The production of ROS and RNS by inflammatory cells is a major component of host antimicrobial defenses (35
). Bacteria produce numerous factors to help defend themselves from the host immune response. One important bacterial factor involved in the detoxification of superoxide radicals is SOD. We have shown here that A. actinomycetemcomitans
produces Cu,Zn SOD and this enzyme interacts with A. actinomycetemcomitans
Superoxide dismutases are a class of enzymes that neutralize superoxide generated as a by-product of aerobic metabolism. Highly reactive superoxide can damage proteins (25
), DNA (38
), and lipids (19
). Superoxide dismutases use metal cofactors (Mn, Fe, Cu, and Zn) to dismutase superoxide to hydrogen peroxide and molecular oxygen (58
). Cu,Zn SOD proteins are widely distributed among bacteria and are located in the periplasm or in the outer membrane (3
). This enzyme may be important for the survival of bacterial pathogens in the host environment by protecting against ROS and RNS produced by host inflammatory cells (3
). This hypothesis is supported by several studies demonstrating that mutants deficient in Cu,Zn SOD production (sodC
mutants) are attenuated in animal models for disease. However, the role that Cu,Zn SOD plays in bacterial virulence is ambiguous—it clearly contributes to disease in some organisms (20
), but not in others (9
). This discrepancy may be due to differences in experimental models.
In addition to protecting bacteria from exogenous superoxide, Cu,Zn SOD may also play an important self-protective role against superoxide formed endogenously. It has recently been found that substantial superoxide is released into the periplasm of E. coli
, apparently due to the spontaneous oxidation of menaquinone (48
). This endogenous superoxide generated during respiration may in fact be the primary substrate for Cu,Zn SOD in gram-negative bacteria.
Herein, we report the physical interaction between A. actinomycetemcomitans
LtxA, an RTX toxin, and Cu,Zn SOD. Toxins of the RTX family contain glycine-rich repeats that are responsible for binding calcium (12
) and are required for toxin activity (17
). Cu,Zn SOD proteins from pathogenic bacteria are characterized by histidine-rich N-terminal extensions that may be involved in metal uptake under conditions of metal starvation in vivo (4
). Thus, one possibility is that Cu,Zn SOD may bind the calcium-rich regions of LtxA through imidazole side chains of histidine. While the molecular mechanism of LtxA-Cu,Zn SOD interaction remains to be investigated, it will be of significant interest to determine if Cu,Zn SOD from other bacteria interact with similar toxins.
Cu,Zn SOD from A. actinomycetemcomitans
has previously not been studied. We found that the sodC
mutant was more sensitive than the wild-type strain to superoxide generated in vitro. This result indicates that Cu,Zn SOD may play an important role in protection against an oxidative burst generated by the host defense system during infection. Macrophages and neutrophils can produce superoxide simultaneously with nitric oxide, yielding significantly more reactive species, such as peroxynitrite (40
). Consistent with previous findings (10
), we found that ROS and RNS generated together were more toxic to A. actinomycetemcomitans
than superoxide alone (data not shown).
Superoxide has been shown to affect primarily proteins containing iron-sulfur clusters such as dehydratases (25
). The action of superoxide can result in peptide bond cleavage, modification of amino acid side chains, and conversion of the protein to derivatives that are highly sensitive to proteolytic degradation (65
). Peroxynitrite and other RNS can nonspecifically oxidize proteins at a variety of sites (39
). Interestingly, it is known that subnanomolar concentrations of LtxA can stimulate an oxidative burst in host inflammatory cells (76
), and our results show that purified LtxA rapidly degrades and is rendered inactive in the presence of ROS and RNS. We demonstrated here that Cu,Zn SOD protected LtxA from ROS- and RNS-induced damage. Therefore, interaction with Cu,Zn SOD may protect both bacteria and secreted LtxA during infection.
To better understand the role of the LtxA-Cu,Zn SOD interaction, it is important to identify the cellular localization of the proteins. We have shown that all A. actinomycetemcomitans
strains examined were able to secrete active LtxA into the culture supernatant (2
). LtxA is also found in the outer membrane (15
) with a portion of it exposed to the extracellular environment (2
). Cu,Zn SOD from gram-negative bacteria is often located in the periplasm (3
); however, it was shown that Cu,Zn SOD from Mycobacterium tuberculosis
is a membrane-associated protein (18
). In addition, Fletcher et al. (24
) reported that A. actinomycetemcomitans
Cu,Zn SOD is a secreted surface-associated protein. To further confirm the location of Cu,Zn SOD in A. actinomycetemcomitans
, we fractionated cells by using a technique based on the differential solubility of membranes in the detergent sarkosyl. Our data show that A. actinomycetemcomitans
Cu,Zn SOD is located in the periplasm and cytosol. In addition, we also detected a small fraction of Cu,Zn COD in the membrane fraction, indicating that Cu,Zn SOD is associated with the cell membrane, consistent with the results of Fletcher et al. (24
). Therefore, it is possible that Cu,Zn SOD and LtxA interact on the surface of bacterial cells. Furthermore, interaction between secreted LtxA and Cu,Zn SOD may occur when Cu,Zn SOD is released from lysed bacterial cells, as would take place when an immune cell, such as a macrophage, attacks invading pathogens.
Iron limitation in vivo is a major obstacle that infecting bacteria must overcome to proliferate and cause disease. It was shown that heme and hemoglobin, but not transferrin and lactoferrin, can be used by A. actinomycetemcomitans
as iron and heme sources (31
). We have recently found that LtxA is able to lyse erythrocytes (1
), and hemolysis may be an important strategy for A. actinomycetemcomitans
. Therefore, hemoglobin and/or heme, produced as a result of the LtxA-mediated hemolysis, may be significant sources of iron during infection (36
). A recent study revealed a new function for Cu,Zn SOD from Haemophilus ducreyi
. It was shown that this enzyme is a heme-binding protein and may serve to accumulate heme from the environment and supply the cell with heme and iron. It was also suggested that under certain conditions, Cu,Zn SOD may protect bacteria from toxic oxyradicals formed from the reaction between heme iron and oxygen (60
). Thus, we suggest that Cu,Zn SOD from A. actinomycetemcomitans
may potentially play a role in iron and heme acquisition, especially during systemic disease.
Based on the data presented here and the results of other studies, we propose the following model for the biological role of A. actinomycetemcomitans
Cu,Zn SOD during infection. LtxA and other A. actinomycetemcomitans
virulence factors stimulate ROS and RNS production in host inflammatory cells (76
). Cu,Zn SOD inactivates superoxide generated by host white blood cells to protect bacteria, while Cu,Zn SOD also interacts with LtxA, either in the extracellular environment or at the cell membrane, to provide protection from protein degradation. In addition, Cu,Zn SOD from A. actinomycetemcomitans
may be involved in heme and iron transport (60
), as LtxA-mediated hemolysis would cause the release of hemoglobin from erythrocytes. The spontaneous and enzymatic oxidation of hemoglobin results in heme accumulation in the environment (28
). In turn, heme may bind to Cu,Zn SOD and serve as a source of iron and heme for A. actinomycetemcomitans
, as occurs for Haemophilus ducreyi
. In further support of this model, we have recently reported that iron represses the secretion of LtxA and consequently results in decreased lysis of erythrocytes (2
). This model describing the interplay between a bacterial SOD and a toxin may represent a new paradigm in bacterial pathogenesis.