scholarly journals A Second Aggregatibacter actinomycetemcomitans Autotransporter Adhesin Exhibits Specificity for Buccal Epithelial Cells in Humans and Old World Primates

2007 ◽  
Vol 75 (9) ◽  
pp. 4440-4448 ◽  
Author(s):  
Gang Yue ◽  
Jeffrey B. Kaplan ◽  
David Furgang ◽  
Keith G. Mansfield ◽  
Daniel H. Fine
Keyword(s):  
Epithelial Cells ◽  
Double Mutant ◽  
Aggregatibacter Actinomycetemcomitans ◽  
Species Specificity ◽  
Targeted Mutagenesis ◽  
Tissue Tropism ◽  
Dependent Manner ◽  
Wild Type ◽  
Old World ◽  
Buccal Epithelial Cells

ABSTRACT Previous work showed that the Aggregatibacter actinomycetemcomitans adhesin Aae demonstrated species specificity and tissue tropism to buccal epithelial cells (BECs) derived from humans and Old World primates, but a second, lower-affinity adhesin was noted. This study was designed to determine if Omp100 (also known as ApiA), a surface-expressed A. actinomycetemcomitans adhesin, is that second adhesin and if so to investigate its tissue tropism and species specificity. A targeted mutagenesis protocol was used to construct an isogenic apiA mutant and an aae apiA double mutant with wild-type A. actinomycetemcomitans. In addition, Escherichia coli strain DH5α was used to express apiA to further assess binding parameters. Results indicated that the apiA mutant strain showed significantly less binding to BECs than its parent strain (P ≤ 0.05). Further, binding mediated by ApiA was specific to BECs from humans and Old World primates, as seen in both wild-type A. actinomycetemcomitans and E. coli expressing ApiA (P ≤ 0.05). Pretreatment of wild-type A. actinomycetemcomitans cells with anti-ApiA antiserum reduced binding in a dose-dependent manner. The aae apiA double mutant completely abrogated A. actinomycetemcomitans binding to both human and Old World primate BECs. Taken together, these studies indicate that ApiA and Aae, in concert, modulate binding of A. actinomycetemcomitans to human BECs. Since the BEC is a prominent reservoir for A. actinomycetemcomitans, identification of this second adhesin could lead to important therapeutic strategies.

scholarly journals The Actinobacillus actinomycetemcomitans Autotransporter Adhesin Aae Exhibits Specificity for Buccal Epithelial Cells from Humans and Old World Primates

2005 ◽  
Vol 73 (4) ◽  
pp. 1947-1953 ◽  
Author(s):  
Daniel H. Fine ◽  
Kabilan Velliyagounder ◽  
David Furgang ◽  
Jeffrey B. Kaplan
Keyword(s):  
Epithelial Cells ◽  
Host Range ◽  
Mutant Strain ◽  
Actinobacillus Actinomycetemcomitans ◽  
Broad Host Range ◽  
Wild Type ◽  
New World Primates ◽  
Old World ◽  
Broad Host Range Plasmid ◽  
Buccal Epithelial Cells

ABSTRACT Cells of the gram-negative periodontopathogen Actinobacillus actinomycetemcomitans express a surface-exposed, outer membrane autotransporter protein, designated Aae, which has been implicated in epithelial cell binding. We constructed a mutant strain of A. actinomycetemcomitans that contained a transposon insertion in the Aae structural gene (aae) and tested the mutant to determine its ability to bind to buccal epithelial cells (BECs) isolated from healthy volunteers. Significantly fewer mutant cells than wild-type cells bound to BECs. A broad-host-range plasmid that contained an intact aae gene driven by a heterologous tac promoter restored the ability of the mutant strain to bind to BECs at wild-type levels. This plasmid also conferred upon Escherichia coli the ability to express the Aae protein on its surface and to bind to human BECs. Aae-expressing E. coli also bound to BECs isolated from six Old World primates but not to BECs isolated from four New World primates or nine other nonprimate mammals, as well as to human gingival epithelial cells but not to human pharyngeal, palatal, tongue, bronchial, or cervical epithelial cells. Our findings indicate that Aae mediates binding of A. actinomycetemcomitans to BECs from humans and Old World primates and that this process may contribute to the host range specificity and tissue tropism exhibited by this bacterium.

scholarly journals Evaluation of the effects of sdiA, a luxR homologue, on adherence and motility of Escherichia coli O157 : H7

Microbiology ◽  
2010 ◽  
Vol 156 (5) ◽  
pp. 1303-1312 ◽  
Author(s):  
Vijay K. Sharma ◽  
Shawn M. D. Bearson ◽  
Bradley L. Bearson
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Mutant Strain ◽  
Regulatory Networks ◽  
Double Mutant ◽  
Negative Regulator ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Expression Of Genes ◽  
Genes Encoding

Quorum-sensing (QS) signalling pathways are important regulatory networks for controlling the expression of genes promoting adherence of enterohaemorrhagic Escherichia coli (EHEC) O157 : H7 to epithelial cells. A recent study has shown that EHEC O157 : H7 encodes a luxR homologue, called sdiA, which upon overexpression reduces the expression of genes encoding flagellar and locus of enterocyte effacement (LEE) proteins, thus negatively impacting on the motility and intimate adherence phenotypes, respectively. Here, we show that the deletion of sdiA from EHEC O157 : H7 strain 86-24, and from a hha (a negative regulator of ler) mutant of this strain, enhanced bacterial adherence to HEp-2 epithelial cells of the sdiA mutant strains relative to the strains containing a wild-type copy of sdiA. Quantitative reverse transcription PCR showed that the expression of LEE-encoded genes ler, espA and eae in strains with the sdiA deletions was not significantly different from that of the strains wild-type for sdiA. Similarly, no additional increases in the expression of LEE genes were observed in a sdiA hha double mutant strain relative to that observed in the hha deletion mutant. While the expression of fliC, which encodes flagellin, was enhanced in the sdiA mutant strain, the expression of fliC was reduced by several fold in the hha mutant strain, irrespective of the presence or absence of sdiA, indicating that the genes sdiA and hha exert opposing effects on the expression of fliC. The strains with deletions in sdiA or hha showed enhanced expression of csgA, encoding curlin of the curli fimbriae, with the expression of csgA highest in the sdiA hha double mutant, suggesting an additive effect of these two gene deletions on the expression of csgA. No significant differences were observed in the expression of the genes lpfA and fimA of the operons encoding long polar and type 1 fimbriae in the sdiA mutant strain. These data indicate that SdiA has no significant effect on the expression of LEE genes, but that it appears to act as a strong repressor of genes encoding flagella and curli fimbriae, and the alleviation of the SdiA-mediated repression of these genes in an EHEC O157 : H7 sdiA mutant strain contributes to enhanced bacterial motility and increased adherence to HEp-2 epithelial cells.

scholarly journals Lysophosphatidic acid receptor 1 modulates lipopolysaccharide-induced inflammation in alveolar epithelial cells and murine lungs

2011 ◽  
Vol 301 (4) ◽  
pp. L547-L556 ◽  
Author(s):  
Jing Zhao ◽  
Donghong He ◽  
Yanlin Su ◽  
Evgeny Berdyshev ◽  
Jerold Chun ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Lysophosphatidic Acid ◽  
Lung Inflammation ◽  
Lung Epithelial Cells ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Wild Type ◽  
Lpa Receptor ◽  
Knock Down ◽  
Lpa Receptors

Lysophosphatidic acid (LPA), a bioactive phospholipid, plays an important role in lung inflammation by inducing the release of chemokines and lipid mediators. Our previous studies have shown that LPA induces the secretion of interleukin-8 and prostaglandin E2 in lung epithelial cells. Here, we demonstrate that LPA receptors contribute to lipopolysaccharide (LPS)-induced inflammation. Pretreatment with LPA receptor antagonist Ki16425 or downregulation of LPA receptor 1 (LPA1) by small-interfering RNA (siRNA) attenuated LPS-induced phosphorylation of p38 MAPK, I-κB kinase, and I-κB in MLE12 epithelial cells. In addition, the blocking of LPA1 also suppressed LPS-induced IL-6 production. Furthermore, LPS treatment promoted interaction between LPA1 and CD14, a LPS coreceptor, in a time- and dose-dependent manner. Disruption of lipid rafts attenuated the interaction between LPA1 and CD14. Mice challenged with LPS increased plasma LPA levels and enhanced expression of LPA receptors in lung tissues. To further investigate the role of LPA receptors in LPS-induced inflammation, wild-type, or LPA1-deficient mice, or wild-type mice pretreated with Ki16425 were intratracheally challenged with LPS for 24 h. Knock down or inhibition of LPA1 decreased LPS-induced IL-6 release in bronchoalveolar lavage (BAL) fluids and infiltration of cells into alveolar space compared with wild-type mice. However, no significant differences in total protein concentration in BAL fluids were observed. These results showed that knock down or inhibition of LPA1 offered significant protection against LPS-induced lung inflammation but not against pulmonary leak as observed in the murine model for lung injury.

scholarly journals Helicobacter pylori perceives the quorum-sensing molecule AI-2 as a chemorepellent via the chemoreceptor TlpB

Microbiology ◽  
2011 ◽  
Vol 157 (9) ◽  
pp. 2445-2455 ◽  
Author(s):  
Bethany A. Rader ◽  
Christopher Wreden ◽  
Kevin G. Hicks ◽  
Emily Goers Sweeney ◽  
Karen M. Ottemann ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Double Mutant ◽  
Soft Agar ◽  
Dependent Manner ◽  
Wild Type ◽  
Environmental Chemical ◽  
Single Mutant ◽  
Autoinducer 2 ◽  
Reduced Frequency ◽  
H Pylori

Helicobacter pylori moves in response to environmental chemical cues using a chemotaxis two-component signal-transduction system. Autoinducer-2 (AI-2) is a quorum-sensing signal produced by the LuxS protein that accumulates in the bacterial environment in a density-dependent manner. We showed previously that a H. pylori luxS mutant was defective in motility on soft agar plates. Here we report that deletion of the luxS gene resulted in swimming behaviour with a reduced frequency of stops as compared to the wild-type strain. Stopping frequency was restored to wild-type levels by genetic complementation of the luxS mutation or by addition of synthetic 4,5-dihydroxy-2,3-pentanedione (DPD), which cyclizes to form AI-2. Synthetic DPD also increased the frequency of stops in wild-type H. pylori, similar to the behaviour induced by the known chemorepellent HCl. We found that whereas mutants lacking the chemoreceptor genes tlpA, tlpC or tlpD responded to an exogenous source of synthetic DPD, the chemoreceptor mutant tlpB was non-responsive to a gradient or uniform distribution of the chemical. Furthermore, a double mutant lacking both tlpB and luxS exhibited chemotactic behaviour similar to the tlpB single mutant, whereas a double mutant lacking both tlpB and the chemotransduction gene cheA behaved like a nonchemotactic cheA single mutant, supporting the model that tlpB functions in a signalling pathway downstream of luxS and upstream of cheA. We conclude that H. pylori perceives LuxS-produced AI-2 as a chemorepellent via the chemoreceptor TlpB.

scholarly journals Myc supercompetitor cells exploit the NMDA receptor to subdue their wild-type neighbours via cell competition

2020 ◽  
Author(s):  
Agnes R. Banreti ◽  
Pascal Meier
Keyword(s):  
Cell Death ◽  
Nmda Receptor ◽  
Cell Growth ◽  
Epithelial Cells ◽  
Fitness Landscape ◽  
Dependent Manner ◽  
Wild Type ◽  
Direct Inhibition ◽  
Cell Competition ◽  
Competitive Behaviour

SUMMARYMyc is a major driver of cell growth in many cancers, but direct inhibition of Myc’s oncogenic activity has been challenging. Interactions between wild-type and Myc-expressing cells cause Myc cells to acquire ‘supercompetitor’ behaviour that increases their fitness and enables them to overtake the tissue by killing their wild-type neighbours through TNF-induced cell death during a process called cell competition. Here we report that the competitive behaviour of Myc, RasV12 cells, and normal epithelial cells, critically depends on the NMDA receptor. Myc cells upregulate NMDAR2 (NR2) to gain supercompetitor status and subdue their wild-type neighbours. Pharmacological inhibition or genetic depletion of NR2 changes the supercompetitor status of oncogenic Myc or RasV12 clones into ‘superlosers’, resulting in their elimination via cell competition by wild-type neighbours in a TNF-dependent manner. Our data demonstrate that that the NMDA receptor (NMDAR) determines cellular fitness during cell competition, and can be targeted to change the fitness landscape of supercompetitive Myc and RasV12 clones, converting them into superlosers.

scholarly journals Mapping the epithelial-cell-binding domain of the Aggregatibacter actinomycetemcomitans autotransporter adhesin Aae

Microbiology ◽  
2010 ◽  
Vol 156 (11) ◽  
pp. 3412-3420 ◽  
Author(s):  
Daniel H. Fine ◽  
Jeffrey B. Kaplan ◽  
David Furgang ◽  
Maribasappa Karched ◽  
Kabilan Velliyagounder ◽  
...  
Keyword(s):  
Amino Acids ◽  
Epithelial Cells ◽  
Aggregatibacter Actinomycetemcomitans ◽  
Binding Domain ◽  
Full Length ◽  
Dependent Manner ◽  
Passenger Domain ◽  
E Coli ◽  
Pre Treatment ◽  
Dose Dependent

The Gram-negative periodontopathogen Aggregatibacter actinomycetemcomitans (Aa) binds selectively to buccal epithelial cells (BECs) of human and Old World primates by means of the outer-membrane autotransporter protein Aae. We speculated that the exposed N-terminal portion of the passenger domain of Aae would mediate binding to BECs. By using a series of plasmids that express full-length or truncated Aae proteins in Escherichia coli, we found that the BEC-binding domain of Aae was located in the N-terminal surface-exposed region of the protein, specifically in the region spanning amino acids 201–284 just upstream of the repeat region within the passenger domain. Peptides corresponding to amino acids 201–221, 222–238 and 201–240 were synthesized and tested for their ability to reduce Aae-mediated binding to BECs based on results obtained with truncated Aae proteins expressed in E. coli. BEC-binding of E. coli expressing Aae was reduced by as much as 50 % by pre-treatment of BECs with a 40-mer peptide (201–240; P40). Aae was also shown to mediate binding to cultured human epithelial keratinocytes (TW2.6), OBA9 and TERT, and endothelial (HUVEC) cells. Pre-treatment of epithelial cells with P40 resulted in a dose-dependent reduction in binding and reduced the binding of both full-length and truncated Aae proteins expressed in E. coli, as well as Aae expressed in Aa. Fluorescently labelled P40 peptides reacted in a dose-dependent manner with BEC receptors. We propose that these proof-of-principle experiments demonstrate that peptides can be designed to interfere with Aa binding mediated by host-cell receptors specific for Aae adhesins.

scholarly journals MgrA regulates interaction of Staphylococcus aureus with mucin

2020 ◽  
Author(s):  
Connor P. Parker ◽  
Nour Akil ◽  
Cullen R. Shanrock ◽  
Patrick D. Allen ◽  
Anna L. Chaly ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Epithelial Cells ◽  
Dose Range ◽  
Microtiter Plate ◽  
Bacterial Attachment ◽  
Surface Proteins ◽  
Respiratory Pathogen ◽  
Dependent Manner ◽  
Bacterial Cells ◽  
Wild Type

AbstractBackgroundTo defend the lungs, mucus adheres to bacterial cells and facilitates their removal by ciliary transport. Our goals were to measure the affinity of mucus for the respiratory pathogen Staphylococcus aureus and identify bacterial genes that regulate this interaction.MethodsS. aureus was added to pig tracheas to determine whether it binds mucus or epithelial cells. To quantify its affinity for mucus, we developed a competition assay in microtiter plates. Mucin was added over a dose range as an inhibitor of bacterial attachment. We then examined how transcriptional regulator MgrA and cell wall transpeptidase sortase (SrtA) affect bacterial interaction with mucin.ResultsIn pig tracheas, S. aureus bound mucus strands from submucosal glands more than epithelial cells. In microtiter plate assays, ΔsrtA failed to attach even in the absence of mucin. Mucin blocked wild type S. aureus attachment in a dose-dependent manner. Higher concentrations were needed to inhibit binding of ΔmgrA. Co-deletion of ebh and sraP, which encode surface proteins repressed by MgrA, suppressed the ΔmgrA binding phenotype. No differences between ΔmgrA and wild type were observed when methylcellulose or heparin sulfate were substituted for mucin, indicating specificity.ConclusionsMucin decreases attachment of S. aureus to plastic, consistent with its physiologic role in host defense. S. aureus deficient in MgrA has decreased affinity for mucin. Ebh and SraP, which are normally repressed by MgrA, may function as inhibitors of attachment to mucin. These data show that specific bacterial factors may regulate the interaction of S. aureus with mucus.

scholarly journals Phenotypic Effect of Isogenic uspA1 anduspA2 Mutations on Moraxella catarrhalis 035E

1998 ◽  
Vol 66 (7) ◽  
pp. 3113-3119 ◽  
Author(s):  
Christoph Aebi ◽  
Eric R. Lafontaine ◽  
Leslie D. Cope ◽  
Jo L. Latimer ◽  
Sheryl L. Lumbley ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Moraxella Catarrhalis ◽  
Parent Strain ◽  
Double Mutant ◽  
Wild Type ◽  
Phenotypic Effect ◽  
In Vitro Systems ◽  
Normal Human ◽  
Wild Type Parent

ABSTRACT The UspA surface antigen of Moraxella catarrhalis was recently shown to be comprised of two different proteins (UspA1 and UspA2) which share an internal region containing 140 amino acids with 93% identity (C. Aebi, I. Maciver, J. L. Latimer, L. D. Cope, M. K. Stevens, S. E. Thomas, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 65:4367–4377, 1997). Isogenic uspA1, uspA2, and uspA1 uspA2 mutants were tested in a number of in vitro systems to determine what effect these mutations, either individually or together, might exert on the phenotype of M. catarrhalis 035E. Monoclonal antibodies specific for UspA1 or UspA2 were used in an indirect antibody accessibility assay to prove that both of these proteins were expressed on the surface of M. catarrhalis. All three mutants grew in vitro at the same rate and did not exhibit autoagglutination or hemagglutination properties that were detectably different from those of the wild-type parent strain. When tested for the ability to adhere to human epithelial cells, the wild-type parent strain and the uspA2 mutant readily attached to Chang conjunctival cells. In contrast, theuspA1 mutant and the uspA1 uspA2 double mutant both attached to these epithelial cells at a level nearly 2 orders of magnitude lower than that obtained with the wild-type parent strain, a result which suggested that expression of UspA1 byM. catarrhalis is essential for attachment to these epithelial cells. Both the wild-type parent strain and theuspA1 mutant were resistant to the bactericidal activity of normal human serum, whereas the uspA2 mutant and theuspA1 uspA2 double mutant were readily killed by this serum. This latter result indicated that the presence of UspA2 is essential for expression of serum resistance by M. catarrhalis.

scholarly journals The Pseudomonas aeruginosa PhoP-PhoQ Two-Component Regulatory System Is Induced upon Interaction with Epithelial Cells and Controls Cytotoxicity and Inflammation

2012 ◽  
Vol 80 (9) ◽  
pp. 3122-3131 ◽  
Author(s):  
Shaan L. Gellatly ◽  
Brittany Needham ◽  
Laurence Madera ◽  
M. Stephen Trent ◽  
Robert E. W. Hancock
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Lipid A ◽  
Regulatory System ◽  
Dependent Manner ◽  
Wild Type ◽  
Content Type ◽  
Regulatory Systems ◽  
Two Component ◽  
Inflammatory Phenotype

ABSTRACTThe adaptation ofPseudomonas aeruginosato its environment, including the host, is tightly controlled by its network of regulatory systems. The two-component regulatory system PhoPQ has been shown to play a role in the virulence and polymyxin resistance ofP. aeruginosaas well as several other Gram-negative species. Dysregulation of this system has been demonstrated in clinical isolates, yet how it affects virulence ofP. aeruginosais unknown. To investigate this, an assay was used whereby bacteria were cocultured with human bronchial epithelial cells. The interaction of wild-type (WT) bacteria that had adhered to epithelial cells led to a large upregulation of the expression of theoprH-phoP-phoQoperon and its target, thearnlipopolysaccharide (LPS) modification operon, in a PhoQ-dependent manner, compared to cells in the supernatant that had failed to adhere. Relative to the wild type, aphoQmutant cocultured on epithelial cells produced less secreted protease and lipase and, like thephoQmutant,piv,lipH, andlasBmutants demonstrated reduced cytotoxicity toward epithelial cells. Mutation inphoQalso resulted in alterations to lipid A and to increased inflammatory LPS. These data indicate that mutation ofphoQresults in a phenotype that is similar to the less virulent but more inflammatory phenotype of clinical strains isolated from chronic-stage cystic fibrosis lung infections.

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