses in an E. coli recombinant background. In the absence of the C genes, neither LktA nor HlyA had their respective cytotoxic activities. When hlyC was provided in trans to MktA, the toxin that was produced had the same activity and target cell specificity as the wild-type leukotoxin; it was leukotoxic for bovine lymphoid cells but not human lymphoblast cells when it was evaluated by a 51Cr-release assay. We also detected a weak hemolytic activity for the active form of LktA against sheep erythrocytes. In contrast, an E. coli strain containing IktC with hlyA produced a form of HlyA which was neither hemolytic nor cytotoxic. A monoclonal antibody (D12) against HlyA which recognized an epitope specific to the active form of HlyA did not cross-react in immunoblots with LktA that was activated by either LktC or HlyC. We conclude that the mechanism for activation of leukotoxin and hemolysin by their respective C proteins (LktC and HlyC) is mechanistically similar but that the exact structural requirements involved in the process are different.

processing of HlyA (16), and it is not the result of phosphorylation or glycosylation (21). The sequence homologies between LktC and HlyC suggest that LktC functions similarly to HlyC, but this has not been thoroughly investigated. The two other genes, B and D, are located downstream of the A genes and are involved in the extracellular secretion of HlyA and LktA (27, 31). Despite the sequence similarities, HlyA and LktA do not have the same target cell specificity. The E. coli hemolysin has a wide range of target cells, including erythrocytes as well as leukocytes and epithelial cells from different species (7, 13). In contrast, the P. haemolytica leukotoxin has a more limited range of target cells on which it is active and only kills ruminant leukocytes (1, 11, 24). The mechanism for target cell recognition by these toxins remains unclear, and the toxin domains that are involved in the recognition processes have not been identified. It is also uncertain whether the C gene product-mediated activation of the respective A proteins is involved in the recognition process. We investigated the role of the C gene products (HlyC and LktC) on both LktA and HlyA proteins by transcomplementation with different subclones of the toxin operons. The cytotoxic effects of these constructs were determined by using erythrocytes and bovine as well as human lymphoid cells. The hemolysin and leukotoxin subclones used in this study are listed in Fig. 1. Recombinant DNA procedures were performed essentially as described by Maniatis et al. (20). The hemolysin assay was performed as described previously (32) by using 10% sheep erythrocytes. Cytotoxicity of the different strains was determined by a 51Cr-release assay (24) with bovine leukemia-derived B lymphocytes

Recent cloning and DNA sequence analyses have revealed that a number of previously unrecognized evolutionary relationships exist among a variety of exotoxins produced by gram-negative pathogens. Lo et al. (19) have discovered a clear homology between the sequence of the Pasteurella haemolytica leukotoxin (LktA) and the Escherichia coli hemolysin (HlyA). Since then, hemolysins from other gramnegative bacteria (Proteus and Morganella species) as well as the Actinobacillus actinomycetemcomitans leukotoxin and the Bordetella pertussis adenyl cyclase have been found to be related to one another (8, 14, 15, 18, 32). P. haemolytica is the principal microorganism associated with bovine pneumonic pasteurellosis, and its leukotoxin has been implicated as a virulence factor in that disease (12, 24). The genetic organization of the P. haemolytica leukotoxin operon is similar to that of E. coli hemolysin and consists of four contiguous genes designated lktCABD and hlyCABD (5, 9, 12, 19, 24). Similarities of 66, 62, 90.5, and 75.6%, respectively, exist between the deduced amino acid sequences of the P. haemolytica and E. coli C, A, B, and D proteins (unpublished data). The toxin structural genes (hlyA and WktA) encode 110-kilodalton (kDa) (HlyA) and 102-kDa (LktA) proteins (3, 4, 19). Although they are slightly different in size and gaps are required to achieve their optimal sequence alignments, HlyA and LktA are clearly of homologous origin. The product of hlyC is a 20-kDa protein (5). In the absence of HlyC, HlyA is synthesized but does not possess hemolytic activity (21, 22). The nature of HlyCmediated modification on HlyA remains unknown. It involves neither N-terminal (4) nor C-terminal proteolytic *

Corresponding author. 828

NOTES

VOL. 58, 1990

genotype

829

reference or source

vector

plasmid

pUC19

pWAM04

32

pUC9

pWAM300

our laboratory

pACYC184

pWAM783

our laboratory

pUC9

pWAM825

this study

pACYC184

pWAM716

6

pBR322

pLKT5

19

pUC1 9

pPH5B

27

pUC19

pWAM785

this study

pUC18

pWAM955

this study

pUC18 Ml 3mpl 8

pWAM827 pWAM956

this study this study

pACYC1 84

pWAM957

this study

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FIG. 1. Restriction maps of the DNA fragment inserts from E. coli hemolysin (A) and P. hemolytica leukotoxin (B) operons. The relative positions of the C, A, B, and D genes are shown; and the vectors used in each construct are indicated, as are the restriction sites that were used.

(BL3 cells) provided by Gary Splitter (University of Wisconsin) or with Daudi (ATCCCCL213) or Raji (ATCC CCL86) cells provided by Gerald Byrne (University of Wisconsin). Briefly, target cells were washed twice and suspended in RPMI 1640 medium (GIBCO Laboratories, Grand Island, N.Y.) to a concentration of 106 cells per ml. A total of 50 ,uCi of [51Cr]sodium chromate (specific activity, 1 mCi/ml; Dupont, NEN Research Products, Boston, Mass.) was added per ml of cell suspension, and the mixture was incubated at 37°C for 1 h on a rocking platform. After incubation and three washes with RPMI 1640 medium, cells were adjusted to a concentration of 106 cells per ml. This suspension (0.1 ml) was mixed with 0.2 ml of bacterial supernatant from a late-log-phase culture in a 96-well tissue culture plate (GIBCO) and incubated at 37°C in 5% CO2 for 1 h. After a 5-min centrifugation (208 x g), 0.1 ml of the supernatant

containing the released 51Cr was transferred to vials and counted in a gamma counter. The presence of toxins in the supernatant was also monitored by performing immunoblots. Trichloroacetic acid (l0o)-precipitated material from the bacterial supernatant was subjected to discontinuous sodium dodecyl sulfatepolyacrylamide gel electrophoresis with a 10% polyacrylamide gel by the method of Laemmli (17). Proteins were transferred to nitrocellulose by the method of Towbin et al. (30). Following transfer, binding sites on nitrocellulose were blocked with 5% skim milk (GIBCO) in Tris-buffered saline (0.02 M Tris [pH 7.5], 0.9% NaCl). The proteins were probed with the appropriate antibody in Tris-buffered saline plus 0.5% skim milk; and after the addition of an anti-immunoglobulin serum conjugated to horseradish peroxidase, colorimetric development was performed by the protocol de-

830

NOTES

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INFECT. IMMUN. 6

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FIG. 2. Immunoblot analyses. Immunoblot analysis of trichloroacetic acid-precipitated material from 0.5 ml of cell-free, late-logphase supernatants of P. haemolytica Al (lane 1), E. coli DH1 (pWAM827) (LktA) x pWAM716 (HlyBD) (lane 2), E. coli DH1 (pWAM957) (LktA) x pWAM825 (HlyCBD) (lane 3), E. coli DH1 (pWAM04) (HlyCABD) (lane 4), E. coli DHl(pWAM783) (HlyABD) (lane 5), and E. coli DH1 (lane 6). The blots were developed with polyclonal anti-P. haemolytica leukotoxin antibody (A) and monoclonal anti-E. coli hemolysin D12 antibody (B). The molecular mass standards are indicated by arrows, and the values are expressed in kilodaltons.

scribed by the manufacturer (Sigma Chemical Co., St. Louis, Mo.). We combined the lktC and lktA genes from pLKT5 with the lktB and lktD genes of pPH5B to obtain the entire leukotoxin operon on one recombinant plasmid. The resulting plasmid, pWAM785, was then transformed into E. coli DH1 [F- recAl endAl gyrA96 thi-J hsdRJ7 (r- m+) supE44]. Secretion of leukotoxin into the culture supernatant was monitored by immunoblotting with a polyclonal

antiserum against the P. haemolytica supernatant (provided by R. Lo, University of Guelph, Guelph, Ontario, Canada) and by cytotoxicity assays. Although pWAM785 harbors the genes necessary for secretion of LktA, little LktA protein or leukotoxic activity was detected in the supernatant (data not shown), indicating that this process is inefficient in E. coli. Insertion of the inducible tac promoter upstream of lktC led to stronger expression of lktA, as judged by immunoblots of whole cells, but there was no concomitant increase in its secretion (data not shown). The apparent inefficiency of the P. haemolytica secretion genes (lktB and 1ktD) in E. coli has also been reported by others (10, 26). Because these secretion proteins are thought to interact with bacterial membranes, it is possible that LktB, LktD, or both differ from HlyB and HlyD in some domain(s) that specifically recognizes membranes. In our study, we chose to use the E. coli hemolysin hlyB and hlyD genes to achieve secretion of both HlyA and LktA. An lktA subclone was constructed by inserting a 3,934base-pair (bp) BglII fragment from pWAM785 into BamHIdigested pUC18. When complemented in trans with hlyB and hlyD (pWAM716) (6), E. coli DH1 which harbored this subclone (pWAM827) secreted LktA into the supernatant, as shown in Fig. 2A. However, this protein did not exhibit any cytotoxic effect on BL3 cells (leukotoxicity) or erythrocytes, indicating that LktC is probably needed for the activation of LktA (Table 1). To study this, a fragment from pWAM785 containing all of lktC and the first 476 bp of lktA was inserted into pUC18 (pWAM955). To achieve a three-vector system, lktA was subcloned on the bacteriophage vector M13mpl8, resulting in pWAM956. This transcomplementation (pWAM716, pWAM955, and pWAM956) was performed in E. coli JM101 (F' supE traD36 proA+B+ lacIq lacZ). As shown in Table 1, the supernatant of this strain exhibited a significant level of toxicity against BL3 cells (46.26%) compared with the percentage of cytotoxicity observed with the control P. haemolytica Al wild-type strain (61.50%) (kindly

TABLE 1. Cytotoxicity of the supernatants of the different strains on erythrocytes and BL3, Daudi, and Raji cells Strain or genotype

Leukotoxicity

Hemolysis

(OD54) (540)

(% 51Cr release)b

D

0.35 ± 0.13

~~~~BL3Daudi 61.50 ± 7.4 0.80 ± 0.0

Raji 0.54 ± 0.0

hlyCABD (pWAM04) lktA (pWAM827) x hlyBD (pWAM716) hlyABD (pWAM783) lktC (pWAM955) x hlyABD (pWAM783)

0.03 8.11 0.03 0.02 0.02

± 0.00

0.56 ± 0.5 71.62 ± 2.9 0.80 ± 0.2 0.05 ± 0.0