scholarly journals The galE Gene of Campylobacter jejuni Is Involved in Lipopolysaccharide Synthesis and Virulence

2000 ◽  
Vol 68 (5) ◽  
pp. 2594-2601 ◽  
Author(s):  
Benjamin N. Fry ◽  
Shi Feng ◽  
Yuen-Yuen Chen ◽  
Diane G. Newell ◽  
Peter J. Coloe ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Parent Strain ◽  
Type Strain ◽  
Essential Role ◽  
Lipid A ◽  
Wild Type Strain ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
Gram Negative ◽  
Important Virulence Factor

ABSTRACT Lipopolysaccharide (LPS) is one of the main virulence factors of gram-negative bacteria. The LPS from Campylobacter spp. has endotoxic properties and has been shown to play a role in adhesion. We previously cloned a gene cluster (wla) which is involved in the synthesis of the Campylobacter jejuni 81116 LPS molecule. Sequence alignment of the first gene in this cluster indicated similarity with galE genes. These genes encode a UDP-glucose 4-epimerase, which catalyzes the interconversion of UDP-galactose and UDP-glucose. A Salmonella galE mutant was transformed with the galE gene from C. jejuni. The LPS analysis of wild-type, galE, and complementedgalE Salmonella strains showed that the C. jejuni galE gene could restore the smooth wild-typeSalmonella LPS. A UDP-glucose 4-epimerase assay was used to demonstrate that the galE gene from C. jejuniencoded this epimerase. We constructed a C. jejuni galEmutant which expressed a lipid A-core molecule of reduced molecular weight that did not react with antiserum raised against the parental strain. These results show an essential role for the galEgene in the synthesis of C. jejuni LPS. ThegalE mutant also showed a reduction in its ability to adhere to and invade INT407 cells. However, it was still able to colonize chickens to the same level as the wild-type strain. The serum resistance and hemolytic activity of this mutant were not changed compared to the parent strain. The ability of the mutant to take up DNA and integrate it in its genome was reduced 20-fold. These results show that LPS of C. jejuni is an important virulence factor.

scholarly journals Effect of rpoS Mutation on the Stress Response and Expression of Virulence Factors in Pseudomonas aeruginosa

1999 ◽  
Vol 181 (13) ◽  
pp. 3890-3897 ◽  
Author(s):  
Sang-Jin Suh ◽  
Laura Silo-Suh ◽  
Donald E. Woods ◽  
Daniel J. Hassett ◽  
Susan E. H. West ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Stress Response ◽  
Parent Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Infection Model ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
Gram Negative

ABSTRACT The sigma factor RpoS (ςS) has been described as a general stress response regulator that controls the expression of genes which confer increased resistance to various stresses in some gram-negative bacteria. To elucidate the role of RpoS inPseudomonas aeruginosa physiology and pathogenesis, we constructed rpoS mutants in several strains of P. aeruginosa, including PAO1. The PAO1 rpoS mutant was subjected to various environmental stresses, and we compared the resistance phenotype of the mutant to that of the parent. The PAO1rpoS mutant was slightly more sensitive to carbon starvation than the wild-type strain, but this phenotype was obvious only when the cells were grown in a medium supplemented with glucose as the sole carbon source. In addition, the PAO1 rpoS mutant was hypersensitive to heat shock at 50°C, increased osmolarity, and prolonged exposure to high concentrations of H2O2. In accordance with the hypersensitivity to H2O2, catalase production was 60% lower in the rpoS mutant than in the parent strain. We also assessed the role of RpoS in the production of several exoproducts known to be important for virulence of P. aeruginosa. TherpoS mutant produced 50% less exotoxin A, but it produced only slightly smaller amounts of elastase and LasA protease than the parent strain. The levels of phospholipase C and casein-degrading proteases were unaffected by a mutation in rpoS in PAO1. The rpoS mutation resulted in the increased production of the phenazine antibiotic pyocyanin and the siderophore pyoverdine. This increased pyocyanin production may be responsible for the enhanced virulence of the PAO1 rpoS mutant that was observed in a rat chronic-lung-infection model. In addition, the rpoSmutant displayed an altered twitching-motility phenotype, suggesting that the colonization factors, type IV fimbriae, were affected. Finally, in an alginate-overproducing cystic fibrosis (CF) isolate, FRD1, the rpoS101::aacCI mutation almost completely abolished the production of alginate when the bacterium was grown in a liquid medium. On a solid medium, the FRD1rpoS mutant produced approximately 70% less alginate than did the wild-type strain. Thus, our data indicate that although some of the functions of RpoS in P. aeruginosa physiology are similar to RpoS functions in other gram-negative bacteria, it also has some functions unique to this bacterium.

scholarly journals Mutation of waaC, Encoding Heptosyltransferase I in Campylobacter jejuni 81-176, Affects the Structure of both Lipooligosaccharide and Capsular Carbohydrate

2006 ◽  
Vol 188 (9) ◽  
pp. 3273-3279 ◽  
Author(s):  
Margaret I. Kanipes ◽  
Erzsebet Papp-Szabo ◽  
Patricia Guerry ◽  
Mario A. Monteiro
Keyword(s):  
Campylobacter Jejuni ◽  
Type Strain ◽  
Insertional Mutagenesis ◽  
Lipid A ◽  
Wild Type Strain ◽  
Capsular Polysaccharide ◽  
Resonance Spectroscopy ◽  
Gas Liquid Chromatography ◽  
Wild Type ◽  
Isolation And Characterization

ABSTRACT Campylobacter jejuni 81-176 lipooligosaccharide (LOS) is composed of two covalently linked domains: lipid A, a hydrophobic anchor, and a nonrepeating core oligosaccharide, consisting of an inner and outer core region. We report the isolation and characterization of the deepest rough C. jejuni 81-176 mutant by insertional mutagenesis into the waaC gene, encoding heptosyltransferase I that catalyzes the transfer of the first l-glycero-d-manno-heptose residue to 3-deoxy-d-manno-octulosonic residue (Kdo)-lipid A. Tricine gel electrophoresis, followed by silver staining, showed that site-specific mutation in the waaC gene resulted in the expression of a severely truncated LOS compared to wild-type strain 81-176. Gas-liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy showed that the waaC LOS species lacked all sugars distal to Kdo-lipid A. Parallel structural studies of the capsular polysaccharides of the wild-type strain 81-176 and waaC mutant revealed loss of the 3-O-methyl group in the waaC mutant. Complementation of the C. jejuni mutant by insertion of the wild-type C. jejuni waaC gene into a chromosomal locus resulted in LOS and capsular structures identical to those expressed in the parent strain. We also report here the presence of O-methyl phosphoramidate in wild-type strain 81-176 capsular polysaccharide.

scholarly journals The Riemerella anatipestiferAS87_01735Gene Encodes Nicotinamidase PncA, an Important Virulence Factor

2016 ◽  
Vol 82 (19) ◽  
pp. 5815-5823 ◽  
Author(s):  
Xiaolan Wang ◽  
Beibei Liu ◽  
Yafeng Dou ◽  
Hongjie Fan ◽  
Shaohui Wang ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Vaccine Candidate ◽  
Wild Type ◽  
Salvage Pathway ◽  
Effective Protection ◽  
Domestic Ducks ◽  
Content Type ◽  
Key Enzyme ◽  
Important Virulence Factor

ABSTRACTRiemerella anatipestiferis a major bacterial pathogen that causes septicemic and exudative diseases in domestic ducks. In our previous study, we found that deletion of theAS87_01735gene significantly decreased the bacterial virulence ofR. anatipestiferstrain Yb2 (mutant RA625). TheAS87_01735gene was predicted to encode a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD+salvage pathway. In this study, theAS87_01735gene was expressed and identified as the PncA-encoding gene, using an enzymatic assay. Western blot analysis demonstrated thatR. anatipestiferPncA was localized to the cytoplasm. The mutant strain RA625 (named Yb2ΔpncAin this study) showed a similar growth rate but decreased NAD+quantities in both the exponential and stationary phases in tryptic soy broth culture, compared with the wild-type strain Yb2. In addition, Yb2ΔpncA-infected ducks showed much lower bacterial loads in their blood, and no visible histological changes were observed in the heart, liver, and spleen. Furthermore, Yb2ΔpncAimmunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2. Our results suggest that theR. anatipestiferAS87_01735gene encodes PncA, which is an important virulence factor, and that the Yb2ΔpncAmutant can be used as a novel live vaccine candidate.IMPORTANCERiemerella anatipestiferis reported worldwide as a cause of septicemic and exudative diseases of domestic ducks. ThepncAgene encodes a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD+salvage pathway. In this study, we identified and characterized thepncA-homologous geneAS87_01735inR. anatipestiferstrain Yb2.R. anatipestiferPncA is a cytoplasmic protein that possesses similar PncA activity, compared with other organisms. Generation of thepncAmutant Yb2ΔpncAled to a decrease in the NAD+content, which was associated with decreased capacity for invasion and attenuated virulence in ducks. Furthermore, Yb2ΔpncAimmunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2. Altogether, these results suggest that PncA contributes to the virulence ofR. anatipestiferand that the Yb2ΔpncAmutant can be used as a novel live vaccine candidate.

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.

scholarly journals Campylobacter jejuni FlpA Binds Fibronectin and Is Required for Maximal Host Cell Adherence

2009 ◽  
Vol 192 (1) ◽  
pp. 68-76 ◽  
Author(s):  
Michael E. Konkel ◽  
Charles L. Larson ◽  
Rebecca C. Flanagan
Keyword(s):  
Amino Acids ◽  
Epithelial Cells ◽  
Campylobacter Jejuni ◽  
Type Strain ◽  
Wild Type Strain ◽  
Major Constituent ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Wild Type ◽  
Type Iii

ABSTRACT Campylobacter jejuni is one of the most frequent bacterial causes of food-borne gastrointestinal disease in developed countries. Previous work indicates that the binding of C. jejuni to human intestinal cells is crucial for host colonization and disease. Fibronectin (Fn), a major constituent of the extracellular matrix, is a ∼250-kDa glycoprotein present at regions of cell-to-cell contact in the intestinal epithelium. Fn is composed of three types of repeating units: type I (∼45 amino acids), type II (∼60 amino acids), and type III (∼90 amino acids). The deduced amino acid sequence of C. jejuni flpA (Cj1279c) contains at least three Fn type III domains. Based on the presence of the Fn type III domains, we hypothesized that FlpA contributes to the binding of C. jejuni to human INT 407 epithelial cells and Fn. We assessed the contribution of FlpA in C. jejuni binding to host cells by in vitro adherence assays with a C. jejuni wild-type strain and a C. jejuni flpA mutant and binding of purified FlpA protein to Fn by enzyme-linked immunosorbent assay (ELISA). Adherence assays revealed the binding of the C. jejuni flpA mutant to INT 407 epithelial cells was significantly reduced compared with that for a wild-type strain. In addition, rabbit polyclonal serum generated against FlpA blocked C. jejuni adherence to INT 407 cells in a concentration-dependent manner. Binding of FlpA to Fn was found to be dose dependent and saturable by ELISA, demonstrating the specificity of the interaction. Based on these data, we conclude that FlpA mediates C. jejuni attachment to host epithelial cells via Fn binding.

scholarly journals Mapping the Role of AcrAB-TolC Efflux Pumps in the Evolution of Antibiotic Resistance Reveals Near-MIC Treatments Facilitate Resistance Acquisition

2020 ◽  
Author(s):  
Ariel M. Langevin ◽  
Imane El Meouche ◽  
Mary J. Dunlop
Keyword(s):  
Antibiotic Resistance ◽  
Genetic Background ◽  
Parent Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Efflux Pumps ◽  
Wild Type ◽  
Antibiotic Concentration ◽  
Drug Concentrations ◽  
Evolution Of Resistance

ABSTRACTAntibiotic resistance has become a major public health concern as bacteria evolve to evade drugs, leading to recurring infections and a decrease in antibiotic efficacy. Systematic efforts have revealed mechanisms involved in resistance; yet, in many cases, how these specific mechanisms accelerate or slow the evolution of resistance remains unclear. Here, we conducted a systematic study of the impact of the AcrAB-TolC efflux pump on the evolution of antibiotic resistance. We mapped how population growth rate and resistance change over time as a function of both the antibiotic concentration and the parent strain’s genetic background. We compared the wild type strain to a strain overexpressing AcrAB-TolC pumps and a strain lacking functional pumps. In all cases, resistance emerged when cultures were treated with chloramphenicol concentrations near the MIC of their respective parent strain. The genetic background of the parent strain also influenced resistance acquisition. The wild type strain evolved resistance within 24 h through mutations in the acrAB operon and its associated regulators. Meanwhile, the strain overexpressing AcrAB-TolC evolved resistance more slowly than the wild type strain; this strain achieved resistance in part through point mutations in acrB and the acrAB promoter. Surprisingly, the strain without functional AcrAB-TolC efflux pumps still gained resistance, which it achieved through upregulation of redundant efflux pumps. Overall, our results suggest that treatment conditions just above the MIC pose the largest risk for the evolution of resistance and that AcrAB-TolC efflux pumps impact the pathway by which chloramphenicol resistance is achieved.IMPORTANCECombatting the rise of antibiotic resistance is a significant challenge. Efflux pumps are an important contributor to drug resistance; they exist across many cell types and can export numerous classes of antibiotics. Cells can regulate pump expression to maintain low intracellular drug concentrations. Here, we explored how resistance emerged depending on the antibiotic concentration, as well as the presence of efflux pumps and their regulators. We found that treatments near antibiotic concentrations that inhibit the parent strain’s growth were most likely to promote resistance. While wild type, pump overexpression, and pump knock out strains were all able to evolve resistance, they differed in the absolute level of resistance evolved, the speed at which they achieved resistance, and the genetic pathways involved. These results indicate that specific treatment regimens may be especially problematic for the evolution of resistance and that the strain background can influence how resistance is achieved.

scholarly journals Riboflavin Biosynthesis Is Associated with Assimilatory Ferric Reduction and Iron Acquisition by Campylobacter jejuni

2007 ◽  
Vol 73 (24) ◽  
pp. 7819-7825 ◽  
Author(s):  
Rachel A. Crossley ◽  
Duncan J. H. Gaskin ◽  
Kathryn Holmes ◽  
Francis Mulholland ◽  
Jerry M. Wells ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Iron Uptake ◽  
Type Strain ◽  
Wild Type Strain ◽  
Iron Acquisition ◽  
Flavin Mononucleotide ◽  
Riboflavin Biosynthesis ◽  
Wild Type ◽  
Low Oxygen ◽  
Riboflavin Production

ABSTRACT One of the pathways involved in the acquisition of the essential metal iron by bacteria involves the reduction of insoluble Fe3+ to soluble Fe2+, followed by transport of Fe2+ to the cytoplasm. Flavins have been implicated as electron donors in this poorly understood process. Ferrous iron uptake is essential for intestinal colonization by the important pathogen Campylobacter jejuni and may be of particular importance under low-oxygen conditions. In this study, the links among riboflavin biosynthesis, ferric reduction, and iron acquisition in C. jejuni NCTC11168 have been investigated. A riboflavin auxotroph was generated by inactivation of the ribB riboflavin biosynthesis gene (Cj0572), and the resulting isogenic ribB mutant only grew in the presence of exogenous riboflavin or the riboflavin precursor diacetyl but not in the presence of the downstream products flavin adenine dinucleotide and flavin mononucleotide. Riboflavin uptake was unaffected in the ribB mutant under iron-limited conditions but was lower in both the wild-type strain and the ribB mutant under iron-replete conditions. Mutation of the fur gene, which encodes an iron uptake regulator of C. jejuni, resulted in an increase in riboflavin uptake which was independent of the iron content of the medium, suggesting a role for Fur in the regulation of the as-yet-unknown riboflavin transport system. Finally, ferric reduction activity was independent of iron availability in the growth medium but was lowered in the ribB mutant compared to the wild-type strain and, conversely, increased in the fur mutant. Taken together, the findings confirm close relationships among iron acquisition, riboflavin production, and riboflavin uptake in C. jejuni.

scholarly journals Novel Toluene Elimination System in a Toluene-Tolerant Microorganism

2000 ◽  
Vol 182 (22) ◽  
pp. 6451-6455 ◽  
Author(s):  
Hideki Kobayashi ◽  
Katsuyuki Uematsu ◽  
Hisako Hirayama ◽  
Koki Horikoshi
Keyword(s):  
Membrane Proteins ◽  
Cell Membrane ◽  
Outer Membrane ◽  
Parent Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Wild Type ◽  
Sensitive Mutant

ABSTRACT In studies of Pseudomonas putida IH-2000, a toluene-tolerant microorganism, membrane vesicles (MVs) were found to be released from the outer membrane when toluene was added to the culture. These MVs were found to be composed of phospholipids, lipopolysaccharides (LPS), and very low amounts of outer membrane proteins. The MVs also contained a higher concentration of toluene molecules (0.172 ± 0.012 mol/mol of lipid) than that found in the cell membrane. In contrast to the wild-type strain, the toluene-sensitive mutant strain 32, which differs from the parent strain in LPS and outer membrane proteins, did not release MVs from the outer membrane. The toluene molecules adhering to the outer membrane are eliminated by the shedding of MVs, and this system appears to serve as an important part of the toluene tolerance system of IH-2000.

scholarly journals Leptospira interrogans lpxDHomologue Is Required for Thermal Acclimatization and Virulence

2015 ◽  
Vol 83 (11) ◽  
pp. 4314-4321 ◽  
Author(s):  
Azad Eshghi ◽  
Jeremy Henderson ◽  
M. Stephen Trent ◽  
Mathieu Picardeau
Keyword(s):  
Outer Membrane ◽  
Type Strain ◽  
Lipid A ◽  
Wild Type Strain ◽  
Infection Model ◽  
Leptospira Interrogans ◽  
Wild Type ◽  
Content Type ◽  
Physiological Temperature ◽  
Animal Infection

ABSTRACTLeptospirosis is an emerging disease with an annual occurrence of over 1 million human cases worldwide. PathogenicLeptospirabacteria are maintained in zoonotic cycles involving a diverse array of mammals, with the capacity to survive outside the host in aquatic environments. Survival in the diverse environments encountered byLeptospiralikely requires various adaptive mechanisms. Little is known aboutLeptospiraouter membrane modification systems, which may contribute to the capacity of these bacteria to successfully inhabit and colonize diverse environments and animal hosts.Leptospirabacteria carry two genes annotated as UDP-3-O-[3-hydroxymyristoyl] glucosamineN-acyltransferase genes (la0512 and la4326 [lpxD1andlpxD2]) that in other bacteria are involved in the early steps of biosynthesis of lipid A, the membrane lipid anchor of lipopolysaccharide. Inactivation of only one of these genes, la0512/lpxD1, imparted sensitivity to the host physiological temperature (37°C) and rendered the bacteria avirulent in an animal infection model. Polymyxin B sensitivity assays revealed compromised outer membrane integrity in thelpxD1mutant at host physiological temperature, but structural analysis of lipid A in the mutant revealed only minor changes in the lipid A moiety compared to that found in the wild-type strain. In accordance with this, anin transcomplementation restored the phenotypes to a level comparable to that of the wild-type strain. These results suggest that the gene annotated aslpxD1inLeptospira interrogansplays an important role in temperature adaptation and virulence in the animal infection model.

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