Exploring bacterial binding to
receptors of the CEACAM family as a basis for translational approaches
Dissertation
Zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) des Fachbereichs
Biologie der Universität Konstanz vorgelegt von
Alexandra Roth Juni 2013
Tag der mündlichen Prüfung: 16.08.2013 1. Referent: Prof. Dr. Christof R. Hauck
2. Referent: Prof Dr. Thomas Brunner
Juni 2013
Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-243247
T
ABLE OFC
ONTENTSTABLE OF CONTENTS ... 3
ABBREVIATIONS ... 5
ACKNOWLEDGMENT (DANKSAGUNG) ... 7
SUMMARY ... 8
ZUSAMMENFASSUNG ... 10
1 GENERAL INTRODUCTION ... 12
1.1 Neisseria gonorrhoeae ... 12
1.2 Gonorrhea - Disease and treatment options ... 12
1.3 Major colonization factors of N. gonorrhoeae ... 15
1.3.1 Pili ……… ... 15
1.3.2 Lipooligosaccharide (LOS) ... 16
1.3.3 Opa proteins ... 17
1.4 Opa-CEACAM interaction ... 18
1.5 Other CEACAM-binding human-restricted pathogens ... 22
2 AIMS OF THE STUDY ... 25
3 CHAPTERI:HEMITAM SIGNALING BY CEACAM3, A HUMAN GRANULOCYTE RECEPTOR RECOGNIZING BACTERIAL PATHOGENS ... 27
3.1 Abstract ... 28
3.2 CEACAM family proteins as bacterial receptors ... 28
3.3 Phosphorylation of the CEACAM3 cytoplasmic domain and CEACAM3 membrane localization ... 31
3.4 CEACAM3-initiated signaling leading to actin rearrangements and phagocytosis ... 33
3.5 CEACAM3-initiated elimination of phagocytosed bacteria ... 36
3.6 Parallels between CEACAM3 signaling and hemITAM mediated phagocytosis by Dectin-1 ... 40
3.7 Conclusions ... 41
3.8 Acknowledgments ... 42
4 CHAPTERII:IDENTIFICATION OF NOVEL CEACAM-BINDING BACTERIA IN MACAQUE INTESTINAL FLORA... 43
4.1 Abstract ... 44
4.2 Introduction ... 45
4.3 Methods ... 47
4.4 Results ... 51
4.5 Discussion ... 56
4.6 Acknowledgments ... 60
5 CHAPTERIII: INNATE RECOGNITION BY NEUTROPHIL GRANULOCYTES DIFFERS BETWEEN NEISSERIA GONORRHOEAE STRAINS CAUSING LOCAL OR DISSEMINATING INFECTIONS ... 61
5.1 Abstract ... 62
5.2 Introduction ... 62
5.3 Material and Methods ... 65
5.4 Results ... 70
5.5 Discussion ... 84
5.6 Acknowledgments ... 89
5.7 Supplementary figures ... 90
6 CHAPTERIV:INDUCTION OF OPSONO-PHAGOCYTOSIS BY CEA-FC FUSION PROTEINS ... 91
6.1 Abstract ... 92
6.2 Introduction ... 92
6.3 Material and Methods ... 95
6.4 Results ... 100
6.5 Discussion ... 111
6.6 Acknowledgments ... 116
7 GENERAL DISCUSSION ... 117
DECLARATION OF AUTHOR´S CONTRIBUTIONS ... 122
8 LIST OF PUBLICATIONS ... 124
8.1 Parts of this thesis are published or ongoing to be submitted for publication ... 124
8.2 Publications not part of this thesis ... 124
REFERENCES ... 125
A
BBREVIATIONS% percent
°C degree Celsius
µm micrometer
µM micromolar
bp basepairs
BSA bovine serum albumin
Ca calcium
CEA carcinoembryonic antigen
CEACAM carcinoembryonic antigen-related cell adhesion molecule CFSE carboxy-fluorescein-succhinimidylester
CFU colony-forming units
CS calf serum
DGI disseminated gonococcal infections DMSO dimethyl sulfoxide
DNA desoxyribonucleic acid
EDTA ethylenediaminetetraacetic acid EGFP enhanced green fluorescent protein et al. et alii; and others
FACS Fluorescence Activated Cell Sorting FcR Fc receptor
FCS fetal calf serum
g gramm
GFP green fluorescent protein
h hour
HEK human embryonic kidney
HSPG heparansulphate proteoglycans HV hypervariable region
ITAM immunoreceptor tyrosine-based activation motif ITIM immunoreceptor tyrosine-based inhibitory motif kDa kilo Dalton
l liter
LOS lipooligosaccharide LPS lipopolysaccharide
M molar
mg milligramm
ml milliliter mM millimolar
MOI multiplicity of infection Ngo Neisseria gonorrhoeae
nm nanometer
NT aminoterminal
opa opacity associated
OpaCEA-a Opa proteins bind to all CEACAMs
OpaCEA-e Opa proteins bind to epithelial CEACAMs PBS phosphate buffered saline
PCR polymerase chain reaction PFA paraformaldehyde
PI3K Phosphatidylinositol 3 kinase PTK protein tyrosine kinase
RT room temperature
s seconds
SD standard deviation SDS sodium dodecylsulfate SFK Src family kinase SH2 Src-homology 2 SH3 Src-homology 3 SV semivariable region
UspA ubiquitous surface protein A WCL whole cell lysate
WT wildtype
A
CKNOWLEDGMENT(D
ANKSAGUNG)
Mein besonderer Dank geht an Prof. Dr. Christof Hauck für seine hervorragende Betreuung, die vielen inspirierenden wissenschaftlichen Gespräche und seinen grenzenlosen Optimismus.
Vielen Dank für die wunderschöne Zeit am Lehrstuhl Zellbiologie!
Des Weiteren möchte ich mich bei Prof. Dr. Thomas Brunner und Prof. Dr. Daniel Legler für die bereitwillige Übernahme des Zweitgutachtens und den Prüfungsvorsitz bedanken.
Ein großer Dank geht an meine Arbeitsgruppe, die über die Jahre wie eine zweite Familie für mich geworden ist und mit der mich auch außerhalb der Arbeit ein freundschaftliches Verhältnis verbunden hat. Vielen Dank an alle ehemaligen und gegenwärtigen Laborkollegen, die meine Zeit hier unvergesslich gemacht haben.
Besonders bedanke ich mich bei Anne Keller, Ruth Hohenberger-Bregger, Petra Zoll-Kiewitz und Susanne Feindler-Boeckh, die oft unbemerkt dafür sorgten, dass alles „läuft wie geschmiert“. Weiterhin danke ich Alexander „Lexi“ Timper für die vielen feucht-fröhlichen
„Mocsow Mule-Abende“ und das „denkwürdige Finale“, Arnaud Kengmo für seine offene und ehrliche Freundschaft, Naja Nyffenegger, die mir gezeigt hat, wie nett Schweizer sein können, Chris Paone für seine stete Hilfsbereitschaft und Ruhe, Petra Muenzner-Voigt für die vielen erheiternden Momente, Julia Delgado Tascon für den gemeinsamen Kampf gegen die
„Anaeroben“ und Alex Buntru für die tiefen wissenschaftlichen sowie nicht-wissenschaftlichen Gespräche. Außerdem danke ich meinem „Bruder“ Thomas Grabinger, meiner Lieblingsmasterstudentin Corinna Mattheis und Katrin Küspert, der besten Betreuerin der Welt.
Ein besonderer Dank geht an meine Labormitbewohnerin Nina Dierdorf, die mit mir von Anfang bis zum Ende die Zeit im „Hauck lab“ verbracht hat. Es wird seltsam sein in Zukunft ohne dich im Labor zu stehen!
Bedanken möchte ich mich auch bei all meinen Freunden, die mich während der neun Jahre in meiner Wahl-Heimat Konstanz begleitet haben. Ganz besonderer Dank hierbei an Nathalie Feiner und Hans Recknagel, für die schönen Stunden als „Vierergespann“!
Von Herzen danke ich meinem Bruder Hannes Roth für die vielen offenen Gespräche und meiner Schwester Nikola Roth für ihre hilfsbereite Art und die lustigen „Perfekte Dinner- Abende“. Ganz besonders möchte ich mich auch bei meinen Eltern bedanken, die mir das alles erst ermöglicht haben. Vielen Dank für eure Unterstützung und Liebe.
Zum Schluss geht mein Dank an Tobi Menzel, der immer an meiner Seite ist, mit mir durch alle Höhen und Tiefen geht und meinem Leben den „entscheidenden Glanz“ verleiht.
S
UMMARYCarcinoembryonic antigen-related cell adhesion molecules (CEACAMs), expressed on human epithelia, are engaged by several Gram-negative human-
restricted pathogens, including Neisseria gonorrhoeae. In detail, N. gonorrhoeae, the causative agent of gonorrhea, expresses several colony
opacity (Opa) proteins that interact with the amino-terminal domain of epithelial CEACAMs (CEACAM1, CEA and CEACAM6). Accordingly, the interaction of gonococcal Opa proteins and CEACAMs enables N. gonorrhoeae to colonize human mucosal surfaces. In contrast to the benefit N. gonorrhoeae gains by binding to epithelial CEACAMs, binding of Opa proteins to the granulocyte receptor CEACAM3, results in bacterial phagocytosis and destruction. In the first part of the study we summarized the CEACAM3-mediated internalization and killing of bacteria and compared the CEACAM3-signaling pathways to the well-studied Fcγ receptors (FcγRs) and Dectin-1.
CEACAM3-mediated elimination of gonococci, might contribute to the fact that most gonococcal infections are local. Though, in rare cases, gonococci enter the bloodstream and cause systemic or disseminating gonococcal infections (DGI). Here we analyzed the CEACAM3-binding capacity of the DGI strain VP1.
Accordingly, we identified the whole repertoire of VP1 opa genes and constitutively expressed them in Escherichia coli. Strikingly, several VP1 Opa proteins interacted with epithelial CEACAMs, but not a single VP1 Opa protein bound to the phagocyte receptor CEACAM3. Thus, E. coli expressing VP1 Opa proteins were not phagocytosed and eliminated by human granulocytes.
Furthermore, the analysis of Opa variants from four additional clinical DGI isolates again demonstrated a lack of CEACAM3-binding. In conclusion, our results suggest that CEACAM3-avoidance of N. gonorrhoeae could be one factor that contributes to DGI.
A major challenge for the treatment of gonorrhea is the emergence of multi-drug resistant gonococcal strains. In line with this, the investigation of additional treatment options is essential. Here, we tested a novel passive immunization approach which is based on the opsonin-dependent, FcγR-mediated phagocytosis of CEACAM-binding gonococci by human granulocytes. Precisely,
we opsonized CEA-binding N. gonorrhoeae strains, including a multi-drug resistant strain, with soluble CEA-Fc fusion proteins. Importantly, we
demonstrated that CEA-Fc opsonized gonococci are taken up into FcγR-expressing human cells as well as into human granulocytes.
Phagocytosis of CEA-Fc opsonized gonococci by granulocytes resulted in an oxidative burst response and bacterial destruction. Thus, CEA-Fc fusion proteins could be a starting point for the development of novel therapeutics against antibiotic-resistant gonococci.
CEACAM orthologues are also expressed in other mammals, but no bacterial ligands have been identified for non-human CEACAMs. Another aim of this study was to search for CEACAM-binding bacteria in rhesus monkey. For that purpose, we generated a soluble construct of the macaque orthologue of human CEA and developed a cultivation-independent screen to enrich a pool of macaque stool samples for macaque CEA-binding bacteria. By 16S rRNA gene pyrosequencing we identified several bacteria, belonging to the genus Prevotella, to be potential macaque CEA-binding bacteria. Biochemical approaches confirmed the interaction of Prevotella and macaque CEA.
Unexpectedly, Prevotella also bound to several human CEACAMs, but not to CEACAMs from more distantly related mammals. Our observations indicate that CEACAM-engagement is species-specific and might contribute to the specific microbiota of different mammalian hosts.
Altogether, the findings obtained in this study allow new insights in gonococcal pathogenicity and provide a basis for novel translational research approaches.
Z
USAMMENFASSUNGMehrere Gram-negative humanspezifische Krankheitserreger, wie zum Beispiel Neisseria gonorrhoeae, interagieren mit Rezeptoren der CEACAM-Familie, welche von humanen Epithelien exprimiert werden. N. gonorrhoeae, Verursacher der Gonorrhö, exprimiert mehrere Opa-Proteine, die mit den aminoterminalen Rezeptordomänen von epithelialen CEACAMs interagieren (CEACAM1, CEA und CEACAM6). Demzufolge ermöglicht die Interaktion mit epithelialen CEACAMs N. gonorrhoeae, die menschliche Schleimhaut zu kolonisieren. Im Gegensatz dazu führt die Bindung von Opa-Proteinen an CEACAM3, das ausschließlich auf Granulozyten exprimiert wird, zur Aufnahme und Abtötung der Bakterien. Im ersten Teil dieser Arbeit wird die CEACAM3- vermittelte Aufnahme und Abtötung von Bakterien zusammengefasst und mit anderen Rezeptoren, wie dem Fcγ-Rezeptor und Dectin-1, verglichen.
Die CEACAM3-vermittelte Eliminierung von Bakterien könnte dazu beitragen, dass Gonokokken meist nur lokale Infektionen auslösen. Allerdings können Gonokokken in seltenen Fällen auch in die Blutbahn gelangen und disseminierte Erkrankungen auslösen. Im Rahmen dieser Arbeit wurde deshalb analysiert, ob der Stamm N. gonorrhoeae VP1, der für disseminierte Krankheitsverläufe verantwortlich ist, an CEACAM3 bindet. Dazu wurden alle Opa-Proteine des Stammes VP1 identifiziert und konstitutiv in Escherichia coli exprimiert. Interessanterweise interagierten mehrere VP1 Opa-Proteine mit epithelialen CEACAMs, aber kein VP1 Opa-Protein mit dem Phagozytoserezeptor CEACAM3. Folglich wurden E. coli, welche VP1 Opa- Proteine exprimierten, auch nicht von Granulozyten aufgenommen und eliminiert. Des Weiteren interagierten Opa-Protein-Varianten vier weiterer Gonokokken-Stämme, die disseminierte Infektionen auslösen, ebenfalls nicht mit CEACAM3. Zusammenfassend unterstützt das Ergebnis dieser Untersuchung die Hypothese, dass Gonokokken, die disseminierte Erkrankungen auslösen, der CEACAM3-basierten Erkennung durch Granulozyten entgehen können.
Eine große Herausforderung bei der Behandlung von Gonorrhö ist das Aufkommen von multiresistenten Stämmen, weshalb die Entwicklung neuer
Behandlungsmethoden essentiell ist. In der vorliegenden Arbeit wurde deshalb ein neuer Ansatz für passive Immunisierung getestet, der auf der opsoninabhängigen, Fcγ-vermittelten Aufnahme von CEACAM-bindenden Gonokokken durch Granulozyten basiert. Folglich wurden CEA-bindende Gonokokken-Stämme, darunter auch ein multiresistenter Stamm, mit löslichen CEA-Fc Fusionsproteinen opsoniert. Interessanterweise führte diese Opsonisierung zur Aufnahme der Gonokokken in Fcγ-exprimierende humane Zellen, sowie in humane Granulozyten. Die Aufnahme in humane Granulozyten löste wiederum das Entstehen reaktiver Sauerstoffspezies (ROS) und letztendlich die Abtötung der Gonokokken aus. Zusammenfassend könnten CEA-Fc Fusionsproteine deshalb einen interessanten Ansatzpunkt für die Entwicklung neuer Behandlungsmethoden gegen antibiotikaresistente Stämme darstellen.
CEACAM-Orthologe werden, neben dem Menschen, auch von anderen Säugetieren exprimiert, allerdings wurden bis jetzt keine Bakterien identifiziert, die mit nicht-menschlichen CEACAMs interagieren. Ein weiteres Ziel dieser Arbeit bestand deshalb darin, CEACAM-bindende Bakterien im Rhesusaffen zu identifizieren. Dazu wurde ein lösliches CEA-Konstrukt des Rhesusaffen hergestellt und eine kultivierungsunabhängige Methode zur Anreicherung von CEACAM-bindenden Bakterien aus einem Pool von Rhesusaffenstuhlproben, entwickelt. Mittels 16S rRNA Pyrosequenzierung konnten mehrere Bakterien der Gattung Prevotella als CEACAM-bindende Bakterien identifiziert werden.
Zusätzlich wurde die Interaktion von verschiedenen Prevotellen und dem Rhesusaffen-CEA durch biochemische Ansätze verifiziert.
Überraschenderweise konnten diese Prevotellen auch mit humanen CEACAMs interagieren, jedoch nicht mit CEACAMs entfernt verwandterer Säugetiere.
Unsere Beobachtungen weisen deshalb daraufhin, dass die CEACAM-Bindung verschiedener Bakterien speziesspezifisch ist und zur spezifischen Mikrobiota verschiedener Säugetierarten beitragen könnte.
Zusammengefasst, liefern die Befunde in der vorliegenden Arbeit neue Einblicke in die Pathogenität von Gonokokken und eröffnen neue Möglichkeiten für translationale Forschungsansätze.
1 G
ENERALI
NTRODUCTION1.1 Neisseria gonorrhoeae
The Gram-negative diplococcus Neisseria gonorrhoeae is a member of the genus Neisseria, which encompasses a large group of Betaproteobacteria, specialized to colonize mucosal epithelia of humans and animals. The genus Neisseria was named after Albert Ludwig Sigesmund Neisser, who in 1879 described for the first time the human-restricted pathogen N. gonorrhoeae, the gonococcus. Besides, N. meningitidis, N. gonorrhoeae is the only pathogen within the genus of Neisseria. N. gonorrhoeae and N. meningitidis, which are the causative agents of the name-giving diseases gonorrhea and meningitis respectively, are closely related, yet display different lifestyles and niche preferences. Mainly due to different modes of transmission, N. meningitidis colonizes the human nasopharynx, whereas N. gonorrhoeae primarily inhabits the human urogenital tract.
1.2 Gonorrhea - Disease and treatment options
Gonorrhea is, besides Chlamydia trachomatys infections, the second most common sexually transmitted disease (STD) on a global scale. Thus, an estimated ~90 million new cases of gonorrhea occur every year (WHO 2011).
N. gonorrhoeae mainly colonizes the urogenital tract, where it can cause symptomatic infections in about 50 - 90% of the cases depending on sex.
Normally, the typical symptoms of gonorrhea show up within one week. These symptoms differ largely between men and women. For women the primary affected area is the cervix, however ascending infections can result in pelvic inflammatory disease (PID), which in turn can lead to ectopic pregnancy or infertility. In addition, vertical transmission from women to their newborn can result in neonatal conjunctivitis, that may lead to blindness of the newborn (Handsfield 1990). In women, a large fraction of colonized persons do not develop symptoms, which is one reason why the pathogen often remains unrecognized and untreated. Accordingly, asymptomatic infections in women largely contribute to the transmission and persistence of the bacteria in the
human population. Unlike in women, gonococcal infections in men are usually symptomatic. Common symptoms in men include purulent penile discharge and dysuria. Ascending infections in men may finally lead to urethritis or in severe cases to epididymitis.
One characteristic of gonococcal infections is that they are poorly controlled by the adaptive immune system. Thus, specific antibody levels after gonococcal infections are generally low. In addition, no immunological memory is established, which is the reason why reinfections with the same gonococcal strain are common (Hedges et al. 1999).
Gonococci have developed multiple mechanisms to escape the antibody- and complement-mediated killing afforded by the human immune system (Virji 2009;
Liu et al. 2011). Accordingly, gonococci are often resistant to normal human serum (NHS), mostly due to lipooligosaccharide (LOS) sialylation or the recruitment of human factor H to PorBIA (Vogel and Frosch 1999). In addition, gonococci are known to secrete an IgA1-specific serine protease (Pohlner et al.
1987). This IgA1 protease not only cleaves human mucosal IgA1, but also lysosomal LAMP1 (lysosome-associated membrane protein1), contributing to extracellular and intracellular survival of gonococci (Pohlner et al. 1987; Hauck and Meyer 1997).
Since gonococci evade recognition by the adaptive immune response, gonococcal infections are mainly cleared by the innate immune system. In line with this, one hallmark of gonorrhea is the massive influx of neutrophil granulocytes to sites of infection. (Rest and Shafer 1989). Owing to fast granulocyte phagocytosis and destruction of gonococci, infections remain mostly locally confined and resolve rapidly. However, in rare cases, gonococci may also disseminate, enter the blood stream and cause more severe disease.
Such systemic or disseminated gonococcal infections (DGI) are usually associated with gonorrheal arthritis and can result in bacterial endocarditis, meningitis, myocarditis or pneumonia.
Given the fact that there is no vaccine against gonococci available, current treatments of gonorrhea mostly depend on antibiotics. However, there is a raising challenge, due to the increasing emergence of antibiotic-resistant strains (Lewis 2010). Antimicrobial resistance of gonococci immediately started after the introduction of the first antibiotics in the 1930s. Over the years gonococcal
strains became resistant to penicillins, sulphonamides, tetracyclines, quinolones and macrolides. Alarming are recent reports about gonococcal resistance to the third-generation cephalosporins, ceftriaxone and cefixime, 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) (Fig. 1.1).
Fig. 1.1: Decreased susceptibility to cefixime (MIC>0.12mg/L) and reported treatment failures in Europe (2010-2013). The rapid increase and spread of decreased susceptibility to cefixime is extremely concerning as, beside ceftriaxone, this is the last remaining treatment option against gonorrhea (updated from European Centre for Disease Prevention and Control (ECDC), “Response plan to control and manage the threat of multidrug-resistant gonorrhea in Europe”).
Since third-generation cephalosporins were the last remaining treatment options, these gonococci can now be considered multidrug-resistant and reach a superbug status. The main mechanisms for resistance to third-generation cephalosporins are alterations in the penA gene of gonococci, encoding penicillin-binding protein2 and mutations in the promotor or coding sequence of the mtrR gene, leading to increased expression of the MtrCDE efflux pump (Lewis 2010; Unemo et al. 2011).
In the light of increasing incidences of antibiotic resistant strains, new treatment options or the development of a vaccine against gonococci become more and more urgent. However, until to date, all potential vaccine candidates against gonococci have failed. Since whole-cell vaccines, consisting of autolyzed bacteria were shown to have no protective effects, most vaccine research was
concentrated on purified gonococcal proteins (Greenberg et al. 1974). However, a major problem in gonococcal vaccine development is the high antigenic variability of gonococcal proteins, hampering the identification of a vaccine, which combats a majority of virulent strains.
1.3 Major colonization factors of N. gonorrhoeae
For the efficient colonization of human mucosal epithelial cells, N. gonorrhoeae has evolved dedicated surface components, including, pili, colony opacity (Opa) proteins, and lipooligosaccharide (LOS). All three components are well-studied virulence factors of these bacteria and undergo phase and/or antigenic variation, allowing gonococci to evade the human immune response (Fig.1.2).
Fig. 1.2: Major virulence factors contributing to gonococcal colonization of epithelial cells. The outer membrane (OM) of gonococci contains the filamentous pili, the integral Opa proteins and LOS. All three virulence factors are associated with host cell attachment and are important for the establishment and maintenance of a gonococcal infection (modified from Virji 2009).
1.3.1 Pili
The initial attachment of gonococci to the apical side of epithelial tissues is mediated by the type IV pili. Hence, studies with human male volunteers demonstrated that the pili is crucial for the establishment of a successful infection of the urethra (Swanson et al. 1987). The pilus fibre is composed of numerous PilE subunits (pilin), which are arranged in a helical configuration.
Additionally, several minor pilus subunits (PilC, PilV and PilX) can be
incorporated into the fiber and modulate its function. Among them, PilC is thought to be the major pilus adhesin. Thus, it was shown that PilC mutants do not adhere to epithelial cells (Rudel et al. 1992).
Pili undergo antigenic variation via several recombinase A-dependent homologous recombination events (Haas and Meyer 1986). Beside the expression locus pilE, which contains the functional pilin gene, a number of silent, promotor-less pilS genes are encoded on the gonococcal chromosome.
Variation of the pilE gene arises from recombination between pilS copies and the pilE locus, generating mosaics of the pilE gene and different numbers of pilS storage copies. In addition, the pilus varies through phase variation of the pilC genes, regulated by insertion or deletion of single nucleotides into a homopolymeric run of G within the coding sequence (Jonsson et al. 1991).
Although, the gonococcal pilus is well studied, its corresponding receptor on human epithelial cells remains elusive. Former investigations identified the transmembrane glycoprotein CD46 as pilus receptor, however no direct interaction of CD46 and PilC has been observed (Kallstrom et al. 1997).
Additionally, further studies could not confirm an essential role of CD46 in pilus- mediated cell adherence (Kirchner et al. 2005).
1.3.2 Lipooligosaccharide (LOS)
Gonococcal LOS is composed of a highly hydrophobic lipid A and an attached hydrophilic nonrepeating oligosaccharide chain. In contrast to the common lipopolysaccharide (LPS) of other Gram-negative bacteria, gonococcal LOS lacks the repeating O-carbohydrate antigen side chain. Variation of gonococcal LOS is mediated by phase variation of enzymes employed in the biosynthesis of LOS (Shafer et al. 2002).
LOS is also thought to be involved in gonococcal cell adherence. Accordingly, it was shown that LOS with a terminal lacto-N-neotetraose group interacts with the asialoglycoprotein receptor expressed on primary urethral epithelial cells (Harvey et al. 2001). In addition, it was demonstrated, that LOS lipid A recruits the complement molecule C3b. Inactive C3b (iC3b), together with gonococcal pili and porin promote complement receptor type 3 (CR3) binding, which allows gonococci to colonize and invade cervical epithelial cells (Edwards and Apicella 2002; Edwards et al. 2002).
1.3.3 Opa proteins
Opa proteins contribute, as the name already indicates, to the opaque phenotype of gonococcal colonies on solid growth media. In total, up to twelve different Opa proteins can be expressed in gonococci. Opa proteins form a β-barrel secondary structure, integrated in the outer membrane of gonococci.
They are composed of eight antiparallel β-strands and four extracellular loops.
Two of these loops are highly variable in their amino acid sequence (HV1 and HV2), one loop is semivariable (SV) and one loop is conserved among different Opa variants. In contrast to the pil loci, the opa gene loci are complete genes, which are constitutively transcribed. However similar to pili and LOS, Opa proteins undergo phase variation (Stern et al. 1986). This is due to a pentameric repeat sequence (CTCTT) within the 5’ coding region of each opa gene. This sequence is variable in length and depending on the number of repeat units the opa gene is in frame and expressed in full-length or owing to a premature stopcodon a truncated non-functional Opa protein version is expressed. The coding repeat variation is based on a RecA-independent DNA slipped strand mispairing mechanism during DNA replication. Phase variation of individual opa genes occurs independently at a frequency of 10-3, resulting in heterogeneous gonococcal populations where single bacteria express none, one, or multiple different Opa proteins. Due to the fact that it is not possible to control, which Opa protein variants are expressed in a gonococcal population, it is not easy to study the function of single Opa proteins. Furthermore, Opa proteins are highly diverse among different gonococcal strains. Thus, a former study revealed that 14 unrelated gonococcal strains had no opa alleles in common (Bilek et al.
2009). Novel gonococcal opa genes arise mainly by homologous recombination events between opa loci of the same chromosome, as well as between different organisms. Thereby, novel alleles are mostly generated by shuffling of already existing HV regions of distinct opa genes. In contrast, gene duplication and de novo amino acid mutations occur much less frequently than changes due to homologous recombinations of different HV regions (Bilek et al. 2009). The diversity of gonococcal Opa proteins is most likely due to the selective pressure of the human immune system and their importance for gonococcal colonization.
Gonococcal Opa proteins play, beside pili, a central role in the establishment and the maintenance of a gonococcal infection. After the initial pili-mediated
attachment, Opa proteins are responsible for a more intimate interaction of gonococci with epithelial cells. The importance of Opa proteins during a gonococcal infection was underscored by a study which showed that the gonococci isolated from urethra of male volunteers, who were previously challenged with piliated, Opa-negative gonococci, were predominantly Opa- positive (Swanson et al. 1988; Jerse et al. 1994). This experiment provides evidence that there is a strong in vivo selection for the opaque phenotype of gonococci in their natural host.
Gonococcal Opa proteins are known to target human cell surface receptors.
Thus, it was shown that gonococci expressing Opa50 interact with heperan- sulphate proteoglycans (HSPGs) expressed on Chang conjunctiva epithelial cells. Interaction of Opa50 and HSPGs finally results in bacterial internalization (Chen et al. 1995; van Putten and Paul 1995). This internalization is dependent on the activation of the phosphatidylcholine-dependent phospholipase C (PC- PLC9), which generates the second messenger diaglycerol (DAG), which in turn activates the acidic sphingomyelinase (ASM). ASM generates ceramide that seems to be involved in actincytoskeleton reorganization and bacterial uptake (Esen et al. 2001). In addition, Opa50-expressing gonococci bind via vitronectin or fibronectin to integrin αvβ5 orα5β1, respectively (Duensing and van Putten 1997; Gomez-Duarte et al. 1997; van Putten et al. 1998). Simultaneous interaction of Opa50 with HSPGs and integrins again triggers gonococcal uptake by epithelial cells.
1.4 Opa-CEACAM interaction
Most Opa proteins do not interact with HSPGs, but with carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) expressed on epithelial, endothelial or immune cells (Virji et al. 1996; Gray-Owen et al. 1997; Gray- Owen et al. 1997). In humans twelve distinct CEACAMs are expressed, however, only four, namely CEACAM1, CEACAM3 CEA (the product of the gene CEACAM5), and CEACAM6 are known to be engaged by N. gonorrhoeae.
All CEACAMs are composed of an amino-terminal immunoglobulin variable (IgV)-like domain and up to six immunoglobulin constant (IgC)-like domains in their extracellular part. CEACAMs are anchored to the membrane by a transmembrane domain (CEACAM1, CEACAM3, CEACAM4) or by a GPI-
anchor (CEA, CEACAM6, CEACAM7, CEACAM8) (Thompson et al. 1991).
Whereas the cytoplasmic domain of CEACAM1 bears an immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequence, the cytoplasmic tail of CEACAM3 and CEACAM4 contain an immunoreceptor tyrosine-based activation motif (ITAM)-like sequence (Chen et al. 2001; Chen et al. 2001) (Fig. 1.3).
Fig. 1.3: The human CEACAM family. Domain structure and predicted glycosylation pattern of the major human CEACAMs. All CEACAMs share an amino-terminal Igv-like domain, followed by different numbers of Igc-like domains in their extracellular part. CEACAMs can be anchored to the membrane via a transmembrane domain or via a GPI anchor (modified from Gray-Owen and Blumberg, 2006).
Although CEACAMs are highly glycosylated, Opa proteins interact with the protein backbone of the amino-terminal CEACAM domain. In particular, the non-glycosylated C’’C’CFG face of the immunoglobulin fold serves as a binding site for N. gonorrhoeae (Popp et al. 1999). Interestingly, amino-terminal domains of CEACAMs, which do not interact with Opa proteins, are up to 80%
identical to Opa-binding CEACAM domains, indicating that the interaction of Opa proteins and certain CEACAMs is highly specific. Accordingly, single amino acids in the C’’C’CFG face, were found to be crucial for Opa engagement (Bos et al. 1999; Virji et al. 1999). Furthermore, it was shown that most Opa proteins display a particular tropism for specific CEACAMs and do not bind to all four CEACAMs, known as pathogen receptors. Accordingly, previous studies
demonstrated that most gonococcal Opa proteins interact with one or several epithelial CEACAMs (CEACAM1, CEA and CEACAM6). In line with this, the binding of Opa proteins to epithelial CEACAMs promotes tight adherence of gonococci and epithelial cells, after the initial pili anchorage (Bos et al. 1997;
Gray-Owen et al. 1997). In addition, the interaction of Opa-expressing gonococci with epithelial CEACAMs is known to enhance the integrin-mediated matrix adhesion of infected epithelial cells and thus block exfoliation of these cells (Muenzner et al. 2010). Moreover, gonococcal engagement of epithelial CEACAMs can lead to the transcytosis of gonococci through epithelial cells and their emergence in the subepithelial spaces (Wang et al. 1998). Binding of gonococci to CEACAM1 expressed on immune cells may impair maturation of dendritic cells or suppress T-cell activation (Kammerer et al. 2001; Boulton and Gray-Owen 2002; Zhu et al. 2012). In contrast to the benefit the gonococci gain by binding to epithelial CEACAMs or to CEACAM1 expressed on immune cells, interaction of Opa proteins with granulocyte-restricted CEACAM3, results in rapid phagocytosis and elimination of the bacteria by human granulocytes (Buntru et al. 2012). Accordingly, granulocyte-phagocytosis belongs to the first line of defense during gonococcal infections. However, some gonococcal strains circumvent granulocyte phagocytosis, disseminate and cause more severe disease (Fig.1.4).
Fig. 1.4: Interaction of Opa-expressing N. gonorrhoeae with human CEACAMs expressed on different cell types. (a) Initial attachment of N.gonorrhoeae to epithelial cells is mediated by the type IV pilus. (b) The interaction of Opa proteins and epithelial CEACAMs promotes a more intimate adherence of gonococci to epithelial cells and enhances epithelial cell matrix adhesion.
(c) Interactions of Opa-expressing gonococci and epithelial CEACAMs may result in transcytosis through epithelial cells. (d) Rarely, gonococci enter endothelial cells and cause systemic disease. (e+f) Binding of gonococci to CEACAM1 expressed on immune cells may impair maturation of dendritic cells or suppress T-cell activation. (g) Interaction of Opa proteins to the phagocyte receptor CEACAM3 results in gonococcal phagocytosis and elimination (modified after Gray-Owen and Blumberg, 2006).
Since individual Opa proteins display distinct tropism for certain CEACAMs, it is plausible that the CEACAM-binding region is located in the variable regions of Opa proteins. Thus, it was shown that the HV-1 and the HV-2 regions of Opa proteins are involved in CEACAM recognition (Rest and Shafer 1989; Virji et al.
1999). In this regard, both HV regions seem to be indispensable for receptor binding. Interestingly, combining HV-1 and HV-2 regions from distinct CEACAM-binding Opa proteins can result in a decrease or loss of CEACAM- binding (Bos et al. 2002). This suggests that HV loops do not contain independent CEACAM-binding motifs, but cooperate to establish CEACAM- binding of gonococci (de Jonge et al. 2003). In contrast to the well- characterized Opa protein binding site on CEACAMs, the exact CEACAM
binding-motif on Opa proteins remains elusive. Hence, the amino acid sequence of Opa proteins, which display the same CEACAM-binding profiles, can be highly dissimilar and, therefore, it is not possible to draw conclusion about the CEACAM-binding properties of Opa proteins by looking at their amino acid sequences.
1.5 Other CEACAM-binding human-restricted pathogens
Beside N. gonorrhoeae, several other human-restricted, Gram-negative bacteria engage CEACAMs for mucosal colonization. Accordingly, N. meningitidis is also known to express CEACAM-binding Opa proteins, which are structurally related to gonococcal Opa proteins (Virji et al. 1996). In contrast to N. gonorrhoeae, N. meningitidis expresses just up to four different Opa proteins. Although, N. meningitidis shares its mucosal lifestyle with N. gonorrhoeae, it inhabits a completely distinct mucosal niche in the human body. Thus, meningococci are frequent colonizer of the upper respiratory tract. Though up to 10% of a population are asymptomatic carrier of N. meningitidis, this bacterium can cause serious blood and brain infections (septicaemia and meningitis). A major difference between gonococci and meningococci is the carbohydrate capsule expressed by most meningococcal strains. According to their capsule structure, meningococci are divided into 13 distinct serogroups. Disease-causing meningococci mainly belong to the serogroups A, B, C, W-135 and Y. Capsules are ideal vaccine candidates, which is the reason why, in contrast to the unencapsulated N. gonorrhoeae, capsule-based vaccines against meningococci of the serogroups A, C, W-135, and Y are on the market. For a long time meningococci of the serogroup B remained a severe problem, since their capsule contained a polysaccharide identical to the polysialic acid present in many human glycoproteins, the reason why it is poorly immunogenic. Recently, the first multicomponent vaccine against meningococci serogroup B (4CMenB;
Bexero) was approved for the market, containing three recombinant meningococcal proteins combined with outer membrane vesicle components of a New Zealand outbreak strain (Tappero et al. 1999).
In addition to the pathogenic Neisseria, commensal Neisseria which share the human respiratory tract with N. meningitidis, express CEACAM-binding Opa adhesins (Toleman et al. 2001). In particular N. lactamica and N. subflava were
shown to express Opa proteins interacting with amino-terminal CEACAM domains (Toleman et al. 2001).
Beside different Neisseria species, several strains of Haemophilus influenzae target epithelial CEACAMs to colonize the human mucosa (Virji et al. 2000).
H. influenzae colonizes the human nasopharynx, thus sharing its mucosal habitat and lifestyle with N. meningitidis. Similar to N. meningitidis, H influenzae can be a commensal colonizer of the human nasopharynx; however, it also can cause localized and systemic disease. H. influenzae is divided into two major groups, depending if they express a capsule (typeable H. influenzae) or if they are unencapsulated (non-typeable H. influenzae). In line with N. gonorrhoeae, there is no vaccine available against non-typeable H. influenzae.
Former studies identified the variable outer membrane protein P5 as CEACAM- binding adhesin of H. influenzae (Hill et al. 2001). Interestingly, P5 displays similarities to neisserial Opa proteins. P5, like Opa proteins, are β-barrels with eight β-stranded transmembrane domains and four extracellular loops, including one conserved loop and three variable loops (Webb and Cripps 1998). In agreement with neisserial Opa proteins, P5 interacts with the C’’C’CFG face of amino-terminal CEACAM domains (Virji et al. 2000; Hill et al. 2001). Like for Opa proteins, the precise CEACAM-binding motif in P5 proteins has not been characterized, yet.
Moraxella catarrhalis represents another commensal inhabitant of the human nasopharynx which interacts with CEACAMs (Hill and Virji 2003). M. catarrhalis can cause infections such as otitis media, purulent conjunctivitis or sinusitis.
Interestingly, the CEACAM-binding adhesin of M. catarrhalis, the ubiquitous surface protein A1 (UspA1), displays a completely different structure compared to the neisserial Opa and the H. influenzae P5 adhesin. Accordingly, it belongs to the family of trimeric autotransporters where the most prominent member is
the YadA adhesin of Yersinia species (Koretke et al. 2006). Hence, the N-terminal region of UspA proteins is considered to form a β-sheet-based
globular head, while the C-terminal region is predicted to be a membrane- spanning β-barrel. The stalk region, connecting the head to the membrane- anchoring domains consists of a coiled-coil structure. Despite the differences regarding their structure, UspA proteins bind to the same motif in the amino- terminal CEACAM domain, as neisserial Opa proteins or P5 of H. influenzae do.
In contrast to Opa proteins and P5, the CEACAM-binding motif of UspA1 could be mapped to a linear peptide sequence in the UspA1 stalk (Hill et al. 2005;
Brooks et al. 2008). Interestingly, a recent study demonstrated that this CEACAM-binding domain can be incorporated into the closely related UspA2 protein, which otherwise lacks CEACAM-binding properties (Hill et al. 2012).
In addition to Neisseria species, H. influenzae and M. catarrhalis, pathogenic E. coli exploit human CEACAMs. These E. coli include diffusely adhering E. coli (DAEC), which are involved in urinary tract infections. Accordingly, DAEC which express a subclass of Afa/Dr fimbriae, recognize CEACAM molecules for mucosal colonization (Guignot et al. 2000; Guignot et al. 2009). Moreover, CEACAMs are engaged by adherent-invasive E. coli (AIEC), associated with Crohn’s disease. Interestingly, it was shown that recent point mutations in the CEACAM-binding adhesin of AIEC, which is the common type 1 pilus adhesin FimH significantly enhanced their CEACAM-binding capacity (Carvalho et al.
2009; Dreux et al. 2013). These finding highlight a mechanism of AIEC pathogenic evolution that involves selection of FimH pathoadaptive mutations, to enhance colonization of human epithelial cells.
In conclusion, the convergent evolution of structurally distinct CEACAM-binding adhesins by several human-restricted pathogens shows the importance of CEACAM-recruitment for these bacteria. Indeed, CEACAM-binding not only promotes mucosal colonization of the bacteria, but also suppresses the detachment of epithelial cells (Muenzner et al. 2010). It is therefore obvious that exploitation of CEACAMs is highly advantageous for bacteria.
2 A
IMS OF THE STUDYMembers of the CEACAM family serve as cellular receptors for several human- restricted pathogens including H. influenzae, M. catarrhalis N. meningitidis and N. gonorrhoeae. In this study, the interaction of microbes with CEACAMs was the basis for the analysis of gonococcal pathogenicity and the development of novel techniques and therapeutics.
I. Several human-restricted pathogens engage epithelial CEACAMs (CEACAM1, CEA, and CEACAM6) to colonize the human mucosa. In contrast, recognition by CEACAM3, a receptor exclusively expressed by human granulocytes, results in uptake and destruction of CEACAM-binding bacteria. This chapter provides an up-to-date review of CEACAM3-dependent bacterial internalization and killing and compares CEACAM3-signaling pathways to other phagocytic receptors, such as Fcγ receptors and Dectin-1.
II. Orthologues of human CEACAMs are also expressed in other primates. Importantly, previous studies demonstrated that human- restricted pathogens selectively interact with human CEACAMs, but not with CEACAMs from rodents or canines. Therefore, we tried to identify and isolate novel CEACAM-binding bacteria from primates. In this chapter, an unbiased cultivation-independent approach was established, which should allow the enrichment and identification of CEACAM-binding bacteria from mixed bacterial populations. Using soluble CEACAM-constructs, a pool of macaque stool samples was enriched for CEACAM-binding bacteria. After next-generation sequencing of 16S rRNA gene libraries and bioinformatics analysis, candidate microbes should be tested in vitro for their CEACAM- binding properties.
III. CEACAM3-mediated uptake and destruction of Opa-expressing N. gonorrhoeae by human granulocytes should limit the spread of gonococci. However, some strains can cause disseminating gonococcal infections (DGI) and it is currently unknown, how these strains escape detection by granulocyte CEACAM3. In this chapter,
our aim was to identify the whole Opa protein repertoire of the DGI strain VP1 and constitutively express them in E. coli. This would allow for the first time detailed functional analysis of the complete Opa protein repertoire of a DGI strain and the comparison to Opa proteins of a non-disseminating strain. The resulting Opa-expressing bacteria should then also be tested with regard to CEACAM3-mediated killing and uptake by primary human granulocytes.
IV. In this study, we developed a novel passive immunization approach, to combat CEACAM-binding pathogens, which avoid CEACAM3-binding.
The aim was to generate soluble Fc-fusion proteins, composed of the amino-terminal part of CEA fused to the Fc part of IgG1. These proteins should then be used to opsonize CEA-binding gonococci to allow their recognition by phagocytes. Accordingly, it was our goal to measure the uptake of CEA-Fc opsonized gonococci into FcγRIIa- expressing human cells and the internalization and destruction by primary human granulocytes.
3 CHAPTER I
HemITAM signaling by
CEACAM3, a human granulocyte receptor recognizing bacterial
pathogens
Alexander Buntru1,2, Alexandra Roth1, Naja Nyffenegger-Jann1 and Christof R.
Hauck1,2
1Lehrstuhl für Zellbiologie, Universität Konstanz, and 2Konstanz Research School Chemical Biology, Universität Konstanz, Germany
Arch Biochem Biophys. 2012 Aug 1;524(1):77-83.
3.1 Abstract
Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) belong to the immunoglobulin superfamily and contribute to cell–cell adhesion and signal modulation in various tissues. In humans, several CEACAMs are targeted by pathogenic bacteria. One peculiar member of this family, CEACAM3, is exclusively expressed by human granulocytes and functions as an opsonin-independent phagocytic receptor for CEACAM-binding bacteria.
Here, we will discuss CEACAM3-dependent processes by summarizing recent insight into the phosphotyrosine-based signaling complex formed upon CEACAM3 engagement. Compared to different well-studied phagocytic receptors, such as Fcγ receptors and Dectin-1, CEACAM3 appears as an example of a hemITAM-containing innate immune receptor, which promotes rapid internalization and intracellular destruction of a diverse group of CEACAM-binding bacteria. The particular efficiency of CEACAM3 arises from the direct coupling of upstream activators and downstream effectors of the small GTPase Rac by the cytoplasmic domain of CEACAM3, which co-ordinates actin cytoskeleton re-arrangements and bactericidal effector mechanisms of granulocytes.
3.2 CEACAM family proteins as bacterial receptors
CEACAM3 is a member of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family. In humans, twelve closely related CEACAMs can be expressed by various tissues, but are predominantly found on cells of epithelial or hematopoietic origin (Kuespert et al. 2006; Muenzner et al. 2010).
All CEACAMs share a characteristic amino-terminal immunoglobulin variable (IgV)-like domain and some members have in addition up to six immunoglobulin constant (IgC)-like domains in their extracellular part. Reflected in their wide tissue distribution, CEACAMs modulate various cellular functions. For instance, epithelial CEACAMs (CEACAM1, CEA and CEACAM6) generally contribute to intercellular adhesion by the formation of homophilic interactions with other CEACAM family members and some CEACAMs bind soluble extracellular proteins. Thereby, CEACAMs can initiate signaling processes that affect the proliferation and differentiation of cells as well as the functional organization of tissues (Nagaishi et al. 2006; Yokoyama et al. 2007; Nouvion et al. 2010; Zheng
et al. 2011). Interestingly, several human-restricted pathogens, namely Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, Moraxella catarrhalis, as well as some strains of pathogenic Escherichia coli, have evolved distinct afimbrial adhesins to engage human CEACAMs (Chen and Gotschlich 1996; Virji et al. 1996; Chen et al. 1997; Gray-Owen et al. 1997;
Hill et al. 2001; Hill and Virji 2003; Barnich et al. 2007). The importance of CEACAM-binding for the bacterial life cycle is underscored by the fact that certain bacterial species seem to have at least two independent means to engage members of this receptor family (Kuespert et al. 2011). Furthermore, human-restricted bacteria selectively recognize CEACAMs in a species-specific manner, as CEACAM1 orthologues from dog, cattle or mouse do not function as receptors for pathogenic Neisseriae or M. catarrhalis (Voges et al. 2010). Most importantly, binding to CEACAMs found on the apical surface of epithelial cells facilitates the bacterial colonization of the human mucosa in several ways. In addition to promote the attachment of the bacteria to the tissue surface, stimulation of epithelial CEACAMs, such as CEACAM1, CEA, or CEACAM6, triggers gene expression events resulting in altered extracellular matrix-binding of the infected cells (Muenzner et al. 2005). In vivo, this interaction between bacteria and epithelial CEACAMs suppresses exfoliation of superficial epithelial cells from the tissue, thereby generating a stable platform for bacterial colonization (Muenzner et al. 2010). It is highly plausible, that the pronounced suppression of exfoliation in a stratified epithelium is the primary driving force behind the convergent evolution of CEACAM-binding adhesins in diverse bacterial species.
In vitro, however, additional CEACAM-dependent cellular responses have been observed. For example, CEACAM-binding results in bacterial internalization and promotes transcytosis of bacteria through an intact polarized epithelial layer (Wang et al. 1998; McCaw et al. 2004; Schmitter et al. 2007; Wang et al. 2007;
Muenzner et al. 2008). Moreover, interaction of bacteria and CEACAM1 in vitro can modulate T-cell responses (Boulton and Gray-Owen 2002) and can increase the expression of pro-inflammatory cytokines as well as expression of the receptor itself by endothelial and epithelial cells (Muenzner et al. 2001;
Muenzner et al. 2002; Griffiths et al. 2007), suggesting that CEACAM- recognition could benefit the bacteria beyond the initial colonization step.
In contrast to the advantage bacteria might gain from binding to epithelial CEACAMs, the expression of a CEACAM-binding adhesin might also predispose bacteria for detection and elimination by human granulocytes. In the case of N. gonorrhoeae, it has long been recognized that certain colony opacity (Opa) protein-expressing variants are readily engulfed by human granulocytes in the absence of opsonizing antibodies or complement factors (Naids and Rest 1991; Belland et al. 1992; Kupsch et al. 1993) indicating the presence of specific Opa protein receptors on human granulocytes. The identification of CEACAMs as receptors for neisserial Opa proteins immediately suggested a major role for these molecules in opsonin-independent phagocytosis of bacteria (Chen and Gotschlich 1996; Gray-Owen et al. 1997; Hauck et al. 1998). It is important to note that human granulocytes express several CEACAM family members including CEACAM1, CEACAM3, CEACAM4, CEACAM6, and CEACAM8. Clearly, each of these receptors is able to individually trigger granulocyte responses such as degranulation or increased integrin-mediated adhesion upon stimulation with CEACAM-specific antibodies (Skubitz et al.
1996; Skubitz and Skubitz 2008). Therefore, a central question was initially, which CEACAM(s) is/are responsible for granulocyte-dependent phagocytosis of CEACAM-binding bacteria? Within the last few years, several independent lines of investigation have pointed to CEACAM3 as the major driver behind this efficient phagocytic process, which is based on large lamellipodial protrusions (Fig. 3.1).
Fig. 3.1: CEACAM3-mediated, opsonin-independent phagocytosis by human granulocytes. Primary human granulocytes were isolated from peripheral blood and infected for 15 min with N. gonorrhoeae expressing a CEACAM3-binding Opa protein. Upon fixation, samples were processed for scanning electron microscopy. Shown is a pseudocolored image of a human granulocyte (dark) in the process of opsonin-independent phagocytosis of multiple
gonococci (orange) via large lamellipodial protrusions (arrowheads). The boxed area is enlarged in the right panel and shows a detail of the phagocyte surface at the site of CEACAM3-mediated phagocytosis of N. gonorrhoeae.
3.3 Phosphorylation of the CEACAM3 cytoplasmic domain and CEACAM3 membrane localization
Besides the highly selective expression in human granulocytes, CEACAM3 is peculiar within the group of bacteria-binding CEACAMs for other reasons. On the one hand, the overall domain structure of this type 1 transmembrane protein differs from epithelial bacteria-binding CEACAMs by the lack of extracellular Igc-like domains (Pils et al. 2008). Furthermore, the CEACAM3 cytoplasmic domain encompasses an immunoreceptor tyrosine-based activation motif (ITAM)-like sequence, which is not present in other bacteria-binding CEACAMs.
In this respect, the CEACAM3 ITAM-like sequence (YxxLx(7)YxxM) shares features with the amino acid sequence of canonical ITAMs (YxxL/Ix(6-12)YxxL/I) found in immune receptors such as the well-studied T-cell receptor (TCR) ζ- chain or the activating Fcγ receptors (FcγRs) (Reth 1989) (Fig. 3.2).
Fig. 3.2: Alignment of consensus ITAM sequences and ITAM-like sequences. The amino acid sequences of cytoplasmic domains of the corresponding human membrane proteins and the acidic region of Vav1 were obtained from NCBI. Tyrosine residues conforming to the consensus ITAM sequence are highlighted in blue, critical neighboring determinants are bold.
CEACAM3 and Dectin-1 tyrosine residues not conforming to the consensus ITAM and their sequence context are marked in red. The orientation of the transmembrane domain (TM) relative to the shown cytoplasmic domains is indicated.
The individual members of the FcγR family are expressed on hematopoietic cells, with FcγRI and FcγRIIa present on human granulocytes (Nimmerjahn and
Ravetch 2008). Upon crosslinking by immune complexes, such as antibody- coated microorganisms, FcγRs are phosphorylated on tyrosine residues within the ITAM by protein tyrosine kinases (PTKs) of the Src-family (Cooney et al.
2001; Cox and Greenberg 2001; Greenberg 2001).
In analogy to FcγRs, the ITAM-like sequence of CEACAM3 was proposed to become tyrosine phosphorylated and to initiate a signaling cascade to promote phagocytosis (Chen et al. 2001; Billker et al. 2002). Indeed, bacterial engulfment via large lamellipodia protrusions, which can be observed during CEACAM3-mediated uptake, is highly reminiscent to that described for ITAM- containing FcγRs (Billker et al. 2002; Schmitter et al. 2004). Correspondingly, previous studies have shown that the two tyrosines Tyr230 and Tyr241 within the ITAM-like sequence of CEACAM3 become phosphorylated and contribute to efficient uptake of the human pathogen N. gonorrhoeae (Chen et al. 2001;
McCaw et al. 2003; Schmitter et al. 2004). In a similar fashion to FcγR, CEACAM3 is tyrosine phosphorylated by Src-family PTKs (in particular the Src family PTKs Hck and Fgr), which are activated in granulocytes in response to CEACAM-binding bacteria (Hauck et al. 1998; Schmitter et al. 2007).
Furthermore, phosphorylation of CEACAM3 as well as bacterial uptake by CEACAM3 transfected cell lines or primary human granulocytes is strongly affected by Src kinase-specific inhibitors, in line with the notion that Src-family kinases are required for the CEACAM-mediated opsonin-independent phagocytosis of bacteria (McCaw et al. 2003; Schmitter et al. 2007). In contrast, bacterial internalization via CEACAM6 is barely affected by Src PTK inhibitors (Schmitter et al. 2007). Though CEACAM6 is more abundant than CEACAM3 on the surface of human granulocytes, the strong inhibitory effect of Src family PTK inhibitors on CEACAM-mediated uptake suggests that CEACAM6 only plays a minor role during CEACAM-mediated opsonin-independent phagocytosis by granulocytes. Furthermore, CEACAM6 is a GPI-anchored protein, which resides in cholesterol- and sphingolipid-rich membrane microdomains (lipid rafts) (Schmitter et al. 2007; Muenzner et al. 2008).
CEACAM6 shares this particular subcellular localization with other epithelial CEACAMs, such as CEA and CEACAM1, which translocate into a lipid raft membrane fraction upon cross-linking (Muenzner et al. 2008). The lipid raft association renders CEACAM6 and CEACAM1-initiated bacterial internalization
sensitive to cholesterol depleting agents such as methyl-β-cyclodextrin (Schmitter et al. 2007; Muenzner et al. 2008). In contrast, CEACAM3 localizes to non-raft, phosphoglycerolipid-rich subdomains of the plasma membrane and this distinct membrane distribution is determined by the transmembrane domain of CEACAM3 (Muenzner et al. 2008). Exchange of the CEACAM1 transmembrane domain for the membrane-spanning amino acid sequence of CEACAM3 can re-direct CEACAM1 into the non-raft membrane fraction and makes CEACAM1-mediated uptake insensitive to cholesterol depletion (Muenzner et al. 2008). It is important to note that CEACAM-mediated, opsonin- independent phagocytosis of bacteria by primary human granulocytes is insensitive to cholesterol depletion (Schmitter et al. 2007) again ruling out a major contribution of CEACAM1 and CEACAM6 to this process. Consistent with the lipid-raft independent uptake by granulocytes, the CEACAM3-mediated bacterial internalization in transfected cell lines is not affected by cholesterol depletion (Schmitter et al. 2007; Muenzner et al. 2008). Together with the effect of Src PTK inhibitors, the independence from lipid rafts provides direct evidence, that CEACAM3-mediated opsonin-independent phagocytosis, despite the presence of considerable amounts of CEACAM1 and CEACAM6, is the major route of uptake upon encounter of non-opsonized, CEACAM-binding bacteria and human granulocytes.
3.4 CEACAM3-initiated signaling leading to actin rearrangements and phagocytosis
Upon phosphorylation of the FcγR cytoplasmic domain, kinases of the Syk- family are recruited to the dually phosphorylated ITAM, where they bind via their tandem Src homology 2 (SH2) domains (Kiefer et al. 1998). In the case of FcγR-mediated phagocytosis, Syk kinase is essential for activation of downstream events, either by phosphorylation and recruitment of adapter proteins such as LAT, Nck, CrkII, SLP-76, and Gab2 or by functioning as an adapter itself, e.g. by binding to the regulatory p85 subunit of type I phosphatidylinositol-3’ kinase (PI3K) or the guanine nucleotide exchange factor (GEF) Vav (Flannagan et al. 2012). Accordingly, Syk-dependent events result in the local recruitment and activation of GEFs, which stimulate small GTPases of the Rho family, such as Rac and Cdc42. In turn, these GTPases orchestrate,
via their effector proteins WASP and WAVE, the actin-cytoskeleton-driven formation of lamellipodial protrusions that form a phagocytic cup (Cox et al.
1997; Greenberg and Grinstein 2002; Swanson and Hoppe 2004).
CEACAM-mediated phagocytosis by primary human granulocytes is also characterized by the formation of f-actin containing lamellipodial structures (Billker et al. 2002; Schmitter et al. 2004). Upon encounter with CEACAM- binding bacteria, a strong increase in GTP-loaded Rac, but not Cdc42, has been observed in human phagocytes (Hauck et al. 1998). Detailed analysis in transfected cell lines demonstrated that engagement of CEACAM3, but not of CEACAM1 or CEACAM6, results in lamellipodial protrusions and enhanced Rac GTP loading. The increased Rac-GTP levels depend on both, the integrity of the CEACAM3 ITAM-like sequence as well as Src PTK activity (Schmitter et al.
2004). Together with the fact that dominant negative Rac, but not dominant- negative Cdc42, interferes with uptake of CEACAM-binding bacteria by primary human granulocytes, these results highlight the stringent connection between CEACAM3 and Rac in promoting this opsonin-independent phagocytosis.
The GEF Vav, which is involved in Rac activation downstream of FcγR phosphorylation, is also the critical GEF mediating CEACAM3-initiated Rac GTP loading. Biochemical and functional evidence established that Vav directly binds via its SH2 domain to the phosphorylated Tyr230 within the ITAM-like sequence of CEACAM3 (Schmitter et al. 2007). This is in striking contrast to FcγRs, where Vav indirectly associates with the activated receptor via an interaction with the ITAM-bound Syk or via binding to the phosphorylated adaptor protein Slp-76 (Deckert et al. 1996; Tuosto et al. 1996; Rouard et al.
1999). The direct, Syk-independent association of Vav with CEACAM3 could help to explain, why Syk is dispensable for the uptake via CEACAM3. Indeed, treatment of primary human granulocytes with the Syk inhibitor piceatannol does not impair CEACAM-mediated internalization (Sarantis and Gray-Owen 2007). Furthermore, Syk overexpression in different cell lines, which lack endogenous Syk expression, does not increase CEACAM3-initiated uptake of N. gonorrhoeae (Sarantis and Gray-Owen 2007) (Pils and Hauck, unpublished observations). These results are unexpected given that FcγR-mediated phagocytosis of IgG-opsonized particles strongly depends on Syk activity (Crowley et al. 1997; Kiefer et al. 1998). The direct association with Vav and the
lack of a contribution of Syk kinases underscore the fact that the CEACAM3 ITAM-like sequence clearly operates in a manner distinct from canonical ITAMs.
The current data support the idea, that the CEACAM3 ITAM-like sequence directly engages downstream components involved in canonical ITAM signaling, thereby circumventing the need for Syk kinases, which coordinate signaling complexes during FcγR signaling. Recent insight has further strengthened the concept that CEACAM3 short wires receptor clustering with re-organization of the actin cytoskeleton for opsonin-independent phagocytosis.
A biochemical screen has revealed that the SH2 domains of the adapter proteins Nck1 and Nck2 bind to the phosphorylated CEACAM3 ITAM-like sequence and Nck transiently co-localizes with CEACAM3 upon bacterial binding (Pils et al. 2012). SH2 domains of other adapter proteins involved in canonical ITAM signaling, such as Grb2, Crk, or SLP-76, do not bind to the receptor. The highly homologous proteins Nck1 and Nck2, which are composed of three SH3 domains and a single SH2 domain and which are co-expressed in most tissues, are often involved in regulating actin cytoskeleton dynamics (Buday and Tompa 2010). In line with this notion, Nck1/Nck2-deficient cells do not form lamellipodia upon CEACAM3 stimulation (Pils et al. 2012). A known binding partner of Nck SH3 domains is the Nck-associated protein 1 (Nap1, also termed NCKAP1 or Hem2). The Rac-effectors Nap1 and the specifically Rac- associated protein 1 (Sra1) are part of the intrinsically inactive WAVE complex and seem to mask the carboxy-terminal VCA-domain (Verprolin-, Cofilin- homology acidic domain) of WAVE thereby preventing WAVE-initiated Arp2/3 complex stimulation (Miki et al. 1998; Derivery and Gautreau 2010). The VCA- domain is released due to activation of the WAVE-complex by multiple stimuli including association with GTP-loaded Rac, binding to phosphoinositides and phosphorylation of WAVE, although the exact mechanism are currently unknown (Leng et al. 2005; Danson et al. 2007; Lebensohn and Kirschner 2009). While the WAVE V-region binds actin-monomers, the CA region associates with the Arp2/3 complex to create a nucleation core for actin polymerisation (Miki and Takenawa 1998; Machesky and Insall 1999; Rohatgi et al. 1999). Nck connects the phosphorylated CEACAM3 ITAM-like sequence with the WAVE complex triggering local f-actin-based lamellipodial structures.
Accordingly, shRNA-mediated knock-down of WAVE complex components or
