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In vitro and in vivo phenotypes resulting from deletion of the high temperature requirement A (htrA) gene from the bovine vaccine strain Brucella abortus S19
veterinary microbiology ELSEWER
Veterinary Microbiology 49 ( 19%) 197-207
III vitro and in vivo phenotypes resulting from deletion of the high temperature requirement A [ htrA) gene from the bovine vaccine strain Brucella abortus S 19 Gregory T. Robertson, Philip H. Elzer ‘, R. Martin Roop II * Department of Microbiology and Immunology. Louisiana State University Medical Center, Shreveport, LA 71130-3932. USA
Received 10 May 1995; accepted 22 September 1995
Abstract An htrA deletion mutant was created in the bovine vaccine strain, B. abortus S19, by replacing the majority of the htrA gene with a kanamycin resistance gene. Antibiotic selection for a double crossover event yielded kanamycin-resistant, ampicillin-sensitive colonies confirmed by Southern and western blot analysis to be HtrA deficient. The B. abortus S19 htrA mutant was significantly more susceptible than the parental strain to killing by H,O, (P < 0.001) and 0; generated by the redox cycling agent paraquat (P < 0.05) in disk sensitivity assays. Deletion of the htrA gene from S19 produced a bimodal effect on the spleen colonization profile of this strain in BALB/c mice. At one week post-infection, the B. abortus S19 hrrA mutant colonized the spleens of experimentally infected BALB/c mice at significantly lower levels (P < 0.01) than the parental strain. Enhanced clearance (P < 0.05) was also observed at later timepoints, i.e. 4 and 7 weeks post infection, however at 2 and 3 weeks post infection, the mutant and parental strains colonized the mice at equivalent levels. The temporal development of specific delayed type hypersensitivity and antibody responses in BALB/c mice infected with the mutant or parental strain were equivalent. These results suggest that the htrA gene product contributes to successful host colonization by S19. However, deletion of this gene does not radically alter the overall, characteristic spleen colonization profile of this vaccine strain in the BALB/c mouse model, nor compromise the
* Corresponding author. Department of Microbiology and Immunology, Louisiana State University Medical Center, 1501 Kings Highway, P.O. Box 33932, Shreveport, LA 71130-3932, USA. Tel: (318) 675-5771; Fax: (3 18) 675-5764, E-mail: [email protected]. ’ Present address: Department of Veterinary Science, Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USA. 0378- 1135/‘96/$15.00 SSDI 0378-l 135
0 1996 Elsevier Science B.V. All rights reserved
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capacity of this strain to elicit Bruceflu experimental host.
Microbiology 49 (1996) 197-207
specific cellular or humoral immune responses
in this
Keywords: Brucella abortus; Genetics; Vaccines; Stress response; Reactive oxygen intermediates
1. Introduction
Live, attenuated strains have been used in conjunction with immune surveillance and quarantine for controlling brucellosis in food animals in many areas of the world (Nicoletti, 1990). The two most widely used vaccines of this type are Brucellu abortus Strain 19 (S19) which is used in cattle, and B. mefitensis Revl, which is used in goats and sheep. These strains have been in use for decades, and have been examined extensively in both the laboratory (Alton, 1990; Nicoletti, 1989) and in natural (Alton, 1990; Nicoletti, 1990) and experimental (Montaraz and Winter, 1986) hosts, but the basis for the attenuation of neither strain is currently known. One of the characteristic properties of B. abortus S19 is its colonization profile in the BALB/c mouse model (Montaraz and Winter, 1986). During the first two weeks of infection, this strain replicates in the spleens and livers of mice to much higher levels than virulent strains of B. abortus. Additionally, while virulent strains generally reach a plateau level and produce chronic infections in these organs which persist for greater than 20 weeks, after 2 weeks S19 begins to be progressively cleared with most animals being free of detectable brucellae by 8 to 12 weeks post infection. Regardless of this accelerated rate of clearance, however, S19 induces protective immunity in BALB/c mice against subsequent challenge with virulent B. abortus, and this protective immunity appears to depend upon both cellular and humoral immune responses (Montaraz and Winter, 1986; Araya et al., 1989). Because the accelerated clearance of S19 and its ability to induce protective immunity in mice parallels the properties of this strain in ruminants, the BALB/c mouse model has been used extensively to evaluate protective immunity in brucellosis. Previous studies have shown that the B. abortus htrA gene encodes a stress response protease which contributes to virulence in the BALB/c mouse model (Elzer et al., 1994c). The increased in vitro sensitivity of a B. abortus htrA mutant to reactive oxygen intermediates (ROIs) and its reduced survival in cultured macrophages (Elzer et al., 1994d) strongly suggest that the B. abortus HtrA protease represents an important defense mechanism against ROI mediated killing in host phagocytes. This type of killing is generally believed to be the primary mechanism by which host phagocytes are able to eventually eliminate intracellular brucellae (Jiang et al., 1993). Previous studies employing cultured macrophages and B. abortus S19 have suggested that increased sensitivity to oxidative killing in activated macrophages compared to virulent strains may be an important component of the attenuation of this vaccine strain (Jones and Winter, 1992; Elzer et al., 1994a, Elzer et al., 1994b). In the present study, the effects of eliminating the htrA gene from B. abortus S19 were evaluated in vitro and in the BALB/c mouse model. The capacity of a B. abortus S19 htrA mutant to elicit B~-~celluspecific immune responses was also compared to that of the parental vaccine strain in BALB/c mice.
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2. Materials and methods 2.1. Bacterial strains The .attenuated bovine vaccine strain B. abortus S19, virulent B. abortus 2308 and the isog,enic 2308 htrA mutant PHEl (Elzer et al., 1994c) were used in these studies. Stock cultures were stored in brucella broth containing 25% glycerol at -80°C. Working cultures were grown on Schaedler agar @A) plates (Difco Laboratories, Detroit, MI) supplemented with 5% defibrinated bovine blood (SBA) at 37°C with 5% CO, and stored at 4°C with monthly transfer. 2.2. Construction
of a B. abortus S19 htrA deletion mutant
The Imajority of the gene encoding the HtrA protein of B. abortus S19 was removed using the gene replacement strategy used previously in our laboratory to construct a B. abortus htrA mutant from virulent strain 2308 (Elzer et al., 1994c). Plasmid pPS1 contains a 5’ flanking region from the B. abortus htrA gene and a short segment of the 3’ end of this gene separated by the kanamycin resistance gene from TnphoA (Manoil and Beckwith, 1985). This pUC-based vector will not replicate in B. abortus. Following introduction of pPS 1 into B. abortus S19 by electroporation, a kanamycin-resistant. ampicillin-sensitive transformant resulting from the replacement of the native S19 htrA by the disrupted, incomplete htrA gene from pPS1 via a double crossover event was selected for further evaluation. Deletion of the htrA gene was confirmed by preparing whole cell lysates and genomic DNA from the putative S19 htrA mutant and the parental strain S19, and performing western immunoblot and Southern analysis by previously described procedures (Elzer et al., 19941~).An htrA-specific nucleotide probe was prepared from a 1.9 kilobase (kb) EcoRI fragment obtained from the htrA coding region in pBA323 (Roop et al., 1994). A kanamycin cartridge-specific probe was prepared from the BamHI-Hind111 fragment containing the TnphoA kanamycin resistance marker used for gene replacement, and a vector-specific probe was created from pUC19 linearized with SatI. Probes were labeled with [ a-“P]dCTP by nick translation procedures (Sambrook et al., 1989). Western immunoblot analysis was performed with a monoclonal antibody specific for the perosamine O-chain of the B. abortus lipopolysaccharide (BRU38) (Schurig et al., 19841, serum from a goat hyperimmunized with the rough mutant B. abortus RB51 (Roop et al., 19921, and antiserum specific for the B. abortus HtrA protein (Elzer et al., 1994c). 2.3. Phenotypic characteriz,ation
of the B. abortus S19 htrA mutant
The (capacity of the B. abortus S19 htrA mutant and the parental strain to grow at elevated. temperatures was measured by testing the ability of these strains to form isolated colonies on SA after 4 days incubation at 40” and 41°C under an atmosphere of 5% co:!. To evaluate the sensitivity of B. abortus strains to killing by various mediators of oxidative damage, strains were grown overnight in brucella broth at 37°C adjusted to an
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optical density (O.D.) of 0.2 at 600 nm [log colony forming units (cfu) ml-’ I in the same medium, and 75 ~1 of the bacterial suspension was spread onto SA plates. An 8-mm filter paper disk was placed in the center of a 85mm diameter plate and saturated with 10 ~1 of either a 25% H,O, solution (Sigma, St. Louis, MO.), a 1 mg ml-’ solution of the redox recycling reagent plumbagin or a 20 mg ml-’ solution of the redox recycling reagent paraquat. The diameter of the zones of inhibition was then determined following growth for 72 h at 37°C in 5% CO,. These same methods were also used to determine the sensitivity of B. abortus strains to puromycin employing disks saturated with 10 ~1 of an 8 mg ml’ stock solution of this antibiotic. 2.4. Experimental infection of BAL.B/c cellular and humoral immune responses
mice and evaluation of Brucella
speci$c
Bacterial strains were grown for 3 days on SA plates supplemented with 5% defibrinated bovine blood (SBA), harvested in 1 ml brucella broth, and adjusted to a 10’ cfu ml-‘. After dilution in standardized O.D.,,, nm equivalent to approximately phosphate-buffered saline (PBS), approximately 2.3 X lo5 cfu per mouse were introduced intravenously into 7 to 8 week old female BALB/c mice in 0.1 ml PBS. Infecting doses were confirmed by serial dilution and plating on SBA and SBA supplemented with 45 pug ml-’ kanamycin (SBAk). At 1, 2, 3, 4, 6, and 7 weeks post infection, five mice per group were exsanguinated via cardiac puncture, spleens were aseptically harvested, and the total number of brucellae per organ determined by serial dilution and plating on SBA. The total number of bacteria were expressed as log cfu per spleen. Statistical comparisons were made using the two-tailed Students t test (Snedecor and Cochran, 1985). The development of delayed type hypersensitivity responses in infected mice was evaluated by injecting 20 ,ul of a B. abortus cell lysate (1 mg ml-’ protein concentration in PBS), prepared as described previously (Roop et al., 19941, into the right hind foot pad 48 h prior to euthanasia. As a control, 20 ~1 of PBS was injected into the left hind foot pad at this timepoint. Foot pad thickness was measured for both hind feet just prior to euthanasia and necropsy. Increases in footpad thickness 2 0.2 mm were considered positive reactions. At necropsy, blood was collected via cardiac puncture, serum removed and stored at - 80°C. The induction of Brucella specific antibodies in infected mice was measured by enzyme linked immunosorbance assay (ELISA) using previously described methods (Elzer et al., 1994a and b). Lyophilized, methanol-killed B. abortus S19 whole cells (Winter et al., 1983) were used as the test antigen at a concentration of 0.5 pug cells in 100 ~1 0.1 M carbonate buffer, pH 9.6 per well. Pooled sera was diluted 1: 100 in 0.02 M Tris-0.15 M NaCl, pH 8.0 supplemented with 0.05% Tween 20 and 2% nonfat dry milk, and reactions were generated using affinity purified, horseradish peroxidase conjugated anti-mouse IgG (Sigma, St. Louis, MO.) diluted 1:500 in the same buffer. Hydrogen peroxide and 2,2-azino-di-(3-ethylbenzthiazoline sulfonic acid) (Sigma, St. Louis, MO.) were used as substrates, and absorbance at 410 nm was measured after 30 min incubation at 37°C employing a Dynatech MR5000 ELISA plate reader.
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2.5. Biological containment and animal use
All procedures involving live Brucella were performed in a Biosafety Level 3 (BL-3) containment facility following Centers for Disease Control/National Institutes of Health guidelines (USDHHS, 1993). In conducting research using animals, the investigator(s) adhered to the “Guide for the Care and Use of Laboratory Animals,” prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (NIH Publication No. 86-23, Revised 1985, USDHHS, 1985).
3. Results The predicted gene replacement in the putative B. abortus S19 htrA mutant was confirmed by Southern blot analysis with the htrA, kanamycin resistance gene and pUC specific probes (data not shown), and absence of HtrA expression in the mutant was verified by western blot analysis with HtrA specific serum (Fig. 1). The mutant produced identical profiles as the parental strain when whole cell lysates were subjected to western blot analysis with Brucella specific hyperimmune goat serum or the lipopolysaccharide 0 side chain-specific monoclonal antibody, BRU38 (data not shown).
Fig. 1. Western immunoblot analysis of rabbit anti-Brucellu HtrA antiserum aborrus S 19 and S I9 hfrA. The arrow designates the HtrA-spedific band.
with whole-cell
extracts
of
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Table 1 Sensitivity of B. abortus strains S19, S19 htrA, 2308. and 2308 htrA (PHEl) to killing by H,O, and the O$generating redox cyclers plumbagin and paraquat on solid growth medium. Values presented represent zone of inhibition sizes in mm. The results presented represent the averages of five determinations Strain Compound
s19
S19 htrA
HA
46.5 f 0.9 44.2k3.0 36.5 f 1.O
60.0f 1.1 * ’ 43.4 f 4.6 39.8+ 1.5 *
plumbagin pamquat
*
2308
2308 htrA (PHE 1)
44.3k2.1 38.3 k 1.4 30.3 f 2.3
64.3f 1.3 * * * 50.6+ 4.6 * * 39.9+3.3 **
*
= P values < 0.05 when compared to parental strain in Student’s two-tailed t test. ** = P values < 0.01 when compared to parental strain in Student’s two-tailed t test. *** = P values < 0.001 when compared to parental strain in Student’s two-tailed t test.
Introduction of the ho-A mutation into S19 resulted in a significant increase in sensitivity to killing by H,O, in disk sensitivity assays, and this increase was comparable to that observed in an hfrA mutant constructed from virulent strain 2308 (Table 1). The effect of this mutation on the 0; sensitivity of S19 was less dramatic, however, and the results obtained were dependent upon the redox cycler used (Table 1). This is in direct contrast to the effect of introducing the hfrA mutation into the B. abortus 2308 genetic background, where it produces a much more dramatic increase in 0; sensitivity (Table 1). However, the parental S19 strain appeared to have a generally higher basal level of sensitivity to OTthan 2308 (Table I), and this likely contributed to the disparate results obtained with these two strains with respect to the effect of an htrA mutation on 0; sensitivity. The S19 hrrA mutant was also compared to its parental strain for sensitivity to puromycin, an antibiotic that promotes premature termination of translation resulting in the intracellular accumulation of incomplete or abnormal peptides (Goldberg, 19721, and growth at elevated temperatures. Increased sensitivity to the accumulation of abnormal peptides and failure to grow at elevated temperatures are well documented characteristics of htrA mutants (Strauch et al., 1989; Lipinska et al., 19901, and both properties are consistent with the proposed function of the hfrA gene product. The S19 htrA mutant showed a slight, but significant increase in sensitivity to puromycin in disk sensitivity assays compared to the parent strain (36.3 f 1.9 mm zone size for S19 htrA vs. 32.3 + 2.6 for S19, P < 0.05). However, the puromycin sensitivity of both the S19 parental strain and the corresponding htrA mutant was significantly greater than that observed with either the parental 2308 strain (11 .O f 0.5 mm zone size) or a 2308 htrA mutant (18.7 f 3.1 mm zone size). Unlike its parental strain, the S19 htrA mutant also demonstrated significantly restricted growth on SBA at 4O”C, and neither S19 or the S19 htrA mutant was capable of forming isolated colonies on this medium at 41°C. This latter observation was particularly interesting, as both virulent B. abortus strain 2308 and virulent B. melitensis 16M grow well and readily form colonies at this latter temperature. The kinetics of growth of S19 hrrA and the parental S19 were evaluated in BALB/c mice over the course of seven weeks (Fig. 2). Like hfrA mutants constructed from
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1
0
2
3
Weeks
4 post
5
6
7
infection
Fig. 2. Spleen colonization of BALB/c mice by B. abortus S19 and S19 htrA. Five mice per group were sacrificed at each time point and the colony forming units (cfu) per spleen were determined by dilution and plating on SBA. Symbols: vertical bars indicate standard deviation; significance values* , P < 0.01; * , P < 0.05. l
0
s19
0
S19
htrA
L 2 = positive
.5
0
I
I
I
1
0
1
2
3
I
I
I
4
5
6
1
Weeks
post
reaction
I
7
infection
Fig. 3. Delayed type hypersensitivity (DTH) responses to B. abortus S19 or S19 htrA over the course of seven weeks infection in BALB/c mice. Data presented represents mean values* standard deviation obtained with five mice per experimental group.
G.T. Robertson et al./ Veterinary Microbiology 49 (1996) 197-207
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0.90
0
1
s19
l S19
htrA
E
5 7 G 2 6
0.60 -
0.45 -
-P : 2 0.30 -
0.15
4 0
1
2
3 Weeks
4 post
5
6
7
infection
Fig. 4. Serologic responses to B. abortus S19 or S19 htrA over the course of seven weeks infection in BALB/c mice. Data presented represents results obtained using pooled serum samples from five mice per experimental group.
virulent B. aborrus and B. melitensis strains (Elzer et al., 1994~; Phillips et al., 1994; Tatum et al., 1994), the S19 htrA mutant colonized the spleens of BALB/c mice at significantly lower levels than the parental strain at one week post infection (P < 0.01). However, unlike other previously described Brucellu htrA mutants, the S19 htrA mutant also showed significant decreases in spleen colonization compared to S 19 at four (P < 0.05) and seven (P < 0.05) weeks post infection. Brucella-specific delayed type hypersensitivity reactions were observed beginning at 3 weeks post infection (Fig. 3) and positive serologic responses were detected beginning at one week post infection (Fig. 4) in mice infected with both S19 and the S19 htrA mutant.
4. Discussion Recent studies employing Salmonella (Johnson et al., 1991; Baumler et al., 1994) and Brucellu (Elzer et al., 1994c, Elzer et al., 1994d; Tatum et al., 1994) strains have linked bacterial stress response proteins of the high temperature requirement A (HtrA) family to the survival and replication of facultative intracellular pathogens in host phagocytes. These proteins appear to function by degrading oxidatively damaged proteins before they accumulate to toxic levels in bacterial cells (Davies and Lin, 1988). Consistent with results obtained with virulent B. abortus and B. melitensis strains, deletion of the htrA gene product from B. abortus S19 results in an increased sensitivity to both oxidative killing in vitro and a significant decrease in the level of spleen
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20.5
colonization in BALB/c mice observed at 1 week post infection. These findings are consistent with the htrA gene product contributing to the initial stages of host colonization by S19, likely due to its contribution to cellular defense against oxidative killing mediated by host phagocytes. In contrast to virulent B. aborrus strains, S 19 shows a distinctive colonization profile in the BALB/c mouse model characterized by initial replication to high numbers in the spleen, followed by clearance beginning at 3 weeks post infection (Montaraz and Winter, 1986). Although mice are usually free of S19 by 6 to 8 weeks post infection, infection with this strain induces specific humoral and cellular immune responses which can provide protective immunity in this model (Araya et al., 1989). Although introduction of the htrA mutation into S19 did lead to decreased colonization of the spleen at early and later timepoints, it did not dramatically alter the overall characteristic colonization profile of this strain in the mouse model, i.e. the early peak at 2 weeks followed by clearance. Deletion of the hfrA gene also did not compromise the capacity of S19 to induce Brucella specific immune responses, as evidenced by the Brucefla specific antibody and delayed type sensitivity responses reported here. Based on these findings, one might predict that the use of S19 hfrA mutants as vaccines in ruminants would allow for sufficient colonization to induce protective immunity while providing a stable genetic marker to distinguish vaccinated versus infected herds, as has been previously suggested by Tatum et al. (1994). In support of this idea, the HtrA protein has been shown to be an immunoreactive protein recognized by sera from naturally infected cattle (Roop et al., 1994). Confirmation of this proposal, however, will require an evaluation of S19 hfrA mutants in ruminants. The basis for the attenuation of S19 in the murine model is presently unknown. This strain does not survive as well as virulent B. abortus strains in cultured macrophages (Jones and Winter, 1992), and increased phagocytosis mediated by IgG opsonization ultimately leading to greater killing in activated macrophages has been put forth as a possible mechanism underlying the enhanced clearance of S 19 from experimentally infected mice compared to virulent strains (Elzer et al., 1994a). The production of toxic ROIs hats been proposed to be the primary mechanism by which host phagocytes eliminate intracellular brucellae (Jiang et al., 19931, but the results obtained in this study seem to suggest that an increased sensitivity to ROI-mediated killing for S19 compared to virulent B. abortus strains is unlikely to be the sole basis for the decreased virulence of this strain in the murine model. Rather, the results presented here suggest that S19 has an as yet undefined defect or defects in its ability to mount an effective stress response, as indicated by its increased sensitivities in vitro to: a) growth at elevated temperatures; and b) the accumulation of abnormal peptides, in comparison to virulent B. abortus 2308. These are characteristic phenotypes associated with previously described bacterial stress response mutants (Strauch et al., 1989; Lipinska et al., 1990). This compromised ability to mount a fully functional stress response could result in the inability of S19 to adequately adapt to the environmental stresses associated with the intracellular environment of host phagocytes, which in turn, could lead to the enhanced clearance from these host cells. A more thorough examination of the genetic defects present in S19 will be necessary before we understand the basis for the attenuation of this strain in natural and experime:ntal hosts.
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Acknowledgements
These studies were supported by grants from National Institutes of Health (Al 28867) and the LSUMC Center for Excellence in Cancer Research, Treatment and Education, and a contract (DAMD17-94-C-4054, contribution number 2004) from the United States Army Medical Research, Development, Acquisition and Logistics Command (Prov.), to R. M. R. II. The views, opinions and/or findings contained in this report are those of the authors and should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation.
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Nicoletti, I’., 1990. Vaccination. In: K. Nielsen and J.R. Duncan (Eds.), Animal brucellosis. CRC Press, Boca Raton, FL. pp. 283-299. Phillips, R.W., Elzer, P.H. and Roop II. R.M., 1994. Virulence of high temperature requirement A deletion (htrA) mutants of Erucella abortus 2308 and Brucella melitensis 16M in BALB/c mice. Abstr. 94th Annu. Meet. Amer. Sot. Microbial., Abstr. B-370, p. 94. Roop, R.MI. II., Price, M.L., DUM, B.E., Boyle, S.M., Sriranganathan, N. and Schurig, G.G., 1992. Molecular cloning and nucleotide sequence analysis of the gene encoding the immunoreactive Brucella abortus Hsp6O protein, BA6OK. Microb. Pathogen., 12: 47-62. Roop, R.M. II, Fletcher, T.W., Sriranganathan, N.M., Boyle, S.M. and Schurig, G. G., 1994. Identification of an immunoreactive Brucella abortus HtrA stress response protein homolog. Infect. Immun., 62: lOOO- 1007. Sambrook, J., Fritsch, E.F. and Maniatis, T., 1989. Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Schurig, G.G., Hammerberg, C. and Finkler, B.R., 1984. Monoclonal antibodies to Brucella surface antigens associated with smooth lipopolysaccharide complex. Am. J. Vet. Res., 45: 967-971. Snedecor, G.W. and Cochran, W.G., 1985. Statistical methods. Iowa State University Press, Ames, IA. .%-such, K.L., Johnson, K. and Beckwith, J., 1989. Characterization of de@, a gene required for proteolysis in the (cell envelope and essential for growth of Escherichia coli at high temperature. J. Bacterial., 171: 2689-2696. Tatum, FM., Cheville, N.F. and Morfitt, D., 1994. Cloning, characterization and construction of htrA and htrA-like mutants of Brucellu abortus and their survival in BALB/c mice. Microb. Pathogen., 16: 23-36. United States Department of Health and Human Services, 1985. Guide for the care and use of laboratory animals. H.H.S. Publication No. (NIH) 86-23, U.S. Government Printing Office, Washington, D.C. United States Department of Health and Human Services, 1993. Biosafety in microbiological and biomedical laborat~ories. H.H.S. Publication No. (CDC) 93-8395, U.S. Government Printing Office, Washington, D.C. Winter, A.J., Verstreate, D.R., Hall, C.E.. Jacobson, R.H., Castleman, W.L., Meredith, M.P. and McLaughlin, CA., 1983. Immune response to porin in cattle immunized with whole cell, outer membrane, and outer membrane protein antigens of Brucella aborrus combined with trchalose dimycolate and mummy1 dipeptkle adjuvants. Infect. Immun., 42: 1159- 1167.
Veterinary Microbiology 49 ( 19%) 197-207
III vitro and in vivo phenotypes resulting from deletion of the high temperature requirement A [ htrA) gene from the bovine vaccine strain Brucella abortus S 19 Gregory T. Robertson, Philip H. Elzer ‘, R. Martin Roop II * Department of Microbiology and Immunology. Louisiana State University Medical Center, Shreveport, LA 71130-3932. USA
Received 10 May 1995; accepted 22 September 1995
Abstract An htrA deletion mutant was created in the bovine vaccine strain, B. abortus S19, by replacing the majority of the htrA gene with a kanamycin resistance gene. Antibiotic selection for a double crossover event yielded kanamycin-resistant, ampicillin-sensitive colonies confirmed by Southern and western blot analysis to be HtrA deficient. The B. abortus S19 htrA mutant was significantly more susceptible than the parental strain to killing by H,O, (P < 0.001) and 0; generated by the redox cycling agent paraquat (P < 0.05) in disk sensitivity assays. Deletion of the htrA gene from S19 produced a bimodal effect on the spleen colonization profile of this strain in BALB/c mice. At one week post-infection, the B. abortus S19 hrrA mutant colonized the spleens of experimentally infected BALB/c mice at significantly lower levels (P < 0.01) than the parental strain. Enhanced clearance (P < 0.05) was also observed at later timepoints, i.e. 4 and 7 weeks post infection, however at 2 and 3 weeks post infection, the mutant and parental strains colonized the mice at equivalent levels. The temporal development of specific delayed type hypersensitivity and antibody responses in BALB/c mice infected with the mutant or parental strain were equivalent. These results suggest that the htrA gene product contributes to successful host colonization by S19. However, deletion of this gene does not radically alter the overall, characteristic spleen colonization profile of this vaccine strain in the BALB/c mouse model, nor compromise the
* Corresponding author. Department of Microbiology and Immunology, Louisiana State University Medical Center, 1501 Kings Highway, P.O. Box 33932, Shreveport, LA 71130-3932, USA. Tel: (318) 675-5771; Fax: (3 18) 675-5764, E-mail: [email protected]. ’ Present address: Department of Veterinary Science, Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USA. 0378- 1135/‘96/$15.00 SSDI 0378-l 135
0 1996 Elsevier Science B.V. All rights reserved
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capacity of this strain to elicit Bruceflu experimental host.
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specific cellular or humoral immune responses
in this
Keywords: Brucella abortus; Genetics; Vaccines; Stress response; Reactive oxygen intermediates
1. Introduction
Live, attenuated strains have been used in conjunction with immune surveillance and quarantine for controlling brucellosis in food animals in many areas of the world (Nicoletti, 1990). The two most widely used vaccines of this type are Brucellu abortus Strain 19 (S19) which is used in cattle, and B. mefitensis Revl, which is used in goats and sheep. These strains have been in use for decades, and have been examined extensively in both the laboratory (Alton, 1990; Nicoletti, 1989) and in natural (Alton, 1990; Nicoletti, 1990) and experimental (Montaraz and Winter, 1986) hosts, but the basis for the attenuation of neither strain is currently known. One of the characteristic properties of B. abortus S19 is its colonization profile in the BALB/c mouse model (Montaraz and Winter, 1986). During the first two weeks of infection, this strain replicates in the spleens and livers of mice to much higher levels than virulent strains of B. abortus. Additionally, while virulent strains generally reach a plateau level and produce chronic infections in these organs which persist for greater than 20 weeks, after 2 weeks S19 begins to be progressively cleared with most animals being free of detectable brucellae by 8 to 12 weeks post infection. Regardless of this accelerated rate of clearance, however, S19 induces protective immunity in BALB/c mice against subsequent challenge with virulent B. abortus, and this protective immunity appears to depend upon both cellular and humoral immune responses (Montaraz and Winter, 1986; Araya et al., 1989). Because the accelerated clearance of S19 and its ability to induce protective immunity in mice parallels the properties of this strain in ruminants, the BALB/c mouse model has been used extensively to evaluate protective immunity in brucellosis. Previous studies have shown that the B. abortus htrA gene encodes a stress response protease which contributes to virulence in the BALB/c mouse model (Elzer et al., 1994c). The increased in vitro sensitivity of a B. abortus htrA mutant to reactive oxygen intermediates (ROIs) and its reduced survival in cultured macrophages (Elzer et al., 1994d) strongly suggest that the B. abortus HtrA protease represents an important defense mechanism against ROI mediated killing in host phagocytes. This type of killing is generally believed to be the primary mechanism by which host phagocytes are able to eventually eliminate intracellular brucellae (Jiang et al., 1993). Previous studies employing cultured macrophages and B. abortus S19 have suggested that increased sensitivity to oxidative killing in activated macrophages compared to virulent strains may be an important component of the attenuation of this vaccine strain (Jones and Winter, 1992; Elzer et al., 1994a, Elzer et al., 1994b). In the present study, the effects of eliminating the htrA gene from B. abortus S19 were evaluated in vitro and in the BALB/c mouse model. The capacity of a B. abortus S19 htrA mutant to elicit B~-~celluspecific immune responses was also compared to that of the parental vaccine strain in BALB/c mice.
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2. Materials and methods 2.1. Bacterial strains The .attenuated bovine vaccine strain B. abortus S19, virulent B. abortus 2308 and the isog,enic 2308 htrA mutant PHEl (Elzer et al., 1994c) were used in these studies. Stock cultures were stored in brucella broth containing 25% glycerol at -80°C. Working cultures were grown on Schaedler agar @A) plates (Difco Laboratories, Detroit, MI) supplemented with 5% defibrinated bovine blood (SBA) at 37°C with 5% CO, and stored at 4°C with monthly transfer. 2.2. Construction
of a B. abortus S19 htrA deletion mutant
The Imajority of the gene encoding the HtrA protein of B. abortus S19 was removed using the gene replacement strategy used previously in our laboratory to construct a B. abortus htrA mutant from virulent strain 2308 (Elzer et al., 1994c). Plasmid pPS1 contains a 5’ flanking region from the B. abortus htrA gene and a short segment of the 3’ end of this gene separated by the kanamycin resistance gene from TnphoA (Manoil and Beckwith, 1985). This pUC-based vector will not replicate in B. abortus. Following introduction of pPS 1 into B. abortus S19 by electroporation, a kanamycin-resistant. ampicillin-sensitive transformant resulting from the replacement of the native S19 htrA by the disrupted, incomplete htrA gene from pPS1 via a double crossover event was selected for further evaluation. Deletion of the htrA gene was confirmed by preparing whole cell lysates and genomic DNA from the putative S19 htrA mutant and the parental strain S19, and performing western immunoblot and Southern analysis by previously described procedures (Elzer et al., 19941~).An htrA-specific nucleotide probe was prepared from a 1.9 kilobase (kb) EcoRI fragment obtained from the htrA coding region in pBA323 (Roop et al., 1994). A kanamycin cartridge-specific probe was prepared from the BamHI-Hind111 fragment containing the TnphoA kanamycin resistance marker used for gene replacement, and a vector-specific probe was created from pUC19 linearized with SatI. Probes were labeled with [ a-“P]dCTP by nick translation procedures (Sambrook et al., 1989). Western immunoblot analysis was performed with a monoclonal antibody specific for the perosamine O-chain of the B. abortus lipopolysaccharide (BRU38) (Schurig et al., 19841, serum from a goat hyperimmunized with the rough mutant B. abortus RB51 (Roop et al., 19921, and antiserum specific for the B. abortus HtrA protein (Elzer et al., 1994c). 2.3. Phenotypic characteriz,ation
of the B. abortus S19 htrA mutant
The (capacity of the B. abortus S19 htrA mutant and the parental strain to grow at elevated. temperatures was measured by testing the ability of these strains to form isolated colonies on SA after 4 days incubation at 40” and 41°C under an atmosphere of 5% co:!. To evaluate the sensitivity of B. abortus strains to killing by various mediators of oxidative damage, strains were grown overnight in brucella broth at 37°C adjusted to an
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optical density (O.D.) of 0.2 at 600 nm [log colony forming units (cfu) ml-’ I in the same medium, and 75 ~1 of the bacterial suspension was spread onto SA plates. An 8-mm filter paper disk was placed in the center of a 85mm diameter plate and saturated with 10 ~1 of either a 25% H,O, solution (Sigma, St. Louis, MO.), a 1 mg ml-’ solution of the redox recycling reagent plumbagin or a 20 mg ml-’ solution of the redox recycling reagent paraquat. The diameter of the zones of inhibition was then determined following growth for 72 h at 37°C in 5% CO,. These same methods were also used to determine the sensitivity of B. abortus strains to puromycin employing disks saturated with 10 ~1 of an 8 mg ml’ stock solution of this antibiotic. 2.4. Experimental infection of BAL.B/c cellular and humoral immune responses
mice and evaluation of Brucella
speci$c
Bacterial strains were grown for 3 days on SA plates supplemented with 5% defibrinated bovine blood (SBA), harvested in 1 ml brucella broth, and adjusted to a 10’ cfu ml-‘. After dilution in standardized O.D.,,, nm equivalent to approximately phosphate-buffered saline (PBS), approximately 2.3 X lo5 cfu per mouse were introduced intravenously into 7 to 8 week old female BALB/c mice in 0.1 ml PBS. Infecting doses were confirmed by serial dilution and plating on SBA and SBA supplemented with 45 pug ml-’ kanamycin (SBAk). At 1, 2, 3, 4, 6, and 7 weeks post infection, five mice per group were exsanguinated via cardiac puncture, spleens were aseptically harvested, and the total number of brucellae per organ determined by serial dilution and plating on SBA. The total number of bacteria were expressed as log cfu per spleen. Statistical comparisons were made using the two-tailed Students t test (Snedecor and Cochran, 1985). The development of delayed type hypersensitivity responses in infected mice was evaluated by injecting 20 ,ul of a B. abortus cell lysate (1 mg ml-’ protein concentration in PBS), prepared as described previously (Roop et al., 19941, into the right hind foot pad 48 h prior to euthanasia. As a control, 20 ~1 of PBS was injected into the left hind foot pad at this timepoint. Foot pad thickness was measured for both hind feet just prior to euthanasia and necropsy. Increases in footpad thickness 2 0.2 mm were considered positive reactions. At necropsy, blood was collected via cardiac puncture, serum removed and stored at - 80°C. The induction of Brucella specific antibodies in infected mice was measured by enzyme linked immunosorbance assay (ELISA) using previously described methods (Elzer et al., 1994a and b). Lyophilized, methanol-killed B. abortus S19 whole cells (Winter et al., 1983) were used as the test antigen at a concentration of 0.5 pug cells in 100 ~1 0.1 M carbonate buffer, pH 9.6 per well. Pooled sera was diluted 1: 100 in 0.02 M Tris-0.15 M NaCl, pH 8.0 supplemented with 0.05% Tween 20 and 2% nonfat dry milk, and reactions were generated using affinity purified, horseradish peroxidase conjugated anti-mouse IgG (Sigma, St. Louis, MO.) diluted 1:500 in the same buffer. Hydrogen peroxide and 2,2-azino-di-(3-ethylbenzthiazoline sulfonic acid) (Sigma, St. Louis, MO.) were used as substrates, and absorbance at 410 nm was measured after 30 min incubation at 37°C employing a Dynatech MR5000 ELISA plate reader.
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2.5. Biological containment and animal use
All procedures involving live Brucella were performed in a Biosafety Level 3 (BL-3) containment facility following Centers for Disease Control/National Institutes of Health guidelines (USDHHS, 1993). In conducting research using animals, the investigator(s) adhered to the “Guide for the Care and Use of Laboratory Animals,” prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (NIH Publication No. 86-23, Revised 1985, USDHHS, 1985).
3. Results The predicted gene replacement in the putative B. abortus S19 htrA mutant was confirmed by Southern blot analysis with the htrA, kanamycin resistance gene and pUC specific probes (data not shown), and absence of HtrA expression in the mutant was verified by western blot analysis with HtrA specific serum (Fig. 1). The mutant produced identical profiles as the parental strain when whole cell lysates were subjected to western blot analysis with Brucella specific hyperimmune goat serum or the lipopolysaccharide 0 side chain-specific monoclonal antibody, BRU38 (data not shown).
Fig. 1. Western immunoblot analysis of rabbit anti-Brucellu HtrA antiserum aborrus S 19 and S I9 hfrA. The arrow designates the HtrA-spedific band.
with whole-cell
extracts
of
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Table 1 Sensitivity of B. abortus strains S19, S19 htrA, 2308. and 2308 htrA (PHEl) to killing by H,O, and the O$generating redox cyclers plumbagin and paraquat on solid growth medium. Values presented represent zone of inhibition sizes in mm. The results presented represent the averages of five determinations Strain Compound
s19
S19 htrA
HA
46.5 f 0.9 44.2k3.0 36.5 f 1.O
60.0f 1.1 * ’ 43.4 f 4.6 39.8+ 1.5 *
plumbagin pamquat
*
2308
2308 htrA (PHE 1)
44.3k2.1 38.3 k 1.4 30.3 f 2.3
64.3f 1.3 * * * 50.6+ 4.6 * * 39.9+3.3 **
*
= P values < 0.05 when compared to parental strain in Student’s two-tailed t test. ** = P values < 0.01 when compared to parental strain in Student’s two-tailed t test. *** = P values < 0.001 when compared to parental strain in Student’s two-tailed t test.
Introduction of the ho-A mutation into S19 resulted in a significant increase in sensitivity to killing by H,O, in disk sensitivity assays, and this increase was comparable to that observed in an hfrA mutant constructed from virulent strain 2308 (Table 1). The effect of this mutation on the 0; sensitivity of S19 was less dramatic, however, and the results obtained were dependent upon the redox cycler used (Table 1). This is in direct contrast to the effect of introducing the hfrA mutation into the B. abortus 2308 genetic background, where it produces a much more dramatic increase in 0; sensitivity (Table 1). However, the parental S19 strain appeared to have a generally higher basal level of sensitivity to OTthan 2308 (Table I), and this likely contributed to the disparate results obtained with these two strains with respect to the effect of an htrA mutation on 0; sensitivity. The S19 hrrA mutant was also compared to its parental strain for sensitivity to puromycin, an antibiotic that promotes premature termination of translation resulting in the intracellular accumulation of incomplete or abnormal peptides (Goldberg, 19721, and growth at elevated temperatures. Increased sensitivity to the accumulation of abnormal peptides and failure to grow at elevated temperatures are well documented characteristics of htrA mutants (Strauch et al., 1989; Lipinska et al., 19901, and both properties are consistent with the proposed function of the hfrA gene product. The S19 htrA mutant showed a slight, but significant increase in sensitivity to puromycin in disk sensitivity assays compared to the parent strain (36.3 f 1.9 mm zone size for S19 htrA vs. 32.3 + 2.6 for S19, P < 0.05). However, the puromycin sensitivity of both the S19 parental strain and the corresponding htrA mutant was significantly greater than that observed with either the parental 2308 strain (11 .O f 0.5 mm zone size) or a 2308 htrA mutant (18.7 f 3.1 mm zone size). Unlike its parental strain, the S19 htrA mutant also demonstrated significantly restricted growth on SBA at 4O”C, and neither S19 or the S19 htrA mutant was capable of forming isolated colonies on this medium at 41°C. This latter observation was particularly interesting, as both virulent B. abortus strain 2308 and virulent B. melitensis 16M grow well and readily form colonies at this latter temperature. The kinetics of growth of S19 hrrA and the parental S19 were evaluated in BALB/c mice over the course of seven weeks (Fig. 2). Like hfrA mutants constructed from
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1
0
2
3
Weeks
4 post
5
6
7
infection
Fig. 2. Spleen colonization of BALB/c mice by B. abortus S19 and S19 htrA. Five mice per group were sacrificed at each time point and the colony forming units (cfu) per spleen were determined by dilution and plating on SBA. Symbols: vertical bars indicate standard deviation; significance values* , P < 0.01; * , P < 0.05. l
0
s19
0
S19
htrA
L 2 = positive
.5
0
I
I
I
1
0
1
2
3
I
I
I
4
5
6
1
Weeks
post
reaction
I
7
infection
Fig. 3. Delayed type hypersensitivity (DTH) responses to B. abortus S19 or S19 htrA over the course of seven weeks infection in BALB/c mice. Data presented represents mean values* standard deviation obtained with five mice per experimental group.
G.T. Robertson et al./ Veterinary Microbiology 49 (1996) 197-207
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0.90
0
1
s19
l S19
htrA
E
5 7 G 2 6
0.60 -
0.45 -
-P : 2 0.30 -
0.15
4 0
1
2
3 Weeks
4 post
5
6
7
infection
Fig. 4. Serologic responses to B. abortus S19 or S19 htrA over the course of seven weeks infection in BALB/c mice. Data presented represents results obtained using pooled serum samples from five mice per experimental group.
virulent B. aborrus and B. melitensis strains (Elzer et al., 1994~; Phillips et al., 1994; Tatum et al., 1994), the S19 htrA mutant colonized the spleens of BALB/c mice at significantly lower levels than the parental strain at one week post infection (P < 0.01). However, unlike other previously described Brucellu htrA mutants, the S19 htrA mutant also showed significant decreases in spleen colonization compared to S 19 at four (P < 0.05) and seven (P < 0.05) weeks post infection. Brucella-specific delayed type hypersensitivity reactions were observed beginning at 3 weeks post infection (Fig. 3) and positive serologic responses were detected beginning at one week post infection (Fig. 4) in mice infected with both S19 and the S19 htrA mutant.
4. Discussion Recent studies employing Salmonella (Johnson et al., 1991; Baumler et al., 1994) and Brucellu (Elzer et al., 1994c, Elzer et al., 1994d; Tatum et al., 1994) strains have linked bacterial stress response proteins of the high temperature requirement A (HtrA) family to the survival and replication of facultative intracellular pathogens in host phagocytes. These proteins appear to function by degrading oxidatively damaged proteins before they accumulate to toxic levels in bacterial cells (Davies and Lin, 1988). Consistent with results obtained with virulent B. abortus and B. melitensis strains, deletion of the htrA gene product from B. abortus S19 results in an increased sensitivity to both oxidative killing in vitro and a significant decrease in the level of spleen
G.T. Robertson er al./ Veterinary Microbiology 49 (1996) 197-207
20.5
colonization in BALB/c mice observed at 1 week post infection. These findings are consistent with the htrA gene product contributing to the initial stages of host colonization by S19, likely due to its contribution to cellular defense against oxidative killing mediated by host phagocytes. In contrast to virulent B. aborrus strains, S 19 shows a distinctive colonization profile in the BALB/c mouse model characterized by initial replication to high numbers in the spleen, followed by clearance beginning at 3 weeks post infection (Montaraz and Winter, 1986). Although mice are usually free of S19 by 6 to 8 weeks post infection, infection with this strain induces specific humoral and cellular immune responses which can provide protective immunity in this model (Araya et al., 1989). Although introduction of the htrA mutation into S19 did lead to decreased colonization of the spleen at early and later timepoints, it did not dramatically alter the overall characteristic colonization profile of this strain in the mouse model, i.e. the early peak at 2 weeks followed by clearance. Deletion of the hfrA gene also did not compromise the capacity of S19 to induce Brucella specific immune responses, as evidenced by the Brucefla specific antibody and delayed type sensitivity responses reported here. Based on these findings, one might predict that the use of S19 hfrA mutants as vaccines in ruminants would allow for sufficient colonization to induce protective immunity while providing a stable genetic marker to distinguish vaccinated versus infected herds, as has been previously suggested by Tatum et al. (1994). In support of this idea, the HtrA protein has been shown to be an immunoreactive protein recognized by sera from naturally infected cattle (Roop et al., 1994). Confirmation of this proposal, however, will require an evaluation of S19 hfrA mutants in ruminants. The basis for the attenuation of S19 in the murine model is presently unknown. This strain does not survive as well as virulent B. abortus strains in cultured macrophages (Jones and Winter, 1992), and increased phagocytosis mediated by IgG opsonization ultimately leading to greater killing in activated macrophages has been put forth as a possible mechanism underlying the enhanced clearance of S 19 from experimentally infected mice compared to virulent strains (Elzer et al., 1994a). The production of toxic ROIs hats been proposed to be the primary mechanism by which host phagocytes eliminate intracellular brucellae (Jiang et al., 19931, but the results obtained in this study seem to suggest that an increased sensitivity to ROI-mediated killing for S19 compared to virulent B. abortus strains is unlikely to be the sole basis for the decreased virulence of this strain in the murine model. Rather, the results presented here suggest that S19 has an as yet undefined defect or defects in its ability to mount an effective stress response, as indicated by its increased sensitivities in vitro to: a) growth at elevated temperatures; and b) the accumulation of abnormal peptides, in comparison to virulent B. abortus 2308. These are characteristic phenotypes associated with previously described bacterial stress response mutants (Strauch et al., 1989; Lipinska et al., 1990). This compromised ability to mount a fully functional stress response could result in the inability of S19 to adequately adapt to the environmental stresses associated with the intracellular environment of host phagocytes, which in turn, could lead to the enhanced clearance from these host cells. A more thorough examination of the genetic defects present in S19 will be necessary before we understand the basis for the attenuation of this strain in natural and experime:ntal hosts.
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Acknowledgements
These studies were supported by grants from National Institutes of Health (Al 28867) and the LSUMC Center for Excellence in Cancer Research, Treatment and Education, and a contract (DAMD17-94-C-4054, contribution number 2004) from the United States Army Medical Research, Development, Acquisition and Logistics Command (Prov.), to R. M. R. II. The views, opinions and/or findings contained in this report are those of the authors and should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation.
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Nicoletti, I’., 1990. Vaccination. In: K. Nielsen and J.R. Duncan (Eds.), Animal brucellosis. CRC Press, Boca Raton, FL. pp. 283-299. Phillips, R.W., Elzer, P.H. and Roop II. R.M., 1994. Virulence of high temperature requirement A deletion (htrA) mutants of Erucella abortus 2308 and Brucella melitensis 16M in BALB/c mice. Abstr. 94th Annu. Meet. Amer. Sot. Microbial., Abstr. B-370, p. 94. Roop, R.MI. II., Price, M.L., DUM, B.E., Boyle, S.M., Sriranganathan, N. and Schurig, G.G., 1992. Molecular cloning and nucleotide sequence analysis of the gene encoding the immunoreactive Brucella abortus Hsp6O protein, BA6OK. Microb. Pathogen., 12: 47-62. Roop, R.M. II, Fletcher, T.W., Sriranganathan, N.M., Boyle, S.M. and Schurig, G. G., 1994. Identification of an immunoreactive Brucella abortus HtrA stress response protein homolog. Infect. Immun., 62: lOOO- 1007. Sambrook, J., Fritsch, E.F. and Maniatis, T., 1989. Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Schurig, G.G., Hammerberg, C. and Finkler, B.R., 1984. Monoclonal antibodies to Brucella surface antigens associated with smooth lipopolysaccharide complex. Am. J. Vet. Res., 45: 967-971. Snedecor, G.W. and Cochran, W.G., 1985. Statistical methods. Iowa State University Press, Ames, IA. .%-such, K.L., Johnson, K. and Beckwith, J., 1989. Characterization of de@, a gene required for proteolysis in the (cell envelope and essential for growth of Escherichia coli at high temperature. J. Bacterial., 171: 2689-2696. Tatum, FM., Cheville, N.F. and Morfitt, D., 1994. Cloning, characterization and construction of htrA and htrA-like mutants of Brucellu abortus and their survival in BALB/c mice. Microb. Pathogen., 16: 23-36. United States Department of Health and Human Services, 1985. Guide for the care and use of laboratory animals. H.H.S. Publication No. (NIH) 86-23, U.S. Government Printing Office, Washington, D.C. United States Department of Health and Human Services, 1993. Biosafety in microbiological and biomedical laborat~ories. H.H.S. Publication No. (CDC) 93-8395, U.S. Government Printing Office, Washington, D.C. Winter, A.J., Verstreate, D.R., Hall, C.E.. Jacobson, R.H., Castleman, W.L., Meredith, M.P. and McLaughlin, CA., 1983. Immune response to porin in cattle immunized with whole cell, outer membrane, and outer membrane protein antigens of Brucella aborrus combined with trchalose dimycolate and mummy1 dipeptkle adjuvants. Infect. Immun., 42: 1159- 1167.