Identification and characterization of a novel family of pneumococcal proteins that are protective against sepsis.

Four pneumococcal genes (phtA, phtB, phtD, and phtE) encoding a novel family of homologous proteins (32 to 87% identity) were identified from the Streptococcus pneumoniae genomic sequence. These open reading frames were selected as potential vaccine candidates based upon their possession of hydrophobic leader sequences which presumably target these proteins to the bacterial cell surface. Analysis of the deduced amino acid sequences of these gene products revealed the presence of a histidine triad motif (HxxHxH), termed Pht (pneumococcal histidine triad) that is conserved and repeated several times in each of the four proteins. The four pht genes (phtA, phtB, phtD, and a truncated version of phtE) were expressed in Escherichia coli. A flow cytometry-based assay confirmed that PhtA, PhtB, PhtD and, to a lesser extent, PhtE were detectable on the surface of intact bacteria. Recombinant PhtA, PhtB, and PhtD elicited protection against certain pneumococcal capsular types in a mouse model of systemic disease. These novel pneumococcal antigens may serve as effective vaccines against the most prevalent pneumococcal serotypes.

Use of a whole genome approach to identify vaccine molecules affording protection against Streptococcus pneumoniae infection.

Microbial targets for protective humoral immunity are typically surface-localized proteins and contain common sequence motifs related to their secretion or surface binding. Exploiting the whole genome sequence of the human bacterial pathogen Streptococcus pneumoniae, we identified 130 open reading frames encoding proteins with secretion motifs or similarity to predicted virulence factors. Mice were immunized with 108 of these proteins, and 6 conferred protection against disseminated S. pneumoniae infection. Flow cytometry confirmed the surface localization of several of these targets. Each of the six protective antigens showed broad strain distribution and immunogenicity during human infection. Our results validate the use of a genomic approach for the identification of novel microbial targets that elicit a protective immune response. These new antigens may play a role in the development of improved vaccines against S. pneumoniae.

Molecular characterization of a human scavenger receptor, human MARCO.

Murine MARCO has been identified recently in subsets of macrophages located in the peritoneum, marginal zone of the spleen, and the medullary cord of lymph nodes, where it has been proposed that it serves as a bacteria-binding receptor. A scavenger receptor family member with an extended collagenous domain, murine MARCO has also been demonstrated in atherosclerotic lesions of susceptible mice. We report here the identification, tissue and chromosomal localization, and pharmacological characterization of human (h)MARCO. hMARCO was identified from a macrophage cDNA library by electronic screening with the murine MARCO sequence. Nucleotide sequence analysis confirmed that the full-length hMARCO clone encoded a 519-amino acid protein sharing 68.5% identity with murine MARCO. RNA blot analysis indicated that the hMARCO transcript is 2.0 kb in length and is predominantly expressed in human lung, liver, and lymph nodes. Radiation hybrid mapping localized hMARCO to chromosome 2q14. Ligand-binding studies of COS cells expressing hMARCO demonstrated significant specific binding of both Escherichia coli and Staphylococcus aureus. In contrast, the hMARCO receptor expressed in COS cells did not specifically bind the scavenger receptor ligand acetylated low-density lipoprotein (LDL), despite its similarity to the elongated collagen-like binding domain of the macrophage scavenger receptor. In addition, acetylated (Ac)LDL and oxidized (Ox)LDL did not inhibit E. coli binding to hMARCO. These data suggest that hMARCO may play an important role in host defense, but it has no obvious role in the accumulation of modified lipoproteins during atherogenesis.

Adhesins as targets for vaccine development.

Blocking the primary stages of infection, namely bacterial attachment to host cell receptors and colonization of the mucosal surface, may be the most effective strategy to prevent bacterial infections. Bacterial attachment usually involves an interaction between a bacterial surface protein called an adhesin and the host cell receptor. Recent preclinical vaccine studies with the FimH adhesin (derived from uropathogenic Escherichia coli) have confirmed that antibodies elicited against an adhesin can impede colonization, block infection, and prevent disease. The studies indicate that prophylactic vaccination with adhesins can block bacterial infections. With recent advances in the identification, characterization, and isolation of other adhesins, similar approaches are being explored to prevent infections, from otitis media and dental caries to pneumonia and sepsis.

Molecular cloning and characterization of the porcine calcitonin gene-related peptide receptor.

Calcitonin gene-related peptide (CGRP) receptors (CGRP-Rs) are widely distributed throughout the central and peripheral nervous systems. A novel CGRP-R was identified from a porcine lung complementary DNA library. Sequence analysis indicated that the CGRP-R is 462 amino acids in length and shares 93% sequence identity with the human CGRP-R. Northern blot analysis indicated a messenger RNA species of 5.4 kilobases, which is abundantly expressed in the lung. Ligand binding studies of the cloned CGRP-R expressed in human embryonic kidney (HEK-293) cells showed the presence of high affinity receptor for CGRP with a Kd of 38.5 pM. The pharmacological profiles of various ligands competing for [125I]CGRP binding to the expressed receptor were in accordance with those for the natural receptor. Binding of [125I]CGRP to the expressed receptor was decreased in the presence of a nonhydrolyzable analog of GTP, guanosine 5' (gamma-thio)-triphosphate. In functional studies, CGRP stimulated the activation of adenylyl cyclase with an EC50 of 2.5 nM. The linear analog of CGRP, diacetoamidomethyl cysteine CGRP, did not affect adenylyl cyclase activity on its own or in the presence of CGRP. Furthermore, the CGRP receptor antagonists, CGRP-(8-37)alpha, inhibited the CGRP-mediated response in a competitive manner. Collectively, the binding and functional data demonstrate that we have cloned a porcine CGRP type 1 receptor. The availability of the CGRP-R complementary DNA will allow us to examine its participation in pathophysiological processes.

Adherence of Streptococcus pneumoniae to human bronchial epithelial cells (BEAS-2B).

Pneumococcal adherence to alveolar epithelial cells and nasopharyngeal epithelial cells has been well characterized. However, the interaction of Streptococcus pneumoniae with bronchial epithelial cells has not been studied. We have now shown that pneumococci bind specifically to a human bronchial epithelial cell line (BEAS-2B cells). Pneumococci adhered to BEAS-2B cells in a time- and dose-dependent manner. These results suggest that the bronchial epithelium may serve as an additional site of attachment for pneumococci and demonstrate the utility of the BEAS-2B cell line for studying mechanisms of pneumococcal infection.

Cloning and functional characterization of a novel human CC chemokine that binds to the CCR3 receptor and activates human eosinophils.

Eotaxin has been found to bind exclusively to a single chemokine receptor, CCR3. Using expression sequence tag screening of an activated monocyte library, a second chemokine has been identified; it was expressed and purified from a Drosophila cell culture system and appears to only activate CCR3. Eotaxin-2, MPIF-2, or CKbeta-6, is a human CC chemokine with low amino acid sequence identity to other chemokines. Eotaxin-2 promotes chemotaxis and Ca2+ mobilization in human eosinophils but not in neutrophils or monocytes. Cross-desensitization calcium mobilization experiments using purified eosinophils indicate that eotaxin and MCP-4, but not RANTES, MIP-1alpha, or MCP-3, can completely cross-desensitize the calcium response to eotaxin-2 on these cells, indicating that eotaxin-2 shares the same receptor used by eotaxin and MCP-4. Eotaxin-2 was the most potent eosinophil chemoattractant of all the chemokines tested. Eotaxin-2 also displaced 125I-eotaxin bound to the cloned CCR3 stably expressed in CHO cells (CHO-CCR3) and to freshly isolated human eosinophils with affinities similar to eotaxin and MCP-4. 125I-Eotaxin-2 binds with high affinity to eosinophils and both eotaxin and cold eotaxin-2 displace the ligand with equal affinity. Eotaxin and eotaxin-2 promote a Ca2+ transient in RBL-2H3 cells stably transfected with CCR3 (RBL-2H3-CCR3) and both ligands cross-desensitized the response of the other but not the response to LTD4. The data indicate that eotaxin-2 is a potent eosinophil chemotactic chemokine exerting its activity solely through the CCR3 receptor.

Molecular characterization of a novel human endothelin receptor splice variant.

Endothelin receptors are widely distributed throughout a number of tissues. A novel ETB receptor splice variant (ETB-SVR) was identified from a human placental cDNA library. Sequence analysis indicated that the ETB-SVR is 436 amino acids long and shares 91% identity to the human ETB-R. Northern blot analysis indicated an mRNA species of 2.7 kilobases, which is expressed in the lung, placenta, kidney, and skeletal muscle. Ligand binding studies of the cloned ETB-SVR and ETB-R receptors expressed in COS cells showed that ET peptides exhibited similar potency in displacing 125I-ET-1 binding. Functional studies showed that ET-1, ET-3, and sarafotoxin 6c displayed similar potencies for inositol phosphates accumulation in ETB-R-transfected COS cells, whereas no increase in inositol phosphate accumulation was observed in ETB-SVR-transfected cells. In addition, exposure of ETB-R-transfected cells to ET-1 caused an increase in the intracellular acidification rate whereas ETB-SVR-transfected cells did not respond to ET-1. These data suggest that the ETB-SVR and ETB-R are functionally distinct and the difference in the amino acid sequences between the two receptors may determine functional coupling. Availability of cDNA clones for endothelin receptors can facilitate our understanding of the role of ET in the pathophysiology of various diseases.

A cDNA encoding the calcitonin gene-related peptide type 1 receptor.

Calcitonin gene-related peptide (CGRP) is a neuropeptide with diverse biological effects including potent vasodilator activity. We report here the cloning of a complementary DNA (cDNA) encoding a human CGRP1 receptor, which shares significant peptide sequence homology with the human calcitonin receptor, a member of the G-protein-coupled receptor superfamily. Northern blot analysis revealed that the messenger RNA for this receptor is predominantly expressed in the lung and heart. In situ studies showed specific localization of the receptor mRNA to alveolar cells in the lung and to cardiac myocytes in the heart. Stable expression of the cDNA in human embryonic kidney 293 (HEK 293) cells produced specific, high affinity binding sites for CGRP that displayed pharmacological and functional properties very similar to native human CGRP1 receptor. Exposure of these cells to CGRP resulted in a 60-fold increase in cAMP production, which was inhibited in a competitive manner by the CGRP1 receptor antagonist, CGRP-(8-37).

Cloning and functional characterization of the human vasoactive intestinal peptide (VIP)-2 receptor.

A full-length clone encoding the human VIP-2 receptor was isolated from a human placenta cDNA library. The 1317-bp cDNA insert encodes a protein of 438 amino acids which is a member of the seven transmembrane domain G-protein-coupled receptor superfamily. Expression of the human VIP-2 receptor in COS-7 cells confered high affinity binding to [125I] VIP (IC50 = 0.93 nM), which was displaced by unlabeled PACAP-38 (IC50 = 6.2 nM). VIP and PACAP-38 were equipotent in stimulating accumulation of cAMP in COS-7 cells transfected with the human VIP-2 receptor. Northern blot analysis revealed two VIP-2 receptor mRNAs of 4.6 kb and 2.3 kb in size which were expressed predominantly in the human skeletal muscle and to a lesser extent in the human brain, heart, pancreas and placenta.

Alteration of translation and stability of mRNA for the poly(A)-binding protein during myogenesis.

The regulation of synthesis of various factors involved in mRNA translation during differentiation of muscle cells was examined. The steady-state levels of mRNAs coding for eukaryotic initiation factor (eIF) 2 alpha, 2 beta and elongation factor (eEF)-1 alpha were measured in both proliferating rat L6 myoblast and differentiated myotubes. The steady-state levels of these mRNAs were not altered during myogenesis. Furthermore, the distribution of these mRNAs between repressed and translated populations remained unchanged. Recent studies suggest a role for poly(A)-binding protein (PABP) in translation initiation. Therefore, we also examined the expression of PABP mRNA during myogenesis. The PABP mRNA was less abundant in myotubes compared to myoblasts. However, the synthesis of PABP remained unchanged. In myoblasts, approximately 50-60% of the total mRNA was associated with polyribosomes, whereas in myotubes more than 80% of the mRNA was associated with polyribosomes. These results, therefore, suggest that the PABP mRNA was more efficiently translated in differentiated myotubes than in the proliferating myoblasts. Measurement of the stability and transcription of PABP mRNA showed that, while transcription was not affected during myogenesis, the stability of the mRNA was reduced in differentiated cells. The t1/2 of PABP mRNA in myoblasts was 13 h compared to 7.5 h in myotubes. This observation suggests that the reduced steady-state level of PABP mRNA in myotube were largely due to the change in stability of this mRNA during myogenesis.