Virulence Factors Of Gram-negative Bacteria Research Articles

Green synthesis of silver nanoparticles using Moringa oleifera and its efficacy against gram-negative bacteria targeting quorum sensing and biofilms

AbstractGram-negative pathogenic bacteria are the leading cause of high morbidity and mortality in humans globally. The majority of such pathogens has gained the status of multidrug resistance and requires the development of new anti-pathogenic agent. Moringa oleifera is a widely distributed and quickly proliferating medicinal plant, making it an easy source for extracting bioactive components. It is rich in bioactive compounds which can act as stabilizing and reducing agents during the synthesis of silver nanoparticles. It is well-recognized for its several medical benefits, including antibacterial action. As a result, it is an excellent option for synthesizing AgNPs with improved antibacterial characteristics. Considering the above properties, less explored Moringa oleifera was used for the green synthesis of nanoparticles. Targeting biofilms and virulence factors of Gram-negative bacteria by green synthesized metal nanoparticles is an alternative approach to combat antimicrobial resistance. Silver nanoparticles (MO-AgNPs) synthesized using Moringa oleifera leaf (MOL) extract were characterized using Ultraviolet–visible spectroscopy (UV–vis spectroscopy), Fourier-transform infrared spectroscopy (FTIR), Scanning electron microscopy-energy dispersive X-ray analysis (SEM–EDX), Transmission Electron Microscopy (TEM), and X-Ray Diffraction analysis (XRD). The absorption spectra of silver nanoparticles showed a band of absorption near 440 nm, associated with spherical silver nanoparticles. At sub-minimum inhibitory concentrations (sub-MICs), the MO-AgNPs inhibit the Quorum Sensing-mediated virulence factors and biofilm formation against three Gram-negative bacteria (Escherichia fergusonii, Serratia marcescens, and Chromobacterium violaceum). QS-mediated virulence factors in test bacteria were reduced by 80.67% (violacein), prodigiosin production (77.45%), exoprotease activity (76.02%), and swarming motility (86.5%). MO-AgNPs also demonstrated broad-spectrum antibiofilm activity against test bacteria, ranging from 77.95 to 82.4% inhibition. Microscopic analysis of biofilms showed significant structural change and inhibition. Our results demonstrate appreciable in vitro activity of MO-AgNPs against the selected pathogens, which could be used as an alternative therapeutic agent for treating infection caused by drug-resistant bacteria and preventing biofilm development by bacteria on medical devices and other surfaces. Graphical abstract

Virulence factors of Gram-negative bacteria from free-ranging Amazon river dolphins (Inia geoffrensis).

Freshwater cetaceans play a significant role as sentinel animals, providing important data on animal species and aquatic ecosystem health. They also may serve as potential reservoirs of emerging pathogens and host virulence genes in their microbiota. In this study, we evaluated virulence factors produced by Gram-negative bacteria recovered from individuals belonging to two populations of free-ranging Amazon river dolphins (Inia geoffrensis). A total of 132 isolates recovered from the oral cavity, blowhole, genital opening and rectum of 21 river dolphins, 13 from Negro River and 8 from Tapajós River, Brazil, were evaluated for the production of virulence factors, such as biofilms and exoproducts (proteases, hemolysins and siderophores), in planktonic and biofilm forms. In planktonic form, 81.1% (107/132) of the tested bacteria of free-ranging Amazon river dolphins were able to produce virulence factors, with 44/132 (33.4%), 65/132 (49,2%) and 54/132 (40,9%) positive for protease, hemolysin and siderophore production, respectively. Overall, 57/132 (43.2%) of the isolates produced biofilms and, under this form of growth, 66/132 (50%), 88/132 (66.7%) and 80/132 (60.6%) of the isolates were positive for protease, hemolysin and siderophore production. In general, the isolates showed a higher release of exoproducts in biofilm than in planktonic form (P < 0.001). The present findings show that Amazon river dolphins harbor potentially pathogenic bacteria in their microbiota, highlighting the importance of monitoring the micro-organisms from wild animals, as they may emerge as pathogens for humans and other animals.

Modeling and Simulation of Bacterial Outer Membranes with Lipopolysaccharides and Capsular Polysaccharides.

Capsule is one of the common virulence factors in Gram-negative bacteria protecting pathogens from host defenses and consists of long-chain capsular polysaccharides (CPS) anchored in the outer membrane (OM). Elucidating structural properties of CPS is important to understand its biological functions as well as the OM properties. However, the outer leaflet of the OM in current simulation studies is represented exclusively by LPS due to the complexity and diversity of CPS. In this work, representative Escherichia coli CPS, KLPS (a lipid A-linked form) and KPG (a phosphatidylglycerol-linked form), are modeled and incorporated into various symmetric bilayers with co-existing LPS in different ratios. All-atom molecular dynamics simulations of these systems have been conducted to characterize various bilayer properties. Incorporation of KLPS makes the acyl chains of LPS more rigid and ordered, while incorporation of KPG makes them less ordered and flexible. These results are consistent with the calculated area per lipid (APL) of LPS, in which the APL of LPS becomes smaller when KLPS is incorporated, whereas it gets larger when KPG is included. Torsional analysis reveals that the influence of the CPS presence on the conformational distributions of the glycosidic linkages of LPS is small, and minor differences are also detected for the inner and outer regions of the CPS. Combined with previously modeled enterobacterial common antigens (ECAs) in the form of mixed bilayers, this work provides more realistic OM models as well as the basis for characterization of interactions between the OM and OM proteins.

LPS-Induced Liver Injury of Magang Geese through Toll-like Receptor and MAPK Signaling Pathway.

Lipopolysaccharide (LPS) is one of the main virulence factors of Gram-negative bacteria. In the process of waterfowl breeding, an inflammatory reaction due to LPS infection is easily produced, which leads to a decline in waterfowl performance. The liver plays a vital role in the immune response and the removal of toxic components. Therefore, it is necessary to study the mechanism of liver injury induced by LPS in goose. In this study, a total of 100 1-day-old goslings were randomly divided into a control group and LPS group after 3 days of pre-feeding. On days 21, 23, and 25 of the formal experiment, the control group was intraperitoneally injected with 0.5 mL normal saline, and the LPS group was intraperitoneally injected with LPS 2 mg/(kg body weight) once a day. On day 25 of the experiment, liver samples were collected 3 h after the injection of saline and LPS. The results of histopathology and biochemical indexes showed that the livers of the LPS group had liver morphological structure destruction and inflammatory cell infiltration, and the levels of ALT and AST were increased. Next, RNA sequencing analysis was used to determine the abundances and characteristics of the transcripts, as well as the associated somatic mutations and alternative splicing. We screened 727 differentially expressed genes (DEGs) with p < 0.05 and |log2(Fold Change)| ≥ 1, as the thresholds; GO and KEGG enrichment analysis showed that LPS-induced liver injury may be involved in the Toll-like receptor signaling pathway, MAPK signaling pathway, NOD-like receptor signaling pathway, FoxO, and PPAR signaling pathway. Finally, we intersected the genes enriched in the key pathway of LPS-induced liver injury with the top 50 key genes in protein−protein interaction networks to obtain 28 more critical genes. Among them, 17 genes were enriched in Toll-like signaling pathway and MAPK signaling pathway. Therefore, these results suggest that LPS-induced liver injury in geese may be the result of the joint action of Toll-like receptor, MAPK, NOD-like receptor, FoxO, and PPAR signaling pathway. Among them, the TLR7-mediated MAPK signaling pathway plays a major role.

Resveratrol ameliorates the defects of meiotic maturation in lipopolysaccharide exposed porcine oocytes

Lipopolysaccharide (LPS), a significant virulence factor of gram-negative bacteria, adversely affects female reproduction, especially the maturation and early embryonic development of oocytes, through inducing of inflammatory and oxidative stress-associated toxic responses. Resveratrol (Res), a potent antioxidant, exhibits many beneficial effects on the maturation and developmental competence of oocytes. However, it is unclear whether Res can restore LPS-induced defects in the maturation of oocytes during meiosis. In this study, we used porcine oocytes to explore the protective effects of Res and its underlying mechanism against the toxic impacts of LPS exposure on meiotic maturation and developmental competence of oocytes during meiosis. The oocytes were randomly assigned to a control, LPS-exposed or Res-supplemented group. Nuclear and cytoplasmic maturation was assessed after 26 h (MI) or 44 h (MII) of in vitro maturation (IVM). Our results showed that 10 µM Res significantly improved the rates of oocyte maturation and blastocyst formation after exposure to 15 µg/mL LPS. In addition, Res preserved the normal spindle/chromosome structure and maintained acetylated tubulin levels, actin polymerization and cortical granules (CGs) distribution. Additionally, Res protected mitochondrial content and function, scavenges reactive oxygen species (ROS), and reduced DNA damage and apoptosis in LPS-exposed oocytes. Furthermore, inhibition of SIRT1 by its specific inhibitor EX527 suppressed the recovery of ROS levels, mitochondrial content, and spindle/chromosome structure by Res supplementation. In summary, this study shows that Res can alleviate the impacts of LPS-induced toxicity on meiosis in porcine oocytes by upregulating SIRT1, which ameliorates oxidative stress and increasing mitochondrial content.

Allicin protects porcine oocytes against LPS-induced defects during maturation in vitro

Lipopolysaccharide (LPS), the main virulence factor of gram-negative bacteria, severely impairs the function of the female reproductive system. It has especially harmful effects on oocytes and subsequent embryonic development. The use of active plant substances to ameliorate the damage caused by LPS exposure is a strategy worthy of attention. In this study, porcine oocytes were used to investigate the protective effects and underlying mechanisms of allicin, an extract derived from garlic, on LPS-exposed oocytes in vitro. Our data indicated that supplementation with 1 μM allicin significantly attenuated the LPS-mediated reductions in the first polar body extrusion rate and the subsequent blastocyst formation rate. Allicin also mitigated the abnormalities in spindle assembly, chromosome alignment, actin polymerization, and cortical granule distribution caused by LPS exposure. Furthermore, allicin restored reactive oxygen species (ROS), early apoptosis and autophagy to normal physiological levels in LPS-exposed oocytes. In conclusion, our findings confirm that allicin can protect oocytes against LPS-induced damage. The results of this study will help promote the application of plant-derived bioactive substances to ameliorate oocyte maturation defects.

Selenium and Taurine Combination Is Better Than Alone in Protecting Lipopolysaccharide-Induced Mammary Inflammatory Lesions via Activating PI3K/Akt/mTOR Signaling Pathway by Scavenging Intracellular ROS.

Mastitis is mainly induced by gram-negative bacterial infections, causing devastating economic losses to the global cattle industry. Both selenium (Se) and taurine (Tau) exhibit multiple biological effects, including reducing inflammation. However, no studies have reported the protective effect of the combined use of Se and Tau against mastitis, and the underlying mechanisms remain unclear. In this study, lipopolysaccharide (LPS), the vital virulence factor of gram-negative bacteria, was used to construct the in vivo and vitro mastitis models. The results of in vivo model showed that Se and Tau combination was more effective than either substance alone in reducing tissue hyperemia, edema, and neutrophil infiltration in the mammary acinar cavity, improving the blood-milk barrier in LPS-induced mice mastitis, and decreasing the expression of proinflammatory factors and the activity of MPO. Moreover, Se and Tau combination significantly increased the levels of LPS-induced reduction in PI3K/Akt/mTOR, but the expressions of TLRs and NLRP3 were not significantly changed in the mammary tissue. In the in vitro experiments, the effects of Se and Tau combination or alone on inflammatory factors, inflammatory mediators, MPO activity, and blood-milk barrier were consistent with those in vivo. The Se and Tau combination has also been found to increase the survival rate of BMECs compared with each substance alone via promoting cellular proliferation and inhibiting apoptosis. Also, it has been confirmed that this combination could restore the LPS-induced inhibition in the PI3K/Akt/mTOR signaling pathway. Inhibition of mTOR by Rapamycin counteracted the combined protection of SeMet and Tau against LPS-induced inflammatory damage, the inhibition of PI3K by LY294002 blocked the activation of mTOR, and the accumulation of ROS by the ROS agonist blocked the activation of PI3K. In conclusion, these findings suggested that Se and Tau combination was better than either substance alone in protecting LPS-induced mammary inflammatory lesions by upregulating the PI3K/Akt/mTOR signaling pathway.

Plumbagin inhibits quorum sensing-regulated virulence and biofilms of Gram-negative bacteria: in vitro and in silico investigations

The global rise in antimicrobial resistance and lack of discovery of new antimicrobials have created serious concerns. Targeting quorum sensing (QS) and biofilms of pathogenic bacteria is considered a promising approach in antimicrobial drug discovery. This study explored the inhibitory effect of plumbagin against biofilms and QS of Chromobacterium violaceum, Serratia marcescens and Pseudomonas aeruginosa. Violacein production in C. violaceum 12472 was reduced by >80%. The virulent traits of P. aeruginosa PAO1 such as pyocyanin, rhamnolipid and proteases were also inhibited at sub-minimum inhibitory concentrations. Moreover, the biofilms of the test bacteria were reduced by 56–70%. Plumbagin reduced the bacterial adherence and colonization on solid surface. Computational studies gave closer insights regarding the possible modes of action. Molecular dynamics simulations revealed that the protein complexes were quite stable under physiological conditions. This study provides both experimental and computational evidence regarding the efficacy of plumbagin against biofilms and the QS-controlled virulence factors of Gram-negative bacteria.

Clonal Clusters, Molecular Resistance Mechanisms and Virulence Factors of Gram-Negative Bacteria Isolated from Chronic Wounds in Ghana.

Wound infections are common medical problems in sub-Saharan Africa but data on the molecular epidemiology are rare. Within this study we assessed the clonal lineages, resistance genes and virulence factors of Gram-negative bacteria isolated from Ghanaian patients with chronic wounds. From a previous study, 49 Pseudomonas aeruginosa, 21 Klebsiella pneumoniae complex members and 12 Escherichia coli were subjected to whole genome sequencing. Sequence analysis indicated high clonal diversity with only nine P. aeruginosa clusters comprising two strains each and one E. coli cluster comprising three strains with high phylogenetic relationship suggesting nosocomial transmission. Acquired beta-lactamase genes were observed in some isolates next to a broad spectrum of additional genetic resistance determinants. Phenotypical expression of extended-spectrum beta-lactamase activity in the Enterobacterales was associated with blaCTX-M-15 genes, which are frequent in Ghana. Frequently recorded virulence genes comprised genes related to invasion and iron-uptake in E. coli, genes related to adherence, iron-uptake, secretion systems and antiphagocytosis in P. aeruginosa and genes related to adherence, biofilm formation, immune evasion, iron-uptake and secretion systems in K. pneumonia complex. In summary, the study provides a piece in the puzzle of the molecular epidemiology of Gram-negative bacteria in chronic wounds in rural Ghana.

Impact of Polymyxin Resistance on Virulence and Fitness among Clinically Important Gram-Negative Bacteria

Humanity is facing an enormous and growing worldwide threat from the emergence of multi-drug-resistant (MDR) Gram-negative bacteria such as Escherichia coli , Klebsiella pneumoniae , and Acinetobacter baumannii . Polymyxin B and E (colistin) constitute the last-line therapies for treating MDR Gram-negative bacteria. Polymyxin is a cationic antibacterial peptide that can destroy the outer membrane of Gram-negative bacteria. With the increasing clinical application of polymyxin, however, there have been many reports of the occurrence of polymyxin-resistant Gram-negative bacteria. This resistance is mainly mediated by the modification or complete loss of lipopolysaccharide (LPS). LPS is also a virulence factor of Gram-negative bacteria, and alterations of LPS may correlate with virulence. Although it is generally believed that the biological costs associated with drug resistance may enable benign susceptible bacteria to overcome resistant bacteria when antibiotic pressure is reduced, some studies have shown that polymyxin-resistant bacteria are associated with higher virulence and greater fitness compared with their susceptible counterparts. To predict the development of polymyxin resistance and evaluate interventions for its mitigation, it is important to understand the relative biological cost of polymyxin resistance compared with susceptibility. The impact of polymyxin resistance mechanisms on the virulence and fitness of these three Gram-negative bacteria are summarized in this review.

Inhibition of autotransporter biogenesis by small molecules.

SummaryDisarming pathogens by targeting virulence factors is a promising alternative to classic antibiotics. Many virulence factors in Gram‐negative bacteria are secreted via the autotransporter (AT) pathway, also known as Type 5 secretion. These factors are secreted with the assistance of two membrane‐based protein complexes: Sec and Bam. To identify inhibitors of the AT pathway, we used transcriptomics analysis to develop a fluorescence‐based high‐throughput assay that reports on the stress induced by the model AT hemoglobin protease (Hbp) when its secretion across the outer membrane is inhibited. Screening a library of 1600 fragments yielded the compound VUF15259 that provokes cell envelope stress and secretion inhibition of the ATs Hbp and Antigen‐43. VUF15259 also impairs β‐barrel folding activity of various outer membrane proteins. Furthermore, we found that mutants that are compromised in outer membrane protein biogenesis are more susceptible to VUF15259. Finally, VUF15259 induces the release of vesicles that appear to assemble in short chains. Taken together, VUF15259 is the first reported compound that inhibits AT secretion and our data are mostly consistent with VUF15259 interfering with the Bam‐complex as potential mode of action. The validation of the presented assay incites its use to screen larger compound libraries with drug‐like compounds.

A Unique Sugar l-Perosamine (4-Amino-4,6-dideoxy-l-mannose) Is a Compound Building Two O-Chain Polysaccharides in the Lipopolysaccharide of Aeromonas hydrophila Strain JCM 3968, Serogroup O6.

Lipopolysaccharide (LPS) is the major glycolipid and virulence factor of Gram-negative bacteria, including Aeromonas spp. The O-specific polysaccharide (O-PS, O-chain, O-antigen), i.e., the surface-exposed part of LPS, which is a hetero- or homopolysaccharide, determines the serospecificity of bacterial strains. Here, chemical analyses, mass spectrometry, and 1H and 13C NMR spectroscopy techniques were employed to study the O-PS of Aeromonas hydrophila strain JCM 3968, serogroup O6. MALDI-TOF mass spectrometry revealed that the LPS of A. hydrophila JCM 3968 has a hexaacylated lipid A with conserved architecture of the backbone and a core oligosaccharide composed of Hep6Hex1HexN1HexNAc1Kdo1P1. To liberate the O-antigen, LPS was subjected to mild acid hydrolysis followed by gel-permeation-chromatography and revealed two O-polysaccharides that were found to contain a unique sugar 4-amino-4,6-dideoxy-l-mannose (N-acetyl-l-perosamine, l-Rhap4NAc), which may further determine the specificity of the serogroup. The first O-polysaccharide (O-PS1) was built up of trisaccharide repeating units composed of one α-d-GalpNAc and two α-l-Rhap4NAc residues, whereas the other one, O-PS2, is an α1→2 linked homopolymer of l-Rhap4NAc. The following structures of the O-polysaccharides were established: O-PS1→3)-α-l-Rhap4NAc-(1→4)-α-d-GalpNAc-(1→3)-α-l-Rhap4NAc-(1→O-PS2→2)-α-l-Rhap4NAc-(1→ The present paper is the first work that reveals the occurrence of perosamine in the l-configuration as a component of bacterial O-chain polysaccharides.

Mutagenesis of DsbAss is Crucial for the Signal Recognition Particle Mechanism in Escherichia coli: Insights from Molecular Dynamics Simulations.

The disulfide bond signal sequence (DsbAss) protein is characterized as an important virulence factor in gram-negative bacteria. This study aimed to analyze the “alanine” alteration in the hydrophobic (H) region of DsbAss and to understand the conformational DsbAss alteration(s) inside the fifty-four homolog (Ffh)-binding groove which were revealed to be crucial for translocation of ovine growth hormone (OGH) to the periplasmic space in Escherichia coli via the secretory (Sec) pathway. An experimental design was used to explore the hydrophobicity and alteration of alanine (Ala) to isoleucine (Ile) in the tripartite structure of DsbAss. As a result, two DsbAss mutants (Ala at positions -11 and -13) with same hydrophobicity of 1.539 led to the conflicting translocation of the active OGH gene. We performed molecular dynamics (MD) simulations and molecular mechanics generalized born surface area (MM-GBSA) binding free energy calculations to examine the interaction energetic and dynamic aspects of DsbAss/signal repetition particle 54 (SRP54) binding, which has a principle role in Escherichia coli Sec pathways. Although both DsbAss mutants retained helicity, the MD simulation analysis evidenced that altering Ala-13 changed the orientation of the signal peptide in the Ffh M binding domain groove, favored more stable interaction energies (MM-GBSA ΔGtotal = −140.62 kcal mol−1), and hampered the process of OGH translocation, while Ala-11 pointed outward due to unstable conformation and less binding energy (ΔGtotal = −124.24 kcal mol−1). Here we report the dynamic behavior of change of “alanine” in the H-domain of DsbAss which affects the process of translocation of OGH, where MD simulation and MM-GBSA can be useful initial tools to investigate the virulence of bacteria.

Investigating the nuclear localisation and proteolytic activity of the meningococcal App and MspA autotransporters

Autotransporter proteins are major secreted virulence factors of Gram-negative bacteria. They are translocated across the inner membrane via the Sec machinery and the outer membrane via the Bam complex and a series of periplasmic chaperones, respectively. The passenger domain may then be proteolytically cleaved and released into the external milieu. The meningococcal autotransporters Adhesion and penetration protein (App) and Meningococcal serine protease A (MspA) are secreted S6-peptidase family autotransporters. Our previous work has shown that FITC-labelled recombinant App or MspA can be taken up by host cells and translocated into the nucleus. App and MspA can also bind to, and cleave recombinant host histones. We furthered investigate the ability of App and MspA (and their inactive derivatives) to cleave recombinant and host-derived histones. Our data demonstrate proteolytic activity of App and MspA on recombinant H3 and Hep-2 cell-derived H3 (which may undergo post-translational modifications that are not applied to the recombinant protein); no cleavage was observed when the histone proteins were treated with proteolytically inactive mutants of the autotransporter proteins. We have also further investigated the nuclear localisation of App and MspA by deleting areas of interest within the meningococcal autotransporters and assessing the impact on nuclear localisation in order to identify the autotransporter motifs required to direct App and MspA to the nuclear compartment. In summary, our results confirm that App and MspA can reach the nuclear compartment of the host cell and clip host-derived histone H3.

Gingival Epithelial Cell Recognition of Lipopolysaccharide.

Gingival epithelium plays a pivotal role in protecting the underlying periodontium from the microbial colonization found in the gingival sulcus. Having an appropriate phenotype displayed by gingival epithelial cells is a critical host component required for protection against bacterial invasion into gingival tissues. In the present study, gingival epithelial homeostasis associated with the CXCL-8/IL-8 chemokine response was investigated in vitro to determine the mechanisms that gingival epithelial cells utilize for sensing gram-positive and gram-negative microorganisms. The findings of this study have demonstrated, by using Fusobacterium nucleatum, a heterogeneity of gingival epithelial cell response by Toll-like receptor (TLR) 2, a lipoprotein sensor. Notably, however, lipopolysaccharide (LPS), a major virulence factor of gram-negative bacteria, is not recognized by gingival epithelial cells unless the LPS is internalized into the cells. Activation of TLR4 in gingival epithelial cells occurs in the endosome, an intracellular event that requires a vesicular acidification to turn on TLR4 signaling, indicating their stringency for fine-tuning a local LPS response. This study has identified a unique LPS sensing mechanism of the oral epithelium to overcome a periodontal infection associated with LPS derived from gram-negative microbes that arises during dysbiosis.

Investigation into Cellular Glycolysis for the Mechanism Study of Energy Metabolism Disorder Triggered by Lipopolysaccharide.

Lipopolysaccharide (LPS) is the main virulence factor of Gram-negative bacteria, which can incite inflammation in tissues by inducing cells to secrete a variety of proinflammatory mediators, including cytokines, chemokines, interleukins, and prostaglandins. Herein, we chose LPS as an inducer to establish an inflammatory model of HeLa cells, and explored the effects of LPS on energy metabolism. We treated HeLa cells with different concentrations (0, 0.4, 1.0, 2.0, 4.0, and 6.0 μg/mL) of LPS for 24 h, and explored its effects on intercellular adenosine triphosphate (ATP) levels, intercellular nitrous oxide (NO) content, mitochondrial functions, and enzyme activities related to energy metabolism. Furthermore, we used metabonomics to study the metabolites that participated in energy metabolism. We found a positive correlation between LPS concentrations and intracellular ATP levels. In addition, LPS increased intracellular NO production, altered mitochondrial functions, strengthened glycolytic enzyme activities, and changed metabolites related to energy metabolism. Hence, in this study, we showed that LPS can strengthen energy metabolism by enhancing glycolysis, which could be used as an early diagnostic biomarker or a novel therapeutic target for inflammation-associated cancers.

Effects of Christensenella minuta lipopolysaccharide on RAW 264.7 macrophages activation

Christensenella minuta (C. minuta) is a gram-negative gastrointestinal bacterium associated with weight loss. However, recent studies have shown that C. minuta might be a potential pathogen and thus limited its application in the control of obesity. Research into the genetic characteristics and pathogenicity of C. minuta remain elusive. As a major virulence factor of gram-negative bacteria, lipopolysaccharide (LPS) can induce various diseases. In this study, we report the complete genome sequence of C. minuta and have also identified some genes related to LPS biosynthesis. The structure of C. minuta LPS, detected by SDS-PAGE, was different from that of Escherichia coli (E. coli) LPS. The incubation of RAW 264.7 macrophages with C. minuta LPS resulted in lower levels of cellular proliferation, phagocytosis and nuclear factor-kappa B (NF-κB) activation as compared to incubation with E. coli LPS. Furthermore, the expression of pro-inflammatory cytokines, as well as nitric oxide and reactive oxygen species production, was induced in C. minuta LPS-treated cells but to a much lower extent than that by E. coli LPS. These findings show that C. minuta LPS acts as a weak agonist for RAW 264.7 macrophages and can only trigger a weak inflammatory response through the NF-κB signalling pathway. In conclusion, these results suggest that the toxicity of C. minuta LPS is significantly attenuated due to its atypical structure and weak agonist activity for RAW 264.7 macrophages.

Biosynthesis of lipopolysaccharides with different length of the O-specific region as a virulence factor of Gram-negative bacteria

The outer membrane of Gram-negative bacteria is a biological structure with a unique composition that significantly contributes to the survival of bacteria in the unfavorable conditions of the host organism. The lipopolysaccharide constitutes about 70% of the external part of the outer membrane. The LPS molecule is composed of three different parts: lipid A, core oligosaccharide and O antigen. Despite the O-specific antigen being one of the most intensely studied surface structures of bacterial polysaccharides, a number of questions regarding the mechanism of the O antigen biosynthesis and its transport to the cell surface are still unanswered. The paper describes the biosynthesis of the lipopolysaccharide molecule, with particular emphasis on the O-specific chain biosynthesis, the mechanism of lipopolysaccharide length regulation and the influence of the type of synthesized O-specific chains on bacterial survival in adverse host organisms.

Interaction of Mannose-Binding Lectin With Lipopolysaccharide Outer Core Region and Its Biological Consequences.

Lipopolysaccharide (LPS, endotoxin), the main surface antigen and virulence factor of Gram-negative bacteria, is composed of lipid A, core oligosaccharide, and O-specific polysaccharide (O-PS) regions. Each LPS region is capable of complement activation. We have demonstrated that LPS of Hafnia alvei, an opportunistic human pathogen, reacts strongly with human and murine mannose-binding lectins (MBLs). Moreover, MBL–LPS interactions were detected for the majority of other Gram-negative species investigated. H. alvei was used as a model pathogen to investigate the biological consequences of these interactions. The core oligosaccharide region of H. alvei LPS was identified as the main target for human and murine MBL, especially l-glycero-d-manno-heptose (Hep) and N-acetyl-d-glucosamine (GlcNAc) residues within the outer core region. MBL-binding motifs of LPS are accessible to MBL on the surface of bacterial cells and LPS aggregates. Generally, the accessibility of outer core structures for interaction with MBL is highest during the lag phase of bacterial growth. The LPS core oligosaccharide–MBL interactions led to complement activation and also induced an anaphylactoid shock in mice. Unlike Klebsiella pneumoniae O3 LPS, robust lectin pathway activation of H. alvei LPS in vivo was mainly the result of outer core recognition by MBL; involvement of the O-PS is not necessary for anaphylactoid shock induction. Our results contribute to a better understanding of MBL–LPS interaction and may support development of therapeutic strategies against sepsis based on complement inhibition.

Effects of Pasteurella multocida lipopolysaccharides on bovine leukocytes

Lipopolysaccharide (LPS) is a major virulence factor of Gram-negative bacteria playing a major role in stimulating protective immune response in mammalian host. However, in many gram-negative bacterial infections, LPS also elicits immunopathology by inducing excessive inflammatory changes. P. multocida (Pm), a gram-negative bacterium, causes acute lung inflammation and fatal septicemic disease in animals. However, the effects of Pm LPS on host cells are little known. In this study, LPS isolated from three different serotypes (B:2, A:1 and A:3) of Pm were individually tested in vitro to assess the response of bovine leukocytes. Pm LPS induced cell proliferation and cell death of leukocytes, in a dose- and time-dependent manner. In these cells, mitochondrial dysfunction and caspase activation mediate cell death.