Molecular Microbiology (1990) 4(2). 283-293

A 59 kiloDalton outer membrane protein of Salmonella typhimurium protects against oxidative intraleukocytic killing due to human neutrophils p. S. Stinavage,* L. E. Martin and J. K. Spitznagel Department of P

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protein production, although the previously described iron-regulated proteins were readily identified (data not shown).

Acid phosphatase production of parent and mutant bacteria In view of recent reports in the literature regarding the effects of phoP and phoN mutants on Salmonella virulence, we compared both constitutive and induced expression of acid phosphatase. Neither induced nor constitutive acid phosphatase production was changed in the JKS400 mutant compared to its LT2 parent (data not shown).

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To determine if restoration of protein production in the mutant would result in wild-type resistance, and to clone the gene of interest closely linked to a readily selectable marker (tetracycline resistance), we transduced JKS400

m Fig. 4. A. Coomassie-blue-stained SDS-PAGE of outer membrane protein preparations of LT2 and JKS400, Lane A: Omp of JKS400. lane B: Omp of LT2, lane C: molecular weight markers. The arrow denotes a difference in outer membrane protein profile. JKS400 clearly lacked a protein migrating at approximately 59000 Daltons. Molecular masses of molecular mass standards are indicated in kiloDaltons. B. Coomassie-blue-stained SDS-PAGE of outer membrane preparations ol the two organisms that have been neither heated nor reduced. Lanes 1 and 2: duplicate lanes of LT2 proteins, tanes 3 and 4: duplicate lanes of JKS400 proteins. The porin OmpA shows its characteristic change in mobility when not heated (denoted by '). The mobility of the 59kD protein (indicated by the arrow) did not change, and still migrated at approximately 59 kD.

Growth regulation of 59kD protein The growth regulation of the protein was examined by comparing Western blots of outer membrane preparations of stationary and logarithmic growth phase organisms. These results are presented in Fig. 5. Lane A shows the Western blot of LT2 outer membrane proteins of stationary phase organisms. Lane B shows the Western biot of JKS400 stationary phase membranes. Lane C shows the Western blot of LT2 logarithmic phase proteins, and lane D shows the Western blot of JKS400 logarithmic phase proteins. It is clear from comparisons of these blots that the protein is more strongly expressed in stationary phase organisms relative to log phase organisms. Membrane proteins were also prepared from organisms grown in iron-limiting conditions. Iron limitation had no effect on

Fig. 5. Westem blot comparing stationary and logarithmic Omp preparations from LT2 and JKS400. Lane A: stationary phase LT2, lane B: stationary phase JKS400, lane C: logarithmic phase LT2 and lane D; togarithmic phase JKS400. It is clear that far more of the protein is produced in stationary phase organisms than in logarithmic phase organisms. Western blot analysis was able to detect some protein produced in stationary phase JKS400 that was not detectable in gels. Much less protein was produced by log phase LT2 and none of the protein was detected in log phase JKS400.

Outer membrane protein protects against H2O2 287 Comparison of transductant and LT2 in phagooytic assay 200

The sensitivity of the transductant was compared to the wild-type using side-by-side phagocytosis. The results of the assay are presented in Fig. 7. It is clear from Fig. 7 that the transductant had returned to the wild-type resistance phenotype with the return of protein production.

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Fig. 6. Comparison ot outer membrane protein profiles ot LT2 and the iransdLfCtant JKSSOt. Lane A: motecu!ar weight markers, lane B: LT2 proteins, and lane C: JKS801 proteins. No differences m the production of the 59kD protein in the transductant compared to the wild-type could be detected. Molecular masses of molecular mass standards are indicated in kiloDaltons.

Sensitivity of parent, mutant, and transductant to hydrogen peroxide We next addressed the question of the sensitivity of this mutant to oxidative killing and the possible role of this protein in the increased resistance of the wild-type organism to killing in PMNs, Since H2O2 is a major secondary product of the oxidative burst in PMNs and has been invoked repeatedly as an important component of oxidative killing, we examined sensitivity to H2O2- Figure 8 compares the killing of organisms by lOmM H2O2 both with and without induction by 60fjLM H2O2 (Christman et ai, 1985). Figure 8A illustrates a typical curve for survival of the wild-type LT2, the JKS400 mutant, and the JKS801 transductant with 10 mM H2O2 with no previous induction. Viable numbers of both LT2 and JKS801 can be detected for the entire 60-minute assay period, while viable numbers of JKS400 fall below detection limits of the assay between the 30- and 60-minute time points. At the 30-minute point, considerably fewer JKS400 mutants survived than either LT2 or JKS801. When the organisms were exposed to 10 mM H2O2 after a 60-minute induction period with 60JAM H2O2, the JKS400 again showed an impaired capacity to survive, as shown in Fig. 8B. Induction did alter the survival of all of the

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with P22HT(int") grown on a lr\10 library of LT2. We selected for tetracycline resistance and, using colony blots (as described above and in the Experimental procedures), probed with monospecific antibody to the protein for protein production. Several tetracyclineresistant colonies that stained positively for protein production were selected. Outer membrane proteins were prepared from the mutants and transductants and compared to the wild-type using SDS-PAGE. The results of this comparison are shown in Fig. 6. The transductant designated JKS801 shows an outer membrane protein migrating at approximately 59 kD, which is similar to the wild-type. Linkage of the Tn 10 and the 59 kD protein in the JKS801 transductant was >90%.

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Time (minutes) Fig. 7. Comparison ot the resistance of JKS801 transductant and LT2 to killing by intact PMNs- This represents data from a typical assay. Symbols:• •, survival of JKSSOt; x — x, survival of LT2. As shown here, no differences in the survival of the two organisms in intact, actively phagocytosing PMNs could be detected. These results indicate thai with the return of the mutant to protein production, the mutant returned to the parental resistance phenotype.

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organisms, but the JKS400 mutant again survived least well, with more than a log difference in the percentage surviving at the 60-minute point. To confirm these results, we compared the resistance of the organisms to 300 |xg of H2O2 on solid media, as described by Christman et al. (1985). These data are summarized in Table 1. It is clear from these data that the JKS400 mutant was more sensitive to the actions of H2O2 than either its parent or its transductant. Resistance of JKS400 and L T2 to granule protein preparations Even though the phagocytosis experiments suggested that the decreased survival of JKS400 was probably not due to 02-independent killing, we attempted to determine whether the difference in resistance to killing by PMNs could be accounted for by increased sensitivity to the

Fig. 8. A. Resistance of JKS801 to lOmM HaO; relative to that of LT2 and JKS400. Symbols: - — * , survival of LT2; O—-O, survival of JKS801 and X . . . . X, survival of JKS400. This assay method clearly indicates that while LT2 and JKS801 have nearly the same sensitivity to H^Oj, JKS400 is much more sensitive to the antimicrobial action of this agent. Detectable viable numbers of JKS400 fall below the detection limits of the assay following the 30mmute time point, while detectable numbers of both LT2 and JKS801 remain present for the enlire 60-minute period. B. Induction of resistance to 60 [iM HjO? failed to restore JKS400 to resistance to lOmM HjO? comparable to that seen in LT2. Symbols: ••—«, survival of LT2; O—C^. survival of JKS801 and X . . . . X, survival of JKS400. Again, JKS400 was clearly more sensitive. The JKS400 mutant seemed to be able to induce to a higher state of resistance, but not to the extent possible for LT2. These results further support the conclusion that the JKS400 mutant is hypersensitive to the actions of hydrogen peroxide.

non-oxidative bactericidal mechanisms. We compared the survival of the two organisms exposed to a crude granule extract of PMN granules in vitro. The extract contained the bactericidal proteins of PMN granules. As shown in Fig. 9A, JKS400 had a sensitivity to these proteins similar to that of its parent, LT2. We then Table 1. Sensitivity to killing by 300M.g H^O?.

Organism

Diameter of killing zone (mm)

LT2 (parent) JKS400 JKS801

23.0 ± 0.0 27.2 ± 1.3 23.7 ± 0.58

The sensitivity of LT2 (parent). JKS400 (mutant), and JKS801 (transductant) to 300 |ig HjOj. The assay was done as described by Christman ef al. (1985). JKS400 was significantly more sensitive to the bactencidal action of H7O? than either its parent or the transductant returned to production of the 59kD outer membrane protein.

Outer membrane protein protects against H2O2 289

IOO Crude granule extract /well Fig, 9. Comparison of the sensitivity of LT2 and JKS400 to crude granule extract of PMNs. Both strains were incubated with various amounts of crude granule extract for 60 minutes. Symbols: x — x. percent survival ot LT2; O—C), percent survival of JKS400. These da!a clearly indicate that ttiere was no difference in the survival ot these organisms when exposed to crude granule extract. If anything, JKS400 may be slightly more resistant to these proteins.

compared the survival of the organisms using a purified granule protein, CAP57, which has been shown previously to have potent bactericidal activity against Salmonella (Farley et ai, 1987). JKS400 was no more sensitive to the actions of this agent than was LT2; if anything, JKS400 may have been slightly more resistant (data not shown). These data indicated that the loss of resistance to killing by PMNs was not due to increased sensitivity to the non-oxidative bactericidal proteins of PMNs.

inserted near the gene for the protein of interest and then to map the location of the marker in the Salmonella chromosome. The tight linkage previously shown between the tetracycline resistance and the protein suggested there was only one Tn 10 insertion. Results of this mapping are shown in Table 2. The map position achieved by this method was determined to be near 96 minutes on the Salmonella chromosome. Figure 10 shows a conventionalized map of the Salmonella typhimurium genome (Sanderson and Roth, 1988) with the 96-minute map location of the 59 kD protein gene, as determined by our mapping. Included on this map are the map positions of other genes implicated in virulence of Salmonella. As indicated in Fig. 10, the gene for the 59 kD protein maps in the area of the phoP-phoO regulon suggested by Miller et ai (1989). Discussion We have shown that expression of a 59 kD outer membrane protein in S. typhimurium LT2 correlates with wild-type resistance to killing by intact PMNs and resistance to H2O2. A mutation that leads to failure to express the protein is closeiy linked to hypersensitivity to oxidative killing in neutrophils and hypersensitivity to H2O2. This defines a virulence factor in S. typhimurium mapping at 96 minutes on the Salmonella chromosome that, to our knowledge, has not been described previously. The available evidence suggests that this protein helps to

Mapping of the 59i