Identification of fibroblast growth factor receptor 3 (FGFR3) as a protein receptor for botulinum neurotoxin serotype A (BoNT/A).

Botulinum neurotoxin serotype A (BoNT/A) causes transient muscle paralysis by entering motor nerve terminals (MNTs) where it cleaves the SNARE protein Synaptosomal-associated protein 25 (SNAP25206) to yield SNAP25197. Cleavage of SNAP25 results in blockage of synaptic vesicle fusion and inhibition of the release of acetylcholine. The specific uptake of BoNT/A into pre-synaptic nerve terminals is a tightly controlled multistep process, involving a combination of high and low affinity receptors. Interestingly, the C-terminal binding domain region of BoNT/A, HC/A, is homologous to fibroblast growth factors (FGFs), making it a possible ligand for Fibroblast Growth Factor Receptors (FGFRs). Here we present data supporting the identification of Fibroblast Growth Factor Receptor 3 (FGFR3) as a high affinity receptor for BoNT/A in neuronal cells. HC/A binds with high affinity to the two extra-cellular loops of FGFR3 and acts similar to an agonist ligand for FGFR3, resulting in phosphorylation of the receptor. Native ligands for FGFR3; FGF1, FGF2, and FGF9 compete for binding to FGFR3 and block BoNT/A cellular uptake. These findings show that FGFR3 plays a pivotal role in the specific uptake of BoNT/A across the cell membrane being part of a larger receptor complex involving ganglioside- and protein-protein interactions.

Botulinum neurotoxin serotype A specific cell-based potency assay to replace the mouse bioassay.

Botulinum neurotoxin serotype A (BoNT/A), a potent therapeutic used to treat various disorders, inhibits vesicular neurotransmitter exocytosis by cleaving SNAP25. Development of cell-based potency assays (CBPAs) to assess the biological function of BoNT/A have been challenging because of its potency. CBPAs can evaluate the key steps of BoNT action: receptor binding, internalization-translocation, and catalytic activity; and therefore could replace the current mouse bioassay. Primary neurons possess appropriate sensitivity to develop potential replacement assays but those potency assays are difficult to perform and validate. This report describes a CBPA utilizing differentiated human neuroblastoma SiMa cells and a sandwich ELISA that measures BoNT/A-dependent intracellular increase of cleaved SNAP25. Assay sensitivity is similar to the mouse bioassay and measures neurotoxin biological activity in bulk drug substance and BOTOX® product (onabotulinumtoxinA). Validation of a version of this CBPA in a Quality Control laboratory has led to FDA, Health Canada, and European Union approval for potency testing of BOTOX®, BOTOX® Cosmetic, and Vistabel®. Moreover, we also developed and optimized a BoNT/A CBPA screening assay that can be used for the discovery of novel BoNT/A inhibitors to treat human disease.

The human immunodeficiency virus type 1 tat protein enhances Cryptosporidium parvum-induced apoptosis in cholangiocytes via a Fas ligand-dependent mechanism.

While Cryptosporidium parvum infection of the intestine has been reported in both immunocompetent and immunocompromised individuals, biliary infection is seen primarily in adult AIDS patients and is associated with development of AIDS cholangiopathy. However, the mechanisms of pathogen-induced AIDS cholangiopathy remain unclear. Since we previously demonstrated that the Fas/Fas ligand (FasL) system is involved in paracrine-mediated C. parvum cytopathicity in cholangiocytes, we also tested the potential synergistic effects of human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (Tat)-mediated FasL regulation on C. parvum-induced apoptosis in cholangiocytes by semiquantitative reverse transcription-PCR, immunoblotting, immunofluorescence analysis, and immunogold electron microscopy. H69 cells do not express CXCR4 and CCR5, which are receptors required for direct HIV-1 viral infection. However, recombinant biologically active HIV-1-associated Tat protein increased FasL expression in the cytoplasm of cholangiocytes without a significant increase in apoptosis. We found that C. parvum-induced apoptosis was associated with translocation of intracellular FasL to the cell membrane surface and release of full-length FasL from infected H69 cells. Tat significantly (P < 0.05) increased C. parvum-induced apoptosis in bystander cells in a dose-dependent manner. Moreover, Tat enhanced both C. parvum-induced FasL membrane translocation and release of full-length FasL. In addition, the FasL neutralizing antibody NOK-1 and the caspase-8 inhibitor Z-IETD-fmk both blocked C. parvum-induced apoptosis in cholangiocytes. The data demonstrated that HIV-1 Tat enhances C. parvum-induced cholangiocyte apoptosis via a paracrine-mediated, FasL-dependent mechanism. Our results suggest that concurrent active HIV replication, with associated production of Tat protein, and C. parvum infection synergistically increase cholangiocyte apoptosis and thus jointly contribute to AIDS-related cholangiopathies.

Cryptosporidium parvum infects human cholangiocytes via sphingolipid-enriched membrane microdomains.

Cryptosporidium parvum attaches to intestinal and biliary epithelial cells via specific molecules on host-cell surface membranes including Gal/GalNAc-associated glycoproteins. Subsequent cellular entry of this parasite depends on host-cell membrane alterations to form a parasitophorous vacuole via activation of phosphatidylinositol 3-kinase (PI-3K)/Cdc42-associated actin remodelling. How C. parvum hijacks these host-cell processes to facilitate its infection of target epithelia is unclear. Using specific probes to known components of sphingolipid-enriched membrane microdomains (SEMs), we detected aggregation of host-cell SEM components at infection sites during C. parvum infection of cultured human biliary epithelial cells (i.e. cholangiocytes). Activation and membrane translocation of acid-sphingomyelinase (ASM), an enzyme involved in SEM membrane aggregation, were also observed in infected cells. Pharmacological disruption of SEMs and knockdown of ASM via a specific small interfering RNA (siRNA) significantly decreased C. parvum attachment (by approximately 84%) and cellular invasion (by approximately 88%). Importantly, knockdown of ASM and disruption of SEMs significantly blocked C. parvum-induced accumulation of Gal/GalNAc-associated glycoproteins at infection sites by approximately 90%. Disruption of SEMs and knockdown of ASM also significantly blocked C. parvum-induced activation of host-cell PI-3K and subsequent accumulation of Cdc42 and actin by up to 75%. Our results suggest an important role of SEMs for C. parvum attachment to and entry of host cells, likely via clustering of membrane-binding molecules and facilitating of C. parvum-induced actin remodelling at infection sites through activation of the PI-3K/Cdc42 signalling pathway.

Multiple TLRs are expressed in human cholangiocytes and mediate host epithelial defense responses to Cryptosporidium parvum via activation of NF-kappaB.

Infection of epithelial cells by Cryptosporidium parvum triggers a variety of host-cell innate and adaptive immune responses including release of cytokines/chemokines and up-regulation of antimicrobial peptides. The mechanisms that trigger these host-cell responses are unclear. Thus, we evaluated the role of TLRs in host-cell responses during C. parvum infection of cultured human biliary epithelia (i.e., cholangiocytes). We found that normal human cholangiocytes express all known TLRs. C. parvum infection of cultured cholangiocytes induces the selective recruitment of TLR2 and TLR4 to the infection sites. Activation of several downstream effectors of TLRs including IL-1R-associated kinase, p-38, and NF-kappaB was detected in infected cells. Transfection of cholangiocytes with dominant-negative mutants of TLR2 and TLR4, as well as the adaptor molecule myeloid differentiation protein 88 (MyD88), inhibited C. parvum-induced activation of IL-1R-associated kinase, p-38, and NF-kappaB. Short-interfering RNA to TLR2, TLR4, and MyD88 also blocked C. parvum-induced NF-kappaB activation. Moreover, C. parvum selectively up-regulated human beta-defensin-2 in directly infected cells, and inhibition of TLR2 and TLR4 signals or NF-kappaB activation were each associated with a reduction of C. parvum-induced human beta-defensin-2 expression. A significantly higher number of parasites were detected in cells transfected with a MyD88 dominant-negative mutant than in the control cells at 48-96 h after initial exposure to parasites, suggesting MyD88-deficient cells were more susceptible to infection. These findings demonstrate that cholangiocytes express a variety of TLRs, and suggest that TLR2 and TLR4 mediate cholangiocyte defense responses to C. parvum via activation of NF-kappaB.

Localized glucose and water influx facilitates Cryptosporidium parvum cellular invasion by means of modulation of host-cell membrane protrusion.

Dynamic membrane protrusions such as lamellipodia and filopodia are driven by actin polymerization and often hijacked by intracellular microbes to enter host cells. The overall rate of membrane protrusion depends on the actin polymerization rate and the increase of localized cell volume. Although the signaling pathways involving actin polymerization are well characterized, the molecular mechanisms regulating local cell volume associated with membrane protrusion are unclear. Cryptosporidium parvum, an intracellular parasite, depends on host-cell membrane protrusion to accomplish cell entry and form the parasitophorous vacuole. Here, we report that C. parvum infection of cholangiocytes recruits host-cell SGLT1, a Na+/glucose cotransporter, and aquaporin 1 (AQP1), a water channel, to the attachment site. SGLT1-dependent glucose uptake occurs at the attachment site. Concordantly, the region of attachment displays localized water influx that is inhibited by either suppression of AQP1 by means of AQP1-small interfering RNA (siRNA) or inhibition of SGLT1 by a specific pharmacologic inhibitor, phlorizin. Inhibition of SGLT1 does not affect actin accumulation but decreases the membrane protrusion at the attachment site. Moreover, functional inhibition of host-cell AQP1 and SGLT1 hampers C. parvum invasion of cholangiocytes. Thus, glucose-driven, AQP-mediated localized water influx is involved in the membrane protrusion during C. parvum cellular invasion, phenomena that may also be relevant to the mechanisms of cell membrane protrusion in general.

Acetic acid induces expression of the Staphylococcus aureus cidABC and lrgAB murein hydrolase regulator operons.

The Staphylococcus aureus lrg and cid operons encode homologous proteins that regulate extracellular murein hydrolase activity and penicillin tolerance in a diametrically opposing manner. Although their specific regulatory functions remain unknown, it has been postulated that the functions of CidA and LrgA are analogous to those of bacteriophage holins and antiholins, respectively, and that these proteins serve as molecular control elements of bacterial programmed cell death. Although these studies demonstrated that cidBC transcription is abundant in sigmaB-proficient strains, cidABC transcription was only minimally expressed under standard growth conditions. In this study, we demonstrate that cidABC and lrgAB transcription in the clinical isolate UAMS-1 is induced by growth in the presence of 35 mM glucose and that this enhances murein hydrolase activity and decreases tolerance to vancomycin and rifampin. The effect of glucose on murein hydrolase activity was not observed in the cidA mutant, indicating that the induction of this activity was dependent on enhanced cidABC expression. Furthermore, we demonstrate that the effects of glucose on cidABC and lrgAB transcription are mediated by the generation of acetic acid produced by the metabolism of this and other carbon sources. These results shed new light on the control of the S. aureus cidABC and lrgAB genes and demonstrate that these operons, as well as murein hydrolase activity and antibiotic tolerance, are responsive to carbohydrate metabolism.

Apical organelle discharge by Cryptosporidium parvum is temperature, cytoskeleton, and intracellular calcium dependent and required for host cell invasion.

The apical organelles in apicomplexan parasites are characteristic secretory vesicles containing complex mixtures of molecules. While apical organelle discharge has been demonstrated to be involved in the cellular invasion of some apicomplexan parasites, including Toxoplasma gondii and Plasmodium spp., the mechanisms of apical organelle discharge by Cryptosporidium parvum sporozoites and its role in host cell invasion are unclear. Here we show that the discharge of C. parvum apical organelles occurs in a temperature-dependent fashion. The inhibition of parasite actin and tubulin polymerization by cytochalasin D and colchicines, respectively, inhibited parasite apical organelle discharge. Chelation of the parasite's intracellular calcium also inhibited apical organelle discharge, and this process was partially reversed by raising the intracellular calcium concentration by use of the ionophore A23187. The inhibition of parasite cytoskeleton polymerization by cytochalasin D and colchicine and the depletion of intracellular calcium also decreased the gliding motility of C. parvum sporozoites. Importantly, the inhibition of apical organelle discharge by C. parvum sporozoites blocked parasite invasion of, but not attachment to, host cells (i.e., cultured human cholangiocytes). Moreover, the translocation of a parasite protein, CP2, to the host cell membrane at the region of the host cell-parasite interface was detected; an antibody to CP2 decreased the C. parvum invasion of cholangiocytes. These data demonstrate that the discharge of C. parvum sporozoite apical organelle contents occurs and that it is temperature, intracellular calcium, and cytoskeleton dependent and required for host cell invasion, confirming that apical organelles play a central role in C. parvum entry into host cells.

The Staphylococcus aureus cidAB operon: evaluation of its role in regulation of murein hydrolase activity and penicillin tolerance.

Recent studies have shown that expression of the Staphylococcus aureus lrgAB operon inhibits murein hydrolase activity and decreases sensitivity to penicillin-induced killing. It was proposed that the lrgAB gene products function in a manner analogous to an antiholin, inhibiting a putative holin from transporting murein hydrolases out of the cell. In the present study the cidAB operon was identified and characterized based on the similarity of the cidA and cidB gene products to the products of the lrgAB operon. Zymographic and quantitative analyses of murein hydrolase activity revealed that mutation of the cidA gene results in decreased extracellular murein hydrolase activity compared to that of S. aureus RN6390, the parental strain. Complementation of cidA expression restored the wild-type phenotype, indicating that expression of the cidAB operon has a positive influence on extracellular murein hydrolase activity. The cidA mutant also displayed a significant decrease in sensitivity to the killing effects of penicillin. However, complementation of the cidA defect did not restore penicillin sensitivity to wild-type levels. Reverse transcriptase PCR also revealed that cidAB is maximally expressed during early exponential growth, opposite of what was previously observed for lrgAB expression. Based on these results, we propose that the cidAB operon encodes the holin-like counterpart of the lrgAB operon and acts in a manner opposite from that of lrgAB by increasing extracellular murein hydrolase activity and increasing sensitivity to penicillin-induced killing.