Homotypic Interactions Research Articles

Structural ordering of the Plasmodium berghei circumsporozoite protein repeats by inhibitory antibody 3D11.

Plasmodium sporozoites express circumsporozoite protein (CSP) on their surface, an essential protein that contains central repeating motifs. Antibodies targeting this region can neutralize infection, and the partial efficacy of RTS,S/AS01 - the leading malaria vaccine against P. falciparum (Pf) - has been associated with the humoral response against the repeats. Although structural details of antibody recognition of PfCSP have recently emerged, the molecular basis of antibody-mediated inhibition of other Plasmodium species via CSP binding remains unclear. Here, we analyze the structure and molecular interactions of potent monoclonal antibody (mAb) 3D11 binding to P. berghei CSP (PbCSP) using molecular dynamics simulations, X-ray crystallography, and cryoEM. We reveal that mAb 3D11 can accommodate all subtle variances of the PbCSP repeating motifs, and, upon binding, induces structural ordering of PbCSP through homotypic interactions. Together, our findings uncover common mechanisms of antibody evolution in mammals against the CSP repeats of Plasmodium sporozoites.

Crystal structure of caspase-11 CARD provides insights into caspase-11 activation

Murine caspase-11 is the centerpiece of the non-canonical inflammasome pathway that can respond to intracellular LPS and induce pyroptosis. Caspase-11 contains two components, an N-terminal caspase recruitment domain (CARD) and a C-terminal catalytic domain. The aggregation of caspase-11 is thought to promote the auto-processing and activation of caspase-11. However, the activation mechanism of caspase-11 remains unclear. In this study, we purified the caspase-11 CARD fused to an MBP tag and found it tetramerizes in solution. Crystallographic analysis reveals an extensive hydrophobic interface formed by the H1–2 helix mediating homotypic CARD interactions. Importantly, mutations of the helix H1–2 hydrophobic residues abolished the tetramerization of MBP-tagged CARD in solution and failed to induce pyroptosis in cells. Our study provides the first evidence of the homotypic interaction mode for an inflammatory caspase by crystal model. This finding demonstrates that the tetramerization of the N-terminal CARD can promote releasing of the catalytic domain auto-inhibition, leading to the caspase-11 activation.

A systems-biology model of the tumor necrosis factor (TNF) interactions with TNF receptor 1 and 2

Clustering enables TNF receptors to stimulate intracellular signaling. The differential soluble ligand-induced clustering behavior of TNF receptor 1 (TNFR1) and TNFR2 was modeled. A structured, rule-based model implemented ligand-independent pre-ligand binding assembly domain (PLAD)-mediated homotypic low affinity interactions of unliganded and liganded TNF receptors. Soluble TNF initiates TNFR1 signaling but not TNFR2 signaling despite receptor binding unless it is secondarily oligomerized. We consider high affinity binding of TNF to signaling-incompetent pre-assembled dimeric TNFR1 and TNFR2 molecules and secondary clustering of liganded dimers to signaling competent ligand-receptor clusters. Published receptor numbers, affinities and measured different activities of clustered receptors validated model simulations for a large range of receptor and ligand concentrations. Different PLAD-PLAD affinities and different activities of receptor clusters explain the observed differences in the TNF receptor stimulating activities of soluble TNF. All scripts and data are in manuscript and supplement at Bioinformatics online. Supplementary data are available at Bioinformatics online.

Regulation of titin-based cardiac stiffness by unfolded domain oxidation (UnDOx)

The relationship between oxidative stress and cardiac stiffness is thought to involve modifications to the giant muscle protein titin, which in turn can determine the progression of heart disease. In vitro studies have shown that S-glutathionylation and disulfide bonding of titin fragments could alter the elastic properties of titin; however, whether and where titin becomes oxidized in vivo is less certain. Here we demonstrate, using multiple models of oxidative stress in conjunction with mechanical loading, that immunoglobulin domains preferentially from the distal titin spring region become oxidized in vivo through the mechanism of unfolded domain oxidation (UnDOx). Via oxidation type-specific modification of titin, UnDOx modulates human cardiomyocyte passive force bidirectionally. UnDOx also enhances titin phosphorylation and, importantly, promotes nonconstitutive folding and aggregation of unfolded domains. We propose a mechanism whereby UnDOx enables the controlled homotypic interactions within the distal titin spring to stabilize this segment and regulate myocardial passive stiffness.

Abstract 4587: Utility of recombinant MUC4 domains as potential therapeutic agents

Abstract Pancreatic cancer (PC) is a highly aggressive malignancy with limited therapeutic options and is characterized by aberrant overexpression of mucins. Mucin 4 (MUC4) is a multi-domain transmembrane mucin which is differentially overexpressed in pancreatic cancer. MUC4 expression increases progressively during PC and high expression correlates with poor prognosis. MUC4 functionally contributes to pathobiology and aggressiveness of the disease by promoting cell proliferation, migration and inducing chemotherapy resistance. MUC4 is putatively cleaved at GDPH site protein resulting in two subunits, MUC4α and MUC4β. MUC4α comprises of highly glycosylated tandem repeat region, followed by NIDO and AMOP domains. The NIDO domain is known to play a role in cancer metastasis and tumor growth by interacting with other proteins. MUC4β comprises of vWD domain, three EGF-like domains, and a transmembrane domain. Following cleavage, MUC4β remains tethered to the cell membrane as a functional protein, and is thus an attractive target for therapeutic intervention. We have cloned and expressed various recombinant domains of MUC4 for the generation of monoclonal antibodies and development of therapeutic vaccine. We observed that recombinant MUC4β (rMUC4β) interacts with several cellular proteins including Ezrin, EGFR, HER2 and HER3. On the basis of these protein-protein interactions, we hypothesized that exogenous soluble MUC4β acts as a competitive inhibitor of endogenous MUC4 and disrupts its tumor-promoting functions. To determine the utility of rMUC4β as a therapeutic agent, we examined its effect on the proliferation, aggregation, migration, apoptosis and downstream signaling pathways in MUC4 expressing (CD18/HPAF) and non-expressing (MiaPaCa) human pancreatic cancer cell lines. We observed that rMUC4β induced alterations in cell morphology, including higher membrane blebbing, detachment from the plate, and disrupted homotypic cell-cell interactions. Further, exogenous MUC4β significantly inhibited (p value=0.0001) the proliferation of MUC4 expressing CD18/HPAF cells in a dose and time dependent manner. In contrast, no significant change was observed in growth rates and cells-cell interactions in MUC4 negative MiaPaCa cells. We also observed that MUC4β induced significantly higher apoptosis (> 15%) in MUC4 expressing cells as compared to MUC4 negative. Additionally, treatment of MUC4 expressing cells with rMUC4β inhibited their migration by up to 30 percent in a dose-dependent manner while no significant effect was observed in MUC4 non-expressing cells. Recombinant MUC4β treatment resulted in decreased phosphorylation of ERK-1 and AKT. Overall these studies suggest that rMUC4β can competitively inhibit some of the functions of endogenous MUC4 and disrupt the growth and migration promoting signaling pathways. Thus, MUC4β is an attractive therapeutic agent that can potentially inhibit tumorigenesis and metastasis in MUC4-positive PC. Citation Format: Mansi Gulati. Utility of recombinant MUC4 domains as potential therapeutic agents [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4587.

Synthetic ECM: Bioactive Synthetic Hydrogels for 3D Tissue Engineering.

The specific microenvironment that cells reside in fundamentally impacts their broader function in tissues and organs. At its core, this microenvironment is composed of precise arrangements of cells that encourage homotypic and heterotypic cell-cell interactions, biochemical signaling through soluble factors like cytokines, hormones, and autocrine, endocrine, or paracrine secretions, and the local extracellular matrix (ECM) that provides physical support and mechanobiological stimuli, and further regulates biochemical signaling through cell-ECM interactions like adhesions and growth factor sequestering. Each cue provided in the microenvironment dictates cellular behavior and, thus, overall potential to perform tissue and organ specific function. It follows that in order to recapitulate physiological cell responses and develop constructs capable of replacing damaged tissue, we must engineer the cellular microenvironment very carefully. Many great strides have been made toward this goal using various three-dimensional (3D) tissue culture scaffolds and specific media conditions. Among the various 3D biomimetic scaffolds, synthetic hydrogels have emerged as a highly tunable and tissue-like biomaterial well-suited for implantable tissue-engineered constructs. Because many synthetic hydrogel materials are inherently bioinert, they minimize unintentional cell responses and thus are good candidates for long-term implantable grafts, patches, and organs. This review will provide an overview of commonly used biomaterials for forming synthetic hydrogels for tissue engineering applications and techniques for modifying them to with bioactive properties to elicit the desired cell responses.

RIP3-mediated necroptosis is regulated by inter-filament assembly of RIP homotypic interaction motif

Necroptosis is mediated by signaling complexes called necrosomes, which contain receptor-interacting protein 3 (RIP3) and upstream effectors, such as RIP1. In necrosomes, the RIP homotypic interaction motif (RHIM) of RIP3 and RIP1 forms amyloidal complex. But how the amyloidal necrosomes control RIP3 activation and cell necroptosis has not been determined. Here, we showed that RIP3 amyloid fibrils could further assemble into large fibrillar networks which presents as cellular puncta during necroptosis. A viral RHIM-containing necroptosis inhibitor M45 could form heteroamyloid with RIP3 in cells and prevent RIP3 puncta formation and cell necroptosis. We characterized mutual antagonism between RIP3–RHIM and M45–RHIM in necroptosis regulation, which was caused by distinct inter-filament interactions in RIP3, M45 amyloids revealed with atomic force microscopy. Moreover, double mutations Asn464 and Met468 in RIP3–RHIM to Asp disrupted RIP3 kinase-dependent necroptosis. While the mutant RIP3(N464D/M468D) could form amyloid as wild type upon necroptosis induction. Based on these results, we propose that RIP3 amyloid formation is required but not sufficient in necroptosis signaling, the ordered inter-filament assembly of RIP3 is critical in RIP3 amyloid mediated kinase activation and cell necroptosis.

Targeting of BCL-2 Family Members during Anticancer Treatment: A Necessary Compromise between Individual Cell and Ecosystemic Responses?

The imbalance between BCL-2 homologues and pro-death counterparts frequently noted in cancer cells endows them with a cell autonomous survival advantage. To eradicate ectopic cells, inhibitors of these homologues (BH3 mimetics) were developed to trigger, during anticancer treatment, full activation of the canonical mitochondrial apoptotic pathway and related caspases. Despite efficiency in some clinical settings, these compounds do not completely fulfill their initial promise. We herein put forth that a growing body of evidence indicates that mitochondrial integrity, controlled by BCL-2 family proteins, and downstream caspases regulate other cell death modes and influence extracellular signaling by committed cells. Moreover, intercellular communications play a key role in spreading therapeutic response across cancer cell populations and in engaging an immune response. We thus advocate that BH3 mimetics administration would be more efficient in the long term if it did not induce apoptosis in all sensitive cells at the same time, but if it could instead allow (or trigger) death signal production by non-terminally committed dying cell populations. The development of such a trade-off strategy requires to unravel the effects of BH3 mimetics not only on each individual cancer cell but also on homotypic and heterotypic cell interactions in dynamic tumor ecosystems.

Amyloid Signaling in Filamentous Fungi and Bacteria.

Amyloids are implicated in many protein misfolding diseases. Amyloid folds, however, also display a range of functional roles particularly in the microbial world. The templating ability of these folds endows them with specific properties allowing their self-propagation and protein-to-protein transmission in vivo. This property, the prion principle, is exploited by specific signaling pathways that use transmission of the amyloid fold as a way to convey information from a receptor to an effector protein. I describe here amyloid signaling pathways involving fungal nucleotide binding and oligomerization domain (NOD)-like receptors that were found to control nonself recognition and programmed cell death processes. Studies on these fungal amyloid signaling motifs stem from the characterization of the fungal [Het-s] prion protein and have led to the identification in fungi but also in multicellular bacteria of several distinct families of signaling motifs, one of which is related to RHIM [receptor-interacting protein (RIP) homotypic interaction motif], an amyloid motif regulating mammalian necroptosis.

Nucleophosmin in Its Interaction with Ligands.

Nucleophosmin (NPM1) is a mainly nucleolar protein that shuttles between nucleoli, nucleoplasm and cytoplasm to fulfill its many functions. It is a chaperone of both nucleic acids and proteins and plays a role in cell cycle control, centrosome duplication, ribosome maturation and export, as well as the cellular response to a variety of stress stimuli. NPM1 is a hub protein in nucleoli where it contributes to nucleolar organization through heterotypic and homotypic interactions. Furthermore, several alterations, including overexpression, chromosomal translocations and mutations are present in solid and hematological cancers. Recently, novel germline mutations that cause dyskeratosis congenita have also been described. This review focuses on NPM1 interactions and inhibition. Indeed, the list of NPM1 binding partners is ever-growing and, in recent years, many studies contributed to clarifying the structural basis for NPM1 recognition of both nucleic acids and several proteins. Intriguingly, a number of natural and synthetic ligands that interfere with NPM1 interactions have also been reported. The possible role of NPM1 inhibitors in the treatment of multiple cancers and other pathologies is emerging as a new therapeutic strategy.

Varicella zoster virus encodes a viral decoy RHIM to inhibit cell death.

Herpesviruses are known to encode a number of inhibitors of host cell death, including RIP Homotypic Interaction Motif (RHIM)-containing proteins. Varicella zoster virus (VZV) is a member of the alphaherpesvirus subfamily and is responsible for causing chickenpox and shingles. We have identified a novel viral RHIM in the VZV capsid triplex protein, open reading frame (ORF) 20, that acts as a host cell death inhibitor. Like the human cellular RHIMs in RIPK1 and RIPK3 that stabilise the necrosome in TNF-induced necroptosis, and the viral RHIM in M45 from murine cytomegalovirus that inhibits cell death, the ORF20 RHIM is capable of forming fibrillar functional amyloid complexes. Notably, the ORF20 RHIM forms hybrid amyloid complexes with human ZBP1, a cytoplasmic sensor of viral nucleic acid. Although VZV can inhibit TNF-induced necroptosis, the ORF20 RHIM does not appear to be responsible for this inhibition. In contrast, the ZBP1 pathway is identified as important for VZV infection. Mutation of the ORF20 RHIM renders the virus incapable of efficient spread in ZBP1-expressing HT-29 cells, an effect which can be reversed by the inhibition of caspases. Therefore we conclude that the VZV ORF20 RHIM is important for preventing ZBP1-driven apoptosis during VZV infection, and propose that it mediates this effect by sequestering ZBP1 into decoy amyloid assemblies.

Self-Association of Purified Reconstituted ER Luminal Spacer Climp63.

Membranes of the endoplasmic reticulum (ER) are shaped into cisternal sheets and cylindrical tubules. How ER sheets are generated and maintained is not clear. ER membrane protein Climp63 is enriched in sheets and routinely used as a marker of this structure. The luminal domain (LD) of Climp63 is predicted to be highly helical, and it may form bridges between parallel membranes, regulating the abundance and width of ER sheets. Here, we purified the LD and full-length (FL) Climp63 to analyze their homotypic interactions. The N-terminal tagged LD formed low-order oligomers in solution, but was extremely aggregation-prone when the GST tag was removed. Purified FL Climp63 formed detectable but moderate interactions with both the FL protein and the LD. When Climp63 was reconstituted into proteoliposomes with its LD facing out, the homotypic interactions were retained and could be competed by soluble LD, though vesicle clustering was not observed. These results demonstrate a direct self-association of Climp63, supporting its role as an ER luminal spacer.

Viral silencing suppressors and cellular proteins partner with plant RRP6-like exoribonucleases.

RNA silencing and RNA decay are functionally interlaced, regulate gene expression and play a pivotal role in antiviral responses. As a counter-defensive strategy, many plant and mammalian viruses encode suppressors which interfere with both mechanisms. However, the protein interactions that connect these pathways remain elusive. Previous work reported that RNA silencing suppressors from different potyviruses, together with translation initiation factors EIF(iso)4E, interacted with the C-terminal region of the tobacco exoribonuclease RRP6-like 2, a component of the RNA decay exosome complex. Here, we investigate whether other viral silencing suppressors and cellular proteins might also bind RRP6-like exoribonucleases. A candidate search approach based on yeast two-hybrid protein interaction assays showed that three other unrelated viral suppressors, two from plant viruses and one from a mammalian virus, bound the C-terminus of the tobacco RRP6-like 2, the full-length of the Arabidopsis RRP6L1 protein and its C-terminal region. In addition, RRP6-like proteins were found to interact with members of the cellular double-stranded RNA-binding protein (DRB) family involved in RNA silencing. The C-terminal regions of RRP6L proteins are engaged in homotypic and heterotypic interactions and were predicted to be disordered. Collectively, these results suggest a protein interaction network that connects components of RNA decay and RNA silencing that is targeted by viral silencing suppressors.

The Zα2 domain of ZBP1 is a molecular switch regulating influenza-induced PANoptosis and perinatal lethality during development

Z-DNA-binding protein 1 (ZBP1) is an innate immune sensor of nucleic acids that regulates host defense responses and development. ZBP1 activation triggers inflammation and pyroptosis, necroptosis, and apoptosis (PANoptosis) by activating receptor-interacting Ser/Thr kinase 3 (RIPK3), caspase-8, and the NLRP3 inflammasome. ZBP1 is unique among innate immune sensors because of its N-terminal Zα1 and Zα2 domains, which bind to nucleic acids in the Z-conformation. However, the specific role of these Zα domains in orchestrating ZBP1 activation and subsequent inflammation and cell death is not clear. Here we generated Zbp1ΔZα2/ΔZα2 mice that express ZBP1 lacking the Zα2 domain and demonstrate that this domain is critical for influenza A virus-induced PANoptosis and underlies perinatal lethality in mice in which the RIP homotypic interaction motif domain of RIPK1 has been mutated (Ripk1mRHIM/mRHIM). Deletion of the Zα2 domain in ZBP1 abolished influenza A virus-induced PANoptosis and NLRP3 inflammasome activation. Furthermore, deletion of the Zα2 domain of ZBP1 was sufficient to rescue Ripk1mRHIM/mRHIM mice from perinatal lethality caused by ZBP1-driven cell death and inflammation. Our findings identify the essential role of the Zα2 domain of ZBP1 in several physiological functions and establish a link between Z-RNA sensing via the Zα2 domain and promotion of influenza-induced PANoptosis and perinatal lethality.

Composition-dependent thermodynamics of intracellular phase separation

Intracellular bodies such as nucleoli, Cajal bodies and various signalling assemblies represent membraneless organelles, or condensates, that form via liquid-liquid phase separation (LLPS)1,2. Biomolecular interactions-particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions-are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems3-6 have led to the concept that a single fixed saturation concentration is a defining feature of endogenous LLPS7-9, and has been suggested as a mechanism for intracellular concentration buffering2,7,8,10. However, the assumption of a fixed saturation concentration remains largely untested within living cells, in which the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed saturation concentration. As the concentration of individual components is varied, their partition coefficients change in a manner that can be used to determine the thermodynamic free energies that underlie LLPS. We find that heterotypic interactions among protein and RNA components stabilize various archetypal intracellular condensates-including the nucleolus, Cajal bodies, stress granules and P-bodies-implying that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA-processing condensates such as the nucleolus, this manifests in the selective exclusion of fullyassembled ribonucleoprotein complexes, providing a thermodynamic basis for vectorial ribosomal RNA flux out of the nucleolus. This methodology is conceptually straightforward and readily implemented, and can be broadly used to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deeper understanding of the thermodynamics of multicomponent intracellular phase behaviour and its interplay with the nonequilibrium activity that is characteristic of endogenous condensates.

Crucial Roles of the RIP Homotypic Interaction Motifs of RIPK3 in RIPK1-Dependent Cell Death and Lymphoproliferative Disease

Receptor-interacting protein kinase 3 (RIPK3) has been identified as an essential regulator of necroptosis, apoptosis, and inflammatory signaling. RIPK3 contains an N-terminal kinase domain and a C-terminal RIP homotypic interaction motif (RHIM). However, the physiological roles of RIPK3 RHIM remain unclear. Here we generate knockin mice endogenously expressing the RIPK3 RHIM mutant, RIPK3V448P. Cells expressing RIPK3V448P are resistant to RIPK1 kinase-dependent apoptosis and necroptosis, and Ripk3V448P/V448P mice rescue embryonic lethality of Fadd-deficient mice by intercrossing. Strikingly, Ripk3V448P/V448PFadd-/- mice display more severe lymphoproliferative disease with a marked increase in abnormal CD3+B220+ lymphocytes compared with Ripk3-/-Fadd-/- mice. More importantly, these inflammatory morbidities in Ripk3V448P/V448PFadd-/- mice are profoundly inhibited by additional deletion of Ripk1. Taken together, these results reveal a previously unidentified physiological function of RHIM of RIPK3 in regulating RIPK1-dependent cell death and lymphoproliferative disease.

Analysis of Homotypic Interactions of Lactococcus lactis Pili Using Single-Cell Force Spectroscopy.

Cell surface proteins of Gram-positive bacteria play crucial roles in their adhesion to abiotic and biotic surfaces. Pili are long and flexible proteinaceous filaments known to enhance bacterial initial adhesion. They promote surface colonization and are thus considered as essential factors in biofilm cohesion. Our hypothesis is that pili mediate interactions between cells and may thereby directly affect biofilm formation. In this study, we use single-cell force spectroscopy (SCFS) to quantify the force of the homotypic pili interactions between individual bacterial cells, using different Lactococcus lactis strains producing pili or not as model bacteria. Moreover the force-distance curves were analyzed to determine the physical and nanomechanical properties of L. lactis pili. The results for pili-devoided strains showed a weak adhesion between cells (adhesion forces and work in the range of 100 pN and 7 × 10-18 J, respectively). On the contrary, the piliated strains showed high adhesion levels with adhesion forces and adhesion work over 200 pN and 50 × 10-18 J, respectively. The force-extension curves showed multiple adhesion events, typical of the unfolding of macromolecules. These unfolding force peaks were fitted using the physical worm-like chain model to get fundamental knowledge on the pili nanomechanical properties. In addition, SCFS applied to a L. lactis isolate expressing both pili and mucus-binding protein at its surface and two derivative mutants revealed the capacity of pili to interact with other surface proteins including mucus-binding proteins. This study demonstrates that pili are involved in L. lactis homotypic interactions and thus can influence biofilm structuring.

Kin discrimination in social yeast is mediated by cell surface receptors of the Flo11 adhesin family.

Microorganisms have evolved specific cell surface molecules that enable discrimination between cells from the same and from a different kind. Here, we investigate the role of Flo11-type cell surface adhesins from social yeasts in kin discrimination. We measure the adhesion forces mediated by Flo11A-type domains using single-cell force spectroscopy, quantify Flo11A-based cell aggregation in populations and determine the Flo11A-dependent segregation of competing yeast strains in biofilms. We find that Flo11A domains from diverse yeast species confer remarkably strong adhesion forces by establishing homotypic interactions between single cells, leading to efficient cell aggregation and biofilm formation in homogenous populations. Heterotypic interactions between Flo11A domains from different yeast species or Saccharomyces cerevisiae strains confer weak adhesive forces and lead to efficient strain segregation in heterogenous populations, indicating that in social yeasts Flo11A-mediated cell adhesion is a major mechanism for kin discrimination at species and sub-species levels. These findings, together with our structure and mutation analysis of selected Flo11A domains, provide a rationale of how cell surface receptors have evolved in microorganisms to mediate kin discrimination.

Neisseria gonorrhoeae Blocks Epithelial Exfoliation by Nitric-Oxide-Mediated Metabolic Cross Talk to Promote Colonization in Mice

Several pathogens suppress exfoliation, a key defense of epithelia against microbial colonization. Common among these pathogens, exemplified by Neisseria gonorrhoeae, is their ability to bind carcinoembryonic antigen-related cell adhesion molecules (CEACAMs). Gonococcal CEACAM engagement triggers the expression of CD105, which is necessary to block epithelial exfoliation, whereas homotypic CEACAM-CEACAM interactions or antibody-mediated CEACAM clustering does not lead to CD105 expression. Here, we show that CEACAM-associated bacteria release nitric oxide (NO) during anaerobic respiration, and membrane-permeable NO initiates a eukaryotic signaling pathway involving soluble guanylate cyclase (sGC), protein kinase G, and the transcription factor CREB to upregulate CD105 expression. A murine vaginal infection model with N.gonorrhoeae reveals this metabolic cross communication allows bacterial suppression of epithelial exfoliation to facilitate mucosal colonization. Disrupting NO-initiated responses in host cells re-establishes epithelial exfoliation and inhibits mouse genital tract colonization by N.gonorrhoeae, suggesting a host-directed approach to prevent bacterial infections.

Critical Role for the Extended N Terminus of Chlamydial MreB in Directing Its Membrane Association and Potential Interaction with Divisome Proteins.

Chlamydiae lack the conserved central coordinator protein of cell division FtsZ, a tubulin-like homolog. Current evidence indicates that Chlamydia uses the actin-like homolog, MreB, to substitute for the role of FtsZ in a polarized division mechanism. Interestingly, we observed MreB as a ring at the septum in dividing cells of Chlamydia We hypothesize that MreB, to substitute for FtsZ in Chlamydia, must possess unique properties compared to canonical MreB orthologs. Sequence differences between chlamydial MreB and orthologs in other bacteria revealed that chlamydial MreB possesses an extended N-terminal region, harboring predicted amphipathicity, as well as the conserved amphipathic helix found in other bacterial MreBs. The conserved amphipathic helix-directed green fluorescent protein (GFP) to label the membrane uniformly in Escherichia coli but the extended N-terminal region did not. However, the extended N-terminal region together with the conserved amphipathic region directed GFP to restrict the membrane label to the cell poles. In Chlamydia, the extended N-terminal region was sufficient to direct GFP to the membrane, and this localization was independent of an association with endogenous MreB. Importantly, mutating the extended N-terminal region to reduce its amphipathicity resulted in the accumulation of GFP in the cytosol of the chlamydiae and not in the membrane. The N-terminal domain of MreB was not required for homotypic interactions but was necessary for interactions with cell division components RodZ and FtsK. Our data provide mechanistic support for chlamydial MreB to serve as a substitute for FtsZ by forming a ringlike structure at the site of polarized division.IMPORTANCEChlamydia trachomatis is an obligate intracellular pathogen, causing sexually transmitted diseases and trachoma. The study of chlamydial physiology is important for developing novel therapeutic strategies for these diseases. Chlamydiae divide by a unique MreB-dependent polarized cell division process. In this study, we investigated unique properties of chlamydial MreB and observed that chlamydial species harbor an extended N-terminal region possessing amphipathicity. MreB formed a ring at the septum, like FtsZ in Escherichia coli, and its localization was dependent upon the amphipathic nature of its extended N terminus. Furthermore, this region is crucial for the interaction of MreB with cell division proteins. Given these results, chlamydial MreB likely functions at the septum as a scaffold for divisome proteins to regulate cell division in this organism.