Previous studies have established that either spaceflights or growth in ground-based spaceflight analog bioreactors induce phenotypic modifications and relevant alterations in gene expression in S.
Typhimurium. In particular, these investigations have revealed that growth under microgravity enhances Salmonella
virulence, suggesting that this well characterized microorganism may represent a suitable model to analyze bacterial pathogens to this environmental condition. Studies on the Salmonella
response to LSMMG may provide useful information on the pathogenic mechanisms of this organism as it encounters low fluid shear areas during the infectious process, such as between brush border microvilli in the intestinal tract [28
During infections, pathogenic bacteria are exposed to high concentrations of reactive oxygen species generated by the respiratory burst of phagocytic cells. To investigate the possibility that microgravity could enhance Salmonella
ability to withstand oxidative stress, we have analyzed the effect of LSMMG on Salmonella enterica
resistance to hydrogen peroxide. Our findings indicate that Salmonella
is significantly more resistant to hydrogen peroxide stress when cultivated in LSMMG than in NG (Fig.
). Although this observation is in disagreement with a previous experiment carried out with the S.
Typhimurium strain SL1344 [6
], our results indicate that this response is common to several S.
Typhimurium strains as well as to other Salmonella
strains belonging to different serotypes. Interestingly enough, a comparable increase in resistance to oxidative stress has been recently observed also in P. aruginosa
To evaluate the possibility that LSMMG might influence the antioxidant apparatus involved in H2O2 detoxification, we monitored the activity of Salmonella catalases in extracts from bacteria grown in LSMMG and NG. Staining of gels for catalase activity revealed changes in the activity of KatN and KatG. In fact, KatN activity was 5-10 folds increased in extracts from S. Typhimurium ATCC14028, DT104 and S. Enteritidis LK5 grown in LSMMG, whereas, under the same conditions, it was possible to appreciate a decrease in the activity of KatG in all the strains except for S. Choleraesuis.
The role of KatN in Salmonella
has been poorly investigated, but a possible involvement of this enzyme in virulence has been suggested by studies showing that overexpression of this catalase increases Salmonella
lethality in flies mutated in the NF-kB pathway and is sufficient to render a non-pathogenic E
strain highly virulent to NF-kB pathway mutant flies [43
]. Expression of katN
is under control of the alternative sigma factor RpoS [33
], although it can be induced independently of rpoS
in the presence of bile [44
]. The absence of a KatN band in the extracts from S
. Typhimurium LT2 can be supposedly attributed the rpoS
mutation characterizing this strain. In support of this view, we have observed that KatN activity can not be detected in extracts from the rpoS
mutant strain PF148 (data not shown). We have not investigated if this gene is expressed in S.
Choleraesuis. Interestingly, the increase in KatN activity in S.
Typhimurium grown in LSMMG is not due to an enhanced intracellular concentration of the enzyme, as we failed to observe a variation in the intracellular accumulation of the 3xFLAG epitope-tagged protein. Therefore, we suggest that the explanation for the higher KatN activity in bacteria grown in microgravity should be looked for in post-translational events. For example, an increase in KatN activity could be related to the efficiency of manganese insertion in the enzyme active site or to microgravity-dependent differences in the susceptibility of this protein to oxidative inactivation.
In contrast, the decrease in KatG activity in bacteria grown under LSMMG, is paralleled by a slight change in protein accumulation (Fig.
), thus suggesting that it is at least partially due to gravity-dependent alterations in gene regulation. No differences were observed in the intracellular accumulation of KatE, nor of other proteins involved in bacterial resistance to oxidative stress, including Dps, SodCI, SodCII, and SodA. The involvement of KatG and KatN in the hydrogen peroxide resistance induced by the LSMMG environment was confirmed by the analysis of single and double mutant strains of S.
Typhimurium ATCC14028 devoid of katE
genes. Although the mutant strains were more sensitive than the wild type strains to hydrogen peroxide, all the single mutants and the double katE/katG
mutant strains displayed higher resistance to hydrogen peroxide when grown in LSMMG. In contrast, the double katG/katN
mutant exhibited an identical susceptibility to killing by H2
either in LSMMG or in NG (Fig.
). Solution assays revealed that the catalase activity of extracts from bacteria grown in LSMMG and NG is very similar, indicating that the higher resistance to hydrogen peroxide of cells grown in LSMMG is not simply due to enhanced catalase activity. In contrast, our results suggest that the higher resistance to hydrogen peroxide killing is associated to variations in the relative pattern of activity of KatG and KatN. This finding suggests that the three Salmonella
catalases, while possessing a similar ability to scavenge hydrogen peroxide, play distinct physiological roles in vivo
. This possibility is further supported by the observation that Salmonella
possesses also other enzymes able to scavenge hydrogen peroxide, including two alkyl hydroperoxide reductases [45
] and the Dps protein [46
]. The presence of such a high number of enzymes with apparently redundant functions is likely justified by the significant toxicity of hydrogen peroxide [45
], but also suggests that each of these proteins plays additional roles which are not completely overlapping with those of the other antioxidant enzymes. It is known that the different enzymes belonging to the catalase family differ significantly in their overall and active-site architecture and in the mechanism of reaction [47
] and recent studies have shown that KatG is also able to catalyze the breakdown of peroxynitrite, suggesting that each catalase could play different roles in the detoxification of oxidative stresses [48
]. In addition, the three catalases could be differently implicated in the mechanisms of adaptation to a variety of stimuli and conditions, as previously proposed to explain other known examples of apparent genetic redundancy of antioxidant enzymes. A well known example is represented by SodCI and SodCII, the two periplasmic Cu,Zn superoxide dismutases of S.
Typhimurium. These enzymes catalyze the same reaction and share a very similar three-dimensional structure, but show well distinct functional roles: SodCI facilitate survival within the infected host by protecting bacteria from the phagocytic oxidative burst [46
], whereas SodCII plays a role in protection from the reactive oxygen species produced endogenously during aerobic growth [39
]. Recent studies have established that structural and regulatory differences accounts for the different role of the two enzymes [49
]. Likewise, the sodA
genes, encoding for a manganese- and an iron-containing cytoplasmic superoxide dismutases respectively, are differently regulated: the former is finely regulated to favor its expression under aerobic and oxidative stress conditions, while the latter is constitutively expressed, even under anaerobic conditions [50
]. In addition, it has been shown that MnSOD can associate with DNA and preferentially localizes with the nucleoid, while FeSOD is concentrated in the periphery of the cells, near the inner membrane [52
]. Whereas KatG possess a peroxidative activity that is not present in KatE, the studies of bacterial superoxide dismutases suggest that additional subtle differences in regulation, catalytic properties or intracellular distribution could provide a rationale for the presence of different catalases in Salmonella
. In this connection, the finding that microgravity induces changes in the relative activity of KatN and KatG, which significantly influence Salmonella
ability to withstand hydrogen peroxide stress, support the hypothesis that the various catalases play roles that are not interchangeable.
We have also evaluated the involvement of the global transcriptional regulators OxyR, RpoE, RpoS and Hfq in the increased resistance to hydrogen peroxide stress of LSMMG-grown cells. OxyR coordinates the induction of genes required for survival to hydrogen peroxide stress, including KatG, the alkyl hydroperoxidase reductase, the Dps protein and glutathione reductase [53
]. RpoE is an alternative sigma factor that regulates genes required for the maintenance of membrane and periplasmic homeostasis in response to extracytoplasmic stress, which controls antioxidant defences and is critically important for S.
Typhimurium virulence [38
]. RpoS, an alternative sigma factor, plays a key role in the survival of bacteria during starvation or exposure to various stress conditions and is required for the expression of many genes in the stationary phase of growth [41
]. It is also known that it is involved in Salmonella
virulence in mice [40
]. Hfq is a RNA chaperone that binds small regulatory RNAs and facilitate bacterial responses to different kinds of stress [57
]. Recent studies have suggested that Hfq plays a central role in the Salmonella
response to reduced gravity [7
]. Also, a regulatory cascade involving RpoE, Hfq and other transcriptional regulators controls the expression of the RpoS stress regulon [58
As expected, we have found that S
. Typhimurium strains deleted of such regulators show increased susceptibility to H2
, both in LSMMG and in NG conditions. In addition, in agreement with a recent study showing that rpoE
is induced by acidic conditions and plays an important role in resistance to low pH in vitro
and in the intracellular vacuole [59
], we have observed that the inactivation of rpoE
reduced the acid stress resistance of cells cultivated in LSMMG. However, our results clearly indicate that OxyR, RpoE, RpoS and Hfq are not directly responsible for the increase of S.
Typhimurium resistance to hydrogen peroxide induced by LSMMG. In fact, the S.
Typhimurium strains lacking these genes were all more resistant to H2
when cultivated in LSMMG than in NG (Fig.
). These observations rule out the possibility that Hfq is the general regulator of all the phenotypic changes of Salmonella
in response to microgravity [7
] and suggest that multiple regulatory pathways are involved in the bacterial adaptation to such condition.
Taken together, our results indicate that microgravity conditions induce alterations in the activity of KatG and KatN that enhance Salmonella ability to withstand H2O2-mediated oxidative stress. Besides contributing to understand the mechanisms that modulate bacterial virulence during spaceflights, our observations suggest that low shear conditions such as those encountered in the intestine between the brush border microvilli may induce changes in the relative activity of catalases, which may favor the natural course of Salmonella infections.