An Investigation into the Effects of Outer Membrane Vesicles and Lipopolysaccharide of Porphyromonas gingivalis on Blood-Brain Barrier Integrity, Permeability, and Disruption of Scaffolding Proteins in a Human in vitro Model.

BACKGROUND: The effects of the key pathogens and virulence factors associated with gum disease such as Porphyromonas gingivalis (P. gingivalis) on the central nervous system is of great interest with respect to development of neuropathologies and hence therapeutics and preventative strategies. Chronic infections and associated inflammation are known to weaken the first line of defense for the brain, the blood-brain barrier (BBB). OBJECTIVE: The focus of this study is to utilize an established human in vitro BBB model to evaluate the effects of P. gingivalis virulence factors lipopolysaccharide (LPS) and outer membrane vesicles (OMVs) on a primary-derived human model representing the neurovascular unit of the BBB. METHODS: Changes to the integrity of the BBB after application of P. gingivalis LPS and OMVs were investigated and correlated with transport of LPS. Additionally, the effect of P. gingivalis LPS and OMVs on human brain microvascular endothelial cells in monolayer was evaluated using immunofluorescence microscopy. RESULTS: The integrity of the BBB model was weakened by application of P. gingivalis LPS and OMVs, as measured by a decrease in electrical resistance and a recovery deficit was seen in comparison to the controls. Application of P. gingivalis OMVs to a monoculture of human brain microvascular endothelial cells showed disruption of the tight junction zona occludens protein (ZO-1) compared to controls. CONCLUSION: These findings show that the integrity of tight junctions of the human BBB could be weakened by association with P. gingivalis virulence factors LPS and OMVs containing proteolytic enzymes (gingipains).

Role of bolA and rpoS genes in biofilm formation and adherence pattern by Escherichia coli K-12 MG1655 on polypropylene, stainless steel, and silicone surfaces.

Escherichia coli has developed sophisticated means to sense, respond, and adapt in stressed environment. It has served as a model organism for studies in molecular genetics and physiology since the 1960s. Stress response genes are induced whenever a cell needs to adapt and survive under unfavorable growth conditions. Two of the possible important genes are rpoS and bolA. The rpoS gene has been known as the alternative sigma (σ) factor, which controls the expression of a large number of genes, which are involved in responses to various stress factors as well as transition to stationary phase from exponential form of growth. Morphogene bolA response to stressed environment leads to round morphology of E. coli cells, but little is known about its involvement in biofilms and its development or maintenance. This study has been undertaken to address the adherence pattern and formation of biofilms by E. coli on stainless steel, polypropylene, and silicone surfaces after 24 h of growth at 37 °C. Scanning electron microscopy was used for direct examination of the cell attachment and biofilm formation on various surfaces and it was found that, in the presence of bolA, E. coli cells were able to attach to the stainless steel and silicone very well. By contrast, polypropylene surface was not found to be attractive for E. coli cells. This indicates that bolA responded and can play a major role in the presence and absence of rpoS in cell attachment.

Analysis of rpoS and bolA gene expression under various stress-induced environments in planktonic and biofilm phase using 2(-ΔΔCT) method.

Genetic adaptation is one of the key features of Escherichia coli (E. coli) that ensure its survival in different hostile environments. E. coli seems to initiate biofilm development in response to specific environmental cues. A number of properties inherent within bacterial biofilms indicate that their gene expression is different from that of planktonic bacteria. Two of the possible important genes are rpoS and bolA. The rpoS gene has been known as the alternative sigma (σ) factor, which controls the expression of a large number of genes, which are involved in responses to a varied number of stresses, as well as transition to stationary phase from exponential form of growth. Morphogene bolA response to stress environment leads to round morphology of E. coli cells, but little is known about its involvement in biofilms and its development or maintenance. The purpose of this study was to understand and analyse the responses of rpoS and bolA gene to sudden change in the environment. In this study, E. coli K-12 MG1655, rpoS, and bolA mutant strains were used and gene expression was studied. Results show that both genes contribute to the ability to respond and adapt in response to various types of stresses. RpoS response to various stress environments was somehow constant in both the planktonic and biofilm phases, whereas bolA responded well under various stress conditions, in both planktonic and biofilm mode, up to 5-6-fold change in the expression was noticed in the case of pH variation and hydrogen peroxide stress (H(2)O(2)) as compared with rpoS.

Contribution of rpoS and bolA genes in biofilm formation in Escherichia coli K-12 MG1655.

Flexibility of gene expression in bacteria permits its survival in varied environments. The genetic adaptation of bacteria through systematized gene expression is not only important, but also clinically relevant in their ability to grow biofilms in stress environments. Stress responses enable their survival under more severe conditions, enhanced resistance and/or virulence. In Escherichia coli (E. coli), two of the possible important genes for biofilm growth are rpoS and bolA gene. RpoS is also called as a master regulator of general stress response. Even though many studies have revealed the importance of rpoS in planktonic cells, little is known about the functions of rpoS in biofilms. In contrast, bolA which is a morphogene in E. coli is overexpressed under stressed environments resulting in round morphology. The hypothesis is that bolA could be implicated in biofilm development. This study reviewed the literature with the aim of understanding the stress tolerance response of E. coli in relation with rpoS and bolA genes in different environmental conditions including heat shock, cold shock, and stress in response to oxidation, acidic condition and in presence of cadmium. Knowledge of the genetic regulation of biofilm formation may lead to the understanding of the factors that drive the bacteria to switch to the biofilm mode of growth.