Induction of
opsono-phagocytosis by CEA-Fc fusion proteins
Alexandra Roth1, Corinna Mattheis1, Mario Hupfeld1, Nora Hartmann1, Magnus Unemo1 and Christof R. Hauck1,3
1 Lehrstuhl für Zellbiologie, Universität Konstanz, 78457 Konstanz, Germany
2 WHO Collaborating Centre for Gonorrhoea and other STIs, Department of Laboratory Medicine, Clinical Microbiology, Örebro University Hospital, Sweden
3 Konstanz Research School Chemical Biology, Universität Konstanz, 78457 Konstanz, Germany
In preparation
6.1 Abstract
Several members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family serve as cellular receptors for different Gram-negative humanspecific pathogens including Neisseria gonorrhoeae. The microbes bind to the amino-terminal domain of epithelial CEACAMs (CEACAM1, CEA and CEACAM6) and this interaction promotes bacterial colonization of the mucosa. In sharp contrast, recognition of gonococci by CEACAM3, which is exclusively expressed on human granulocytes, results in phagocytosis and elimination of the bacteria. In principle, CEACAM3-mediated uptake by innate immune cells should limit the spread of CEACAM-binding microbes. However, some gonococcal strains selectively interact with epithelial CEACAMs, but avoid CEACAM3-binding, a feature that makes them more likely to disseminate and cause severe disease. Due to the fact that there is still no vaccine available against N. gonorrhoeae, current treatments are based on antibiotics, however, multi-drug resistant strains are on the race. In this study we investigated a novel passive immunization approach, which relies on the Fcγ receptor (FcγR)-mediated opsono-phagocytosis of CEACAM-binding bacteria by primary human granulocytes. To re-direct gonococcal uptake via the FcγR, we opsonized bacteria with soluble Fc fusion proteins comprising the amino-terminal Igv-like plus the first Igc-like domain of CEA, fused to the Fc part of human IgG1 (CEA-Fc). Our results demonstrate the efficient uptake of CEA-Fc opsonized gonococci by FcγRIIa-expressing human cells. In addition, we show that CEA-Fc opsonized gonococci are phagocytosed by primary human granulocytes and trigger an oxidative burst response, which results in their complete degradation. In summary, opsonization of CEACAM-binding bacteria with soluble CEA-Fc fusion proteins could be a useful passive immunization strategy and an alternative to antibiotics, to treat disseminating infections caused by CEACAM-binding bacteria.
6.2 Introduction
The Gram-negative pathogen N. gonorrhoeae is highly adapted to humans as its sole natural host. Colonization with gonococci often remains asymptomatic and most symptomatic cases remain locally confined, indicating that the
immune system can contain these bacteria in most cases. However,
N. gonorrhoeae employs multiple molecular mechanisms to circumvent the innate and adaptive immune system, such as antigenic variation of virulence factors, serum resistance, or secretion of IgA proteases. Hence, some strains of N. gonorrhoeae are associated with disseminated forms of the disease which can lead to severe complications such as pelvic inflammatory disease (PID), infertility or bacterial meningitis. Moreover, gonococcal infections are one of the significant cofactors for HIV transmission (Fleming and Wasserheit 1999).
Despite the fact, that gonorrhea remains a public health concern and the long recognition of N. gonorrhoeae as one of the major sexually transmitted pathogens, no vaccine exists against gonococci (Zhu et al. 2011). This is in part due to the fact that gonococci modulate their surface antigenic structure with remarkable speed (Stern et al. 1986). Accordingly, several gonococcal adhesins e.g. pili, lipooligosaccharides (LOS) or colony opacity proteins (Opa), are subjected to antigenic and/or phase variation, making the most important surface factors unsuitable as vaccine candidates (Callaghan et al. 2011). In line with this, a pilin target, which was a promising vaccine candidate, elicited a broad antibody response, however failed to protect against a heterologous gonococcal strain, most likely due to the antigenic variation of the pili (McChesney et al. 1982).
Since no vaccine against gonorrhea exists, current treatments are based on antibiotics, but there is an alarming surge of multi-drug resistant strains (Lewis 2010). During the last 70-80 years, N. gonorrhoeae acquired resistance against virtually the complete set of antibiotics introduced for the treatment of the disease. In the 1930s, the antimicrobial treatment started with the use of sulfonamides, which were replaced by penicillin, followed by tetracycline, fluoroquinolones and nowadays the first-line drugs are the third-generation cephalosporins (cefixime and ceftriaxone). However in recent years, first treatment failures with cefixime were reported in Japan, the United States, South Africa and several European countries (Yokoi et al. 2007; Unemo et al.
2010; Unemo et al. 2011; Unemo et al. 2012; Lewis et al. 2013). With the emergence of the first ceftriaxone resistant strains, the last remaining treatment option seems to fall and gonorrhea may eventually become untreatable (Unemo et al. 2012). Thus, N. gonorrhoeae is on its way to reach a superbug status and
novel treatment options are urgently needed. One valid possibility might be to target specific colonization or virulence factors of N. gonorrhoeae.
Gonococci engage carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) expressed on human epithelial cells to successfully colonize their human host. In detail, gonococcal Opa proteins interact with the amino-terminal domain of epithelial CEACAMs, such as CEACAM1, CEA and CEACAM6, to promote bacterial attachment or transcytosis through epithelial cells (Bos et al.
1997; Gray-Owen et al. 1997; Wang et al. 1998). In addition, binding of Opa proteins to epithelial CEACAMs, alters gene expression in the infected cells resulting in enhanced integrin activity (Muenzner et al. 2005). In turn, enhanced integrin activity promotes increased binding of the infected cells to the underlying extracellular matrix and counteracts the exfoliation of superficial epithelial cells (Muenzner et al. 2010). Clearly, by reducing the turnover of the infected tissue surface, the bacteria manage to generate a stable platform for further host colonization. In contrast to the benefit the gonococci might gain from binding to epithelial CEACAMs, interaction of Opa proteins with CEACAM3, an innate immune receptor exclusively expressed on granulocytes, results in fast phagocytosis and bacterial degradation (Pils et al. 2008; Buntru et al. 2012; Roth et al. 2013). Indeed, one hallmark of gonorrhea is the massive recruitment of granulocytes to sites of infections. Although, CEACAM1 and CEACAM6 are also expressed on granulocytes, CEACAM3 has been identified to be the major receptor responsible for bacterial engulfment and destruction (Schmitter et al. 2007). In line with this notion, gonococci that fail to bind to CEACAM3, but interact with epithelial CEACAMs are not readily phagocytosed and killed by human granulocytes. Recently, we could demonstrated that the gonococcal strain VP1, isolated from a patient with disseminated disease, expresses an Opa protein repertoire that interacts with epithelial CEACAMs, however does not express Opa proteins which bind to granulocyte receptor CEACAM3 (Roth et al. 2013). Escaping the CEACAM3-mediated immune recognition could therefore be one factor that contributes to the dissemination of gonococcal infections.
In addition to the opsonin-independent, CEACAM3-mediated phagocytosis of gonococci, granulocytes use opsonin-dependent mechanisms to detect and clear bacterial pathogens. A major opsonin-mediated uptake relies on
immunoglobulins and their recognition by Fc receptors (FcRs). (Nimmerjahn and Ravetch 2008; Buntru et al. 2012). Immunoglobulin G (IgG) is a modular, tetrameric protein with several Ig domains involved in antigen recognition, whereas distinct Ig domains of the two heavy chains, the so-called “Fragment crystallizing” (Fc) part, mediate binding to FcR and trigger phagocytosis of particles. We speculated that a chimeric molecule consisting of the gonococci-binding amino-terminal domain(s) of epithelial CEACAMs (e.g. human CEA) fused to the Fc part of human IgG, should allow opsonization and FcR-mediated phagocytosis of bacteria. Accordingly, we fused the amino-terminal and the A1-domain of human CEA to the Fc part of human IgG1, which binds with moderate to high affinity to FcγRs present on granulocytes.
Here we demonstrate that CEA-Fc fusion proteins strongly bind to Opa protein-expressing N. gonorrhoeae. Moreover, CEA-Fc fusion proteins were efficiently internalized by FcγR-expressing human cells. Upon incubation with CEA-Fc, but not with control Fc fusion proteins, gonococci are recognized, taken up and degraded by primary human granulocytes. Similarly, multi-drug resistant strains of gonococci are readily eliminated by human granulocytes upon incubation with CEA-Fc. In summary, our study indicates, that the induction of opsono-phagocytosis by CEA-Fc fusion proteins might provide an access point for the development of novel therapeutics to combat antibiotic-resistant gonococci.
6.3 Material and Methods
Cell culture and transfection
The human embryonic kidney cell line 293T (293 cells) was grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% calf serum at 37°C, 5% CO2 and subcultured every 2-3 days. 293 cells were transfected by standard calcium-phosphate co-precipitation using 5 - 8 µg of plasmid DNA for each 10 cm culture dish.
Neisserial strains and growth conditions
Opa-expressing, non-piliated N. gonorrhoeae MS11-B2.1 N306 (Opa59), N309 (Opa52), N311 (Opa54), non-piliated, non-opaque gonococci MS11-B2.1 (N302), and wildtype N. gonorrhoeae VP1 (N131) were kindly provided by T.F. Meyer
(Max-Planck Institut für Infektionsbiologie, Berlin, Germany). The antibiotic-resistant gonococcal strain 231 was obtained from Magnus Unemo (WHO Collaborating Centre for Gonorrhoea and other STIs, Department of Laboratory Medicine, Clinical Microbiology, Örebro University Hospital, Sweden) and recently isolated from a patient with ceftriaxone treatment failure of pharyngeal gonorrhea in Slovenia (Unemo et al. 2012). Neisseria were cultured as described previously (Schmitter et al. 2004). Briefly, Neisseria were grown on GC agar plates (Difco BRL, Paisley, UK) supplemented with vitamins at 37°C, 5% CO2 and subcultured daily. For infection or pulldown assays over-night grown bacteria were taken from GC agar plates, suspended in PBS, and colony forming units (cfu) were estimated by OD550 readings according to a standard curve.
Recombinant plasmid constructs
Mammalian expression plasmid encoding soluble GFP-tagged amino-terminal domains of CEACAM1, CEACAM3, CEA or CEACAM6 were described previously (Kuespert et al. 2007). FcγRIIa was amplified from cDNA with the
primers FcγRIIa-IF-sense
5’-GAAGTTATCAGTCGACACCATGACTATGGAGACCCAAATG-3’ and FcγRIIa-IF-antisense 5’-ATGGTCTAGAAAGCTTGCGTTATTACTGTTGACATGGTCG-3’. The resulting PCR fragment was cloned into pDNR-Dual using the In-Fusion PCR Cloning Kit (Clontech, Mountain View, CA) and transferred by Cre-mediated recombination into pLPS-3′EGFP (Clontech) resulting in GFP fused to the carboxy-terminus of the expressed proteins. The Fc-part of human IgG1 was amplified from full-length IgG1 cDNA with the primers Fc-AgeI-sense 5’-GCTAGCACCGGTCGCGGAGCCCAAATCTTGTGACAAAAC -3’ and
Fc-NotI-antisense 5’-ATTTACGCGGCCGCACTCATTTACCCGGAGACAG-3’.
The resulting PCR fragment was subcloned into AgeI-NotI-digested pLPS-3’EGFP replacing EGFP with the Fc-fragment (pLPS-3’Fc). The soluble Fc fusion proteins CEA-NA1-Fc and CEACAM8-N-Fc were generated by Cre-mediated recombination of pDNR-Dual-CEA-NA1and pDNR-Dual-CEACAM8-N with pLPS-3’Fc. The plasmid pDNR-Dual-CEACAM8-N was described previously (Kuespert et al. 2007). CEA-NA1 was amplified from full-length CEA
with the primers CEANA1-IF-sense
5’-GAAGTTATCAGTCGACACCATGGAGTCTCCCTCGGCC-3’ and CEANA1-IF-antisense 5’-ATGGTCTAGAAAGCTTGCCGGGCCATAGAGGACATTCAGG-3’
and cloned into pDNR-Dual.
Cell lysis and Western blotting
Cell lysis and Western blotting were performed as described (Hauck et al. 2001) using a monoclonal antibody (clone JL-8, Clontech) against GFP-fused CEACAMs or a monoclonal antibody against Opa proteins (clone 4B12/C11;
generous gift of Mark Achtman, MPI für Infektionsbiologie, Berlin, Germany).
Rabbit antiserum against N. gonorrhoeae and N. meningitidis (IG-511) was custom-made by Immunoglobe (Himmelstadt, Germany). Soluble CEACAM-Fc fusion proteins were detected with a Biotin-SP-conjugated goat anti-human IgG antibody and HRP-Avidin. Secondary antibodies were from Jackson ImmunoResearch (West Grove, PA).
Binding studies of the different pathogens
Binding studies of the different pathogens with the soluble N-terminal domains of human CEACAMs were performed as described (Kuespert et al. 2007).
Briefly, 2 x 107 bacteria were added to CEACAM-N-domain containing cell culture supernatants in a total volume of 1 ml and incubated for 1 h. After incubation, bacteria were washed twice with PBS++ and boiled in SDS sample buffer prior to SDS-PAGE and Western blotting.
Opsonization of bacteria with Fc-fusion proteins
2 x 107 bacteria were incubated with 1 ml of CEACAM-Fc fusion proteins, or as a negative control in PBS or OptiMEM, respectively, for 1 h under constant rotation at 4°C. As a positive control, bacteria were incubated with 500 µl of rabbit antiserum against Neisseria (heat-inactivated for 1 h at 56°C) diluted 1:2 in PBS. After incubation the bacteria were washed and resuspended in 1 ml PBS + 2 µl rabbit anti-human IgG antibody for 30 min at 4°C under constant rotation. Prior to infection, bacteria were washed once more with PBS.
Gentamicin protection assay
Gentamicin protection assays were conducted as described (Schmitter et al.
2004). Briefly, 4 x 105 293 cells were seeded in gelatine-coated 24-well plates.
The cells were infected with a multiplicity of infection (MOI) of 40 bacteria per cell for 1 h. Afterwards, the medium was replaced with DMEM containing 50 µg/ml gentamicin to kill extracellular bacteria. After 45 min of incubation in gentamicin-containing medium, cells were lysed by the addition of 1% saponin in PBS for 15 min. Suitable dilutions were plated in triplicates on GC agar to determine the number of recovered viable bacteria.
Immunofluorescence staining
For microscopic analysis of bacterial opsonization with soluble CEACAM-Fc fusion proteins, 1 x 108 bacteria were seeded onto gelatine-coated glass-coverslips in 24-well plates. Samples were fixed with 4% paraformaldehyde in PBS, washed and incubated with 2% BSA in PBS for 1 h. Subsequently, samples were incubated with the soluble CEACAM-Fc fusion proteins for 2 h.
After three washes the samples were incubated for 1 h with Biotin-SP-conjugated goat anti-human IgG antibody (Jackson ImmunoResearch), washed again and detected with FITC-Streptavidin (MP Biomedicals). The samples were then embedded in mounting medium (Dako, Glostrup, Denmark). For microscopic analysis of 293 cells, 5 x 104 cells were seeded onto poly-L-lysine- and fibronectin-coated (10 mg/ml and 4 mg/ml, respectively, in PBS) glass-coverslips in 24-well plates. For microscopic analysis of granulocytes, granulocytes were separated from whole blood, resuspended in phagocytosis buffer (PB; 1 x PBS, 0.9 mM CaCl2, 0.5 mM MgCl2, 5 mM glucose, 1% heat-inactivated calf serum) and seeded in 24-well plates. 293 cells were infected (MOI 40) for 1 h with the different bacterial strains at 37°C. The granulocytes were infected (MOI 20) with the different bacterial strains for 15 min at 37 °C.
Samples were washed three times with PBS, fixed with 4% paraformaldehyde in PBS and washed again three times with PBS prior to incubation in blocking buffer (PBS, 10% fetal calf serum) for 20 minutes. Samples were stained with polyclonal anti-Neisseria serum for 45 min. After three washes, samples were incubated with Cy5-coupled goat anti-rabbit antibodies. Following three washes, samples were permeabilized with 0.2% saponin in PBS for 10 min, washed
three times and blocked with blocking solution. For differentiating between extra- and intracellular bacteria, samples were again treated with the antibodies as described above, using a Cy3-coupled goat anti-rabbit as a secondary antibody, resulting in Cy3-labeled intracellular and Cy5/Cy3-labeld extracellular bacteria. The granulocyte nuclei were stained with DAPI for 10 min. The samples were then embedded in mounting medium (Dako, Glostrup, Denmark).
All samples were viewed with a TCS SP5 confocal laser scanning microscope (Leica Wetzlar, Germany). Fluorescence signals of triply labeled specimens were serially recorded with appropriate excitation and emission filters to avoid spectral bleed-through. Images were digitally processed with Image J (NIH, Bethesda, MD) and merged to yield pseudocolored pictures.
Quantification of granulocyte phagocytosis
Phagocytosis was determined by flow cytometry as described previously (Pils et al. 2006). Briefly, 1 x 106 granulocytes were infected with 2 x 107 CFSE-labeled bacteria in 1 ml of phagocytosis buffer (PB; 1 x PBS, 0.9 mM CaCl2, 0.5 mM MgCl2, 5 mM glucose, 1% heat-inactivated calf serum) for 15-30 min at 37 °C.
Phagocytosis was stopped by the addition of ice-cold PB and samples were washed with PBS. Finally, samples were taken up in PBS, containing 1% heat-inactivated FCS. To quench signals derived from extracellular bacteria, trypan blue was added to a final concentration of 0.2% directly before flow cytometric analysis (LSR II; BD Biosciences). The percentage of CFSE-positive cells was multiplied by the mean fluorescence of these cells to obtain an estimate of the total amount of phagocytosed bacteria (uptake index).
Oxidative burst measurements
2 x 105 granulocytes were suspended in chemiluminescence buffer (8 g/l of NaCl, 0.2 g/l of KCl, 0.62 g/l of KH2PO4, 1.14 g/l of Na2HPO4, 1 g/l of glucose, 50 mg/l of bovine serum albumin, pH7.2) with luminol (20 µg/ml). Granulocytes were transferred to a 96-well plate in triplicate and infected with 1 x 107 bacteria or left uninfected. Chemiluminescence was determined every 2 min at 37 °C with a Varioskan Flash fluorescence reader.
Bacterial degradation assay
1 x 106 granulocytes were suspended in phagocytosis buffer and infected with 1 x 106 or 5 x 106 bacteria for 90 and 180 min at 37°C under constant rotation.
Samples were centrifuged and cells were resuspended in 2 x SDS sample buffer followed by gel electrophoresis and Western blot analysis.
6.4 Results
Opa protein-expressing gonococci are opsonized with CEA-Fc fusion proteins
The granulocyte receptor CEACAM3 is responsible for the efficient uptake and killing of bacteria expressing a CEACAM-binding adhesin. However, former studies demonstrated that some gonococcal strains escape the CEACAM3-based detection by granulocytes while still binding to epithelial CEACAMs (Roth et al. 2013). Therefore, we first analyzed the CEACAM-binding profile of several gonococcal strains including strain MS11 lacking Opa protein expression (Opa-), strain MS11 expressing Opa54, strain MS11 expressing Opa59 or N. gonorrhoeae strain VP1. These gonococcal strains were incubated with GFP-fused, soluble amino-terminal domains of CEACAM1, CEA, CEACAM3, CEACAM6, or CEACAM8. After pelleting the bacteria and several washes, bacteria-associated GFP-fusion proteins were detected by Western blotting with GFP antibodies (bacterial pulldown assay, Fig 6.1B). Clearly, N. gonorrhoeae strain MS11 Opa54 and strain VP1 interacted with epithelial CEACAM1 and CEA, whereas N. gonorrhoeae strain MS11 Opa59 selectively bound to CEA (Fig. 6.1B). As expected, Opa-negative gonococci did not interact with any tested CEACAM domain. Interestingly, none of the Opa-expressing strains bound to granulocyte receptor CEACAM3, indicating that they are all able to ecscape the CEACAM3-mediated recognition and subsequent elimination by granulocytes (Fig. 6.1B).
Fig. 6.1: CEA-Fc fusion proteins can opsonize Opa protein-expressing gonococci which lack CEACAM3 recognition. (A) Opa protein expression of the indicated gonococcal strains, verified by Western blotting using a monoclonal anti-Opa antibody. (B) The indicated GFP-fused, soluble CEACAM-domains were expressed in 293 cells and the cell culture supernatants were employed in a pulldown assay with the indicated gonococcal strains. Precipitates (Pull-down) were probed with a monoclonal anti-GFP antibody to detect bacteria-CEACAM interaction. Supernatants (Supe) were probed with a monoclonal anti-GFP antibody to demonstrate equal amounts of the GFP-fusion proteins in the used cell culture supernatants.
(C) CEACAM-Fc fusion proteins were produced in 293 cells and similar amounts were used in a pulldown assay together with the indicated gonococcal strains. Precipitates (Pull-down) were probed with a Biotin-conjugated goat anti-human IgG antibody followed by HRP-Avidin to detect opsonization of the bacteria. Supernatants (Supe) were probed in a similar manner to demonstrate equal amounts of the Fc fusion proteins in the used cell culture supernatants (D) Opa59-expressing and Opa-negative gonococci were fixed and incubated with CEACAM-Fc fusion proteins. Samples were then stained with Biotin-conjugated goat anti-human IgG antibody followed by incubation with Streptavidin. Using confocal microscopy, FITC-labeled bacteria were detected. Bars represent 5 µm.
On the basis of this result, we tested a novel approach to induce the granulocyte phagocytosis of Neisseria which are not recognized by granulocyte receptor CEACAM3, but interact with epithelial CEACAM1 or CEA. Our strategy was to opsonize CEACAM-binding bacteria with recombinant CEA-Fc fusion proteins and thus allow their Fcγ receptor-mediated internalization and destruction. To generate soluble CEA-Fc fusion proteins, we fused the Fc part
of human IgG1 to the amino-terminal domain (N) and the first immunoglobulin constant domain (A1) of CEA and expressed the proteins in human 293 cells (Fig. 6.1C). As a control, the amino-terminal domain of CEACAM8, which is known not to interact with gonococci, was fused to the Fc part of IgG1 and expressed in human 293 cells (Fig. 6.1C). Similar amounts of the CEACAM-Fc fusion proteins were used for a pulldown assay with the same gonococcal strains employed as before (Fig. 6.1C). In line with the previous pulldown, N. gonorrhoeae MS11 Opa54, N. gonorrhoeae MS11 Opa59 and N. gonorrhoeae VP1 were opsonized with CEA-Fc fusion proteins, but not with the control protein CEACAM8-Fc (Fig. 6.1C). To further demonstrate that these gonococci are CEA-Fc opsonized, we employed confocal laser scanning microscopy.
of human IgG1 to the amino-terminal domain (N) and the first immunoglobulin constant domain (A1) of CEA and expressed the proteins in human 293 cells (Fig. 6.1C). As a control, the amino-terminal domain of CEACAM8, which is known not to interact with gonococci, was fused to the Fc part of IgG1 and expressed in human 293 cells (Fig. 6.1C). Similar amounts of the CEACAM-Fc fusion proteins were used for a pulldown assay with the same gonococcal strains employed as before (Fig. 6.1C). In line with the previous pulldown, N. gonorrhoeae MS11 Opa54, N. gonorrhoeae MS11 Opa59 and N. gonorrhoeae VP1 were opsonized with CEA-Fc fusion proteins, but not with the control protein CEACAM8-Fc (Fig. 6.1C). To further demonstrate that these gonococci are CEA-Fc opsonized, we employed confocal laser scanning microscopy.