scholarly journals The Sialylated Lipooligosaccharide Outer Core in Campylobacter jejuni Is an Important Determinant for Epithelial Cell Invasion

2008 ◽  
Vol 76 (10) ◽  
pp. 4431-4438 ◽  
Author(s):  
Rogier Louwen ◽  
Astrid Heikema ◽  
Alex van Belkum ◽  
Alewijn Ott ◽  
Michel Gilbert ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Campylobacter Jejuni ◽  
Outer Core ◽  
Wild Type ◽  
Epithelial Cell Lines ◽  
Bacterial Gastroenteritis ◽  
Important Virulence Factor ◽  
Epithelial Invasion ◽  
Type Strains ◽  
Formalin Fixed

ABSTRACT Campylobacter jejuni is a frequent cause of bacterial gastroenteritis worldwide. Lipooligosaccharide (LOS) has been identified as an important virulence factor that may play a role in microbial adhesion and invasion. Here we specifically address the question of whether LOS sialylation affects the interaction of C. jejuni with human epithelial cells. For this purpose, 14 strains associated with Guillain-Barré syndrome (GBS), 34 enteritis-associated strains, the 81-176 reference strain, and 6 Penner serotype strains were tested for invasion of two epithelial cell lines. C. jejuni strains expressing sialylated LOS (classes A, B, and C) invaded cells significantly more frequently than strains expressing nonsialylated LOS (classes D and E) (P < 0.0001). To further explore this observation, we inactivated the LOS sialyltransferase (Cst-II) via knockout mutagenesis in three GBS-associated C. jejuni strains expressing sialylated LOS (GB2, GB11, and GB19). All knockout strains displayed significantly lower levels of invasion than the respective wild types. Complementation of a Δcst-II mutant strain restored LOS sialylation and reset the invasiveness to wild-type levels. Finally, formalin-fixed wild-type strains GB2, GB11 and GB19, but not the isogenic Δcst-II mutants that lack sialic acid, were able to inhibit epithelial invasion by viable GB2, GB11, and GB19 strains. We conclude that sialylation of the LOS outer core contributes significantly to epithelial invasion by C. jejuni and may thus play a role in subsequent postinfectious pathologies.

scholarly journals Actin Is a Component of the Compensation Mechanism in Pseudorabies Virus Virions Lacking the Major Tegument Protein VP22

2005 ◽  
Vol 79 (13) ◽  
pp. 8614-8619 ◽  
Author(s):  
T. del Rio ◽  
C. J. DeCoste ◽  
L.W. Enquist
Keyword(s):  
Epithelial Cell ◽  
Cell Lines ◽  
Fusion Proteins ◽  
Pseudorabies Virus ◽  
Tegument Protein ◽  
Compensation Mechanism ◽  
Wild Type ◽  
Epithelial Cell Lines ◽  
Del Rio ◽  
Protein Vp22

ABSTRACT Despite being a major component of the pseudorabies virus tegument, VP22 is not required for PRV replication, virulence, or neuroinvasion (T. del Rio, H. C. Werner, and L. W. Enquist, J. Virol. 76:774-782, 2002). In the absence of VP22, tegument assembly compensates in a limited fashion with increased incorporation of cellular actin. Infection of epithelial cell lines expressing fluorescent actin fusion proteins resulted in the incorporation of filamentous and nonfilamentous actin into individual virions that were predominately light, noninfectious particles. We conclude that cellular actin is incorporated in the tegument of wild-type virions and is part of a compensation mechanism for VP22-null virions.

scholarly journals Long-Term Survival of Campylobacter jejuni at Low Temperatures Is Dependent on Polynucleotide Phosphorylase Activity

2009 ◽  
Vol 75 (23) ◽  
pp. 7310-7318 ◽  
Author(s):  
Nabila Haddad ◽  
Christopher M. Burns ◽  
Jean Michel Bolla ◽  
Hervé Prévost ◽  
Michel Fédérighi ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Low Temperatures ◽  
Survival Rates ◽  
Polynucleotide Phosphorylase ◽  
Wild Type ◽  
Term Survival ◽  
Poultry Products ◽  
Bacterial Gastroenteritis

ABSTRACT Campylobacter jejuni is a leading cause of bacterial gastroenteritis worldwide. Infection generally occurs after ingestion of contaminated poultry products, usually conserved at low temperatures. The mechanisms promoting survival of C. jejuni in the cold remain poorly understood despite several investigations. The present study provides insight into the survival mechanism by establishing the involvement of polynucleotide phosphorylase (PNPase), a 3′-5′ exoribonuclease with multiple biological functions in cold survival. The role of PNPase was demonstrated genetically using strains with altered pnp genes (which encode PNPase) created in C. jejuni F38011 and C. jejuni 81-76 backgrounds. Survival assays carried out at low temperatures (4 and 10°C) revealed a difference of 3 log CFU/ml between the wild-type and the pnp deletion (Δpnp) strains. This did not result from a general requirement for PNPase because survival rates of the strains were similar at higher growth temperatures (37 or 42°C). trans-Complementation with plasmid pNH04 carrying the pnp gene under the control of its natural promoter restored the cold survival phenotype to the pnp deletion strains (at 4 and 10°C) but not to the same level as the wild type. In this study we demonstrate the role of PNPase in low-temperature survival of C. jejuni and therefore attribute a novel biological function to PNPase directly related to human health.

scholarly journals Survival of Campylobacter jejuni under Conditions of Atmospheric Oxygen Tension with the Support of Pseudomonas spp.

2010 ◽  
Vol 76 (17) ◽  
pp. 5911-5917 ◽  
Author(s):  
Friederike Hilbert ◽  
Manuela Scherwitzel ◽  
Peter Paulsen ◽  
Michael P. Szostak
Keyword(s):  
Enterococcus Faecalis ◽  
Oxygen Tension ◽  
Campylobacter Jejuni ◽  
Micrococcus Luteus ◽  
Atmospheric Oxygen ◽  
Human Infection ◽  
Chicken Meat ◽  
Citrobacter Freundii ◽  
Wild Type ◽  
Type Strains

ABSTRACT Campylobacter jejuni is a major food-borne pathogen. Despite causing enteritis in humans, it is a well-adapted intestinal microorganism in animals, hardly ever generating disease symptoms. Nevertheless, as a true microaerophilic microorganism it is still puzzling how Campylobacter cells can survive on chicken meat, the main source of human infection. In this study, we demonstrate that C. jejuni is able to withstand conditions of atmospheric oxygen tension when cocultured with Pseudomonas species, major food-spoiling bacteria that are frequently found on chicken meat in rather high numbers. Using an in vitro survival assay, interactions of 145 C. jejuni wild-type strains and field isolates from chicken meat, broiler feces, and human clinical samples with type strains and food isolates of Pseudomonas spp., Proteus mirabilis, Citrobacter freundii, Micrococcus luteus, and Enterococcus faecalis were studied. When inoculated alone or in coculture with Proteus mirabilis, Citrobacter freundii, Micrococcus luteus, or Enterococcus faecalis type strains, Campylobacter cells were able to survive ambient oxygen levels for no more than 18 h. In contrast, Campylobacter bacteria inoculated with type strains or wild-type isolates of Pseudomonas showed a prolonged aerobic survival of up to >48 h. This microbial commensalism was diverse in C. jejuni isolates from different sources; isolates from chicken meat and humans in coculture with P seudomonas putida were able to use this survival support better than fecal isolates from broilers. Scanning electron microscopy revealed the development of fiberlike structures braiding P. putida and C. jejuni cells. Hence, it seems that microaerophilic C. jejuni is able to survive ambient atmospheric oxygen tension by metabolic commensalism with Pseudomonas spp. This bacterium-bacterium interaction might set the basis for survival of C. jejuni on chicken meat and thus be the prerequisite step in the pathway toward human infection.

scholarly journals Infectious Epstein-Barr Virus Lacking Major Glycoprotein BLLF1 (gp350/220) Demonstrates the Existence of Additional Viral Ligands

2000 ◽  
Vol 74 (21) ◽  
pp. 10142-10152 ◽  
Author(s):  
Annette Janz ◽  
Muhsin Oezel ◽  
Christian Kurzeder ◽  
Josef Mautner ◽  
Dagmar Pich ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Target Cells ◽  
Wild Type ◽  
Viral Glycoprotein ◽  
Barr Virus ◽  
Viral Mutant ◽  
Epithelial Cell Lines ◽  
Epstein Barr

ABSTRACT The binding of the viral major glycoprotein BLLF1 (gp350/220) to the CD21 cellular receptor is thought to play an essential role during infection of B lymphocytes by the Epstein-Barr virus (EBV). However, since CD21-negative cells have been reported to be infectible with EBV, additional interactions between viral and cellular molecules seem to be probable. Based on a recombinant genomic EBV plasmid, we deleted the gene that encodes the viral glycoprotein BLLF1. We tested the ability of the viral mutant to infect different lymphoid and epithelial cell lines. Primary human B cells, lymphoid cell lines, and nearly all of the epithelial cell lines that are susceptible to wild-type EBV infection could also be successfully infected with the viral mutant in vitro, although the efficiency of infection with BLLF1-negative virus was clearly lower than the one observed with wild-type EBV. Our studies show that the interaction between BLLF1 and CD21 is not absolutely required for the infection of lymphocytes and epithelial cells, indicating that viral molecules other than BLLF1 can mediate the binding of EBV to its target cells. In this context, our results further suggest the hypothesis that additional cellular molecules, apart from CD21, allow virus entry into these cells.

Expression of caveolin-1 and polarized formation of invaginated caveolae in Caco-2 and MDCK II cells

1998 ◽  
Vol 111 (6) ◽  
pp. 825-832 ◽  
Author(s):  
U. Vogel ◽  
K. Sandvig ◽  
B. van Deurs
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Epithelial Cell ◽  
Basolateral Membrane ◽  
Apical Surface ◽  
Wild Type ◽  
Caveolin 1 ◽  
Endogenous Level ◽  
Epithelial Cell Lines ◽  
Different Levels

We have studied caveolin-1 expression and the frequency and distribution of typical invaginated caveolae as they are identified by electron microscopy in the polarized epithelial cell lines MDCK II and Caco-2. In wild-type MDCK II cells caveolin expression is high and more than 400 caveolae/mm filter were observed at the basolateral membrane. No caveolae were found at the apical surface. By contrast, wild-type Caco-2 cells do not express caveolin-1 and have extremely few, if any caveolae. Caco-2 cells were stably transfected with the gene for caveolin-1 in order to investigate if the formation of caveolae is polarized also in these cells. We have isolated Caco-2 clones expressing different levels of caveolin-1, where the level of expression varies from 10–100% of the endogenous level in MDCK II cells. Caveolin-1 expression in Caco-2 cells gives rise to a marked immunofluorescense labeling mainly at the lateral plasma membrane. By electron microscopy an increase from less than 4 caveolae/mm filter in wild-type Caco-2 cells to 21–76 caveolae/mm filter in Caco-2 clones transfected with caveolin-1 was revealed and these caveolae were exclusively localized to the basolateral membrane. Thus expression of heterologous caveolin-1 in Caco-2 cells leads to polarized formation of caveolae, but there is a lack of correlation between the amount of caveolin expressed in the cells and the number of caveolae, suggesting that factors in addition to caveolin are required for generation of caveolae.

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 Cytomegalovirus UL131-128 Products Promote gB Conformational Transition and gB-gH Interaction during Entry into Endothelial Cells

2007 ◽  
Vol 81 (20) ◽  
pp. 11479-11488 ◽  
Author(s):  
Marco Patrone ◽  
Massimiliano Secchi ◽  
Eleonora Bonaparte ◽  
Gabriele Milanesi ◽  
Andrea Gallina
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Epithelial Cell ◽  
Conformational Change ◽  
Human Cytomegalovirus ◽  
Receptor Binding ◽  
Conformational Transition ◽  
Wild Type ◽  
Epithelial Cell Lines ◽  
A Chain

ABSTRACT Herpesviruses use gB and gH-gL glycoproteins to execute fusion. Other virus-specific glycoproteins are required for receptor binding and fusion activation. The human cytomegalovirus (HCMV) UL131-128 proteins are essential for the infection of leukocytes, endothelial cells (ECs), and many epithelial cell lines. Here we show that UL131-128 play a role in a chain of events involving gB and gH during HCMV entry into ECs. An HCMV strain bearing the wild-type (wt) UL131-128 locus exhibited a gB transition from a protease-resistant to protease-sensitive form, a conformational change that was suppressed by a thiourea inhibitor of fusion (WY1768); in contrast, gH was susceptible to proteolysis throughout entry. Moreover, gB and gH transiently interacted, and a lipid mixing assay showed that the wt strain had carried out fusion by 60 min postinfection. However, these events were greatly altered when UL131-128-defective strains were used for infection or when there was an excess of soluble pUL128 during wt infection: the gB conformational change became WY1768 resistant, the gB-gH complex was no longer observed, and fusion was prevented. Both gB and gH in this case showed late protease resistance, related to their endocytic uptake. Our data point to the involvement of UL131-128 proteins in driving gB through a WY1768-sensitive fold transition, thus promoting a short-lived gB-gH complex and fusion; they also suggest that HCMV fuses with the EC plasma membrane and that endocytosis ensues only when the virus cannot trigger UL131-128-dependent steps.

scholarly journals The Klebsiella pneumoniae wabG Gene: Role inBiosynthesis of the Core Lipopolysaccharide andVirulence

2003 ◽  
Vol 185 (24) ◽  
pp. 7213-7221 ◽  
Author(s):  
Luis Izquierdo ◽  
Núria Coderch ◽  
Nuria Piqué ◽  
Emiliano Bedini ◽  
Maria Michela Corsaro ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Animal Models ◽  
Klebsiella Pneumoniae ◽  
Gene Cluster ◽  
Capsular Polysaccharide ◽  
Outer Core ◽  
Wild Type ◽  
The Core ◽  
Hydrophobic Compounds ◽  
Type Strains

ABSTRACT To determine the function of the wabG gene in the biosynthesis of the core lipopolysaccharide (LPS) of Klebsiella pneumoniae, we constructed wabG nonpolar mutants. Data obtained from the comparative chemical and structural analysis of LPS samples obtained from the wild type, the mutant strain, and the complemented mutant demonstrated that the wabG gene is involved in attachment toα -l-glycero-d-manno-heptopyranose II (l,d-HeppII) at the O-3 position of anα -d-galactopyranosyluronic acid (α-d-GalAp) residue. K. pneumoniae nonpolar wabG mutants were devoid of the cell-attached capsular polysaccharide but were still able to produce capsular polysaccharide. Similar results were obtained with K. pneumoniae nonpolar waaC and waaF mutants, which produce shorter LPS core molecules than do wabG mutants. Other outer core K. pneumoniae nonpolar mutants in the waa gene cluster were encapsulated. K. pneumoniae waaC, waaF, and wabG mutants were avirulent when tested in different animal models. Furthermore, these mutants were more sensitive to some hydrophobic compounds than the wild-type strains. All these characteristics were rescued by reintroduction of the waaC, waaF, and wabG genes from K. pneumoniae.

scholarly journals Genetic Inactivation of an Extracellular Cysteine Protease (SpeB) Expressed by Streptococcus pyogenes Decreases Resistance to Phagocytosis and Dissemination to Organs

1998 ◽  
Vol 66 (2) ◽  
pp. 771-776 ◽  
Author(s):  
Slawomir Lukomski ◽  
Eugene H. Burns ◽  
Philip R. Wyde ◽  
Andreas Podbielski ◽  
Jacqueline Rurangirwa ◽  
...  
Keyword(s):  
Streptococcus Pyogenes ◽  
Cysteine Protease ◽  
Inflammatory Responses ◽  
Wild Type ◽  
Genetic Inactivation ◽  
Streptococcal Pyrogenic Exotoxin B ◽  
Mutant Derivative ◽  
Important Virulence Factor ◽  
Type Strains ◽  
Insight Into

ABSTRACT Streptococcal pyrogenic exotoxin B (SpeB), a conserved cysteine protease expressed by virtually all Streptococcus pyogenes strains, has recently been shown to be an important virulence factor (S. Lukomski, S. Sreevatsan, C. Amberg, W. Reichardt, M. Woischnik, A. Podbielski, and J. M. Musser, J. Clin. Invest. 99:2574–2580, 1997). Genetic inactivation of SpeB significantly decreased the lethality of a serotype M49 strain for mice and abolished the lethality of a serotype M3 strain after intraperitoneal (i.p.) injection. In the present study, a wild-type M3 isolate and an M3 speB mutant derivative were used to investigate the mechanism responsible for altered virulence. Following i.p. injection, the mutant and wild-type strains induced virtually identical cellular inflammatory responses, characterized largely by an influx of polymorphonuclear leukocytes (PMNs). In addition, the mutant and wild-type strains rapidly entered the blood and were recovered from all organs examined. However, significantly fewer (P < 0.05) CFUs of the isogenic mutant derivative than of the wild-type parent strain were recovered from blood and organs. PMNs effectively cleared the M3 speB mutant from the peritoneum by 22 h, thereby sparing the host. In contrast, the wild-type M3 strain continued to replicate intraperitoneally and had the ability to kill phagocytes. This process allowed the wild-type strain to continuously disseminate, resulting in host death. Our results indicate that genetic inactivation of the cysteine protease decreased the resistance of the mutant to phagocytosis and impaired its subsequent dissemination to organs. These results provide insight into the detrimental effect of SpeB inactivation on virulence.

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