Role of extracytoplasmic function sigma factors in biofilm formation of Porphyromonas gingivalis

The anaerobic Gram-negative bacterium Porphyromonas gingivalis is considered one of the important etiological agents of periodontal disease [1]. To colonize and survive in the gingival crevice, P. gingivalis must be capable of sensing and responding to the prevailing environmental conditions, including variations in temperature, oxygen tension, pH, nutrient availability and the presence of other bacterial cells. P. gingivalis possesses transcriptional regulators that have been implicated in protection against heat shock stress or oxidative stress, such as RprY [2, 3] and OxyR [4]. In addition, this bacterium and other bacteria form biofilms to protect against environmental stress [5]. Dental plaque, a multispecies biofilm, is organized on the tooth surface and periodontal tissues of the human oral cavity [6]. Oral bacteria in the biofilms survive in the gingival crevice for a long period of time, leading to gingivitis that can eventually progress into periodontitis. Understanding how bacteria escape environmental stress is very important for the prevention of periodontal disease.

Extracytoplasmic function (ECF) sigma factors serve as bacterial transcriptional regulators in the response to various stresses. The wild-type P. gingivalis 33277 genome encodes six ECF sigma factors (PGN_0274, PGN_0319, PGN_0450, PGN_0970, PGN_1108 and PGN_1740; GenBank: AP009380) [7]. PGN_1108 (W83 ORF number: PG1318) plays a role in the regulation of mutation frequency in the bacterium [8]. PGN_0274 (W83 ORF number: PG0162) and PGN_0450 (W83 ORF number: PG1660) may be involved in the post-transcriptional regulation of gingipain [9], and PGN_1740 (W83 ORF number: PG1827) is required for survival of the bacterium in the presence of oxygen and oxidative stress, hemin uptake and virulence [9, 10].

In this study, to analyze the role of ECF sigma factors in P. gingivalis biofilm formation, disruption of the ECF sigma factors, except PGN_1108, was performed. The PGN_1108-defective mutant may have a mutator phenotype, and we therefore excluded it from our experiments in this study [8]. The PGN_0274 and PGN_1740-defective mutants exhibited enhanced biofilm formation, but the complemented strains did not. These results suggest that the PGN_0274 and PGN_1740 ECF sigma factors are involved in the regulation of biofilm formation in the bacterium.

Bacteria sometimes encounter an environment unfavorable to their survival. The human oral microbiota is also often influenced by various stresses; hence, it must possess the ability to defend itself. Two principal regulatory mechanisms interact with cytoplasmic and extracytoplasmic regions via alternative ECF sigma factors and phosphorylation-dependent response regulators (two-component systems, TCSs) [16, 17]. ECF sigma factors have been shown to regulate cell envelope-related processes (involving maintenance of the membrane/periplasmic architecture), such as secretion, synthesis of exopolysaccharides, iron export and efflux synthesis of extracellular proteases [18]. Bacterial core RNA polymerase (composed of two α subunits, β subunit and β’ subunit) binds sigma factors. Multiple sigma factors are the bacterial transcription initiation factors that enable specific binding of RNA polymerase to gene promoters. In contrast, TCSs typically consist of a membrane-bound histidine kinase that senses a specific environmental stimulus and a corresponding response regulator that mediates the cellular response, mostly through differential expression of target genes [19]. Interestingly, a transcriptional regulator in Methylobacterium extorquens, PhyR, has been identified and determined to combine domains of both systems [20]. Taken together, ECF sigma factors and TCS are essential factors that protect bacteria from environmental stress.

Several P. gingivalis ECF sigma factors have been previously described. Nevertheless, there is no information on the ECF sigma factors that may operate in this bacterium in response to biofilm formation. In Bacillus subtilis and Pseudomonas aeruginosa, ECF sigma factors are involved in regulating biofilm development [21, 22]. In this study, we investigated whether biofilm formation of P. gingivalis is regulated by ECF sigma factors. This study demonstrated that PGN_0274 and PGN_1740 mutants yielded higher biofilm formation than that obtained with the wild-type or the other ECF sigma factor mutants. The inactivation of PGN_1740 also increased the expression of fimS at the transcriptional level [9]. Fimbriae and minor fimbriae influence monospecies biofilms [23]. The transcriptional level of fimS was examined using RT-PCR, which showed the fimS expression was downregulated (see Additional file 3). The results showed FimS may not be involved in controlling biofilm formation. Further work is needed to clarify this point.

The biofilm assay revealed that ethanol did not completely dissolve the biofilm mass and extract the crystal violet stain for the PGN_0274 mutant biofilm (see Additional file 1). Therefore, we dissolved the biofilm mass with SDS and measured the resulting crystal violet present in the sample. The need for a more stringent solvent suggested that the biofilm matrix around the mutant is composed partly of a protein component. The biofilm extracellular polymeric substances (EPS), composed of exopolysaccharides, proteins, nucleic acids and lipids, play a role as a defense structure, protecting bacteria from the host immune system and antimicrobial therapy [24]. Protein is a major component of EPS [25]. As the metabolic pathways of the PGN_0274 mutant are changed by the loss of the PGN_0274 ECF sigma factor, the protein yields in the PGN_0274 mutant are more abundant than those in the wild-type and the other mutants. Therefore, we examined the protein profile of the ECF sigma mutants compared with the wild-type (see Additional file 4). The degradation of 75-k–250-k Da proteins were demonstrated in the wild-type, PGN_0319, PGN_0450, PGN_0970 and PGN_1740 mutants, but not in the PGN_0274 mutant. This alteration was not observed in the presence of the proteinase inhibitors TLCK and leupeptin. The protein profiles of the wild-type and ECF sigma mutants were almost identical. Taken together, these results suggest this apparent difference in solubility might be explained by the decrease of Kgp and Rgp activity in the PGN_0274 mutant [9]. Kgp suppresses biofilm formation and Rgp controls microcolony morphology [26]. The sinR ortholog PGN_0088, a transcriptional regulator, acts as a negative regulator of exopolysaccharide accumulation in wild-type P. gingivalis[27]. PGN_0274 may control different metabolic pathways than PGN_0088 and act as a negative regulator of protein accumulation.

In conclusion, we have identified that PGN_0274 and PGN_1740 play a key role in P. gingivalis biofilm formation. These results show for the first time that P. gingivalis ECF sigma factors are involved in biofilm formation. PGN_0274 is involved in the post-transcriptional regulation of gingipains [8]. Gingipain is a very important virulence factor in P. gingivalis, because gingipains destroy periodontal tissue, immunoglobulins and complement factors [28, 29]. As PGN_1740 plays a significant role in oxidative stress responses in the bacterium [8, 9], the survival of the PGN_1740 mutant was reduced in the presence of host cells [9]. We also observed this in Ca9-22 cells (data not shown). Taken together, these results show that ECF sigma factors PGN_0274 and PGN_1740 are involved in the virulence of P. gingivalis. Further studies on the roles of the P. gingivalis ECF sigma factors, PGN_0274 and PGN_1740, will help us understand the ability of P. gingivalis to colonize and survive in the gingival crevice, and therefore act as a human pathogen.