KatA, the Major Catalase, Is Critical for Osmoprotection and Virulence in Pseudomonas aeruginosa PA14

ABSTRACT We demonstrate that among the three monofunctional catalases of Pseudomonas aeruginosa PA14, KatA and, to a lesser extent, KatB, but not KatE, are required for resistance to peroxide and osmotic stresses. KatA is crucial for adaptation to H2O2 stress and full virulence in both Drosophila melanogaster and mice. This dismantling of catalase roles represents a specialized catalytic system primarily involving KatA in responses to adverse environmental conditions.

in response to environmental stresses and pathogenic interactions as well.
Construction of catalase mutants of P. aeruginosa PA14 and their resistance to H 2 O 2 . The genome sequence of the P. aeruginosa reference strain PAO1 reveals three monofunctional catalase genes, katA, katB, and katE, of different evolutionary origins, clade 3, clade 1, and clade 2, respectively (13,14). No homologue of bifunctional catalases has been found in P. aeruginosa thus far. Isolation of the gene encoding a manganese-containing nonheme pseudocatalase from lactic acid bacteria (12) and compilation of its homologous sequences from the bacterial genome sequence databases revealed a homologous gene (PA2185 or katM after Mn-catalase) on the 12th variable segment of the PAO1 genome (21).
We have verified all the catalase mutants through genetic structure analyses by PCR and Southern hybridization ( Fig. 1) (15), expression profiles by total catalase activity staining (15), and growth inhibition on plates containing 100 M H 2 O 2 ( Fig.  2A). All the mutants exhibited doubling times similar to that of the wild type (15). The growth of the katA and to a lesser extent katB but not katE mutants was inhibited by H 2 O 2 . The contribution of KatB to H 2 O 2 resistance was more evident in the katAB mutant ( Fig. 2A).
The H 2 O 2 sensitivity of the katA mutant was completely restored by introducing the pUCP18 (22)-derived plasmid containing appropriate full-length catalase constructs (Fig. 2B) (1 mM) for 30 min before being exposed to the killing concentration of H 2 O 2 (100 mM). A 30-min treatment time was chosen to investigate the steady-state response rather than the early and acute responses. The viability of cells was determined at 5-min intervals. Less than 0.1% of the unadapted or naive cells remained viable 10 min after exposure to 100 mM H 2 O 2 . In contrast, when cells were pretreated with 1 mM H 2 O 2 , survival was enhanced more than 1,000-fold (Fig. 3A). The sublethal pretreatment affected the cells' growth and/or survival compared to the control (ϳ60% survival as shown in Fig. 3B) and is slightly harsher than those previously described in other bacteria (7,8,9,17).
We analyzed all the catalase mutants in the adaptation experiment to determine whether catalases participate in the adaptive response to H 2 O 2 . The katA mutant was more sensitive to 1 mM H 2 O 2 than the wild type was. Moreover, the katAB mutant was even more sensitive (Ͻ10 Ϫ4 viability) to the pretreatment. Therefore, the residual survival (ϳ25%) of the katA mutant bacteria by the pretreatment may be attributed to KatB, which is in a good agreement with the results on solid agar culture ( Fig. 2A). The H 2 O 2 pretreatment enhanced the cells' resistance and viability against the killing concentration of H 2 O 2 , which was completely abolished in the katA mutant.
Killing of the pretreated katB and katE mutant bacteria by 1 mM H 2 O 2 was discernible ( Fig. 3B and data not shown). This result suggests that the basal and/or inducible expression of KatA, but not KatB, is responsible for the adaptation to H 2 O 2 , despite the rapid induction of katB by H 2 O 2 in the presence of functional KatA (19) (data not shown).
It is clear, however, that KatA and KatB have overlapping but distinct roles in oxidative stress responses, since the multicopy KatB failed to fully compensate for the absence of KatA in terms of H 2 O 2 resistance and adaptation (data not shown). The catalytic functions involving both KatA and KatB during normal growth and oxidative stress remain to be further deciphered by combining this result with detailed and systematic gene expression analyses in each catalase mutant background with or without oxidative challenge.
KatA is preponderantly required for osmoprotection in P. aeruginosa PA14. A minor catalase (CatB) from the actinomycete S. coelicolor is known to be required for resistance to osmotic stress and differentiation (5). We tested whether P. aeruginosa catalase mutants are susceptible to osmotic stresses. As shown in Fig. 4A, KatA was critical in the resistance to KCl treatments (0.8 M and 0.9 M), whereas deletion of katB or katE had no significant effect on salt resistance. However, the different KCl sensitivities of the katA and katAB mutants, depending on the KCl concentration, suggest that KatB may play a minor role in osmoresistance as in H 2 O 2 resistance (Fig. 2).
Since KCl increases ionic strength as well as osmotic strength, we used a nonionic osmolyte, sucrose, with comparable amounts of KCl (23). As shown in Fig. 4B, sucrose treatments at 32% (ϳ0.89 M) and 34% (ϳ0.94 M) exhibited similar results as observed in KCl treatments, uncovering the involvement of KatA in sucrose resistance, although the responses to the two different concentrations were more subtle than those in the KCl treatments, especially in the katA and katAB mutants, indicating the minor role of KatB in this condition.
FIG. 1. Creation of catalase mutants. Based on the PAO1 sequences, PCR deletions of each monofunctional catalase were generated and used to create three single (katA, katB, and katE), three double (katAB, katAE, and katBE), and a triple (katABE) mutant in wild-type PA14 (WT) via homologous recombination followed by sacB-dependent segregation as summarized in Table 1. Multiplex PCR using three sets of primers was used to verify the predicted genetic structures of the mutants. The PCR product sizes of the intact genes (designated by the solid arrowhead on the left) for katA, katB, and katE were 2.1, 2.8, and 2.5 kb, respectively, whereas those of deletions (designated by the empty arrowhead on the right) were 1.5, 2.1, and 0.4 kb, respectively. Because the PCR products from the intact katA gene and the deleted katB gene are almost the same size, only two bands were observed for katB and katBE (lanes 3 and 7, respectively).  The sensitive phenotype of the katA mutant was restored by trans complementation with the pUCP18-derived plasmid expressing KatA (Fig. 4C). Unlike H 2 O 2 sensitivity, however, multicopy KatB could not restore growth of the katA mutant on salt-containing media, which may imply differential functions and/or regulations of KatA and KatB in response to osmotic stress.
It is intriguing that the cell-free culture supernatant from the wild-type culture in the stationary growth phase could restore the KCl sensitivity of the katA mutant, although we were not sure whether or not the supplied activities absent in the culture supernatant of the katA mutant were working extracellularly. Further experimentation is needed to unravel how catalases such as P. aeruginosa KatA and S. coelicolor CatB protect against osmotic stresses. Considering that the general stress responses likely require alternative sigma factors (3, 6), it will be of special interest to analyze the gene expression in response to specific and general stress conditions.
KatA is required for virulence in P. aeruginosa PA14. The in vitro oxidative and osmotic stress phenotypes of catalase mutants are most likely related to the survival pathways, and therefore likely implicated in virulence due to unfavorable conditions P. aeruginosa may encounter in the host environment. We examined whether the P. aeruginosa catalases play a role in host infection using the D. melanogaster model, since it was a simple alternative model host to evaluate P. aeruginosa virulence potentials, as measured by fly mortality and in vivo proliferation of P. aeruginosa (16,27). D. melanogaster infection was performed by pricking 2-to 5-day-old adult flies with 50 to 200 CFU of PA14 cells as described previously (16). Mortality was monitored at 25°C for up to 54 h postinfection (Fig. 5). Four catalase mutants (katA, katAB, katAE, and katABE) commonly deficient in KatA exhibited significant virulence attenuations in terms of delayed death kinetics (by more than 10 h) and lower mortality, whereas the remaining three mutants (katB, katE, and katBE) were as virulent as the wild type (Fig. 5A). Reintroduction of the full-length katA gene restored the attenuated virulence of the katA mutant to the wild-type level, whereas katA mutant cells harboring a multicopy plasmid expressing either KatB or KatE were still avirulent (Fig. 5B).
The virulence attenuation of the katA mutant was verified by bacterial proliferation in D. melanogaster (Fig. 6). PA14 cells proliferate almost exponentially in flies, as described by Lee et al. (16), where the linear regression analyses from the 57 data points (from live flies) gave a slope of 0.1734, which is statistically significant (r 2 ϭ 0.897). The slope corresponds to a doubling time of 1.736 h. However, not all katA cells proliferate exponentially in flies, unlike the wild type. The bacterial proliferations from 78 live flies were delayed about 6 h, and some infected flies completely cleared the bacteria (Fig. 6B).
The involvement of KatA in virulence was further verified in mammalian hosts, using the mouse peritonitis model as described previously (24). The mice were monitored from 6 to 64 h after intraperitoneal challenge with 5 ϫ 10 6 CFU of bacterial cells and regarded as dead when they displayed ruffled fur, evidence of dehydration, and nonresponsiveness to stimuli. More than 90% of the mice that had been infected with the wild-type cells died within 36 h in our experimental conditions (Fig. 7). As in D. melanogaster, the katA, katAB, katAE, and katABE mutants were less virulent in the mouse peritonitis model, with ϳ40% mice surviving the infection, exhibiting delayed killing (by more than 20 h). Conclusion. These phenotypic analyses of the three monofunctional catalases (KatA, KatB, and KatE) in P. aeruginosa PA14 suggest that the catalytic system of KatA is crucial for oxidative and osmotic stress responses. KatA is also required for adaptation to peroxide stress and for virulence of this bacterium, which is intuitively understandable in that it is critical for stress responses as well as adaptations in vitro that may resemble unfavorable host environments. It is also explainable in part by the regulation of katA, which involves quorumsensing circuits (11).
The pivotal roles of KatA in virulence mechanisms can be further authenticated and generalized, by investigating its involvement in virulence of other P. aeruginosa strains such as PAO1, since the multifactorial nature of virulence pathways is related with the genetic backgrounds that accounts for different virulence potentials, and its expression and regulation in conjunction with related enzymes and regulators such as RpoS and OxyR.