Characterization of a waaF mutant of Helicobacter pylori strain 26695 provides evidence that an extended lipopolysaccharide structure has a limited role in the invasion of gastric cancer cells

2007 ◽  
Vol 85 (5) ◽  
pp. 582-590 ◽  
Author(s):  
Vandana Chandan ◽  
Susan M. Logan ◽  
Blair A. Harrison ◽  
Evgenii Vinogradov ◽  
Annie Aubry ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Mutant Strain ◽  
Lipid A ◽  
Inner Core ◽  
Cell Model ◽  
Critical Factor ◽  
Gastric Cancer Cells ◽  
Level Of Invasion ◽  
Insertional Inactivation

An ld-heptosyltransferase gene, HP1191 (waaF), involved in biosynthesis of the inner-core region of Helicobacter pylori strain 26695 lipopolysaccharide (LPS), has been cloned and its function established by complementation of Salmonella enterica serovar Typhimurium waaF mutant strain, strain 3789. Insertional inactivation of the HP1191 open reading frame in strain 26695 resulted in the formation of a deeply truncated LPS molecule, as observed using SDS–PAGE. Subsequent compositional and fatty acid analyses, followed by capillary electrophoresis – mass spectrometry and nuclear magnetic resonance studies established its structure as the following: PE→7)-l-α-d-Hepp-(1→5)-α-Kdop-(2→6)-Lipid A, where PE represents a phosphoethanolamine group, ld-Hep represents l-glycero-d-manno-heptose, and Kdo represents 3-deoxy-d-manno-oct-2-ulosonic acid. This structural analysis identifies the activity of HP1191 as a heptosyltransferase and a waaF homolog. In vitro invasion assays using human cultured gastric adenocarcinoma cells as a host cell model confirmed that the level of invasion was unaffected for an H. pylori HP1191::Kan deep-rough mutant strain compared with the wild-type strain 26695 expressing the O-chain polysaccharide, providing evidence that LPS is not a critical factor for invasion.

scholarly journals A Novel 3-Deoxy-d-manno-Octulosonic Acid (Kdo) Hydrolase That Removes the Outer Kdo Sugar of Helicobacter pylori Lipopolysaccharide

2005 ◽  
Vol 187 (10) ◽  
pp. 3374-3383 ◽  
Author(s):  
Christopher Stead ◽  
An Tran ◽  
Donald Ferguson ◽  
Sara McGrath ◽  
Robert Cotter ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Lipid A ◽  
In Vitro Assay ◽  
Spectrometric Analysis ◽  
Vitro Assay ◽  
The Core ◽  
H Pylori

ABSTRACT The lipid A domain anchors lipopolysaccharide (LPS) to the outer membrane and is typically a disaccharide of glucosamine that is both acylated and phosphorylated. The core and O-antigen carbohydrate domains are linked to the lipid A moiety through the eight-carbon sugar 3-deoxy-d-manno-octulosonic acid known as Kdo. Helicobacter pylori LPS has been characterized as having a single Kdo residue attached to lipid A, predicting in vivo a monofunctional Kdo transferase (WaaA). However, using an in vitro assay system we demonstrate that H. pylori WaaA is a bifunctional enzyme transferring two Kdo sugars to the tetra-acylated lipid A precursor lipid IVA. In the present work we report the discovery of a Kdo hydrolase in membranes of H. pylori capable of removing the outer Kdo sugar from Kdo2-lipid A. Enzymatic removal of the Kdo group was dependent upon prior removal of the 1-phosphate group from the lipid A domain, and mass spectrometric analysis of the reaction product confirmed the enzymatic removal of a single Kdo residue by the Kdo-trimming enzyme. This is the first characterization of a Kdo hydrolase involved in the modification of gram-negative bacterial LPS.

scholarly journals Lipooligosaccharide Structure Contributes to Multiple Steps in the Virulence of Neisseria meningitidis

2006 ◽  
Vol 74 (2) ◽  
pp. 1360-1367 ◽  
Author(s):  
Laura Plant ◽  
Johanna Sundqvist ◽  
Susu Zughaier ◽  
Lena Lövkvist ◽  
David S. Stephens ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mouse Model ◽  
Neisseria Meningitidis ◽  
Type Strain ◽  
Lipid A ◽  
Wild Type Strain ◽  
Inner Core ◽  
Wild Type ◽  
Proinflammatory Response

ABSTRACT Lipooligosaccharide (LOS) of Neisseria meningitidis has been implicated in meningococcal interaction with host epithelial cells and is a major factor contributing to the human proinflammatory response to meningococci. LOS mutants of the encapsulated N. meningitidis serogroup B strain NMB were used to further determine the importance of the LOS structure in in vitro adherence and invasion of human pharyngeal epithelial cells by meningococci and to study pathogenicity in a mouse (CD46 transgenic) model of meningococcal disease. The wild-type strain [NeuNAc-Galβ-GlcNAc-Galβ-Glcβ-Hep2 (GlcNAc, Glcα) 3-deoxy-d-manno-2-octulosonic acid (KDO2)-lipid A; 1,4′ bisphosphorylated], although poorly adherent, rapidly invaded an epithelial cell layer in vitro, survived and multiplied early in blood, reached the cerebrospinal fluid, and caused lethal disease in the mouse model. In contrast, the Hep2 (GlcNAc) KDO2-lipid A (pgm) mutant, which was highly adherent to cultured epithelial cells, caused significantly less bacteremia and mortality in the mouse model. The Hep2-KDO2-lipid A (rfaK) mutant was shown to be moderately adherent and to cause levels of bacteremia and mortality similar to those caused by the wild-type strain in the mouse model. The KDO2-lipid A (gmhB) mutant, which lacks the heptose disaccharide in the inner core of LOS, avidly attached to epithelial cells but was otherwise avirulent. Disease development correlated with expression of specific LOS structures and was associated with lower adherence but rapid meningococcal passage to and survival in the bloodstream, induction of proinflammatory cytokines, and the crossing of the blood-brain barrier. Taken together, the results of this study further define the importance of the LOS structure as a virulence component involved in multiple steps in the pathogenesis of N. meningitidis.

In vitro sensitivity of human gastric cancer cells (HGC‐27) to Helicobacter pylori cytotoxin

2003 ◽  
Vol 38 (12) ◽  
pp. 1228-1234 ◽  
Author(s):  
A. Pessina ◽  
M. Bayo ◽  
C. Croera ◽  
F. Meringolo ◽  
M. G. Neri ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Helicobacter Pylori ◽  
Cancer Cells ◽  
Human Gastric Cancer ◽  
Gastric Cancer Cells ◽  
In Vitro Sensitivity

scholarly journals Two Distinct Antigenic Types of the Polysaccharide Chains of Helicobacter pylori Lipopolysaccharides Characterized by Reactivity with Sera from Humans with Natural Infection

2000 ◽  
Vol 68 (1) ◽  
pp. 151-159 ◽  
Author(s):  
Shin-Ichi Yokota ◽  
Ken-Ichi Amano ◽  
Yoshiko Shibata ◽  
Mizuho Nakajima ◽  
Miyuki Suzuki ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Lipid A ◽  
Inner Core ◽  
Antigenic Variation ◽  
Natural Infection ◽  
Clinical Isolates ◽  
Antigenic Epitope ◽  
Lewis Antigen ◽  
Human Sera ◽  
H Pylori

ABSTRACT We have purified lipopolysaccharides (LPS) from 10Helicobacter pylori clinical isolates which were selected on the basis of chemotype and antigenic variation. Data from immunoblotting of the purified LPS with sera from humans with H. pylori infection and from absorption of the sera with LPS indicated the presence of two distinct epitopes, termed the highly antigenic and the weakly antigenic epitopes, on the polysaccharide chains. Among 68 H. pylori clinical isolates, all smooth strains possessed either epitope; the epitopes were each carried by about 50% of the smooth strains. Thus, H. pylori strains can be classified into three types on the basis of their antigenicity in humans: those with smooth LPS carrying the highly antigenic epitope, those with smooth LPS carrying the weakly antigenic epitope, and those with rough LPS. Sera from humans with H. pylori infection could be grouped into three categories: those containing immunoglobulin G (IgG) antibodies against the highly antigenic epitope, those containing IgG against the weakly antigenic epitope, and those containing both specific IgGs; these groups made up about 50%, less than 10%, and about 40%, respectively, of all infected sera tested. In other words, IgG against the highly antigenic epitope were detected in more than 90% of H. pylori-infected individuals with high titers. IgG against the weakly antigenic epitope were detected in about 50% of the sera tested; however, the antibody titers were low. The two human epitopes existed independently from the mimic structures of Lewis antigens, which are known to be an important epitope ofH. pylori LPS. No significant relationship between the reactivities toward purified LPS of human sera and a panel of anti-Lewis antigen antibodies was found. Moreover, the reactivities of the anti-Lewis antigen antibodies, but not human sera, were sensitive to particular α-l-fucosidases. The human epitopes appeared to be located on O-polysaccharide chains containing endo-β-galactosidase-sensitive galactose residues as the backbone. Data from chemical analyses indicated that all LPS commonly contained galactose, glucosamine, glucose, and fucose (except one rough strain) as probable polysaccharide components, together with typical components of inner core and lipid A. We were not able to distinguish between the differences of antigenicity in humans by on the basis of the chemical composition of the LPS.

scholarly journals Inhibition of Helicobacter pylori and Gastric Cancer Cells by Lipid Aldehydes from Viburnum opulus (Adoxaceae)

2007 ◽  
Vol 2 (10) ◽  
pp. 1934578X0700201
Author(s):  
Maria Teresa Laux ◽  
Manuel Aregullin ◽  
Eloy Rodriguez
Keyword(s):  
Gastric Cancer ◽  
Helicobacter Pylori ◽  
Gastric Cancer Cell Line ◽  
Cancer Cell Line ◽  
Peptic Ulcers ◽  
Viburnum Opulus ◽  
Gastric Cancer Cells ◽  
H Pylori ◽  
Lipid Aldehydes

A unique group of bioactive, naturally occurring lipid aldehydes were isolated from the fruits of Viburnum opulus, (family Adoxaceae). The natural occurrences of these fatty acid derived aldehydes are reported here for the first time. Helicobacter pylori is a prevalent gastroduodenal pathogen, a causal agent of chronic gastritis and peptic ulcers and an important co-factor in gastric cancer development. We investigated the chemistry and bioactivity of these active constituents by evaluating their ability to inhibit the growth of H. pylori and to induce apoptosis in a gastric cancer cell line (CRL-5971) in vitro.

scholarly journals Antagonistic and Immunomodulant Effects of Two Probiotic Strains of Lactobacillus on Clinical Strains of Helicobacter pylori

2020 ◽  
Vol 9 ◽  
pp. 1794
Author(s):  
Somayyeh Taghizadeh ◽  
Tahereh Falsafi ◽  
Rouha Kasra Kermanshahi ◽  
Reihaneh Ramezani
Keyword(s):  
Helicobacter Pylori ◽  
Cell Model ◽  
Host Cells ◽  
Macrophage Cell ◽  
Cell Free Supernatant ◽  
Probiotic Strains ◽  
Ifn Γ ◽  
Probiotic Lactobacilli ◽  
H Pylori

Background: The present study aimed to evaluate the in vitro and in situ antagonistic effects of Lactobacillus probiotic strains on clinical strains of Helicobacter pylori . Also to investigate their immunomodulation effects on a macrophage cell model. Materials and Methods: Anti-microbial effects of probiotic lactobacilli against H. pylori was assessed using the well and disk diffusion methods. Effects of lactobacilli probiotics strains, as well as their cell-free supernatant on adhesion of H. pylori to MKN-45 gastric epithelial cells, were examined in their presence and absence. Immunomodulation effects of probiotic lactobacilli were performed using the U937 macrophage cell model. Incubation of host cells with probiotics and their cell-free supernatants with cultured host cells was performed in different optimized conditions. The supernatant of host cells cultured in their presence and absence was used for cytokines measurement. Results: Two probiotics‏, Lactobacillus acidophilus ATCC4356, and Lactobacillus rhamnosus PTCC1607, could inhibit the growth of clinical H. pylori in vitro. They could also inhibit attachment of H. pylori to MKN-45 cells. Cell-free supernatant of L. acidophilus had a stimulating effect on the production of Interferon-gamma (IFN-γ) by U937 cells. Conclusion: The present study demonstrates that, L. acidophilus ATCC4356 and L. rhamnosus PTCC1607 probiotic strains can inhibit the growth of clinical H. pylori in vitro. Treatment of U937 with alive H. pylori plus cell-free supernatant of L. acidophilus, have a significantly higher capacity to stimulate IFN-γ production than H. pylori alone. So, the metabolite (s) of this probiotic may have an immunomodulatory effect in immune response versus H. pylori. [GMJ.2020;9:e1794]

scholarly journals Toxic SOD1 trimers are off-pathway in the formation of amyloid-like fibrils in ALS

2021 ◽  
Author(s):  
Brianna L Hnath ◽  
Nikolay V Dokholyan
Keyword(s):  
Amyloid Fibrils ◽  
Cell Model ◽  
Limited Proteolysis ◽  
Critical Factor ◽  
Fibril Formation ◽  
Size Exclusion ◽  
Therapeutic Approaches ◽  
Critical Determinant ◽  
Exclusion Chromatography

Accumulation of insoluble amyloid fibrils is widely studied as a critical factor in the pathology of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. Misfolded Cu, Zn superoxide dismutase (SOD1) was the first protein linked to ALS, and non-native SOD1 trimeric oligomers were recently linked to cytotoxicity, while larger oligomers were protective to cells. The balance between trimers and larger aggregates in the process of SOD1 aggregation is, thus, a critical determinant of potential therapeutic approaches to treat ALS. Yet, it is unknown whether these trimeric oligomers are a necessary intermediate for larger aggregate formation or a distinct off-pathway species competing with fibril formation. Depending on the on- or off-pathway scenario of trimer formation, we expect drastically different therapeutic approaches. Here, we show that the toxic SOD1 trimer is an off-pathway intermediate competing with protective fibril formation. We design mutant SOD1 constructs that remain in a trimeric state (super stable trimers) and show that stabilizing the trimeric SOD1 prevents formation of fibrils in vitro and in a motor neuron like cell model (NSC-34). Using size exclusion chromatography we track the aggregation kinetics of purified SOD1 and show direct competition of trimeric SOD1 with larger oligomer and fibril formation. Finally, we show the trimer is structurally independent of both larger soluble oligomers and insoluble fibrils using circular dichroism spectroscopy and limited proteolysis.

scholarly journals The Lipid A 1-Phosphatase of Helicobacter pylori Is Required for Resistance to the Antimicrobial Peptide Polymyxin

2006 ◽  
Vol 188 (12) ◽  
pp. 4531-4541 ◽  
Author(s):  
An X. Tran ◽  
Judy D. Whittimore ◽  
Priscilla B. Wyrick ◽  
Sara C. McGrath ◽  
Robert J. Cotter ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Antimicrobial Peptide ◽  
Lipid A ◽  
Mass Spectrometry Analysis ◽  
Cationic Antimicrobial Peptide ◽  
Vitro Assay ◽  
Chloramphenicol Resistance ◽  
Gram Negative ◽  
H Pylori

ABSTRACT Modification of the phosphate groups of lipid A with amine-containing substituents, such as phosphoethanolamine, reduces the overall net negative charge of gram-negative bacterial lipopolysaccharide, thereby lowering its affinity to cationic antimicrobial peptides. Modification of the 1 position of Helicobacter pylori lipid A is a two-step process involving the removal of the 1-phosphate group by a lipid A phosphatase, LpxEHP (Hp0021), followed by the addition of a phosphoethanolamine residue catalyzed by EptAHP (Hp0022). To demonstrate the importance of modifying the 1 position of H. pylori lipid A, we generated LpxEHP-deficient mutants in various H. pylori strains by insertion of a chloramphenicol resistance cassette into lpxEHP and examined the significance of LpxE with respect to cationic antimicrobial peptide resistance. Using both mass spectrometry analysis and an in vitro assay system, we showed that the loss of LpxEHP activity in various H. pylori strains resulted in the loss of modification of the 1 position of H. pylori lipid A, thus confirming the function of LpxEHP. Due to its unique lipid A structure, H. pylori is highly resistant to the antimicrobial peptide polymyxin (MIC > 250 μg/ml). However, disruption of lpxEHP in H. pylori results in a dramatic decrease in polymyxin resistance (MIC, 10 μg/ml). In conclusion, we have characterized the first gram-negative LpxE-deficient mutant and have shown the importance of modifying the 1 position of H. pylori lipid A for resistance to polymyxin.

scholarly journals Diversification of the AlpB Outer Membrane Protein of Helicobacter pylori Affects Biofilm Formation and Cellular Adhesion

2016 ◽  
Vol 199 (6) ◽  
Author(s):  
Hideo Yonezawa ◽  
Takako Osaki ◽  
Toshiyuki Fukutomi ◽  
Tomoko Hanawa ◽  
Satoshi Kurata ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Outer Membrane ◽  
Mutant Strain ◽  
Biofilm Formation ◽  
Molecular Mechanisms ◽  
Bacterial Biofilm ◽  
Content Type ◽  
Successful Infection ◽  
H Pylori

ABSTRACT Helicobacter pylori is one of the most common causes of bacterial infection in humans, and it forms biofilms on human gastric mucosal epithelium as well as on in vitro abiotic surfaces. Bacterial biofilm is critical not only for environmental survival but also for successful infection. We previously demonstrated that strain TK1402, which was isolated from a Japanese patient with duodenal and gastric ulcers, has high biofilm-forming ability in vitro relative to other strains. In addition, we showed that outer membrane vesicles (OMV) play an important role in biofilm formation. The aim of this study was to analyze which protein(s) in the OMV contributes to biofilm formation in TK1402. We obtained a spontaneous mutant strain derived from TK1402 lacking biofilm-forming ability. The protein profiles of the OMV were compared between this mutant strain and the wild type, and it was found that AlpB, an outer membrane protein in the OMV of the mutant strain, was markedly decreased compared to that of the wild type. Restoration of TK1402 alpB to the mutant strain fully recovered the ability to form biofilm. However, restoration with alpB from other strains demonstrated incomplete recovery of biofilm-forming ability. We therefore inferred that the variable region of AlpB (amino acid positions 121 to 146) was involved in TK1402 biofilm formation. In addition, diversification of the AlpB sequence was shown to affect the ability to adhere to AGS cells. These results demonstrate a new insight into the molecular mechanisms of host colonization by H. pylori. IMPORTANCE Bacterial biofilm is critical not only for environmental survival but also for successful infection. The mechanism of Helicobacter pylori adherence to host cells mediated by cell surface adhesins has been the focus of many studies, but little is known regarding factors involved in H. pylori biofilm formation. Our study demonstrated that AlpB plays an important role in biofilm formation and that this property depends upon the specific sequence of alpB. This in turn was shown to be important in the ability to adhere to gastric cells. We anticipate that these results will provide new insight into the molecular mechanisms of H. pylori colonization.

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