Lipopolysaccharide Preparations Research Articles

Влияние липополисахаридов грамотрицательных бактерий различных таксономических групп на морфогенетическую активность каллусов мягкой пшеницы

In vitro tissue culture is actively used in modern plant breeding for genetic transformation, effective selection, rapid production and propagation of genetically homogeneous lines. For many crops, the low yield of regenerated plants is limiting for the use of calluses in the breeding process. Some bacterial lipopolysaccharides are able to increase the morphogenetic activity of plant calluses, increasing the efficiency of in vitro technology. In this work, we investigated the effect of lipopolysaccharides (LPS) from the rhizobacterial strains Ensifer adhaerens T1Ks14, Ochrobactrum quorumnocens T1Kr02, and Pseudomonas chlororaphis K3 on the processes of callusogenesis, the formation of morphogenic calluses and regenerant plants of common wheat (Triticum aestivum L.) of a low-morphogenic line with the Rht-B1a dwarfing gene, obtained on the basis of the cultivar Saratovskaya 29. It was found that LPS preparations at a concentration of 10 μg/ml had different effects on wheat tissue cultures. The yield of calluses from explants obtained from immature embryos significantly increased by 2.5% when exposed to LPS of the O. quorumnocens strain T1Kr02 and decreased by 4% in the variant with LPS from P. chlororaphis K3. The yield of morphogenic calluses increased under the influence of LPS from E. adhaerens T1Ks14 (+57%) and P. chlororaphis K3 (+41%). LPS strain O. quorumnocens T1Kr02 had a negative effect on the yield of regenerated plants, while the other two LPS preparations did not differ from the control in this parameter. As a result, the effectiveness of using the LPS preparations from explants to regenerants was significantly higher only in the variant of the E. adhaerens strain T1Ks14 (+64%). The data obtained indicate significant differences in the response of plant tissue cultures to LPS of various rhizosphere bacteria.

Characteristics of Stenotrophomonas rhizophila Lipopolysaccharide, a Representative of Antarctica

Characteristics of Stenotrophomonas rhizophila Lipopolysaccharide, a Representative of Antarctica

Humoral immune response and changes in peritoneal cell populations in rats immunized against two Leptospira serovars; serovar patoc and serovar pyrogenes

BackgroundLeptospirosis is a zoonotic disease caused by Leptospira species. Variations in lipopolysaccharide (LPS) structure in Leptospira are known to be associated with the serovar diversity and antigenicity. Development of immunodiagnostics for early detection of leptospirosis based on immune responses against different pathogenic antigens as well as development of vaccines are important. Hence, this study has assessed the immune response generated against leptospiral LPS and whole antigen preparations of pathogenic and saprophytic Leptospira and specific changes in peritoneal cells was also studied to elucidate the cellular responses associated with immune response of Wistar rats.MethodsDuring the study, immune response induced by two types of Leptospira antigen preparations of two selected serovars was compared. Changes in the specific peritoneal cell subpopulations following immunizations of rats were analyzed using flow cytometry.ResultsOf the two antigen preparations tested, the LPS extract induced a higher IgM immune response as opposed to the sonicated antigen preparation. Of the two serovars tested, L. interrogans serovar Pyrogenes had induced a higher IgM response compared to that by L. biflexa serovar Patoc. Considering the IgG titers, equivalent responses were observed with all four antigen preparations. Significant increases in lymphocytes were observed following immunization with LPS of both serovars. Interestingly, the B2 cell percentages increased significantly during the immunization period. Further, significant correlations were observed with both IgM and IgG responses and percentage of B2 cells in the peritoneal cavity (PC).ConclusionLPS extract of L. interrogans serovar Pyrogenes induced higher IgM response while the IgG response was equivalent among the four antigen preparations tested. Significant increase of B2 cell percentage in the peritoneal cavity during the immunization reflects the accumulation of B2 cells in the PC which may play considerable role in generating humoral response against Leptospira antigens.

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Bacterial wetwood, bacterial dropsy, or bacterial slime is a common disease caused by Lelliottia nimipressuralis, which affects the central core of many conifers and deciduous trees. Representatives of this species have been isolated from a variety of trees showing symptoms of the disease, as well as from water and, less commonly, from clinical samples. Important aspects of pathogenesis is the process of pathogen recognition and the protection mechanisms of bacterial cells from plant’s antimicrobial substances. It is known that lipopolysaccharides (LPS) take an active part in these processes. They provide the barrier function of the outer membrane, helping to protect bacteria from plant antimicrobial compounds, and the attachment of bacteria to plant cells. Therefore, the aim of the work was to study the peculiarities of the chemical composition and functional and biological characteristics of Lelliottia nimipressuralis F9a1 LPS obtained by different methods. Methods. LPS was isolated from dry bacterial mass by phenol-water method (LPS I), extraction method with 0.85% NaCl solution (LPS II), and phenol-water extraction of LPS insoluble in NaCl solution (LPS III). The carbohydrates were analyzed by Dubois method, nucleic acids ‒ by Spirin, protein content ‒ by Lowry and 2-keto-3-deoxyoctonic acid (KDO) ‒ by Osborn. The identification of monosaccharides and fatty acids in LPS preparations was carried out on an Agilent 6890N/5973 inert chromato-mass spectrometry system. The pyrogenicity of LPS was tested keeping the rules of bioethics in rabbits. Serological studies were performed by the Ouchterlony method. Results. LPS II of L. nimipressuralis F9а1 was characterized by low relative yield (2.12%), low content of carbohydrates (9.16%) and nucleic acids (3.7%), and high protein content (26.44%), while the studied preparations of LPS I and LPS III were characterized by a high yield, a rather high content of carbohydrates (46.68 and 38.4%, respectively), an insignificant amount of protein (up to 6.72%) and nucleic acids (up to 4.06%). All LPSs contained up to 0.27% KDO, which is a specific component of the LPS of gramnegative bacteria. The monosaccharide composition indicates that the LPS of the studied L. nimipressuralis strains turned out to be heterogeneous. At the same time, such monosaccharides as fucose, galactose, and glucose were recorded in the LPS of all tested strains. The fatty acid composition of LPS was represented by the presence of fatty acids containing from 12 to 18 carbon atoms. Нydroxylated, saturated, and monounsaturated acids were found. In LPS I and LPS III, the dominant fatty acid was 14:0 (3-OH), which is a kind of marker for the entire family of Enterobacteriaceae. In addition to the marker acid, a 16:1 acid was also predominant in LPS III, whereas in LPS II, 16:1 (32.7%) and 16:0 (22.6%) fatty acids dominated. The pyrogenic effect of L. nimipressuralis LPS studied showed that LPS solutions are pyrogenic. The serological studies showed that tested LPS in homologous systems exhibits antigenic activity. Antisera to L. nimipressuralis F9a1 react with LPS strains IMV 8791, LGK1, and L14b, which may indicate the presence of common antigenic determinants and belonging of these strains to the same serogroup. Conclusions. The LPSs of L. nimipressuralis F9a1 were heterogeneous in both monosaccharide and fatty acid composition, which is explained by the use of different methods for their isolation. To isolate LPS from L. nimipressuralis cells, the water-phenol method is better than sodium chloride extraction since with using the latter, the LPS yield is very low and very contaminated with proteins. At the same time, the isolation method does not affect the serological activity of the studied LPS. The results received during these biological-functional studies of L. nimipressuralis LPS contribute to the biological characteristics of this species.

Characterization of Azospirillum brasilense Lipopolysaccharides

Azospirillum brasilense is a gram-negative, nitrogen-fixing bacterium that colonizes the rhizosphere of various types of grasses and cereals. Lipopolysaccharides (LPS) are a class of complex glycolipids present in the cell membrane of gramnegative bacteria and mediate plant-bacteria interactions. Although the effects of LPS of pathogenic plant bacteria on the induction of plant defense mechanisms have been characterized, the role of LPS of beneficial rhizobacteria on plant growth is less clear. Therefore, a very important point is the study of the chemical, biological, and functional activities of A. brasilense LPS, which was the aim of this work. Methods. A. brasilense LPSs were isolated from dry bacterial mass by the phenol-water method. The carbohydrates were analyzed by the Dubois method, nucleic acids — by Spirin, protein content — by Lowry and 2-keto-3-deoxyoctonic acid (KDO) — by Osborn. Pyrogenicity of LPS was tested observing the rules of bioethics in rabbits. Serological studies were performed by the Ouchterlony method. The identification of monosaccharides and fatty acids in LPS preparations was carried out on an Agilent 6890N/5973 inert chromatomass spectrometry system. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAAG electrophoresis) was performed according to Laemmli. Results. LPS of 3 strains of A. brasilense were isolated from dry bacterial mass and purifi ed from nucleic acids by ultracentrifugation. The purified LPSs were characterized by different relative yields from 2.44% to 4.75%, which is slightly higher than other strains of the A. brasilense (1—3%). The studied preparations were characterized by a rather high content of carbohydrates from 50.1% to 72.1%. All LPS contained up to 0.17% KDO, which is a specific component of the LPS of gram-negative bacteria. Analysis of the monosaccharide composition indicates that the LPSs of the studied A. brasilense strains turned out to be heterogeneous. At the same time, such monosaccharides as mannose, galactose, glucose, and heptose were recorded in the LPS of all tested strains. The study of the fatty acid composition of LPS showed the presence of fatty acids containing from 14 to 18 carbon atoms. Нydroxylated, saturated, and monounsaturated acids and their cis isomers were found. In the investigated LPS, the dominant fatty acids were 16:0, 18:1, 14:0(3-OH), and 16:0(3-OH), which coincides with the literature data. The research of the pyrogenic effect of LPS of A. brasilense studied strains showed that LPS solutions are apyrogenic. The double immunodiffusion reaction in Ouchterlon agar showed that all tested LPS in homologous systems exhibited ancultitigenic activity. Serological cross-reactions can be used as an approach in classifying different bacteria. Thus, we found that antisera to A. brasilense 18-2 and 61 react with all LPSs of the studied strains, which may indicate the presence of common antigenic determinants in them and that these strains belong to the same serogroup. The electrophoretic distribution data indicate that A. brasilense produces S-forms of LPS which differ in the length of O-specifi c polysaccharide chains. Conclusions. For the first time, LPS were isolated from cells of A. brasilense 10/1, 18-2 and 61. A characteristic feature of these LPS is their heterogeneity in monosaccharide and fatty acid composition, all of them were apyrogenic. The results obtained during biological-functional studies of three strains of A. brasilense LPS contribute to the biological characteristics of this species.

Influence of pregravid preparation on the structural reorganization of the endometrium in women with chronic endometritis in infertility

Aim. To reveal the peculiarities of structural reorganization of the endometrium in various variants of pregravid preparation in women with chronic endometritis (CE) and infertility.
 Materials and methods. We examined 90 women aged 1840 who were treated for CE. For all patients, examination with microbiological (cultural) examination of the cervical canal to be separated for aerobic and facultative anaerobic microorganisms with the determination of sensitivity to antibiotics, ultrasound examination (ultrasound) of the pelvic organs and morphological examination of endometrial biopsy samples before and after treatment. All women of the study group were divided into 2 subgroups. Patients of the 1st subgroup (n=30) received standard treatment for CE antibacterial and non-steroidal anti-inflammatory drugs; in the 2nd (n=60) an additional course of immunostimulating therapy with a bacterial lipopolysaccharide preparation was prescribed.
 Results. According to the cultural method, repeated cultures from the cervical canal after treatment turned out to be sterile more often in women of the 2nd subgroup. Ultrasound diffuse-focal changes in the subendometrial zone of the myometrium occurred in all patients before and after treatment. In the 2nd subgroup, after treatment, significant changes in the endometrium were noted in the form of an increase in its thickness and a 4-fold decrease in the number of hyperechoic inclusions in the basal layer, as well as the disappearance of the irregularity of the endometrial closure line. In the endometrial biopsies of women of the 2nd subgroup, a significant decrease in the specific area of the inflammatory infiltrate, the absence of foci of fibrosis, and an increase in the thickness of the vessel wall were noted.
 Conclusion. The addition of a bacterial lipopolysaccharide to the standard CE treatment regimen not only improves the microbiological environment of the female genital tract, but also significantly affects the morphofunctional state of the endometrium.

Anti-TMV Activities of Pantoea agglomerans Lipopolysaccharides in vitro

Today there are no antiviral drugs of chemical nature that can completely cure virus-infected plants. The fact that their effect is limited to minimizing the pathogenic effect of viruses motivates many researchers to look for alternatives. In recent years it has been shown that lipopolysaccharides (LPS) of some bacteria, in particular representatives of the Pseudomonas genus were active against Tobacco mosaic virus (TMV). Therefore, we were interested in the additional study of LPS of phytopathogenic bacteria Pantoea agglomerans as a possible drug acting as antiviral agent. The aim of current study was to evaluate the antiviral activities of LPS obtained from phytopathogenic bacteria P. agglomerans against TMV in vitro. Methods. The antiviral activity of LPS preparations was investigated in vitro and assessed according to the inhibition percentage towards the number of local lesions in Datura stramonium leaves. P. agglomerans LPS was isolated from dry bacterial mass by phenol-water method. LPS mild acid degradation allowed to separate O-specific polysaccharide (OPS) and lipid A, which structures were identified by us earlier. The analysis of TMV and LPS interactions was carried out using a JEM 1400 transmission electron microscope (Jeol, Japan) at an accelerating voltage of 80 kV. Results. The most active were LPS preparations from P. agglomerans P324 and 8488. In vitro inhibitory efficacies of TMV infection by these LPS preparations was 59 and 60% respectively. LPS preparations of P. agglomerans 7969, 7604 and 9637, on the contrary, were inactive. Comparative analysis of the antiviral activity of LPS structural components of two P. agglomerans P324 and 7604 strains showed that the greatest inhibitory effect on the infectivity of TMV was exhibited by P. agglomerans P324 lipid A, the antiviral activity of which practically did not differ from the activity of the LPS molecule (it was lower by 7%). At the same time, the inhibitory effect of P. agglomerans 7604 core oligosaccharide (OG-core) against TMV was slightly higher compared to the effect of the whole LPS molecule. It can be assumed that the OG-core stimulated the defense mechanisms of plants and prevented the development of viral infection. Electron microscopic dates have shown that P. agglomerans P324 LPS at the concentration of 1 mg/ml influenced on freely located virions in the control causing “sticking” thus forming dense clusters, complexes or “bundles” of the virus. The individual structural components of P. agglomerans P324 LPS (lipid A and OG-core) did not have the same effect as a whole molecule. Conclusions. The study of the antiviral activity of LPS in the model system TMV – Datura stramonium L. plants showed that the most active were LPS preparations of only two strains of P. agglomerans (P324 and 8488) while the other seven strains were inactive. Individual structural components: lipid A from P. agglomerans P324 and OG-core from P. agglomerans 7604 decreased the infectivity of TMV by 7 and 15% higher than the initial LPS molecule. According to electron microscopy data the virions sticked together forming the dense clusters in case of the direct LPS-virus contacting in vitro whereas in the control it was observed just a single free virus particles. A more detailed study of the effect of individual structural components will help to understand the regularities of the LPS structure effect on TMV infectivity.

Lipopolysaccharide Preparation Derived From Porphyromonas gingivalis Induces a Weaker Immuno-Inflammatory Response in BV-2 Microglial Cells Than Escherichia coli by Differentially Activating TLR2/4-Mediated NF-κB/STAT3 Signaling Pathways.

Alzheimer’s disease (AD) is a degenerative disease of the central nervous system with unclear etiology and pathogenesis. In recent years, as the infectious theory and endotoxin hypothesis of AD has gained substantial attention, several studies have proposed that Porphyromonas gingivalis (P. gingivalis), one of the main pathogenic bacteria of chronic periodontitis, and the lipopolysaccharide (LPS) of P. gingivalis may lead to AD-like pathological changes and cognition impairment. However, research on the relationship between P. gingivalis-LPS and neuroinflammation is still lacking. Our study aimed to investigate the effects of P. gingivalis-LPS preparation on immuno-inflammation in microglial cells and further compared the differential inflammatory response induced by P. gingivalis-LPS and Escherichia coli (E. coli) LPS preparations. The results showed that P. gingivalis-LPS could upregulate the gene expression and release of pro-inflammatory factors in BV-2 microglial cells, including IL-1β, IL-6, TNF-α, IL-17, and IL-23. We also observed an increase in the level of Toll-like receptor 2/4 (TLR2/4) and NF-κB/STAT3 signaling. Moreover, the changes mentioned above were more significant in the E. coli-LPS group and the effects of both kinds of LPS could be differentially reversed by the administration of the TLR2 inhibitor C29 and TLR4 inhibitor TAK-242. The molecular simulation showed that the binding affinity of P. gingivalis-lipid A to TLR4-MD-2 was weaker than E. coli-lipid A, which was probably due to the presence of fewer acyl chains and phosphate groups of P. gingivalis-lipid A than E. coli-lipid A. We conclude that P. gingivalis-LPS could activate TLR2/4-mediated NF-κB/STAT3 signaling pathways, which ultimately resulted in an immune-inflammatory response in BV-2 microglia. In contrast to E. coli-LPS, P. gingivalis-LPS is a weaker TLR2/4 agonist and NF-κB/STAT3 signaling activator. Furthermore, the different fatty acid chains and phosphate groups between P. gingivalis-lipid A and E. coli-lipid A may be the reason for the weaker activating properties of P. gingivalis-LPS.

Response of Human Mesenchymal Stromal Cells from Periodontal Tissue to LPS Depends on the Purity but Not on the LPS Source.

Human periodontal ligament stromal cells (hPDLSCs) and gingival mesenchymal stromal cells (hGMSCs) are resident mesenchymal stromal cells (MSCs) of the periodontal tissue. The lipopolysaccharide (LPS) from Porphyromonas gingivalis is structurally distinct from that of other Gram-negative bacteria, and earlier studies linked this structural difference to a distinct virulence activity and the ability to activate toll-like receptor 2 (TLR-2), besides TLR-4 as commonly occurring upon LPS challenge. Later studies, in contrast, argue that TLR-2 activation by P. gingivalis LPS is due to lipoprotein contamination. In the present study, we aimed to define the influence of structure versus purity of P. gingivalis LPS on the immune response of hPDLSCs and hGMSCs. Cells were stimulated with commercially available “standard” P. gingivalis LPS, “ultrapure” P. gingivalis LPS, or “ultrapure” Escherichia coli LPS, and the expression of interleukin- (IL-) 8, IL-6, monocyte chemoattractant protein- (MCP-) 1, TLR-2, and TLR-4 was evaluated. The contribution of TLR-4 to the LPS-induced response was assessed using the specific TLR-4 inhibitor TAK-242. “Standard” P. gingivalis LPS induced significantly higher IL-8, IL-6, and MCP-1 production compared to the “ultrapure” LPS preparations, with no significant difference detectable for “ultrapure” LPS from P. gingivalis and E. coli. By using TAK-242, the response of hPDLSCs and hGMSCs to “ultrapure” LPS preparations was effectively inhibited to the levels comparable to those of nonstimulated controls. In contrast, high levels of response to “standard” LPS were observed, even in the presence of TAK-242. Our data show that the response of MSCs from periodontal tissue to LPS depends more on the purity of the LPS preparation than on the LPS source. Even a small amount of contaminating lipoproteins can drastically enhance the hPDLSCs' and hGMSCs; responsiveness to P. gingivalis LPS, which might also contribute to the progression of periodontal disease.

Stress Response Simulated by Continuous Injection of ACTH Attenuates Lipopolysaccharide-Induced Inflammation in Porcine Adrenal Gland.

On modern farms, animals are at high risk of bacterial invasion due to environmental stress factors. The adrenal gland is the terminal organ of the stress response. The crosstalk between adrenal endocrine stress and innate immune response is critical for the maintenance of immune homeostasis during inflammation. Thus, it's important to explore whether stresses play a pivotal role in lipopolysaccharide (LPS)-induced inflammatory response in the porcine adrenal gland. Thirty-days-old Duroc × Landrace × Large White crossbred piglets (12 ± 0.5 kg) were randomly allocated into four groups in a 2 × 2 factorial arrangement of treatments, including ACTH pretreatment (with or without ACTH injection) and LPS challenge (with or without LPS injection). Each group consisted of six male piglets. The results showed that our LPS preparation alone induced mRNA expressions of IL-1β, IL-6, TNF-α, IL-10, COX-2, TLR2, TLR4, and GR (P < 0.05). ACTH pretreatment downregulated the TLR2 mRNA and IL-6 protein level induced by our LPS preparation significantly (P < 0.05) by one-way ANOVA analysis. Treatment with LPS alone extremely significantly decreased ssc-miR-338 levels (P < 0.01). Interaction of ACTH × LPS was significant for cNOS level (P = 0.011) and ssc-miR-338 expression (P = 0.04) by two-way ANOVA analysis. The LPS treatment significantly downregulated cNOS levels (P < 0.01), which was significantly attenuated by ACTH pretreatment (P < 0.05). Lipopolysaccharide alone did not affect ssc-miR-146b expression levels compared to that in the vehicle group. However, ACTH pretreatment in combination with LPS significantly increased this micro-RNA expression (P < 0.05). TLRs 1–10 were all expressed in adrenal tissue. The LPS challenge alone induced remarkable compensatory mitochondrial damages at the ultrastructural level, which was alleviated by ACTH pretreatment. Accordingly, ACTH pretreatment was able to block LPS-induced secretion of local adrenal cortisol (P < 0.05). Taken together, our results demonstrate that ACTH pretreatment seems to attenuate LPS-induced mitochondria damage and inflammation that decreased cNOS activity in the adrenal gland and ultimately returned local adrenal cortisol to basal levels at 6 h post LPS injection.

Construction of Polymeric Antigenic Diagnosticum Based on &lt;i&gt;Vibrio cholera&lt;/i&gt; О1 Lipopolysaccharide

Representatives of the genus Vibrio cholerae differ in the structure of lipopolysaccharide, in particular, its O-polysaccharide chains (O-antigen), which determines the serological specificity of vibrios. Currently, the water-phenolic method is used to obtain the lipopolysaccharide preparation. However, this technique relates to harsh chemical methods, leads to a change in original molecular organization of biopolymer, violating its structure and biological properties. Modern technologies in the development of diagnostic kits for the immunosuspension reaction of volume agglomeration allow for obtaining synthetic carriers with different reaction groups on the particle surface capable to bind antigens/antibodies. The aim of this study was to construct cholera antigenic polymeric diagnostic kit based on the lipopolysaccharide of Vibrio cholerae O1 serogroup. Materials and methods . The lipopolysaccharide was used as a sensitizer obtained through the author's modification of enzymatic purification from the cell membranes of Vibrio cholerae using ultrasonic disintegration. Results and discussion . The resulting sensitin contains small impurities of protein (1.5 %) and nucleic acids (0.1 %). Diagnosticum is characterized by high analytical sensitivity in agglomeration reaction with commercial and experimental rabbit serum to Vibrio cholerae O1 serogroup (1:640 - 1:5120) and analytical specificity (the diagnosticum does not interact with heterologous sera, with serums to pathogens of acute intestinal infections, as well as with sera from healthy donors). A polymeric antigenic cholera diagnosticum designed to detect antibodies to lipopolysaccharide of Vibrio cholerae in the blood serum of patients who were ill, suspected of the disease or vaccinated people has been constructed.

Preparation and characterization of lipopolysaccharide (LPS) monolayers for investigating their role in bacterial adhesion

This study was conducted to determine the role played by the length variations and heterogeneous spatial distribution of lipopolysaccharide (LPS) molecules, in gram-negative cells, in mediating adhesion to surfaces. We used extracted LPS molecules from four representative bacterial strains, prepared single molecule thick LPS monolayers using Langmuir-Blodgettry on hydrophobic substrates and used them as model surfaces for AFM studies using nano probes. The prepared LPS monolayers were uniformly distributed over the substrates and enabled preliminary force investigations. Such model monolayers are better alternative to whole-cell analysis and are expected to enable the formulation of an interfacial model for bacterial adhesion.

Effect of ultrapure lipopolysaccharides derived from diverse bacterial species on the modulation of platelet activation

Platelets are small circulating blood cells that play essential roles in the maintenance of haemostasis via blood clotting. However, they also play critical roles in the regulation of innate immune responses. Inflammatory receptors, specifically Toll-like receptor (TLR)-4, have been reported to modify platelet reactivity. A plethora of studies have reported controversial functions of TLR4 in the modulation of platelet function using various chemotypes and preparations of its ligand, lipopolysaccharide (LPS). The method of preparation of LPS may explain these discrepancies however this is not fully understood. Hence, to determine the impact of LPS on platelet activation, we used ultrapure preparations of LPS from Escherichia coli (LPSEC), Salmonella minnesota (LPSSM), and Rhodobacter sphaeroides (LPSRS) and examined their actions under diverse experimental conditions in human platelets. LPSEC did not affect platelet activation markers such as inside-out signalling to integrin αIIbβ3 or P-selectin exposure upon agonist-induced activation in platelet-rich plasma or whole blood whereas LPSSM and LPSRS inhibited platelet activation under specific conditions at supraphysiological concentrations. Overall, our data demonstrate that platelet activation is not largely influenced by any of the ultrapure LPS chemotypes used in this study on their own except under certain conditions.

Substrate structure-activity relationship reveals a limited lipopolysaccharide chemotype range for intestinal alkaline phosphatase

Lipopolysaccharide (LPS) from the Gram-negative bacterial outer membrane potently activates the human innate immune system. LPS is recognized by the Toll-like receptor 4/myeloid differentiation factor-2 (TLR4/MD2) complex, leading to the release of pro-inflammatory cytokines. Alkaline phosphatase (AP) is currently being investigated as an anti-inflammatory agent for detoxifying LPS through dephosphorylating lipid A, thus providing a potential treatment for managing both acute (sepsis) and chronic (metabolic endotoxemia) pathologies wherein aberrant TLR4/MD2 activation has been implicated. Endogenous LPS preparations are chemically heterogeneous, and little is known regarding the LPS chemotype substrate range of AP. Here, we investigated the activity of AP on a panel of structurally defined LPS chemotypes isolated from Escherichia coli and demonstrate that calf intestinal AP (cIAP) has only minimal activity against unmodified enteric LPS chemotypes. Pi was only released from a subset of LPS chemotypes harboring spontaneously labile phosphoethanolamine (PEtN) modifications connected through phosphoanhydride bonds. We demonstrate that the spontaneously hydrolyzed O-phosphorylethanolamine is the actual substrate for AP. We found that the 1- and 4'-lipid A phosphate groups critical in TLR4/MD2 signaling become susceptible to hydrolysis only after de-O-acylation of ester linked primary acyl chains on lipid A. Furthermore, PEtN modifications on lipid A specifically enhanced hTLR4 agonist activity of underacylated LPS preparations. Computational binding models are proposed to explain the limitation of AP substrate specificity imposed by the acylation state of lipid A, and the mechanism of PEtN in enhancing hTLR4/MD2 signaling.

Polysaccharide Chain Length of Lipopolysaccharides From Salmonella Minnesota Is a Determinant of Aggregate Stability, Plasma Residence Time and Proinflammatory Propensity in vivo.

Lipopolysaccharides (LPS) originate from the outer membrane of Gram-negative bacteria and trigger an inflammatory response via the innate immune system. LPS consist of a lipid A moiety directly responsible for the stimulation of the proinflammatory cascade and a polysaccharide chain of variable length. LPS form aggregates of variable size and structure in aqueous media, and the aggregation/disaggregation propensity of LPS is known as a key determinant of their biological activity. The aim of the present study was to determine to which extent the length of the polysaccharide chain can affect the nature of LPS structures, their pharmacokinetics, and eventually their proinflammatory properties in vivo. LPS variants of Salmonella Minnesota with identical lipid A but with different polysaccharide moieties were used. The physical properties of LPS aggregates were analyzed by zetametry, dynamic light scattering, and microscopy. The stability of LPS aggregates was tested in the presence of plasma, whole blood, and cultured cell lines. LPS pharmacokinetics was performed in wild-type mice. The accumulation in plasma of rough LPS (R-LPS) with a short polysaccharidic chain was lower, and its hepatic uptake was faster as compared to smooth LPS (S-LPS) with a long polysaccharidic chain. The inflammatory response was weaker with R-LPS than with S-LPS. As compared to S-LPS, R-LPS formed larger aggregates, with a higher hydrophobicity index, a more negative zeta potential, and a higher critical aggregation concentration. The lower stability of R-LPS aggregates could be illustrated in vitro by a higher extent of association of LPS to plasma lipoproteins, faster binding to blood cells, and increased uptake by macrophages and hepatocytes, compared to S-LPS. Our data indicate that a long polysaccharide chain is associated with the formation of more stable aggregates with extended residence time in plasma and higher inflammatory potential. These results show that polysaccharide chain length, and overall aggregability of LPS might be helpful to predict the proinflammatory effect that can be expected in experimental settings using LPS preparations. In addition, better knowledge and control of LPS aggregation and disaggregation might lead to new strategies to enhance LPS detoxification in septic patients.

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation.

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

LptG contributes to changes in membrane permeability and the emergence of multidrug hypersusceptibility in a cystic fibrosis isolate of Pseudomonas aeruginosa.

PurposeIn the lungs of cystic fibrosis patients, Pseudomonas aeruginosa is exposed to a myriad of antibiotics leading to alterations in antibiotic susceptibility. This study identifies mutations resulting in hypersusceptibility in isogenic mutants of a P. aeruginosa clinical isolate, PA34.MethodsPA34 was exposed to subinhibitory concentrations of doripenem or meropenem during growth to mid‐log phase. Antibiotic susceptibility of surviving colonies was determined by agar dilution. Two carbapenem‐resistant colonies hypersusceptible to non‐carbapenem antibiotics were selected for further analysis. Antibiotic resistance gene expression was evaluated by RT‐rtPCR and OprD production by SDS‐PAGE. PA34 and isogenic mutants were evaluated with whole genome sequencing. Sequence variants were confirmed by Sanger sequencing, and cognate genes in eight carbapenem‐resistant clinical isolates hypersusceptible to non‐carbapenem antibiotics were sequenced. Lipopolysaccharide preparations of PA34 and hypersusceptible mutants were evaluated with ProQ‐Emerald stain.ResultsIsogenic mutants showed 4‐ to 8‐fold MIC increase for imipenem, meropenem, and doripenem. However, they were hypersusceptible (≥4‐fold MIC decrease) to aminoglycosides, fluoroquinolones, and non‐carbapenem β‐lactams. Expression of ampC or mex‐opr efflux pumps was unchanged, but OprD production was decreased. Mutations causing Q86H AlgU and G77C LptG amino acid substitutions and nonsense mutations within OprD were observed in both mutants. Lipopolysaccharide modifications were observed between isogenic mutants and PA34. Non‐synonymous mutations in LptF or LptG were observed in 6/8 hypersusceptible clinical isolates resistant to carbapenem antibiotics.ConclusionEvaluation of hypersusceptible mutants identified the association between lptG and a hypersusceptible phenotype. Modifications in lipopolysaccharide profiles suggests LptG modification interferes with lipopolysaccharide transport and contributes to hypersusceptibility.

In Vitro Studies of Lipopolysaccharide-Mediated DNA Release of Podovirus HK620.

Gram-negative bacteria protect themselves with an outermost layer containing lipopolysaccharide (LPS). O-antigen-specific bacteriophages use tailspike proteins (TSP) to recognize and cleave the O-polysaccharide part of LPS. However, O-antigen composition and structure can be highly variable depending on the environmental conditions. It is important to understand how these changes may influence the early steps of the bacteriophage infection cycle because they can be linked to changes in host range or the occurrence of phage resistance. In this work, we have analyzed how LPS preparations in vitro trigger particle opening and DNA ejection from the E. coli podovirus HK620. Fluorescence-based monitoring of DNA release showed that HK620 phage particles in vitro ejected their genome at velocities comparable to those found for other podoviruses. Moreover, we found that HK620 irreversibly adsorbed to the LPS receptor via its TSP at restrictive low temperatures, without opening the particle but could eject its DNA at permissive temperatures. DNA ejection was solely stimulated by LPS, however, the composition of the O-antigen dictated whether the LPS receptor could start the DNA release from E. coli phage HK620 in vitro. This finding can be significant when optimizing bacteriophage mixtures for therapy, where in natural environments O-antigen structures may rapidly change.

Single-Cell Force Spectroscopy of Interaction of Lipopolysaccharides from Yersinia pseudotuberculosis and Yersinia pestis with J774 Macrophage Membrane Using Optical Tweezers

In order to investigate quantitatively the role of lipopolysaccharides (LPS) from outer bacterial membrane at the initial state of bacterium adhesion to a host cell membrane, a model system for single cell force spectroscopy was developed and used. The system comprised of an LPS-coated microsphere placed into optical trap and a J774 macrophage being approached the microsphere to initiate their binding and then moved back to rupture the bond. An “object shadow” phenomenon was discovered, manifested as large-scale variations of the signal of photodetector registering the trapped microsphere displacement, such variations emerging long before the actual interaction between the macrophage and microsphere. The theory and the measurements technique were developed for registration of the force required for detachment of bounded microsphere from the object investigated by means of optical tweezers under the “object shadow” conditions. Characteristic spectra of binding force between J774 macrophage and microspheres functionalized with various LPS, as well as LPS plus complementary antibodies preparations were obtained at the rate of detachment force application of 3–6 pN/s. Force spectrum characteristic of Yersinia pseudotuberculosis LPS possessing O-antigen had a maximum at ~14 pN with half-width of ~23 pN. The treatment of O-antigen with complementary antibodies resulted in transformation of this spectrum into a spectrum with maximum at ~10 pN and half-width of ~14 pN, being almost identical to the spectrum of Y. pestis LPS devoid of O-antigen, with a maximum at ~9 pN and half-width of ~13 pN. A possible mechanism of force spectra formation has been proposed under assumptions of nonspecific binding of O-antigen and probable receptor-type binding of LPS core region to the macrophage surface. The elastic modulus of macrophage envelope, as estimated using analysis of displacement of the contacting microsphere as an indenter, was ≈0.17 pN/nm.

Structural analysis of the O-polysaccharide from the lipopolysaccharide of Pseudomonas putida BIM B-1100

Two specific polysaccharides, together with an →4)-α-d-Glcp-(1→ glucan (bacterial glycogen), were obtained from a lipopolysaccharide preparation isolated from the bacterium Pseudomonas putida BIM B-1100 by phenol/water extraction. The following structures of the polysaccharides were established by composition analysis, Smith degradation, ESI-MS, and 1D and 2D NMR spectroscopy: [Display omitted]