Interaction between Leukotoxin and Cu,Zn Superoxide Dismutase in Aggregatibacter actinomycetemcomitans

Abstract

Aggregatibacter (Actinobacillus) actinomycetemcomitans is a gram-negative oral pathogen that is the etiologic agent of localized aggressive periodontitis and systemic infections. A. actinomycetemcomitans produces leukotoxin (LtxA), which is a member of the RTX (repeats in toxin) family of secreted bacterial toxins and is known to target human leukocytes and erythrocytes. To better understand how LtxA functions as a virulence factor, we sought to detect and study potential A. actinomycetemcomitans proteins that interact with LtxA. We found that Cu,Zn superoxide dismutase (SOD) interacts specifically with LtxA. Cu,Zn SOD was purified from A. actinomycetemcomitans to homogeneity and remained enzymatically active. Purified Cu,Zn SOD allowed us to isolate highly specific anti-Cu,Zn SOD antibody and this antibody was used to further confirm protein interaction. Cu,Zn SOD-deficient mutants displayed decreased survival in the presence of reactive oxygen and nitrogen species and could be complemented with wild-type Cu,Zn SOD in trans. We suggest that A. actinomycetemcomitans Cu,Zn SOD may protect both bacteria and LtxA from reactive species produced by host inflammatory cells during disease. This is the first example of a protein-protein interaction involving a bacterial Cu,Zn SOD.

Leukotoxin (Leukothera®) Targets Active Leukocyte Function Antigen-1 (LFA-1) Protein and Triggers a Lysosomal Mediated Cell Death Pathway

“…Interestingly, we previously reported that LtxA interacts with Cu,Zn superoxide dismutase from A. actinomycetemcomitans (32). In this publication, we proposed that the Cu,Zn superoxide dismutase may prevent damage to LtxA and bacterial cells from oxygen radicals released from dying WBCs.…”

Cytotoxic effects of Kingella kingae outer membrane vesicles on human cells

“…The immunoreactivity of antiserum was detected by enzyme-linked immunosorbent assay (ELISA), the antibody titer was estimated as 1:500,000. The antibody were purified from the final rabbit bleed using AminoLink plus kit (Pierce, Rockford, IL) as described previously [71]. …”

“…The membranes were subjected to Western blot analysis with 9D4 antibody (1:1000 dilution) (Santa Cruse, Campbell, CA) or anti-OMV antibody as previously described [71]. …”

Aggregatibacter actinomycetemcomitans Leukotoxin

“…Alternatively, SOD may be released from cells that have undergone lysis. SOD is also found associated with the membrane in A. actinomycetemcomitans (Fletcher et al, 2001;Balashova et al, 2007), and so it is possible that LtxA interacts with SOD at the cell surface.…”