Regulatory and structural properties differentiating the chromosomal and the bacteriophage-associated Escherichia coli O157:H7 Cu, Zn Superoxide Dismutases

<p>Abstract</p> <p>Background</p> <p>Highly virulent enterohemorrhagic <it>Escherichia coli </it>O157:H7 strains possess three <it>sodC </it>genes encoding for periplasmic Cu, Zn superoxide dismutases: <it>sodC</it>, which is identical to the gene present in non-pathogenic <it>E. coli </it>strains, and <it>sodC</it>-F1 and <it>sodC</it>-F2, two nearly identical genes located within lambdoid prophage sequences. The significance of this apparent <it>sodC </it>redundancy in <it>E. coli </it>O157:H7 has not yet been investigated.</p> <p>Results</p> <p>We report that strains deleted of one or more <it>sodC </it>genes are less resistant than the wild type strain to a challenge with hydrogen peroxide, thus confirming their involvement in the bacterial antioxidant apparatus. To understand if the different <it>sodC </it>genes have truly overlapping functions, we have carried out a comparison of the functional, structural and regulatory properties of the various <it>E. coli </it>O157:H7 SodC enzymes. We have found that the chromosomal and prophagic <it>sodC </it>genes are differentially regulated <it>in vitro</it>. <it>sodC </it>is exclusively expressed in aerobic cultures grown to the stationary phase. In contrast, <it>sodC</it>-F1 and <it>sodC</it>-F2 are expressed also in the logarithmic phase and in anaerobic cultures. Moreover, the abundance of SodC-F1/SodC-F2 increases with respect to that of SodC in bacteria recovered from infected Caco-2 cells, suggesting higher expression/stability of SodC-F1/SodC-F2 in intracellular environments. This observation correlates with the properties of the proteins. In fact, monomeric SodC and dimeric SodC-F1/SodC-F2 are characterized by sharp differences in catalytic activity, metal affinity, protease resistance and stability.</p> <p>Conclusion</p> <p>Our data show that the chromosomal and bacteriophage-associated <it>E. coli </it>O157:H7 <it>sodC </it>genes have different regulatory properties and encode for proteins with distinct structural/functional features, suggesting that they likely play distinctive roles in bacterial protection from reactive oxygen species. In particular, dimeric SodC-F1 and SodC-F2 possess physico-chemical properties which make these enzymes more suitable than SodC to resist the harsh environmental conditions which are encountered by bacteria within the infected host.</p

Efficient zinc uptake is critical for the ability of Pseudomonas aeruginosa to express virulence traits and colonize the human lung

We have recently shown that Pseudomonas aeruginosa, an opportunistic pathogen that chronically infects the lungs of patients with cystic fibrosis (CF) and other forms of lung disease, is extremely efficient in recruiting zinc from the environment and that this capability is required for its ability to cause acute lung infections in mice. To verify that P. aeruginosa faces zinc shortage when colonizing the lungs of human patients, we analyzed the expression of three genes that are highly induced under conditions of zinc deficiency (wiriA, dksA2 and rpmE2), in bacteria in the sputum of patients with inflammatory lung disease. All three genes were expressed in all the analyzed sputum samples to a level much higher than that of bacteria grown in zinc-containing laboratory medium, supporting the hypothesis that P. aeruginosa is under zinc starvation during lung infections. We also found that the expression of several virulence traits that play a central role in the ability of P. aeruginosa to colonize the lung is affected by disruption of the most important zinc importing systems. Virulence features dependent on zinc intake include swarming and swimming motility and the ability to form biofilms. Furthermore, alterations in zinc assimilation interfere with the synthesis of the siderophore pyoverdine, suggesting that zinc recruitment could modulate iron uptake and affect siderophore-mediated cell signaling. Our results reveal that zinc uptake is likely to play a key role in the ability of P. aeruginosa to cause chronic lung infections and strongly modulates critical virulence traits of the pathogen. Taking into account the recent discovery that zinc uptake in P. aeruginosa is promoted by the release of a small molecular weight molecule showing high affinity for zinc, our data suggest novel and effective possibilities to control lung infections by these bacteria

Prosurvival AMBRA1 turns into a proapoptotic BH3-like protein during mitochondrial apoptosis

Autophagy and apoptosis are 2 stress-response mechanisms that are closely interconnected. However, the molecular interplays between these 2 pathways remain to be clarified. Here we report that the crucial proautophagic factor AMBRA1 can act as a positive mediator of mitochondrial apoptosis. Indeed, we show that, in a proapoptotic positive feedback loop, the C-terminal part of AMBRA1, generated by CASP/ CASPASE cleavage upon apoptosis induction, inhibits the antiapoptotic factor BCL2 by a direct binding through its BH3-like domain. The mitochondrial AMBRA1-BCL2 complex is thus at the crossroad between autophagy and cell death and may represent a novel target in development of therapeutic approaches in clinical diseases

Unique features of the sodC-encoded superoxide dismutase from Mycobacterium tuberculosis, a fully functional copper-containing enzyme lacking zinc in the active site.

The sodC-encoded Mycobacterium tuberculosis superoxide dismutase (SOD) shows high sequence homology to other members of the copper/zinc-containing SOD family. Its three-dimensional structure is reported here, solved by x-ray crystallography at 1.63-A resolution. Metal analyses of the recombinant protein indicate that the native form of the enzyme lacks the zinc ion, which has a very important structural and functional role in all other known enzymes of this class. The absence of zinc within the active site is due to significant rearrangements in the zinc subloop, including deletion or mutation of the metal ligands His115 and His123. Nonetheless, the enzyme has a catalytic rate close to the diffusion limit; and unlike all other copper/zinc-containing SODs devoid of zinc, the geometry of the copper site is pH-independent. The protein shows a novel dimer interface characterized by a long and rigid loop, which confers structural stability to the enzyme. As the survival of bacterial pathogens within their host critically depends on their ability to recruit zinc in highly competitive environments, we propose that the observed structural rearrangements are required to build up a zinc-independent but fully active and stable copper-containing SOD