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Introduction: The innate host response system is designed to detect and facilitate adaptive immune responses to microbial components, such as bacterial polysaccharides-lipopolysaccharide (LPS). To enable this to occur, innate systems contain multiple pattern recognition receptors (Toll-like receptors -TLR’s), which identify certain features within bacterial LPS that are foreign to the host, as well as uniquely specific for bacteria. Innate host identification of unique bacterial components, therefore, relies on the inability of bacteria to alter these essential or critical components dramatically. LPS is an essential outer membrane molecule containing both a highly variable outer region (O-segment) as well as a relatively conserved inner region (lipid A). However, over the last decade, new evidence has emerged, revealing that increased natural diversity or heterogeneity within specific components of LPS, such as lipid A resulting in minor to moderate changes in lipid A structure can produce dramatic host responses. Porphyromonas gingivitis is a pathogen that is linked to the development of periodontitis in humans. In periodontitis, infection with the gram-negative bacterium that resides in the oral cavity, P.gingivalis included results in a chronic inflammatory response. The end result is gingival damage and bone resorption resulting in tooth loss.
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The aim of the Study: The study by Darveau et al reports the modulation of this lipid A structure in LPS isolated from P.gingivalis via Hemin. The authors study the role of hemin on lipid A, as hemin is found in the microenvironment of the periodontal cavity and its concentration is increased following ulceration or infection within the teeth cavity. Another quality of hemin that stimulates the genesis of this study is that hemin binds to iron and is the major system via which the gram-negative bacterium P.gingivalis acquires its nutritional iron. The author’s design a unique in vitro system to test the hypothesis that hemin added to the culture medium of P.gingivalis can alter the structure of lipid A in its LPS. The study is unique and differs from other previously performed studies in the sense that the authors isolate lipid A from LPS obtained directly from P.gingivalis.
Research Design Review: The research design is simplistic in nature. The authors grow out bacterial cultures of P.gingivalis gram-negative bacterium, isolate the LPS from P.gingivalis via two extraction methods namely phenol-water extraction and Tri-Reagent procedure that involves obtaining LPS in lyophilized form. To answer the critique of previous existing endotoxin contamination the authors mainly chose the phenol-water extraction technique for it allows them to test the endotoxin levels in the bacterial culture media. The authors are also thorough in looking for phosphor or glycolipid contamination in the extracted LPS and they do this via a mass spectrometric analysis of the same lipid entities in the LPS extract.
For the analysis of lipid A structure within the extracted LPS from P.gingivalis, both in the presence and absence of hemin in the culture media, the authors use the MALDI-TOF analysis. It is important to understand this technique for it allows for an easy interpretation of the results further on during the course of the discussion and critique of the paper. MALDI-TOF stands for Matrix Associated Laser Desorption Ionization- Time of Flight Mass Spectrometry. The cartoon below helps us to better understand how this analysis works:
The matrix-assisted laser desorption ionization (MALDI) technique developed in 1987, has increased the upper mass limit for mass spectrometric analyses of biomolecules to over 300,000 Da and enabled the analyses of large biomolecules by mass spectrometry. An attractive feature of the time-of-flight (TOF) mass spectrometer is its simple instrumental design. TOF mass spectrometers operate on the principle that when a temporally and spatially well-defined group of ions of differing mass/charge ratios are subjected to the same applied electric field and allowed to drift in a region of constant electric field, they will traverse this region in a time which depends on upon their mass/charge ratios. The authors in the paper use this very technique to quantify the lipid A structure within the LPS and how the presence of hemin affects the mass/ charge ratio of lipid A.
Results Discussion: The main figure in the paper is Figure3. In this figure, the authors compare the effect of hemin directly on lipid A structure in LPS isolated from P.gingivalis. The gram-negative bacteria are grown on culture media containing either hemin and the corresponding negative control where the culture are allowed in the absence of hemin. The big critique, however, is that the authors do not dose-response the hemin in their in vitro culture system. It would have been nice to observe what the effect of increasing amounts of hemin has on the lipid A structure. Still, the authors randomly chose two doses of Hemin at 1ug/ml and at 10ug/ml. At the lower concentration of hemin, the authors find that the lipid structure has a peak in the mass spectrometric analysis that resembles closely to that of bacterial cultures grown in the absence of any hemin, these analyses are demonstrated in the previous figure (figure2) in the paper. Interestingly enough at the high concentration of hemin (10ug/ml), the authors find that the structure of lipid A differs and there is more heterogeneity in the peaks observed via mass spectrometry.
Another question that can be raised to the authors is why they don’t test out different gram-negative bacteria that inhabit the oral cavity for the effect of hemin on their respective lipid A structures within the LPS. Next, the authors go on to test whether the extraction process of LPS has any role in influencing the altering of lipid A structure within the LPS. The authors compare the effect of hemin in culture and its subsequent analysis of lipid A with the phenol extraction procedure of LPS (Figure4). They note that the phenol extraction procedure demonstrates the heterogeneity but is relatively inept in isolating lipid in excess of a mass/charge ratio of 1,770, for when they compare this analysis to the previous one they find those peaks missing in the mass spectrometric analysis.
The next experiment conducted by the authors is also very interesting, they hypothesize for this study that it is the increase in hemin concentration and not the increase in iron concentration in the medium that plays a role in altering the lipid A structure within the LPS extracted from P.gingivalis. In these studies, the authors culture the gram-negative bacterium with increasing amounts of Ferrous chloride (FeCl2). They notice that none of the concentrations of the iron salt can alter the structure the lipid A structure, as a result providing evidence to their claim that it is the hemin concentration that is the major role-player.
The next experiment by the author’s tests the effect of hemin on different strains of P.gingivalis. This is an interesting experiment it is essential to not limit the finding and scope of the study to one particular strain of a bacterium found in the oral cavity. The authors use multiple strains of P.gingivalis, culture them with hemin 10ug/ml and then do a mass spectrometric analysis of their structures. The finding reflects that all strains respond to the presence of hemin in their growing cultures and change their lipid A structures. What should be noticed and the authors fail to answer this in the discussion section or their interpretation of the data is the responsiveness of these strains to lower concentration of hemin in the culture. Both the shown strains alter the lipid A structure to the dose of 1ug/ml, which previously had not been eliciting a change (Figure3). Also, there is variability in the intensity of the peaks obtained at both the doses of hemin of the different strains. The authors fail to comment on this aspect of the experiment.
Review of Discussion Section: The discussion section is brief and basically summarizes the results section. It does not stimulate the reader to think what are the alternative experiments that could have been performed or lay out the plan for future experiments and the importance of these findings. It will be interesting to note whether the LPS extracted after growing bacterial cultures in the presence of hemin have a greater capability in triggering off the innate immune response by stimulating TLR4 to a greater extent. These experiments need to be performed by the authors, maybe the strains lose the ability to tackle TLR4 and hence the presence of hemin is a deterrent in the microenvironment or maybe they promote increased TLR2/4 stimulation that increases the intensity of the immune response.
What also needs to be done is that different gram-negative bacteria should be isolated from the gingival cavity and see if the hemin plays a similar role in altering the lipid A structure of that bacterium. The other aspect left unexplored is the role of hemin on the other component of LPS isolated from P.gingivalis. Highly variable outer regions (O-segment) exist within the extracted LPS that are known to play a role in the ability of LPS to stimulate an immune response. It will be prudent to investigate the effect of hemin concentration on these structures.
Overall the study is thorough and exhaustive in nature, the authors are experts in their field and utilize a technique that has a lot of credibility in the field of biochemistry. Using this approach the authors bring forward an integrated view of previous findings that were loosely characterized, the author suggests that these contradictory findings are due to the nature of the extraction process of LPS, which makes a lot of sense. They also convincingly show that hemin has a role in altering the lipid A structure of LPS isolated from P.gingivalis.
The studies set the tone for future experiments to research the role of hemin on the synthesis of lipid A and its subsequent modification as the infection grows in the periodontal cavity. This study has important translation findings for patients with periodontitis.
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