The sinonasal region of the top respiratory system is segregated from the trachea by the oropharynx and larynx

Within a biofilm, the bacteria exert a significantly increased Temozolomide virulence and resistance to the host immune defences. Therefore, “traditional” experimental models that simply study single individual bacterial species might not be optimal to acknowledge the role of oral biofilms in periodontal diseases. To understand the role of the oral biofilms in disease, it is necessary to unravel the relationships between their constituent species. Based on co-aggregation experiments, it is estimated that there can be numerous interactions between various microbial species of the human oral cavity. Such aggregations reflect the formation of biofilms, both by defining the early colonizing events of the tooth surfaces, and generating optimal microenvironments for the later colonizing species. Interestingly, in a multispecies biofilm model similar to the one employed in this study, it is shown that in the absence of the “early colonizing” species, the “late colonizing” species form different structures within the biofilm. A number of experimental studies were also performed to investigate the detailed effects of virulence factors of one species to another, within a multi-species biofilm community. For example, BspA protein from Tannerella forsythia favours the co-aggregation with Fusobacterium nucleatum, whereas the lysine and arginine gingipains of Porphyromonas gingivalis regulated the growth of T. forsythia and of Treponema denticola, respectively; Aggregatibacter actinomycetemcomitans utilizes L-lactate from Streptococcus gordonii as energy source. However, not all relationships within biofilms are synergistic. For example, streptococcal arginine deiminase inhibits the expression of fimA from Porphyromonas gingivalis and thus abrogates colonization ; AI-2 of A. actinomycetemcomitans inhibits biofilm formation of Candida albicans. Still, most of the models used to investigate inter-species associations involve pair-wise bacterial comparisons, and the obtained data might be an oversimplified version of the reality. Using multi-species biofilm models may be closer to the in vivo situation, and may allow for the extrapolation of biological data that is more clinically relevant. In recent years, a 10-species in vitro “subgingival” biofilm model has been established and optimized in order to address such issues. In the present study, this model was evolved further to incorporate A. actinomycetemcomitans, a highly leukotoxic species that is strongly associated with aggressive forms of periodontitis occurring among young individuals. The many virulence factors of A. actinomycetemcomitans identified are its putative “weapons” against the host immune armament, including polymorphonuclear leukocytes, T-lymphocytes and macrophages. In a biofilm environment, these functions may not only be favouring A. actinomycetemcomitans itself, but might also support all other species in the biofilm in escaping the host immune system. Besides, this bacterium affects other species commonly found in subgingival biofilms, including P. gingivalis and F. nucleatum. As suggested by Hajishengallis et al, a keystone pathogen for periodontal infection might not actually be the dominant species within the biofilm, but may induce changes in other constituent species. Deciphering the protein regulations across the biofilm could therefore be crucial in understanding the role of the individual species in the integrity and function of the biofilm. Yet, most studies have addressed the role of only one or a handful of proteins, rather than the overall protein profile in a biofilm. Proteomics provide an important novel approach to extract detailed information of cellular regulatory mechanisms on the protein level at a large scale. With the utilization of mass spectrometry- based technologies, it is possible to identify and quantify thousands of proteins from complex biological samples in one run. In a biofilm environment, this tool could not only support the identification of regulatory proteins, but also evaluate the trend of their regulation at a universal level. Based on the above considerations, a label-free quantitative proteomic approach was employed to quantify the protein expressions and cluster their functions, in an 11-species in vitro “subgingival” biofilm, or its 10-species variant lacking A. actinomycetemcomitans. Hence, by inference, the relative regulatory roles of A. actinomycetemcomitans in the biofilm were deduced. With the increasing numbers of identified bacteria in oral biofilms, there are increasing needs in understanding their individual or communal functions.

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