Oral Presentation BACPATH 2017

Environmental control of OMV cargo in Porphyromonas gingivalis (#20)

Paul D Veith 1 , Caroline Luong 1 , Kheng H Tan 1 , Stuart G Dashper 1 , Eric C Reynolds 1
  1. Oral Health Cooperative Research Centre, Melbourne Dental School, BIO21 Institute, University of Melbourne, Parkville, Victoria, Australia

Porphyromonas gingivalis is a Gram negative, anaerobic bacterium strongly associated with adult periodontitis. P. gingivalis naturally produces outer membrane vesicles (OMVs) that consist almost entirely of proteins associated with the outer membrane (OM) or periplasm. P. gingivalis has an obligate requirement for iron which it prefers to take up in the form of heme. In this study we explored the effect of changing the growth conditions on the selective packaging of OMV cargo. P. gingivalis was grown in continous culture in chemostats, in heme limitation and heme excess. The conditions were carefully designed such that the OD only increased by about 25% in the heme excess medium. Culture samples were collected from the waste tubing, from which whole cell lysates (WCLs) and OMV samples were prepared. Four biological replicates were digested with trypsin and analysed by tandem mass spectrometry and label free quantitation using MaxQuant software. In total, 122 proteins were quantified from the OMV samples and 1180 proteins were quantified from the WCL samples. In heme limitation, 41 and 20 proteins increased in abundance more than 1.5 fold (with p<0.05) in the WCLs and OMVs respectively while in heme excess, 67 and 29 proteins increased in abundance using the same criteria. For proteins quantified in both WCLs and OMVs, the results generally correlated well indicating that most OM proteins are consistently passed on to OMVs. For example, several TonB-dependent receptors were upregulated to a similar degree in cells and OMVs. However, a few proteins were found to be differentially enriched in OMVs with significant p-values suggesting that P. gingivalis may be able to modulate its packaging mechanisms in response to heme availability. In summary, we present an accurate quantitation of OM proteins and demonstrate that their selective packaging into OMVs can be altered in response to environmental stimuli.