Velusamy Senthil Kumar, Sampathkumar Vandana, Godboley Dipti, Fine Daniel H
Department of Oral Biology, Rutgers School of Dental Medicine, 185 South Orange Ave, Newark, New Jersey, United States of America.
PLoS One. 2016 Mar 15;11(3):e0151361. doi: 10.1371/journal.pone.0151361. eCollection 2016.
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 salivary-coated 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 previous in 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.
白细胞毒素（Ltx）是伴放线聚集杆菌产生的一种重要毒力因子，该口腔微生物与侵袭性牙周炎高度相关。Ltx通过改变中性粒细胞、淋巴细胞和巨噬细胞的活力来损害宿主反应性。此前，我们开发了一种恒河猴（Rh）定植模型，旨在确定毒力基因突变对伴放线聚集杆菌定植的影响。出乎意料的是，伴放线聚集杆菌白细胞毒素（ltxA）突变体（RhAa-VS2）在Rh模型中未能定植。此前没有文献表明Ltx与伴放线聚集杆菌与牙齿表面的结合有关。这些结果促使我们在体外探索ltxA突变的广泛影响。结果表明，RhAa-VS2菌株中LtxA活性完全丧失，而与RhAa-VS2菌株相比，互补作用显著（P<0.0001）恢复了白细胞毒性。ltx基因表达的RT-PCR分析排除了极性效应。此外，与野生型RhAa3菌株相比，RhAa-VS2与唾液包被的羟基磷灰石（SHA）的结合显著减少（P<0.0001）。对RhAa-VS2中与SHA结合相关基因的实时RT-PCR分析表明，与野生型RhAa3相比，与结合相关的基因下调[rcpA（P = 0.018）、rcpB（P = 0.02）、tadA（P = 0.002）]。RhAa-VS2的生物膜深度（P = 0.008）和胞外多糖产量也降低（P<0.0001）。RhAa-VS2与颊上皮细胞（BEC）的结合未受影响。用ltxA互补可恢复与SHA的结合（P<0.002），但与RhAa3相比，对生物膜形成没有影响。总之，ltxA突变在体外减少了硬组织结合，这有助于解释之前ltxA基因敲除在体内未能定植于Rh口腔的原因。这些结果表明：1）一个特定基因的敲除（在本案例中为ltxA）可能会影响其他看似无关的基因（如rcpA、rcpB、tadA等），2）在竞争性体内环境中解释靶向基因突变的影响时应谨慎。
