Investigation of the role of Treponema denticola motility and uncharacterized protein TDE0659 in synergistic biofilm formation with Porphyromonas gingivalis
Year: 2018
Type: dissertation
Abstract: Chronic periodontitis has a polymicrobial biofilm etiology and interactions between key oral bacterial species such as Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia contribute to disease progression.P. gingivalis and T. denticola both have a set of virulence factors that are believed to contribute to the initiation and development of disease.It has been previously shown that P. gingivalis and T. denticola exhibit strong synergy in growth, biofilm formation and virulence in an animal model of disease.The motility of T. denticola, although not considered as a classic virulence factor, is likely to be involved in synergistic biofilm development between P. gingivalis and T. denticola.In order to investigate this, an optimized protocol for the transformation of T. denticola was developed and used to produce a number of T. denticola mutants targeting the motility machinery.The resulting mutants lacked periplasmic flagella (∆flgE) or possessed periplasmic flagella that were either non-functional (∆motA and ∆motB) or non-regulatable (∆cheY).∆cheY contained a large genomic excision and was thus omitted from the study, however analyses of ∆flgE, ∆motA and ∆motB showed that they were impaired in motility and growth.Quantitative proteomic analyses of mutant strains showed that the inactivation of these motility-associated genes, especially motA and motB, have far reaching effects beyond motility.The inactivation of motA and motB activated a cellular stress response in the mutants and directly or indirectly impacted the growth of the mutants through the change in abundance of a number of proteins.T. denticola motility mutant and WT strains were grown as mono-and dual-species biofilms with P. gingivalis.Results showed that T. denticola motility and/or spiral morphology are required for monospecies biofilm formation and T. denticola periplasmic flagella are essential for synergistic biofilm formation with P. gingivalis.Zones of clearing were observed between T. denticola sibling colonies grown close to one another on agar, similar to the sibling killing phenomenon observed in Paenibacillus dendritiformis.Given that a sibling killing phenomenon could have a considerable effect on biofilm formation, this phenomenon was investigated further in T. denticola.Although T. denticola was found to possess a homologue (TDE0659) of the dendritiformis sibling bacteriocin (DfsB) produced by P. dendritiformis, the protein did not undertake the same function in T. denticola.Construction and analysis of a T. denticola mutant strain lacking TDE0659 showed that the loss of this gene product from T. denticola did not prevent PREFACE Several members of the Oral Health CRC, Melbourne Dental School and Bio21 Institute, The University of Melbourne contributed to the work contained within this thesis.Cloning and propagation of plasmid pCF382, a kind gift from Dr Christopher Fenno (School of Dentistry, University of Michigan), in E. coli hosts were carried out by Dr Kheng Hui Tan.Genome sequencing of T. denticola ATCC 33520, ∆flgE33520, ∆motA33520 and ∆motB33520 using the Ion Torrent Personal Genome Machine (PGM) was performed by Ms. Brigitte Hoffmann and the resulting sequencing reads were analyzed by Dr Catherine Butler (Section 2.12.1).Samples of T. denticola ATCC 35405, ATCC 33520, HL51, ∆flgE33520, ∆motA33520 and ∆motB33520 were prepared for cryo-EM and imaged by Dr Yu-Yen Chen (Section 2.12.4).Label-free proteomics by MaxQuant was carried out by Dr Paul Veith (Section 2.12.8).Statistical analysis of the data from the static biofilm assays of T. denticola ATCC 33520, ∆flgE33520, ∆motA33520 and ∆motB33520 using Kruskal-Wallis with Conover-Imam test was performed by Mr. Yong Kai Wong from RMIT University (Section 2.12.9).Real-time PCR for the enumeration of bacterial cells was conducted with the help of Ms. Lin Xin Kin (Section 2.12.10).Flow cell biofilm assays, imaging of biofilms and analysis of the confocal data sets with COMSTAT software were performed by Ms. Sze Wei Liu (Section 2.12.11 and Section 2.12.12).T. denticola HL51 was obtained from Dr Howard Kuramitsu.Part of the Introduction (Chapter 1) to this thesis and Figure 1.3 have been published as the review article, "Ng,
Author: Hong Min Ng
Language: English
Subfield: Periodontics
Field: Dentistry
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2903823567