Co-Culture of P. gingivalis and F. nucleatum Synergistically Elevates IL-6 Expression via TLR4 Signaling in Oral Keratinocytes.

Periodontitis, characterized by persistent inflammation in the periodontium, is intricately connected to systemic diseases, including oral cancer. Bacteria, such as Porphyromonas gingivalis and Fusobacterium nucleatum, play a pivotal role in periodontitis development because they contribute to dysbiosis and tissue destruction. Thus, comprehending the interplay between these bacteria and their impacts on inflammation holds significant relevance in clinical understanding and treatment advancement. In the present work, we explored, for the first time, their impacts on the expressions of pro-inflammatory mediators after infecting oral keratinocytes (OKs) with a co-culture of pre-incubated P. gingivalis and F. nucleatum. Our results show that the co-culture increases IL-1ß, IL-8, and TNF-α expressions, synergistically augments IL-6, and translocates NF-kB to the cell nucleus. These changes in pro-inflammatory mediators-associated with chronic inflammation and cancer-correlate with an increase in cell migration following infection with the co-cultured bacteria or P. gingivalis alone. This effect depends on TLR4 because TLR4 knockdown notably impacts IL-6 expression and cell migration. Our study unveils, for the first time, crucial insights into the outcomes of their co-culture on virulence, unraveling the role of bacterial interactions in polymicrobial diseases and potential links to oral cancer.

The Anti-Oxidant Curcumin Solubilized as Oil-in-Water Nanoemulsions or Chitosan Nanocapsules Effectively Reduces Helicobacter pylori Growth, Bacterial Biofilm Formation, Gastric Cell Adhesion and Internalization.

The bacterium Helicobacter pylori (H. pylori) represents a major risk factor associated with the development of gastric cancer. The anti-oxidant curcumin has been ascribed many benefits to human health, including bactericidal effects. However, these effects are poorly reproducible because the molecule is extremely unstable and water insoluble. Here we solubilized curcumin as either nanoemulsions or chitosan nanocapsules and tested the effects on H. pylori. The nanoemulsions were on average 200 nm in diameter with a PdI ≤ 0.16 and a negative zeta potential (-54 mV), while the nanocapsules were 305 nm in diameter with a PdI ≤ 0.29 and a positive zeta potential (+68 mV). Nanocapsules were safer than nanoemulsions when testing effects on the viability of GES-1 gastric cells. Also, nanocapsules were more efficient than nanoemulsions at inhibiting H. pylori growth (minimal inhibitory concentration: 50 and 75 µM, respectively), whereby chitosan contributed to this activity. Importantly, both formulations effectively diminished H. pylori's adherence to and internalization by GES-1 cells, as well as biofilm formation. In summary, the demonstrated activity of the curcumin nanoformulations described here against H. pylori posit them as having great potential to treat or complement other therapies currently in use against H. pylori infection.

Porphyromonas gingivalis-Helicobacter pylori co-incubation enhances Porphyromonas gingivalis virulence and increases migration of infected human oral keratinocytes.

Background: Porphyromonas gingivalis is part of the subgingival biofilm and a keystone species in the development of periodontitis. Interactions between P.gingivalis and other bacteria in biofilms have been shown to affect bacterial virulence. Helicobacter pylori also inhabits the subgingival biofilm, but the consequences of interactions there with P.gingivalis remain unknown. Here, we investigated how the pre-incubation of P.gingivalis with H.pylori affects P.gingivalis virulence. Methods: We assayed P.gingivalis internalization by oral keratinocytes (OKs), hemagglutination and biofilm formation to identify alterations in virulence after pre-incubation with H. pylori. Also, we evaluated viability and migration of OKs infected with P. gingivalis, as well as the role of toll-like receptor 4 (TLR4).   In addition, we quantified the mRNA of genes associated with P.gingivalis virulence. Results: Pre-incubation of P.gingivalis with H.pylori enhanced P.gingivalis biofilm formation, bacterial internalization into OKs and hemagglutination. Infection with pre-incubated P.gingivalis increased OK migration in a manner dependent on the O-antigen and linked to  increased expression of the gingipain RgpB. Also, OK TLR4 participates in these events, because upon TLR4 knock-down, pre-incubated P.gingivalis no longer stimulated OK migration. Discussion: We provide here for the first time insight to the consequences of direct interaction between P.gingivalis and H.pylori. In doing so, we shed light on the mechanism by which H. pylori presence in the oral cavity increases the severity or progression of periodontitis.

Variability in Genomic and Virulent Properties of Porphyromonas gingivalis Strains Isolated From Healthy and Severe Chronic Periodontitis Individuals.

Porphyromonas gingivalis has been extensively associated with both the onset and progression of periodontitis. We previously isolated and characterized two P. gingivalis strains, one from a patient exhibiting severe chronic periodontitis (CP3) and another from a periodontally healthy individual (H3). We previously showed that CP3 and H3 exhibit differences in virulence since H3 showed a lower resistance to cationic peptides compared with CP3, and a lower ability to induce proliferation in gingival epithelial cells. Here, we aimed to determine whether differences in virulence between these two strains are associated with the presence or absence of specific genes encoding virulence factors. We sequenced the whole genomes of both P. gingivalis CP3 and H3 and conducted a comparative analysis regarding P. gingivalis virulence genetic determinants. To do so, we performed a homology search of predicted protein sequences in CP3 and H3 genomes against the most characterized virulence genes for P. gingivalis available in the literature. In addition, we performed a genomic comparison of CP3 and H3 with all the 62 genomes of P. gingivalis found in NCBI's RefSeq database. This approach allowed us to determine the evolutionary relationships of CP3 and H3 with other virulent and avirulent strains; and additionally, to detect variability in presence/absence of virulence genes among P. gingivalis genomes. Our results show genetic variability in the hemagglutinin genes. While CP3 possesses one copy of hagA and two of hagC, H3 has no hagA and only one copy of hagC. Experimentally, this finding is related to lower in vitro hemmaglutination ability of H3 compared to CP3. Moreover, while CP3 encodes a gene for a major fimbrium subunit FimA type 4 (CP3_00160), H3 possess a FimA type 1 (H3_01400). Such genetic differences are in agreement with both lower biofilm formation ability and less intracellular invasion to oral epithelial cells exhibited by H3, compared with the virulent strain CP3. Therefore, here we provide new results on the genome sequences, comparative genomics analyses, and phenotypic analyses of two P. gingivalis strains. The genomics comparison of these two strains with the other 62 genomes included in the analysis provided relevant results regarding genetic determinants and their association with P. gingivalis virulence.

Chronic Inflammation as a Link between Periodontitis and Carcinogenesis.

Periodontitis is characterized by a chronic inflammation produced in response to a disease-associated multispecies bacterial community in the subgingival region. Although the inflammatory processes occur locally in the oral cavity, several studies have determined that inflammatory mediators produced during periodontitis, as well as subgingival species and bacterial components, can disseminate from the oral cavity, contributing therefore, to various extraoral diseases like cancer. Interestingly, carcinogenesis associated with periodontal species has been observed in both the oral cavity and in extra oral sites. In this review, several studies were summarized showing a strong association between orodigestive cancers and poor oral health, presence of periodontitis-associated bacteria, tooth loss, and clinical signs of periodontitis. Proinflammatory pathways were also summarized. Such pathways are activated either by mono- or polymicrobial infections, resulting in an increase in the expression of proinflammatory molecules such as IL-6, IL-8, IL-1ß, and TNF-α. In addition, it has been shown that several periodontitis-associated species induce the expression of genes related to cell proliferation, cell cycle, apoptosis, transport, and immune and inflammatory responses. Intriguingly, many of these pathways are linked to carcinogenesis. Among them, the activation of Toll-like receptors (TLRs) and antiapoptotic pathways (such as the PI3K/Akt, JAK/STAT, and MAPK pathways), the reduction of proapoptotic protein expression, the increase in cell migration and invasion, and the enhancement in metastasis are addressed. Considering that periodontitis is a polymicrobial disease, it is likely that mixed species promote carcinogenesis both in the oral cavity and in extra oral tissues and probably-as observed in periodontitis-synergistic and/or antagonistic interactions occur between microbes in the community. To date, a good amount of studies has allowed us to understand how monospecies infections activate pathways involved in tumorigenesis; however, more studies are needed to determine the combined effect of oral species in carcinogenesis.

Helicobacter pylori in human health and disease: Mechanisms for local gastric and systemic effects.

Helicobacter pylori (H. pylori) is present in roughly 50% of the human population worldwide and infection levels reach over 70% in developing countries. The infection has classically been associated with different gastro-intestinal diseases, but also with extra gastric diseases. Despite such associations, the bacterium frequently persists in the human host without inducing disease, and it has been suggested that H. pylori may also play a beneficial role in health. To understand how H. pylori can produce such diverse effects in the human host, several studies have focused on understanding the local and systemic effects triggered by this bacterium. One of the main mechanisms by which H. pylori is thought to damage the host is by inducing local and systemic inflammation. However, more recently, studies are beginning to focus on the effects of H. pylori and its metabolism on the gastric and intestinal microbiome. The objective of this review is to discuss how H. pylori has co-evolved with humans, how H. pylori presence is associated with positive and negative effects in human health and how inflammation and/or changes in the microbiome are associated with the observed outcomes.

Changes in lipopolysaccharide profile of Porphyromonas gingivalis clinical isolates correlate with changes in colony morphology and polymyxin B resistance.

Virulence factors on the surface of Porphyromonas gingivalis constitute the first line of interaction with host cells and contribute to immune modulation and periodontitis progression. In order to characterize surface virulence factors present on P. gingivalis, we obtained clinical isolates from healthy and periodontitis subjects and compared them with reference strains. Colony morphology, aggregation in liquid medium, surface charge, membrane permeability to bactericidal compounds, novobiocin and polymyxin B resistance, capsule presence and lipopolysaccharide (LPS) profiles were evaluated. By comparing isolates from healthy and periodontitis subjects, differences in colony morphology and aggregation in liquid culture were found; the latter being similar to two reference strains. These differences were not a consequence of variations in bacterial surface charge. Furthermore, isolates also presented differences in polymyxin B and novobiocin resistance; isolates from healthy subjects were susceptible to polymyxin B and resistant to novobiocin and, in contrast, isolates from periodontitis subjects were resistant to polymyxin B and susceptible to novobiocin. These changes in antimicrobial resistance levels correlate with variations in LPS profiles, since -unlike periodontitis isolates-isolates from healthy samples synthesize LPS molecules lacking both O-antigen moieties and anionic polysaccharide. Additionally, this phenotype correlated with the absence of O-antigen ligase activity. Altogether, our results reveal novel variations on surface components of P. gingivalis isolates obtained from healthy and periodontitis subjects that could be associated with differences in bacterial virulence and periodontitis progression.