TY - JOUR
T1 - Profound effects of aggregatibacter actinomycetemcomitans leukotoxin mutation on adherence properties are clarified in in vitro experiments
AU - Velusamy, Senthil Kumar
AU - Sampathkumar, Vandana
AU - Godboley, Dipti
AU - Fine, Daniel H.
N1 - Funding Information:
DHF would like to acknowledge support derived from grant 1R21DE021172-01A1 from the National Institute of Dental and Craniofacial Research (NIDCR) to support the work performed in this study. Authors would like to thank Drs. David H Figurski and Scott C Kachlany for providing plasmids; Luke Fritzky, Manager of the Confocal Imaging Facility at NJMS, Rutgers for assistance with the imaging and Drs. Narayanan Ramasubbu and Mayilvahanan Shanmugam for critically reading the manuscript.
Publisher Copyright:
© 2016 Velusamy et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2016/3
Y1 - 2016/3
N2 - Leukotoxin (Ltx) is a prominent virulence factor produced by Aggregatibacter actinomycetemcomitans, an oral microorganism highly associated with aggressive periodontitis. Ltx compromises host responsiveness by altering the viability of neutrophils, lymphocytes, and macrophages. Previously, we developed a Rhesus (Rh) monkey colonization model designed to determine the effect of virulence gene mutations on colonization of A. actinomycetemcomitans. Unexpectedly, an A. actinomycetemcomitans leukotoxin (ltxA) mutant (RhAa-VS2) failed to colonize in the Rh model. No previous literature suggested that Ltx was associated with A. actinomycetemcomitans binding to tooth surfaces. These results led us to explore the broad effects of the ltxA mutation in vitro. Results: indicated that LtxA activity was completely abolished in RhAa-VS2 strain, while complementation significantly (P<0.0001) restored leukotoxicity compared to RhAa-VS2 strain. RT-PCR analysis of ltx gene expression ruled out polar effects. Furthermore, binding of RhAa-VS2 to salivarycoated hydroxyapatite (SHA) was significantly decreased (P<0.0001) compared to wild type RhAa3 strain. Real time RT-PCR analysis of the genes related to SHA binding in RhAa-VS2 showed that genes related to binding were downregulated [rcpA (P = 0.018), rcpB (P = 0.02), tadA (P = 0.002)] as compared to wild type RhAa3. RhAa-VS2 also exhibited decreased biofilm depth (P = 0.008) and exo-polysaccharide production (P<0.0001). Buccal epithelial cell (BEC) binding of RhAa-VS2 was unaffected. Complementation with ltxA restored binding to SHA (P<0.002) but had no effect on biofilm formation when compared to RhAa3. In conclusion, mutation of ltxA diminished hard tissue binding in vitro, which helps explain the previousin vivo failure of a ltxA knockout to colonize the Rh oral cavity. These results suggest that; 1) one specific gene knockout (in this case ltxA) could affect other seemingly unrelated genes (such as rcpA, rcpB tadA etc), and 2) some caution should be used when interpreting the effect attributed to targeted gene mutations when seen in a competitive in vivo environment.
AB - Leukotoxin (Ltx) is a prominent virulence factor produced by Aggregatibacter actinomycetemcomitans, an oral microorganism highly associated with aggressive periodontitis. Ltx compromises host responsiveness by altering the viability of neutrophils, lymphocytes, and macrophages. Previously, we developed a Rhesus (Rh) monkey colonization model designed to determine the effect of virulence gene mutations on colonization of A. actinomycetemcomitans. Unexpectedly, an A. actinomycetemcomitans leukotoxin (ltxA) mutant (RhAa-VS2) failed to colonize in the Rh model. No previous literature suggested that Ltx was associated with A. actinomycetemcomitans binding to tooth surfaces. These results led us to explore the broad effects of the ltxA mutation in vitro. Results: indicated that LtxA activity was completely abolished in RhAa-VS2 strain, while complementation significantly (P<0.0001) restored leukotoxicity compared to RhAa-VS2 strain. RT-PCR analysis of ltx gene expression ruled out polar effects. Furthermore, binding of RhAa-VS2 to salivarycoated hydroxyapatite (SHA) was significantly decreased (P<0.0001) compared to wild type RhAa3 strain. Real time RT-PCR analysis of the genes related to SHA binding in RhAa-VS2 showed that genes related to binding were downregulated [rcpA (P = 0.018), rcpB (P = 0.02), tadA (P = 0.002)] as compared to wild type RhAa3. RhAa-VS2 also exhibited decreased biofilm depth (P = 0.008) and exo-polysaccharide production (P<0.0001). Buccal epithelial cell (BEC) binding of RhAa-VS2 was unaffected. Complementation with ltxA restored binding to SHA (P<0.002) but had no effect on biofilm formation when compared to RhAa3. In conclusion, mutation of ltxA diminished hard tissue binding in vitro, which helps explain the previousin vivo failure of a ltxA knockout to colonize the Rh oral cavity. These results suggest that; 1) one specific gene knockout (in this case ltxA) could affect other seemingly unrelated genes (such as rcpA, rcpB tadA etc), and 2) some caution should be used when interpreting the effect attributed to targeted gene mutations when seen in a competitive in vivo environment.
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U2 - 10.1371/journal.pone.0151361
DO - 10.1371/journal.pone.0151361
M3 - Article
C2 - 26977924
AN - SCOPUS:84962281646
SN - 1932-6203
VL - 11
JO - PloS one
JF - PloS one
IS - 3
M1 - e0151361
ER -