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 A Deep-Rough Mutant of Campylobacter jejuni 81-176 Is Noninvasive for Intestinal Epithelial Cells

2004 ◽  
Vol 72 (4) ◽  
pp. 2452-2455 ◽  
Author(s):  
Margaret I. Kanipes ◽  
Lindsay C. Holder ◽  
Adrian T. Corcoran ◽  
Anthony P. Moran ◽  
Patricia Guerry
Keyword(s):  
Epithelial Cells ◽  
Campylobacter Jejuni ◽  
Type Strain ◽  
Wild Type Strain ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Wild Type ◽  
Increased Sensitivity ◽  
Rough Mutant

ABSTRACT A waaF mutant of Campylobacter jejuni 81-176 showed decreased invasion of INT407 cells in vitro and increased sensitivity to some antibiotics compared to what was seen with the wild-type strain.

scholarly journals Evidence for a Type III Secretion System in Aeromonas salmonicida subsp. salmonicida

2002 ◽  
Vol 184 (21) ◽  
pp. 5966-5970 ◽  
Author(s):  
Sarah E. Burr ◽  
Katja Stuber ◽  
Thomas Wahli ◽  
Joachim Frey
Keyword(s):  
Type Iii Secretion ◽  
Type Strain ◽  
Wild Type Strain ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Aeromonas Salmonicida ◽  
Broad Host Range ◽  
Wild Type ◽  
Secretion Systems ◽  
Type Iii

ABSTRACT Aeromonas salmonicida subsp. salmonicida, the etiological agent of furunculosis, is an important fish pathogen. We have screened this bacterium with a broad-host-range probe directed against yscV, the gene that encodes the archetype of a highly conserved family of inner membrane proteins found in every known type III secretion system. This has led to the identification of seven open reading frames that encode homologues to proteins functioning within the type III secretion systems of Yersinia species. Six of these proteins are encoded by genes comprising a virA operon. The A. salmonicida subsp. salmonicida yscV homologue, ascV, was inactivated by marker replacement mutagenesis and used to generate an isogenic ascV mutant. Comparison of the extracellular protein profiles from the ascV mutant and the wild-type strain indicates that A. salmonicida subsp. salmonicida secretes proteins via a type III secretion system. The recently identified ADP-ribosylating toxin AexT was identified as one such protein. Finally, we have compared the toxicities of the wild-type A. salmonicida subsp. salmonicida strain and the ascV mutant against RTG-2 rainbow trout gonad cells. While infection with the wild-type strain results in significant morphological changes, including cell rounding, infection with the ascV mutant has no toxic effect, indicating that the type III secretion system we have identified plays an important role in the virulence of this pathogen.

scholarly journals Evidence for HrpXo-Dependent Expression of Type II Secretory Proteins in Xanthomonas oryzae pv. oryzae

2004 ◽  
Vol 186 (5) ◽  
pp. 1374-1380 ◽  
Author(s):  
Ayako Furutani ◽  
Seiji Tsuge ◽  
Kouhei Ohnishi ◽  
Yasufumi Hikichi ◽  
Takashi Oku ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Gene ◽  
Secretion System ◽  
Xanthomonas Oryzae ◽  
Nucleotide Sequence Analysis ◽  
Secretory Proteins ◽  
Type Ii ◽  
Wild Type ◽  
Type Iii

ABSTRACT Xanthomonas oryzae pv. oryzae is a causal agent of bacterial leaf blight of rice. Recently, an efficient hrp-inducing medium, XOM2, was established for this bacterium. In this medium, more than 10 proteins were secreted from the wild-type strain of X. oryzae pv. oryzae. Many of these proteins disappeared or decreased in amount in culture on XOM2 when incubated with the strain that has a mutation in the hrp regulatory gene. Interestingly, the secretory protein profile of a mutant lacking a type III secretion system (TTSS), components of which are encoded by hrp genes, was similar to that of the wild-type strain except that a few proteins had disappeared. This finding suggests that many HrpXo-dependent secretory proteins are secreted via systems other than the TTSS. By isolating mutant strains lacking a type II secretion system, we examined this hypothesis. As expected, many of the HrpXo-dependent secretory proteins disappeared or decreased when the mutant was cultured in XOM2. By determining the N-terminal amino acid sequence, we identified one of the type II secretory proteins as a cysteine protease homolog, CysP2. Nucleotide sequence analysis revealed that cysP2 has an imperfect plant-inducible-promoter box, a consensus sequence which HrpXo regulons possess in the promoter region, and a deduced signal peptide sequence at the N terminus. By reverse transcription-PCR analysis and examination of the expression of CysP2 by using a plasmid harboring a cysP2::gus fusion gene, HrpXo-dependent expression of CysP2 was confirmed. Here, we reveal that the hrp regulatory gene hrpXo is also involved in the expression of not only hrp genes and type III secretory proteins but also some type II secretory proteins.

scholarly journals Salmonella enterica subspecies enterica serovar Enteritidis Salmonella pathogenicity island 2 type III secretion system: role in intestinal colonization of chickens and systemic spread

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2770-2781 ◽  
Author(s):  
Amanda L. S. Wisner ◽  
Taseen S. Desin ◽  
Birgit Koch ◽  
Po-King S. Lam ◽  
Emil M. Berberov ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Salmonella Enterica ◽  
Type Strain ◽  
Wild Type Strain ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Host Cells ◽  
Wild Type ◽  
Type Iii ◽  
Systemic Spread

Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) has been identified as a significant cause of salmonellosis in humans. Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) each encode a specialized type III secretion system (T3SS) that enables Salmonella to manipulate host cells at various stages of the invasion/infection process. For the purposes of our studies we used a chicken isolate of S. Enteritidis (Sal18). In one study, we orally co-challenged 35-day-old specific pathogen-free (SPF) chickens with two bacterial strains per group. The control group received two versions of the wild-type strain Sal18: Sal18 attTn7 : : tet and Sal18 attTn7 : : cat, while the other two groups received the wild-type strain (Sal18 attTn7 : : tet) and one of two mutant strains. From this study, we concluded that S. Enteritidis strains deficient in the SPI-1 and SPI-2 systems were outcompeted by the wild-type strain. In a second study, groups of SPF chickens were challenged at 1 week of age with four different strains: the wild-type strain, and three other strains lacking either one or both of the SPI-1 and SPI-2 regions. On days 1 and 2 post-challenge, we observed a reduced systemic spread of the SPI-2 mutants, but by day 3, the systemic distribution levels of the mutants matched that of the wild-type strain. Based on these two studies, we conclude that the S. Enteritidis SPI-2 T3SS facilitates invasion and systemic spread in chickens, although alternative mechanisms for these processes appear to exist.

scholarly journals Role of Lung Epithelial Cells in Defense against Klebsiella pneumoniae Pneumonia

2002 ◽  
Vol 70 (3) ◽  
pp. 1075-1080 ◽  
Author(s):  
Guadalupe Cortés ◽  
Dolores Álvarez ◽  
Carles Saus ◽  
Sebastián Albertí
Keyword(s):  
Epithelial Cells ◽  
Klebsiella Pneumoniae ◽  
Type Strain ◽  
Pathogenic Bacteria ◽  
Wild Type Strain ◽  
Capsular Polysaccharide ◽  
Defense Mechanism ◽  
Lung Epithelial Cells ◽  
Wild Type ◽  
Lung Epithelial

ABSTRACT The airway epithelium represents a primary site for the entry of pathogenic bacteria into the lungs. It has been suggested for many respiratory pathogens, including Klebsiella pneumoniae, that adhesion and invasion of the lung epithelial cells is an early stage of the pneumonia process. We observed that poorly encapsulated K. pneumoniae clinical isolates and an isogenic unencapsulated mutant invaded lung epithelial cells more efficiently than highly encapsulated strains independent of the K type. By contrast, the unencapsulated mutant was completely avirulent in a mouse model of pneumonia, unlike the wild-type strain, which produced pneumonia and systemic infection. Furthermore, the unencapsulated mutant bound more epithelially produced complement component C3 than the wild-type strain. Our results show that lung epithelial cells play a key role as a host defense mechanism against K. pneumoniae pneumonia, using two different strategies: (i) ingestion and control of the microorganisms and (ii) opsonization of the microorganisms. Capsular polysaccharide avoids both mechanisms and enhances the virulence of K. pneumoniae.

scholarly journals The Stringent Response Is Required for Amino Acid and Nitrate Utilization, Nod Factor Regulation, Nodulation, and Nitrogen Fixation in Rhizobium etli

2005 ◽  
Vol 187 (15) ◽  
pp. 5075-5083 ◽  
Author(s):  
Arturo Calderón-Flores ◽  
Gisela Du Pont ◽  
Alejandro Huerta-Saquero ◽  
Horacio Merchant-Larios ◽  
Luis Servín-González ◽  
...  
Keyword(s):  
Amino Acids ◽  
Type Strain ◽  
Wild Type Strain ◽  
Stringent Response ◽  
Insertion Mutant ◽  
Rhizobium Etli ◽  
Wild Type ◽  
Nod Factor ◽  
Truncated Protein ◽  
Carbon And Nitrogen

ABSTRACT A Rhizobium etli Tn5 insertion mutant, LM01, was selected for its inability to use glutamine as the sole carbon and nitrogen source. The Tn5 insertion in LM01 was localized to the rsh gene, which encodes a member of the RelA/SpoT family of proteins. The LM01 mutant was affected in the ability to use amino acids and nitrate as nitrogen sources and was unable to accumulate (p)ppGpp when grown under carbon and nitrogen starvation, as opposed to the wild-type strain, which accumulated (p)ppGpp under these conditions. The R. etli rsh gene was found to restore (p)ppGpp accumulation to a ΔrelA ΔspoT mutant of Escherichia coli. The R. etli Rsh protein consists of 744 amino acids, and the Tn5 insertion in LM01 results in the synthesis of a truncated protein of 329 amino acids; complementation experiments indicate that this truncated protein is still capable of (p)ppGpp hydrolysis. A second rsh mutant of R. etli, strain AC1, was constructed by inserting an Ω element at the beginning of the rsh gene, resulting in a null allele. Both AC1 and LM01 were affected in Nod factor production, which was constitutive in both strains, and in nodulation; nodules produced by the rsh mutants in Phaseolus vulgaris were smaller than those produced by the wild-type strain and did not fix nitrogen. In addition, electron microscopy revealed that the mutant bacteroids lacked poly-β-hydroxybutyrate granules. These results indicate a central role for the stringent response in symbiosis.

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 Mutation in the peb1A Locus ofCampylobacter jejuni Reduces Interactions with Epithelial Cells and Intestinal Colonization of Mice

1998 ◽  
Vol 66 (3) ◽  
pp. 938-943 ◽  
Author(s):  
Zhiheng Pei ◽  
Christophe Burucoa ◽  
Bernadette Grignon ◽  
Shahida Baqar ◽  
Xiao-Zhe Huang ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Type Strain ◽  
Wild Type Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Kanamycin Resistance ◽  
Intestinal Colonization ◽  
Wild Type ◽  
Cell Binding ◽  
Binding Factor

ABSTRACT Campylobacter jejuni is one of the leading causes of bacterial diarrhea throughout the world. We previously found that PEB1 is a homolog of cluster 3 binding proteins of bacterial ABC transporters and that a C. jejuni adhesin, cell-binding factor 1 (CBF1), if not identical to, contains PEB1. A single protein migrating at approximately 27 to 28 kDa was recognized by anti-CBF1 and anti-PEB1. To determine the role that the operon encoding PEB1 plays inC. jejuni adherence, peb1A, the gene encoding PEB1, was disrupted in strain 81-176 by insertion of a kanamycin resistance gene through homologous recombination. Inactivation of this operon completely abolished expression of CBF1, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. In comparison to the wild-type strain, the mutant strain showed 50- to 100-fold less adherence to and 15-fold less invasion of epithelial cells in culture. Mouse challenge studies showed that the rate and duration of intestinal colonization by the mutant were significantly lower and shorter than with the wild-type strain. In summary, PEB1 is identical to a previously identified cell-binding factor, CBF1, in C. jejuni, and the peb1A locus plays an important role in epithelial cell interactions and in intestinal colonization in a mouse model.

scholarly journals Spa32 Regulates a Switch in Substrate Specificity of the Type III Secreton of Shigella flexneri from Needle Components to Ipa Proteins

2002 ◽  
Vol 184 (13) ◽  
pp. 3433-3441 ◽  
Author(s):  
Juana Magdalena ◽  
Abderrahman Hachani ◽  
Mustapha Chamekh ◽  
Noureddine Jouihri ◽  
Pierre Gounon ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Congo Red ◽  
Type Strain ◽  
Wild Type Strain ◽  
Physical Contact ◽  
Target Cells ◽  
Virulence Plasmid ◽  
Wild Type ◽  
Secretion Systems ◽  
Type Iii

ABSTRACT Type III secretion systems (TTSS) are essential virulence determinants of many gram-negative bacteria and serve, upon physical contact with target cells, to translocate bacterial proteins directly across eukaryotic cell membranes. The Shigella TTSS is encoded by the mxi/spa loci located on its virulence plasmid. By electron microscopy secretons are visualized as tripartite with an external needle, a transmembrane domain, and a cytoplasmic bulb. In the present study, we generated a Shigella spa32 mutant and studied its phenotype. The spa32 gene shows low sequence homology to Salmonella TTSS1 invJ/spaN and to flagellar fliK. The spa32 mutant, like the wild-type strain, secreted the Ipas and IpgD, which are normally secreted via the TTSS, at low levels into the growth medium. However, unlike the wild-type strain, the spa32 mutant could neither be induced to secrete the Ipas and IpgD instantaneously upon addition of Congo red nor penetrate HeLa cells in vitro. Additionally, the Spa32 protein is secreted in large amounts by the TTSS during exponential growth but not upon Congo red induction. Interestingly, electron microscopy analysis of the spa32 mutant revealed that the needle of its secretons were up to 10 times longer than those of the wild type. In addition, in the absence of induction, the spa32 mutant secreted normal levels of MxiI but a large excess of MxiH. Taken together, our data indicate that the spa32 mutant presents a novel phenotype and that the primary defect of the mutant may be its inability to regulate or control secretion of MxiH.

Effects of Acuc on the Growth Development and Spinosad Biosynthesis of Saccharopolyspora Spinosa

2021 ◽  
Author(s):  
Zhudong Liu ◽  
Jie Xiao ◽  
Jianli Tang ◽  
Yang Liu ◽  
Ling Shuai ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Type Strain ◽  
Wild Type Strain ◽  
Shuttle Vector ◽  
Type I ◽  
Wild Type ◽  
Saccharopolyspora Spinosa ◽  
Fermentation Products ◽  
Overexpression Strain ◽  
Mutant Strains

Abstract Background: The interaction between acuC and spinosad biosynthesis is complex. In this study, acetoin utilization protein (acuC) was characterized. It is a type I histone deacetylase that is highly conserved in bacteria. This study first explored the effect of acuC on the growth and development of secondary metabolites of S. spinosa. Results: The knockout strain and overexpression strain were constructed separately with the shuttle vector pOJ260. The overexpression of the acuC gene affects the growth and phenotype of S. spinosa. Moreover, the spore production ability of the S. spinosa-acuC strain on solid medium was weaker than that of the wild-type strain. HPLC analysis of the fermentation products for the wild-type and mutant strains demonstrated that the yield of the overexpression strain was 87% higher than that of the wild-type strain. Conclusions: We concluded that the overexpression of acuC positively regulated the biosynthesis of spinosad and affected the acetylation pathway and the growth of S. spinosa. A comparative proteomic analysis between the wild-type and overexpression strains revealed related genes in different metabolic pathways that were affected. We envision that these results can be extended to other actinomycetes for secondary metabolite improvement.

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