calculated by the method of Reed and Muench (33). Animals inoculated .... genes for hydroperoxidase enzymes (katE and katG), hktE appears to be the only ...
Vol. 62, No. 11
INFECTION AND IMMUNITY, Nov. 1994, P. 4855-4860
0019-9567/94/$04.00+0 Copyright C 1994, American Society for Microbiology
Characterization and Virulence Analysis of Catalase Mutants of Haemophilus influenzae WILLIAM R. BISHAI, 12* NATHAN S. HOWARD,' JERRY A. WINKELSTEIN,3 AND HAMILTON 0. SMITH'
Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, 1 and Division of Infectious Diseases, Department of Medicine, and Division of Immunology, Department of Pediatics, 3 Johns Hopkins Hospital, Baltimore, Maryland 21205 Received
5
May 1994/Returned for modification 12 July 1994/Accepted 19 August 1994
In addition to detoxifying peroxides generated by aerobic metabolism, the catalases of pathogenic bacteria have also been hypothesized to serve as virulence factors by enabling microorganisms to resist the oxidative bursts of host inflammatory cells. Using transposon mutagenesis of the hktE gene, encoding the Haemophilus influenzae structural gene for catalase, we constructed defined catalase mutants of H. influenzae strains Rdand Eagan b+. These mutants show no detectable catalase production during exponential or stationary phases or following induction with hydrogen peroxide or ascorbic acid, indicating that hktE is the only functional hydroperoxidase gene present in these two strains of H. influenzae. Exponential-phase cultures of hktE mutants are 8- to 25-fold more sensitive to hydrogen peroxide than the wild type. Using the infant rat model, hktE mutants of strain Eagan b+ were 2.3-fold less virulent than the wild type following intraperitoneal inoculation (P = 0.07). When administered intranasally, the Eagan b+ hktE mutant produced wild-type levels of bacteremia and nasal colonization. The results of this study show that while the H. influenzae hktE gene is important for survival in the presence of peroxides, deletion of the gene produces only a modest reduction in ability to cause lethal sepsis following parenteral challenge and no change in ability to colonize following intranasal inoculation in the infant rat model of infection. enzymes such as catalase, which metabolize reactive intermediates, might also protect from the oxidative burst of host phagocytes (5, 11). It is therefore possible that the H. influenzae catalase gene might prove important for the processes of colonization and invasive infection. To test this hypothesis, we constructed H. influenzae mutants containing an insertion in the catalase structural gene hktE (7) and evaluated their properties in vitro and in vivo in the infant rat model of H. influenzae infection. Our results show that while hktE is important for resistance to exogenous peroxides in vitro, mutants of invasive strains which lack the gene have a reduced ability to produce lethal sepsis but are fully capable of producing persistent nasopharyngeal colonization.
disease,
Catalases (hydroperoxidases) are found in most aerobic organisms, where they are believed to detoxify the peroxides that arise in the course of aerobic metabolism. As enzymes which protect against oxidative damage, they have received considerable attention in view of the association between oxidative stress and aging (1). Indeed, catalase and superoxide dismutase (SOD) have recently been shown to play a direct role in aging in fruit flies (32). Among bacterial pathogens, catalase has been proposed as a potential virulence factor, since the ability to detoxify peroxide might protect organisms from the oxidative bursts of neutrophils and other inflammatory cells of the immune response (5). Experimental associations between catalase production and virulence have, in fact, been demonstrated in certain isolates of Mycobacterium tuberculosis and Staphylococcus aureus (27, 29). Yet despite the growing number of bacterial catalase genes which have been cloned and sequenced, the role of catalases in the virulence of upper respiratory pathogens has not been addressed using genetically defined mutants. Haemophilus influenzae is a strictly human commensal organism found, as unencapsulated species, in the upper respiratory tracts of up to 80% of healthy adults and, as encapsulated species, in 3 to 5% of normal individuals (30). Encapsulated strains are capable of invasive infections, including meningitis, pneumonia, and epiglottitis. Because it is restricted to existence in the human upper respiratory tract with occasional episodes of invasive disease, one might expect that its ability to withstand the relatively high oxygen tensions of the nasopharynx would facilitate the colonization ability and pathogenicity of this organism. Moreover, during invasive
oxygen
MATERIALS AND METHODS Strains and plasmids. The strains and plasmids used in this study are listed in Table 1. H. influenzae strains were grown in BHI medium supplemented with hemin and NAD (sBHI) as described (4), and Escherichia coli strains were grown in LB medium (28). H. influenzae growth rates were monitored by turbidity measurements with a UV160 spectrophotometer (Shimadzu, Kyoto, Japan) set at a wavelength of 600 nm. When necessary, H. influenzae was selected with chloramphenicol at 2.5 jig/ml, vancomycin at 25 jig/ml, and streptomycin at 250 jLg/ml. E. coli strains were selected with chloramphenicol at 25 ,ug/ml, ampicillin at 50 ,ig/ml, and tetracycline at 10 ,ug/ml. Plasmids were isolated and analyzed by standard protocols (28) with restriction enzymes obtained from Gibco-BRL (Gaithersburg, Md.) or New England Biolabs (Beverly, Mass.) used according to the manufacturers' recommendations. Catalase activity assays. Catalase activity was measured in washed whole-cell preparations with a Clark oxygen electrode, using bovine liver catalase (Sigma Chemical Co., St. Louis, Mo.) as the standard as described (7, 34). Cultures were grown
* Corresponding author. Mailing address: PCTB Rm. 505, Johns Hopkins School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Phone: (410) 955-3651. Fax: (410) 550-6718.
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BISHAI ET AL.
INFECr. IMMUN.
TABLE 1. Bacterial strains, phage, and plasmids used in this study Strain, phage, or plasmid
E. coli DH5ot
Relevant characteristicsa
Source
Reference
F- recA1 hsdR17 thi-1 gyrA96 supE44 endAl relAl recAl deoR A(lacZYAargF)U169 (480 lacZAM15) W3110 lacIqL8
Gibco/BRL
6
Lab strain
9
Rd- wild type Rd Kmr Nl' Nvr Spr Str Svr Vir Ers KW20 hktE7::mini-TnlOCm Type b+, clinical isolate Eagan Str ES hktE7::mini-TnlOCm
Lab strain Lab strain This study R. Moxon This study This study
40 10 2
Bacteriophage XNK1324
X b522 c1857 Pam80 ninS mini-TnlOCm
G. Barcak
21
Plasmids pNK2882 pBR322-2 pWB5 pWB7
pACYC184 fl Ptac ATS transposase, Tcr hktE-containing 2.8-kb Sau3AI fragment in BamHI site of pBR322, Apr hktE-containing 2.3-kb PstI-SalI fragment from pBR322-2 in pBAD102, Cmr pBR322-2 with hktE7::mini-TnlOCm transposon insertion mutation, Apr Cmr
G. Barcak Lab plasmid Lab plasmid This study
21 7 7
RB791
H. infiuenzae KW20 MAP7 AB2593 Ea an ES ES2593
a Abbreviations: Ap, ampicillin; Cm, chloramphenicol; Er, erythromycin; Km, kanamycin; Nl, nalidixic acid; Nv, novobiocin; Sp, spectinomycin; St, streptomycin; Sv, streptovaricin; Tc, tetracycline; Vi, viomycin. b Constructed by two rounds of serial transformation of the Str allele from MAP7 into strain Eagan b+.
an A600 Of 0.3 (exponential phase) or 2.5 (stationary phase) in sBHI at 37°C and then washed and concentrated fivefold by centrifugation and resuspension in PBS at 4°C before catalase activity was measured. For ascorbate induction, exponentialphase cultures in sBHI were grown in the presence of 10 mM ascorbic acid for 30 min at 37°C before the cells were harvested as above. Peroxide sensitivity assays. Cultures of H. influenzae were grown to the early exponential phase (A600 = 0.2 to 0.5); 250-,ul aliquots were mixed with dilutions of hydrogen peroxide in microtiter wells and allowed to sit at room temperature for 30 min. Tenfold dilutions were then spotted onto sBHI plates in 3-,ul portions. After overnight incubation, the colonies within each spot were counted. The ratio of survivors from treated and untreated cultures was calculated for each concentration of hydrogen peroxide tested. Virulence studies. On the day of inoculation, fresh H. influenzae cultures were grown in sBHI to an A600 of 0.5, washed twice in sterile phosphate-buffered saline containing 0.1% gelatin (PBSG), diluted in PBSG, and titered by plating dilutions. Five-day-old Sprague-Dawley infant rats (Charles River, Inc., Wilmington, Mass.) obtained in litters of 12 to 13 were randomized before inoculation. Intraperitoneal injections (0.2 ml) with a 25-gauge insulin syringe or 0.01-ml intranasal inoculations via a 24-gauge angiocath connected to a Hamilton syringe were administered as previously described (31, 36). Animals inoculated intraperitoneally were monitored every 12 to 24 h for death for 7 days, and 50% lethal doses (LD50s) were calculated by the method of Reed and Muench (33). Animals inoculated intranasally were assessed on day-of-life 7, 11, and 19 for levels of bacteremia and nasal colonization by quantitative cultures as described before (31) on sBHI plates containing streptomycin and vancomycin. For nasal colonization assessment, 25 ,ul of sterile PBSG was instilled intranasally, and 5 ,u1 was removed for dilution and plating; results are reported as CFU per microliter of this 5-,ul sample. All animal experiments were performed in accordance with veterinary protocols approved by the Johns Hopkins Animal Care and Use Committee.
RESULTS
to
Construction of insertion mutations in the hktE gene. The gene from H. influenzae Rd- was recently cloned and sequenced (7), and the cloned gene is carried on a 4.8-kb plasmid called pWB5. E. coli RB791, cotransformed with pWB5 and pNK2882 (Tetr, carrying the TnlO transposase gene), was infected with XNK1324 (carrying mini-TnlOCm) as described before (21). After allowing transposition to occur, plasmid DNA was prepared and transformed into E. coli DH5a. Ampr Cmr Tets transformants were selected and screened by the hydrogen peroxide bubble assay (25) for loss of vigorous catalase activity. This procedure yielded several hundred insertion mutations of the hktE gene; the mutation in one clone, called pWB7 (mutant allele hktE7), was mapped as shown in Fig. 1, and this clone was used for subsequent genetic manipulations. Construction of H. influenzae hktE mutant strains. Plasmid pWB7 was linearized by restriction digestion at its unique BsaI site and naturally transformed into H. influenzae Rd- strain KW20 by the MIV method (20). Cmr H. influenzae transfor-
hktE
AccI P lu BamHI
BamHI NdeI
PstI
KpnI
ii
_
* OC hIktEE iEminiTnlOCm
100
ktE
20
3000
FIG. 1. Genetic map of the hktE7 mini-TnlOCm insertion mutation. The positions of the mini-TnlOCm transposon relative to the restriction map of the hktE gene (7) are shown. The scale at the base is in base pairs.
H. INFLUENZAE CATALASE MUTANTS
VOL. 62, 1994
4857
10
-l
E
C=
CD
in
C)
cD c"i a-) PI---)
Ln
0000
CZ
CD
CD
KW20
oCD oOo r
AB2593
0.1-
ES2593
0.01 Time (minutes) FIG. 2. Relative growth rates of wild-type and hktE7 mutant strains of H. influenzae. Cultures were aeration at 37°C, and turbidity measurements were taken at 600 nm at 30-min intervals.
mants were grown to mid-exponential phase in liquid sBHI
cultures and screened for bubble-negative phenotypes by the hydrogen peroxide bubble assay. A typical clone containing the hktE7 mutation was selected and designated strain AB2593. Chromosomal DNA prepared from AB2593 was used to transform H. influenzae ES b+ by the MIV method, and bubble-negative, Cmr transformants were selected. To minimize the possibility of cotransformation of other genes from strain AB2593 to strain ES, chromosomal DNA from the a first-round ES hktE7 mutant was isolated and retransformed into wild-type strain ES. A typical, second-round, bubblenegative, Cmr transformant was isolated and named strain ES2593. Characterization of hktE mutant strains. As shown in Fig. 2, strain pairs KW20 and AB2593 and strains ES and ES2593 have identical growth rates in rich medium. The hktE7 mutants are also transformable at the same frequency as their parent strains (data not shown). Additionally, strains ES and ES2593 make equal quantities of type b capsule, as assessed by the limiting-dilution capsular agglutination assay (data not shown). Since these properties are dependent on the expression of multiple genes, the demonstration of identical phenotypes for growth rate, transformability, and capsular production supports the assertion that the hktE7 mutants are otherwise isogenic to their parent strains. Next, the levels of catalase production were assessed in detail for strains KW20, AB2593, ES, and ES2593. As shown in Table 2, the hktE7 mutant of KW20 failed to make detectable quantities of catalase activity in the exponential or stationary phase or following ascorbate or peroxide induction. Similar results were obtained for the Eagan type b+-derived strains ES and ES2593 (data not shown). Thus, unlike E. coli and Salmonella typhimurium, which have two unrelated structural genes for hydroperoxidase enzymes (katE and katG), hktE appears to be the only functional hydroperoxidase gene present in these strains of H. influenzae Rd- and type b+. Sensitivity of H. influenzae hktE mutants to exogenous hydrogen peroxide. The sensitivity of wild-type and hktE7
grown in sBHI medium with
vigorous
mutants of H. influenzae to exogenous hydrogen peroxide was determined by exposing exponentially growing bacteria to various concentrations of hydrogen peroxide at room temperature for 30 min and then plating for survivors. As shown in Fig. 3, the hktE7 mutation in an Rd- strain background leads to an 8- to 25-fold-greater sensitivity to peroxide than seen in the wild type, while the presence of hktE on a multicopy plasmid imparts a 5-fold-greater resistance to peroxide to the same wild-type strain (KW20). Similar results were found for the hktE7 mutation in an Eagan b+ strain background (data not shown). Virulence analysis of the H. influenzae hktE mutant. While unencapsulated H. influenzae strains such as Rd- are essentially avirulent in the infant rat model, encapsulated strains, particularly those with type b capsule, are highly virulent (42). Thus, we were able to use this animal model to test the role of the hktE gene in virulence with strains ES and ES2593. We first assessed the lethality of the Eagan hktE7 mutant by intraperitoneal inoculation and LD50 analysis; these data are summarized in Table 3. In two sets of experiments, the hktE mutant consistently showed a modest reduction in virulence (LD50s 2.9- and 2.4-fold higher than that of the wild-type strain; P = 0.098 and 0.072, respectively), although not statistically significant at the 5% level. In these experiments, the
TABLE 2. Comparison of catalase activity under various culture conditions Catalase activity' (U/108 CFU) Strain
Exponential phase phase
KW20 (wild type) AB2593 (hk;tE7)
5.7
~
Exponential phase withinduction ascorbate
17.7
