Petra Muenzner

Carcinoembryonic Antigen Family Receptor Specificity of Neisseria meningitidis Opa Variants Influences Adherence to and Invasion of Proinflammatory Cytokine-Activated Endothelial Cells

ABSTRACT The carcinoembryonic antigen (CEA) family member CEACAM1 (previously called biliary glycoprotein or CD66a) was previously shown to function as a receptor that can mediate the binding of Opa protein-expressing Neisseria meningitidis to both neutrophils and epithelial cells. Since neutrophils and polarized epithelia have both been shown to coexpress multiple CEACAM receptors, we have now extended this work to characterize the binding specificity of meningococcal Opa proteins with other CEA family members. To do so, we used recombinant Escherichia coli expressing nine different Opa variants from three meningococcal strains and stably transfected cell lines expressing single members of the CEACAM family. These infection studies demonstrated that seven of the nine Opa variants bound to at least one CEACAM receptor and that binding to each of these receptors is sufficient to trigger the Opa-dependent bacterial uptake by these cell lines. The other two Opa variants do not appear to bind to either CEACAM receptors or heparan sulfate proteoglycan receptors, which are bound by some gonococcal Opa variants, thus implying a novel class of Opa proteins. We have also extended previous studies by demonstrating induction of CEACAM1 expression after stimulation of human umbilical vein endothelial cells with the proinflammatory cytokine tumor necrosis factor alpha, which is present in high concentrations during meningococcal disease. This induced expression of CEACAM1 leads to an increased Opa-dependent bacterial binding and invasion into the primary endothelia, implying that these interactions may play an important role in the pathogenesis of invasive meningococcal disease.

Pathogenic Neisseria trigger expression of their carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1; previously CD66a) receptor on primary endothelial cells by activating the immediate early response transcription factor, nuclear factor-kappa B

Neisseria gonorrhoeae express opacity-associated (Opa) protein adhesins that mediate binding to various members of the carcinoembryonic antigen-related cellular adhesion molecule (CEACAM; previously CD66) receptor family. Although human umbilical vein endothelial cells express little CEACAM receptor in vitro, we found neisserial infection to induce expression of CEACAM1, CEACAM1-3L, and CECAM1-4L splice variants. This mediates an increased Opa52-dependent binding of gonococci by these cells. The induced receptor expression did not require bacterial Opa expression, but it was more rapid with adherent bacteria. Because the time course of induction was similar to that seen for induced proinflammatory cytokines, we tested whether CEACAM1 expression could be controlled by a similar mechanism. Gonococcal infection activated a nuclear factor-κB (NF-κB) heterodimer consisting of p50 and p65, and inhibitors that prevent the nuclear translocation of activated NF-κB complex inhibited CEACAM1 transcript expression. Each of these effects could be mimicked by using culture filtrates or purified lipopolysaccharide instead of intact bacteria. Together, our results support a model whereby the outer membrane “blebs” that are actively released by gonococci trigger a Toll-like receptor-4-dependent activation of NF-κB, which up-regulates the expression of CEACAM1 to allow Opa52-mediated neisserial binding. The regulation of CEACAM1 expression by NF-κB also implies a broader role for this receptor in the general inflammatory response to infection.

Human-Restricted Bacterial Pathogens Block Shedding of Epithelial Cells by Stimulating Integrin Activation

Here to Stay For systemic infection, bacterial pathogens must breach the mucosal epithelial barrier. Our bodies have developed a variety of strategies to protect the mucosa, including rapid turnover of epithelial cells. Muenzner et al. (p. 1197; see the cover) show how invasive bacteria overcome this host defense in a humanized mouse model susceptible to Neisseria gonorrhoeae urogenital infection. The bacteria bind to a host-cell surface protein called carcinoembryonic antigen (CEA), which triggers a cascade of changes modulating the cell adhesion properties of the targeted epithelium to prevent the cells from being shed. Bacterial colonization of the mucosa is facilitated if the microbes engage a human receptor that counteracts epithelial exfoliation. Colonization of mucosal surfaces is the key initial step in most bacterial infections. One mechanism protecting the mucosa is the rapid shedding of epithelial cells, also termed exfoliation, but it is unclear how pathogens counteract this process. We found that carcinoembryonic antigen (CEA)–binding bacteria colonized the urogenital tract of CEA transgenic mice, but not of wild-type mice, by suppressing exfoliation of mucosal cells. CEA binding triggered de novo expression of the transforming growth factor receptor CD105, changing focal adhesion composition and activating β1 integrins. This manipulation of integrin inside-out signaling promotes efficient mucosal colonization and represents a potential target to prevent or cure bacterial infections.

The anti-inflammatory compound curcumin inhibits Neisseria gonorrhoeae-induced NF-kappa B signaling, release of proinflammatory cytokines/chemokines and attenuates adhesion in late infection

Abstract Neisseria gonorrhoeae (Ngo) is a Gram-negative pathogenic bacterium responsible for an array of diseases ranging from urethritis to disseminated gonococcal infections. Early events in the establishment of infection involve interactions between Ngo and the mucosal epithelium, which induce a local inflammatory response. Here we analyzed the molecular mechanism involved in the Ngo-induced induction of the proinflammatory cytokines tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and IL-8. We identified the immediate early response transcription factor nuclear factor κB (NF-κB) as a key molecule for the induction of cytokine release. Ngo-induced activation of direct upstream signaling molecules was demonstrated for IκB kinase α and β (IKKα and IKKβ) by phosphorylation of IκBα as a substrate and IKK autophosphorylation. Using dominant negative cDNAs encoding kinase-dead IKKα, IKKβ, and NF-κB-inducing kinase (NIK), Ngo-induced NF-κB activity was significantly inhibited. Curcumin, the yellow pigment derived from Curcuma longa, inhibited IKKα, IKKβ and NIK, indicating its strong potential to block NF-κB-mediated cytokine release and the innate immune response. In addition to the inhibition of Ngo-induced signaling, curcumin treatment of cells completely abolished the adherence of bacteria to cells in late infection, underlining the high potential of curcumin as an anti-microbial compound without cytotoxic side effects.

Exploitation of integrin function by pathogenic microbes

Numerous pathogens express adhesive proteins to directly or indirectly associate with integrins. It is well established that by targeting integrins, microbes not only establish an intimate contact with host tissues, but also trigger cellular responses including bacterial internalization. This review will summarize current knowledge about the role of these integrin-dependent processes during infection and how bacteria assure that they efficiently connect to integrins for host cell invasion or translocation of effector molecules. Furthermore, we will discuss recent insight demonstrating that bacteria can harness the physiological, matrix-binding function of integrins for promoting host colonization. From these combined studies, it is becoming evident that integrins are a common nexus for the manipulation of cellular functions by bacterial pathogens. Approaches to disrupt this connection might be an appropriate means to obtain broad-acting tools to modulate a spectrum of infectious diseases.

The CEACAM1 transmembrane domain, but not the cytoplasmic domain, directs internalization of human pathogens via membrane microdomains.

Several bacterial pathogens exploit carcinoembryonic antigen‐related cell adhesion molecules (CEACAMs) to promote attachment and uptake into eukaryotic host cells. The widely expressed isoform CEACAM1 is involved in cell–cell adhesion, regulation of cell proliferation, insulin homeostasis, and neo‐angiogenesis, processes that depend on the cytoplasmic domain of CEACAM1. By analysing the molecular requirements for CEACAM1‐mediated internalization of bacteria, we surprisingly find that the CEACAM1 cytoplasmic domain is completely obsolete for bacterial uptake. Accordingly, CEACAM1‐4L as well as a CEACAM1 mutant with a complete deletion of the cytoplasmic domain (CEACAM1 ΔCT) promote equivalent internalization of several human pathogens. CEACAM1‐4L‐ and CEACAM1 ΔCT‐mediated uptake proceeds in the presence of inhibitors of actin microfilament dynamics, which is in contrast to CEACAM3‐mediated internalization. Bacteria‐engaged CEACAM1‐4L and CEACAM1 ΔCT, but not CEACAM3, localize to a gangliosid GM1‐ and GPI‐anchored protein‐containing portion of the plasma membrane. In addition, interference with cholesterol‐rich membrane microdomains severely blocks bacterial uptake via CEACAM1‐4L and CEACAM1 ΔCT, but not CEACAM3. Similar to GPI‐anchored CEACAM6, both CEACAM1‐4L as well as CEACAM1 ΔCT partition into a low‐density, Triton‐insoluble membrane fraction upon receptor clustering, whereas CEACAM3 is not detected in this fraction. Bacterial uptake by truncated CEACAM1 or chimeric CEACAM1/CEACAM3 molecules reveals that the transmembrane domain of CEACAM1 is responsible for its association with membrane microdomains. Together, these data argue for a functional role of lipid rafts in CEACAM1‐mediated endocytosis that is promoted by the transmembrane domain of the receptor and that might be relevant for CEACAM1 function in physiologic settings.

Uropathogenic E. coli Exploit CEA to Promote Colonization of the Urogenital Tract Mucosa

Attachment to the host mucosa is a key step in bacterial pathogenesis. On the apical surface of epithelial cells, members of the human carcinoembryonic antigen (CEA) family are abundant glycoproteins involved in cell-cell adhesion and modulation of cell signaling. Interestingly, several gram-negative bacterial pathogens target these receptors by specialized adhesins. The prototype of a CEACAM-binding pathogen, Neisseria gonorrhoeae, utilizes colony opacity associated (Opa) proteins to engage CEA, as well as the CEA-related cell adhesion molecules CEACAM1 and CEACAM6 on human epithelial cells. By heterologous expression of neisserial Opa proteins in non-pathogenic E. coli we find that the Opa protein-CEA interaction is sufficient to alter gene expression, to increase integrin activity and to promote matrix adhesion of infected cervical carcinoma cells and immortalized vaginal epithelial cells in vitro. These CEA-triggered events translate in suppression of exfoliation and improved colonization of the urogenital tract by Opa protein-expressing E. coli in CEA-transgenic compared to wildtype mice. Interestingly, uropathogenic E. coli expressing an unrelated CEACAM-binding protein of the Afa/Dr adhesin family recapitulate the in vitro and in vivo phenotype. In contrast, an isogenic strain lacking the CEACAM-binding adhesin shows reduced colonization and does not suppress epithelial exfoliation. These results demonstrate that engagement of human CEACAMs by distinct bacterial adhesins is sufficient to blunt exfoliation and to promote host infection. Our findings provide novel insight into mucosal colonization by a common UPEC pathotype and help to explain why human CEACAMs are a preferred epithelial target structure for diverse gram-negative bacteria to establish a foothold on the human mucosa.

Innate Recognition by Neutrophil Granulocytes Differs between Neisseria gonorrhoeae Strains Causing Local or Disseminating Infections

ABSTRACT Members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family serve as cellular receptors for Neisseria gonorrhoeae. More specifically, neisserial colony opacity (OpaCEA) proteins bind to epithelial CEACAMs (CEACAM1, CEA, CEACAM6) to promote bacterial colonization of the mucosa. In contrast, recognition by CEACAM3, expressed by human granulocytes, results in uptake and destruction of OpaCEA-expressing bacteria. Therefore, CEACAM3-mediated uptake might limit the spread of gonococci. However, some strains can cause disseminating gonococcal infections (DGIs), and it is currently unknown how these strains escape detection by granulocyte CEACAM3. Therefore, the opa gene loci from N. gonorrhoeae strain VP1, which was derived from a patient with disseminated gonococcal disease, were cloned and constitutively expressed in Escherichia coli. Similar to Opa proteins of the nondisseminating strain MS11, the majority of Opa proteins from strain VP1 bound epithelial CEACAMs and promoted CEACAM-initiated responses by epithelial cells. In sharp contrast to the Opa proteins of strain MS11, the Opa proteins of strain VP1 failed to interact with the human granulocyte receptor CEACAM3. Accordingly, bacteria expressing VP1 Opa proteins were not taken up by primary human granulocytes and did not trigger a strong oxidative burst. Analysis of Opa variants from four additional clinical DGI isolates again demonstrated a lack of CEACAM3 binding. In summary, our results reveal that particular N. gonorrhoeae strains express an Opa protein repertoire allowing engagement of epithelial CEACAMs for successful mucosal colonization, while avoiding recognition and elimination via CEACAM3-mediated phagocytosis. A failure of CEACAM3-mediated innate immune detection might be linked to the ability of gonococci to cause disseminated infections.