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 gingivalis is a pathogen that is linked to the development of peridontitis in humans.
In peridontitis, infection with 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. 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 phospho 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 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 affect 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 analysis 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.