CEACAM1 recognition by bacterial pathogens is species-specific

<p>Abstract</p> <p>Background</p> <p>Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), an immunoglobulin (Ig)-related glycoprotein, serves as cellular receptor for a variety of Gram-negative bacterial pathogens associated with the human mucosa. In particular, <it>Neisseria gonorrhoeae</it>, <it>N. meningitidis</it>, <it>Moraxella catarrhalis</it>, and <it>Haemophilus influenzae </it>possess well-characterized CEACAM1-binding adhesins. CEACAM1 is typically involved in cell-cell attachment, epithelial differentiation, neovascularisation and regulation of T-cell proliferation, and is one of the few CEACAM family members with homologues in different mammalian lineages. However, it is unknown whether bacterial adhesins of human pathogens can recognize CEACAM1 orthologues from other mammals.</p> <p>Results</p> <p>Sequence comparisons of the amino-terminal Ig-variable-like domain of CEACAM1 reveal that the highest sequence divergence between human, murine, canine and bovine orthologues is found in the β-strands comprising the bacteria-binding CC'FG-face of the Ig-fold. Using GFP-tagged, soluble amino-terminal domains of CEACAM1, we demonstrate that bacterial pathogens selectively associate with human, but not other mammalian CEACAM1 orthologues. Whereas full-length human CEACAM1 can mediate internalization of <it>Neisseria gonorrhoeae </it>in transfected cells, murine CEACAM1 fails to support bacterial internalization, demonstrating that the sequence divergence of CEACAM1 orthologues has functional consequences with regard to bacterial recognition and cellular invasion.</p> <p>Conclusions</p> <p>Our results establish the selective interaction of several human-restricted bacterial pathogens with human CEACAM1 and suggest that co-evolution of microbial adhesins with their corresponding receptors on mammalian cells contributes to the limited host range of these highly adapted infectious agents.</p

Extracellular IgC2 Constant Domains of CEACAMs Mediate PI3K Sensitivity during Uptake of Pathogens

Several pathogenic bacteria utilize receptors of the CEACAM family to attach to human cells. Binding to different members of this receptor family can result in uptake of the bacteria. Uptake of Neisseria gonorrhoeae, a gram-negative human pathogen, via CEACAMs found on epithelial cells, such as CEACAM1, CEA or CEACAM6, differs mechanistically from phagocytosis mediated by CEACAM3, a CEACAM family member expressed selectively by human granulocytes.We find that CEACAM1- as well as CEACAM3-mediated bacterial internalization are accompanied by a rapid increase in phosphatidylinositol-3,4,5 phosphate (PI(3,4,5)P) at the site of bacterial entry. However, pharmacological inhibition of phosphatidylinositol-3' kinase (PI3K) selectively affects CEACAM1-mediated uptake of Neisseria gonorrhoeae. Accordingly, overexpression of the PI(3,4,5)P phosphatase SHIP diminishes and expression of a constitutive active PI3K increases CEACAM1-mediated internalization of gonococci, without influencing uptake by CEACAM3. Furthermore, bacterial uptake by GPI-linked members of the CEACAM family (CEA and CEACAM6) and CEACAM1-mediated internalization of N. meningitidis by endothelial cells require PI3K activity. Sensitivity of CEACAM1-mediated uptake toward PI3K inhibition is independent of receptor localization in cholesterol-rich membrane microdomains and does not require the cytoplasmic or the transmembrane domain of CEACAM1. However, PI3K inhibitor sensitivity requires the Ig(C2)-like domains of CEACAM1, which are also present in CEA and CEACAM6, but which are absent from CEACAM3. Accordingly, overexpression of CEACAM1 Ig(C2) domains blocks CEACAM1-mediated internalization.Our results provide novel mechanistic insight into CEACAM1-mediated endocytosis and suggest that epithelial CEACAMs associate in cis with other membrane receptor(s) via their extracellular domains to trigger bacterial uptake in a PI3K-dependent manner

Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems

CRISPR/Cas9 technology is currently considered the most advanced tool for targeted genome engineering. Its sequence-dependent specificity has been explored for locus-directed transcriptional modulation. Such modulation, in particular transcriptional activation, has been proposed as key approach to overcome silencing of dormant HIV provirus in latently infected cellular reservoirs. Currently available agents for provirus activation, so-called latency reversing agents (LRAs), act indirectly through cellular pathways to induce viral transcription. However, their clinical performance remains suboptimal, possibly because reservoirs have diverse cellular identities and/or proviral DNA is intractable to the induced pathways. We have explored two CRISPR/Cas9-derived activator systems as targeted approaches to induce dormant HIV-1 proviral DNA. These systems recruit multiple transcriptional activation domains to the HIV 5'long terminal repeat (LTR), for which we have identified an optimal target region within the LTR U3 sequence. Using this target region, we demonstrate transcriptional activation of proviral genomes via the synergistic activation mediator complex in various in culture model systems for HIV latency. Observed levels of induction are comparable or indeed higher than treatment with established LRAs. Importantly, activation is complete, leading to production of infective viral particles. Our data demonstrate that CRISPR/Cas9-derived technologies can be applied to counteract HIV latency and may therefore represent promising novel approaches in the quest for HIV elimination

Internalization of pathogens in mammalian cells via CEACAM1

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) belongs to the immunoglobulin superfamily and has several physiological functions in the body, in-cluding cell-cell adhesion, angiogenesis, insulin metabolism, and regulation of immune cell functions. Within the CEACAM family, CEACAM1 has the widest tissue distribution and is expressed on endothelial, epithelial, and hematopoietic cells. It also serves as receptor for human specific pathogens like Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, Moraxella catarrhalis, and Escherichia coli together with CEACAM3, CEA and CEACAM6. These pathogens express distinct adhesins to associate with the CC´FG interface of the N-terminal domain of CEACAMs; in addition we could demonstrate the selective recognition of human CEACAMs, which presumably explains the absence of non-human hosts. Engagement of CEACAM1 by pathogens has several advantages, based on its physiological functions. Receptor binding leads also to internalization and transcytosis of the bacteria in mammalian cells, causing increased survival of the pathogens in the host. During invasion of the bacteria via the epithelial CEACAMs (CEACAM1, CEA and CEACAM6) the actin cytoskeleton plays only a minor role, but it is highly dependent on receptor relocalization into lipid rafts. Also, the cytoplasmic domain of CEACAM1 is not involved in uptake process. In contrast, bind-ing of the pathogens to the granulocyte specific CEACAM3 induces fast and highly efficient phagocytosis and killing of the bacteria, leading to the suggestion that CEACAM3 might function as immune receptor to get rid of these highly adapted pathogens. For CEACAM3-mediated uptake of pathogens, the uptake is regulated by phosphoryla-tion of two tyrosines in the ITAM-like motif of the cytoplasmic domain of CEACAM3 by Src kinases. The guanine nucleotide exchange factor Vav and the adaptor protein Nck are recruited to these phosphorylated tyrosines. Vav catalyzes the nucleotide exchange of the small GTPase Rac1, which activates the Nck-associated WAVE complex, inducing massive actin cytoskeleton rearrangements and efficient uptake of CEACAM3-bound pathogens. The activity of PI3Ks is not required during CEACAM3-mediated uptake of pathogens, which is surprising because of the involvement during Fcγ receptor mediated uptake in granulocytes. One of the major part of this dissertation was to examine the role of PI(3,4,5)P during pathogen uptake via the epithelial CEACAMs in comparison to CEACAM3. We could clearly demonstrate an important role of this phosphoinositide during the endocytotic process. Further investigations revealed the participation of the extracellular immunoglobulin constant type 2-like (IgC2-like) domains of the epithelial CEACAMs for PI3K dependent uptake of pathogens. This led us to the suggestion, that another membrane protein might be involved in the signaling process as co-receptor, responsible for signal transduction into the mammalian cell. Another important observation was the involvement of the small GTPase RhoG in endocytotic processes via epithelial CEACAMs in contrast to CEACAM3, regulating the uptake upstream of PI3K. Furthermore, we could identify Ephexin4 as responsible guanine nucleotide exchange factor for RhoG after CEACAM engagement by pathogens and we investigated the involvement of the known upstream regulator of Ephexin4, the receptor tyrosine kinase EphA2. Finally, we could clearly demonstrate the important role of EphA2 during CEACAM1-mediated endocytosis of pathogens, confirming our hypothesis of a possible co-receptor in this signaling process. To sum up, the data obtained from this study provide new mechanistic insight in the barley clarified pathogen uptake via epithelial CEACAMs and the involvement of another membrane protein together with CEACAM1 during uptake could be shown for the first time

Abolishing HIV-1 infectivity using a polypurine tract-specific G-quadruplex-forming oligonucleotide

BACKGROUND: HIV is primarily transmitted by sexual intercourse and predominantly infects people in Third World countries. Here an important medical need is self-protection for women, particularly in societies where condoms are not widely accepted. Therefore, availability of antiviral microbicides may significantly reduce sexual HIV transmission in such environments. METHODS: Here, we investigated structural characteristics and the antiviral activity of the polypurine tract (PPT)-specific ODN A, a 54-mer oligodeoxynucleotide (ODN) that has been previously shown to trigger the destruction of viral RNA genomes by prematurely activating the retroviral RNase H. The stability of ODN A and mutants thereof was tested at various storage conditions. Furthermore, antiviral effects of ODN A were analyzed in various tissue culture HIV-1 infection models. Finally, circular dichroism spectroscopy was employed to gain insight into the structure of ODN A. RESULTS: We show here that ODN A is a powerful tool to abolish HIV-1 particle infectivity, as required for a candidate compound in vaginal microbicide applications. We demonstrate that ODN A is not only capable to prematurely activate the retroviral RNase H, but also prevents HIV-1 from entering host cells. ODN A also exhibited extraordinary stability lasting several weeks. Notably, ODN A is biologically active under various storage conditions, as well as in the presence of carboxymethylcellulose CMC (K-Y Jelly), a potential carrier for application as a vaginal microbicide. ODN A’s remarkable thermostability is apparently due to its specific, guanosine-rich sequence. Interestingly, these residues can form G-quadruplexes and may lead to G-based DNA hyperstructures. Importantly, the pronounced antiviral activity of ODN A is maintained in the presence of human semen or semen-derived enhancer of virus infection (SEVI; i.e. amyloid fibrils), both known to enhance HIV infectivity and reduce the efficacy of some antiviral microbicides. CONCLUSIONS: Since ODN A efficiently inactivates HIV-1 and also displays high stability and resistance against semen, it combines unique and promising features for its further development as a vaginal microbicide against HIV

Bacterial uptake via several epithelial CEACAMs requires PI3K activity.

<p>(<b>A</b>) 293 cells were transfected with empty vector (pcDNA) or constructs encoding CEACAM1, CEA, or CEACAM6. CEACAM expression was confirmed by Western blotting of whole cell lysates (WCLs) with α-CEACAM antibody (upper panel). Equal loading of the samples was verified by α-tubulin blot (lower panel). (<b>B, C</b>) Cells transfected as in (A) were pretreated for 30 min with 200 nM wortmannin (WM) or left untreated. After infection for 2 h with Opa_CEA-expressing gonococci, total cell-associated bacteria (B) or recovered intracellular bacteria (C) were quantified. Bars represent mean ± SEM of three independent experiments done in triplicate (n = 9). Numbers are expressed relative to cells expressing the respective receptor without WM treatment. Significance was tested using an unpaired, two-sided Student's t-test; ***, p<0.001.</p

PI(3,4,5)P and PI(4,5)P are generated during CEACAM1- and 3- mediated bacterial entry.

<p>(<b>A</b>) 293 cells were cotransfected with constructs encoding mKate-tagged CEACAM1-4L or CEACAM3 together with GFP-tagged Btk-PH domain (which binds specifically to PI(3,4,5)P). Cells were left uninfected or were infected with Pacific Blue labelled Opa_CEA-expressing <i>N. gonorrhoeae</i> for 60 min, fixed and analysed by confocal microscopy. Arrowheads highlight bacteria associated with CEACAMs and the Btk-PH domain. Insets show enlargement of the highlighted area. Bars represent 5 µm. (<b>B</b>) Cells were cotransfected with CEACAM constructs as in A) together with the GFP-tagged PLC-δ-PH domain (which specifically binds to PI(4,5)P). Cells were infected and analyzed as in (A). Arrowheads highlight bacteria associated with CEACAMs and the PLC-δ-PH domain. Insets show enlargement of the highlighted area. Bars represent 5 µm.</p

Constitutive active PI3K increases, whereas expression of SHIP decreases uptake of Opa_CEA-expressing gonococci via CEACAM1.

<p>(<b>A</b>) 293 cells were cotransfected with constructs encoding CEACAM1-HA or CEACAM3-HA together with cerulean-tagged PI3K or cerulean alone. Cells were infected with fluorescein-labelled Opa_CEA- expressing gonococci for 2 h. Samples were analysed using a flow cytometer by gating on cerulean-positive cells. Dot blot shows a representative gate used to detect the cerulean-positive cell population. (<b>B</b>) The fluorescein signal derived from cerulean-positive cells in (A) was quantified in the presence of trypan blue, which quenches fluorescence derived from extracellular gonococci. Bars represent mean values ± S.E.M of a representative experiment done in triplicate. (<b>C–E</b>) 293 cells were cotransfected with CEACAM1-HA or CEACAM3-HA together with cerulean, cerulean-tagged SHIP1 phosphatase domain (SHIP), or an inactive form of the SHIP1 phosphatase domain (SHIP D675G), respectively. (C) Expression of cerulean, SHIP1, or SHIP1 D675G was analysed by flow cytometry and histograms show representative samples. (D and E) Cells were infected with fluorescein-stained Opa_CEA-expressing gonococci for 2 h and the fluorescein signal derived from cerulean-positive cells was detected in the absence (D) or presence (E) of trypan blue. This allows quantification of total cell-associated bacteria (D) or internalized gonococci (E). Bars represent mean values ± S.E.M of three independent experiments. Numbers are expressed relative to cells co-expressing cerulean and the respective receptor.</p

CEACAM1-mediated uptake of Opa_CEA-expressing <i>N. meningitidis</i> by endothelial cells depends on PI3K activity.

<p>(<b>A</b>) Human brain microvascular endothelial cells (HBMEC) were transduced with GFP-encoding control lentivirus (control) or a CEACAM1-GFP-encoding lentivirus. CEACAM1 expression in transduced HBMEC is analysed by Western blotting of whole cell lysates (WCL) using monoclonal α-CEACAM antibody (upper panel) and equal loading of the samples was demonstrated by α- tubulin antibody (lower panel). (<b>B</b>) Stable CEACAM1-GFP or control GFP expressing HBMECs were infected with biotin- and AlexaFluor647-labelled Opa_CEA-expressing meningococci for 60 min. Upon fixation and and staining of extracellular bacteria with streptavidin-rhodamine, samples were analyzed by confocal microscopy. Arrows highlight intracellular bacteria, whereas arrowheads point to extracellular bacteria. Bars represent 5 µm. (<b>C and D</b>) CEACAM1-GFP or GFP expressing HBMECs were pretreated with the indicated concentrations of wortmannin for 30 min or left untreated. Cells were infected for 3 h with Opa_CEA-expressing meningococci and total cell-associated (C) or viable intracellular bacteria (D) were quantified. Bars represent mean values ± S.E.M of three independent experiments done in triplicate (n = 9). Numbers are expressed relative to CEACAM1-GFP cells without PI3K inhibitor treatment. Significance was tested using an unpaired, two-sided Student's t-test; ***, p<0.001, *, p<0.05.</p

Inhibition of PI3K activity decreases uptake of Opa_CEA-expressing gonococci via CEACAM1.

<p>(<b>A</b>) 293 cells were transfected with empty vector pcDNA, CEACAM1-4L or CEACAM3 WT. CEACAM expression was verified by Western blotting of whole cell lysates (WCL) using α-HA antibody. (<b>B</b>) Cells were transfected as in (A) and pretreated for 30 min with 50 µM of the PI3K inhibitor LY294002 (LY). After infection for 2 h with Opa_CEA-expressing gonococci, the number of total cell-associated bacteria was determined. Bars represent mean values ± S.E.M of three independent experiments done in triplicate. Total cell associated bacteria are shown relative to cells expressing the respective receptor without PI3K inhibitor treatment. (<b>C</b>) Cells were transfected and infected as in (B). Viable intracellular bacteria were determined in gentamicin protection assays. Bars represent mean values ± S.E.M of three independent experiments done in triplicate. Recovered bacteria are shown relative to cells expressing the respective receptor without PI3K inhibitor treatment. (<b>D</b>) 293 cells were cotransfected with mKate-tagged CEACAM1 and the GFP-tagged Btk-PH domain. 30 min before infection with Pacific Blue labelled Opa_CEA-expressing gonococci, cells were treated with 200 nM wortmannin or left untreated. Fixed samples were analyzed by confocal microscopy. Arrowheads highlight CEACAM- recruitment to cell-associated bacteria. Insets show enlargement of the highlighted area. Bars represent 5 µm.</p