Drug discovery in the new millennium: the pivotal role of biotechnology.

Biotechnology has made, and will continue to make, a major contribution to the health of mankind by providing new insights into disease and acting as a pivotal enabler for the drug-discovery process. The available techniques are diverse and changing rapidly: selecting and integrating the best approach is the key to success.

Streptococcus pneumoniae produces a second haemolysin that is distinct from pneumolysin.

Pneumococci are described as alpha-haemolytic but under certain circumstances they produce zones of beta-haemolysis on blood-containing medium. This observation was investigated using wild type strains and a genetically-modified strain unable to produce the haemolytic toxin, pneumolysin. beta-haemolysis was produced by all pneumococci tested. It was not inhibited by anti-pneumolysin antibody but could be inactivated by cholesterol. These data confirm that pneumococci elaborate a second haemolysin, distinct from pneumolysin.

Region 2 of the Escherichia coli K5 capsule gene cluster encoding proteins for the biosynthesis of the K5 polysaccharide.

The nucleotide sequence of region 2 of the Escherichia coli K5 capsule gene cluster has been determined. This region, essential for the biosynthesis of the K5 polysaccharide, contained four genes, termed kfiA-D. The G + C ratio was 33.4%, which was lower than the typical G + C ratio for E. coli and that of the flanking regions 1 and 3 in the K5 capsule gene cluster. Three major RNA transcripts were detected within region 2 by Northern blotting and three promoters located by transcript mapping. Promoter activity was confirmed by promoter-probe analysis. The predicted amino acid sequence of KfiC had homology to a number of glycosyl transferase enzymes and overexpression of the KfiC gene resulted in increased K5 transferase activity. The predicted amino acid sequence of KfiD had homology to a number of NAD-dependent dehydrogenase enzymes and was demonstrated to be a UDP-glucose dehydrogenase that catalyses the information of UDP-glucuronic acid from UDP-glucose.

The role of pneumolysin and autolysin in the pathology of pneumonia and septicemia in mice infected with a type 2 pneumococcus.

Mice were infected intranasally with a serotype 2 pneumococcus, a pneumolysin-negative derivative (PLN-A), or an autolysin-negative derivative (AL-2). Numbers of wild type pneumococci were seen in the lung from approximately 12 h after infection and were first detected in the blood around this time. Immunofluorescent staining of lung sections showed that pneumolysin was produced in vivo. Pneumococcal infection resulted in alteration of the composition of the blood but not the bone marrow. Some of the hematologic changes did not occur after PLN-A. PLN-A had a slower growth rate in the lung and bacteremia was delayed. AL-2 was rapidly cleared from the lungs and was not detected in the blood. These events paralleled the pattern of histology in the lung, with the severity of inflammation reduced with PLN-A and no inflammation or hematologic changes with AL-2.

Growth and virulence of a complement-activation-negative mutant of Streptococcus pneumoniae in the rabbit cornea.

Our previous work has demonstrated the importance of pneumolysin in the virulence of S. pneumoniae in a rabbit intracorneal model. This was accomplished by showing that deletion of the gene encoding pneumolysin resulted in reduced virulence, whereas restoration of the wild-type gene resulted in restoration of the virulent phenotype. To assess the importance of a particular domain in the pneumolysin molecule, we have now constructed a strain which produces a pneumolysin molecule which is hemolytic but which bears a site-specific mutation in the domain known to be associated with the complement-activating properties of this molecule. Comparison of the virulence of this strain with that of a strain bearing the wild-type gene showed statistically significantly lower total slit lamp examination (SLE) scores at 12, 18, 24, and 36 h (particularly with respect to fibrin formation), but no difference at 48 h. Determination of colony forming units (CFU) in eyes infected with the two strains showed approximately 10(6) bacteria per cornea until 36 h. Between 36 and 48 h, the bacteria were almost completely cleared with very few bacteria recoverable at the later time point. The loss of virulence observed with this mutation in the complement-activation domain of pneumolysin, though less than that observed with the gene deletion mutant, suggests that complement activation by pneumolysin has a significant role in the pathology observed in this model of corneal infection.

A neuraminidase from Streptococcus pneumoniae has the features of a surface protein.

A gene from Streptococcus pneumoniae (nanA), with features entirely consistent with a neuraminidase gene, has been sequenced. High levels of neuraminidase activity were obtained after cloning of this gene, without flanking sequences, into a high-expression vector. RNA hybridization studies have shown that the gene is transcribed by a virulent pneumococcus strain. The predicted molecular weight of the protein and certain amino acid sequences are typical of other neuraminidases. NanA contains the four copies of the sequence SXDXGXTW that is present in all the bacterial neuraminidases previously described. Kyte and Doolittle analysis showed that NanA is a hydrophilic protein with hydrophobic domains at the N terminus and the C terminus. A putative signal peptide was found in the N terminus of this protein, indicating that the protein is exported from the pneumococcus. The C terminus has the features of the anchor motif found in other surface proteins from gram-positive bacteria. Electron microscopy studies showed the presence of neuraminidase associated with the cell surface of the pneumococcus.

A role in cell-binding for the C-terminus of pneumolysin, the thiol-activated toxin of Streptococcus pneumoniae.

Cell binding of pneumolysin to target cells is an important step in the lysis of cells by this toxin. We sought to locate the cell-binding region of pneumolysin. Deletion of the six C-terminal amino acids decreased cell-binding activity by 98%. Furthermore, mutagenesis of an amino acid near the C-terminus decreased the cell-binding activity of full-length pneumolysin by 90%. The C-terminus of pneumolysin has an important role in cell-binding activity.

Molecular cloning and analysis of genes for sialic acid synthesis in Neisseria meningitidis group B and purification of the meningococcal CMP-NeuNAc synthetase enzyme.

The gene encoding for the CMP-NeuNAc synthetase enzyme of Neisseria meningitidis group B was cloned by complementation of a mutant of Escherichia coli defective for this enzyme. The gene (neuA) was isolated on a 4.1-kb fragment of meningococcal chromosomal DNA. Determination of the nucleotide sequence of this fragment revealed the presence of three genes, termed neuA, neuB, and neuC, organized in a single operon. The presence of a truncated ctrA gene at one end of the cloned DNA and a truncated gene encoding for the meningococcal sialyltransferase at the other confirmed that the cloned DNA corresponded to region A and part of region C of the meningococcal capsule gene cluster. The predicted amino acid sequence of the meningococcal NeuA protein was 57% homologous to that of NeuA, the CMP-NeuNAc synthetase encoded by E. coli K1. The predicted molecular mass of meningococcal NeuA protein was 24.8 kDa, which was 6 kDa larger than that formerly predicted (U. Edwards and M. Frosch, FEMS Microbiol. Lett. 96:161-166, 1992). Purification of the recombinant meningococcal NeuA protein together with determination of the N-terminal amino acid sequence confirmed that this 24.8-kDa protein was indeed the meningococcal CMP-NeuNAc synthetase. The predicted amino acid sequences of the two other encoded proteins were homologous to those of the NeuC and NeuB proteins of E. coli K1, two proteins involved in the synthesis of NeuNAc. These results indicate that common steps exist in the biosynthesis of NeuNAc in these two microorganisms.

Development of a PCR probe test for identifying Pseudomonas aeruginosa and Pseudomonas (Burkholderia) cepacia.

AIMS: To develop a system of species specific polymerase chain reaction (PCR) and DNA hybridisation based on 16s ribosomal RNA sequences for the identification of Pseudomonas aeruginosa and Pseudomonas (Burkholderia) cepacia in sputum from children with cystic fibrosis. METHODS: Most of the 16s rRNA sequences from strains of Ps aeruginosa, Ps (Burkholderia) cepacia, and Ps putida were determined. PCR primers and DNA probes were synthesised from suitable sequences and then evaluated on bacterial cultures and sputum samples. RESULTS: About 1000 bases of sequence was obtained from strains of Ps aeruginosa, Ps (Burkholderia) cepacia, and Ps putida. PCR of bacterial cultures was species specific, but PCR on sputum resulted in some non-specific amplification products. The subsequent hybridisation reaction was species specific. CONCLUSION: A species specific system of PCR and DNA hybridisation based on 16s rRNA sequences is applicable in clinical practice, and may aid the early diagnosis of respiratory tract infection with small numbers of Ps aeruginosa and Ps (Burkholderia) cepacia in patients with cystic fibrosis.

Synthesis of the K5 (group II) capsular polysaccharide in transport-deficient recombinant Escherichia coli.

The genes directing the expression of group II capsules in Escherichia coli are organized into three regions. The central region 2 is type specific and thought to determine the synthesis of the respective polysaccharide, whilst the flanking regions 1 and 3 are common to all group II gene clusters and direct the surface expression of the capsular polysaccharide. In this communication we analyze the involvement of region 1 and 3 genes in the synthesis of the capsular KS polysaccharide. Recombinant E. coli strains harboring all KS specific region 2 genes and having various combinations of region 1 and 3 genes were studied using immunoelectron microscopy. Membranes from these bacteria were incubated with UDP[14C]GlcA and UDPGlcNAc in the absence or presence of KS polysaccharide as an exogenous acceptor. It was found that recombinant strains with only gene region 2 did not produce the K5 polysaccharide. Membranes of such strains did not synthesize the polymer and did not elongate K5 polysaccharide added as an exogenous acceptor. An involvement of genes from region 1 (notably kpsC and kpsS) and from region 3 (notably kpsT) in the K5 polysaccharide synthesis was apparent and is discussed.

Molecular analysis of region 1 of the Escherichia coli K5 antigen gene cluster: a region encoding proteins involved in cell surface expression of capsular polysaccharide.

The nucleotide sequence of region 1 of the K5 antigen gene cluster of Escherichia coli was determined. This region is postulated to encode functions which, at least in part, participate in translocation of polysaccharide across the periplasmic space and onto the cell surface. Analysis of the nucleotide sequence revealed five genes that encode proteins with predicted molecular masses of 75.7, 60.5, 44, 43, and 27 kDa. The 27-kDa protein was 70.7% homologous to the CMP-2-keto-3-deoxyoctulosonic acid synthetase enzyme encoded by the E. coli kdsB gene, indicating the presence of a structural gene for a similar enzyme within the region 1 operon. The 43-kDa protein was homologous to both the Ctrb and BexC proteins encoded by the Neisseria meningitidis and Haemophilus influenzae capsule gene clusters, respectively, indicating common stages in the expression of capsules in these gram-negative bacteria. However, no homology was detected between the 75.7, 60.5-, and 44-kDa proteins and any of the proteins so far described for the H. influenzae and N. meningitidis capsule gene clusters.

Expression of the capsular K5 polysaccharide of Escherichia coli: biochemical and electron microscopic analyses of mutants with defects in region 1 of the K5 gene cluster.

The gene cluster of the capsular K5 polysaccharide, a representative of group II capsular antigens of Escherichia coli, has been cloned previously, and three regions responsible for polymerization and surface expression have been defined (I.S. Roberts, R. Mountford, R. Hodge, K. B. Jann, and G. J. Boulnois, J. Bacteriol. 170:1305-1330, 1988). Region 1 has now been sequenced, and five open reading frames (kpsEDUCS) have been defined (C. Pazzani, C. Rosenow, G. J. Boulnois, D. Bronner, K. Jann, and I. S. Roberts, J. Bacteriol. 175:5978-5983, 1993). In this study, we characterized region 1 mutants by immunoelectron microscopy, membrane-associated polymerization activity, cytoplasmic CMP-2-keto-3-deoxyoctonate (KDO) synthetase activity, and chemical analysis of their K5 polysaccharides. Certain mutations within region 1 not only effected polysaccharide transport (lack of region 1 gene products) but also impaired the polymerization capacity of the respective membranes, reflected in reduced amounts of polysaccharide but not in its chain length. KDO and phosphatidic acid (phosphatidyl-KDO) substitution was found with extracellular and periplasmic polysaccharide and not with cytoplasmic polysaccharide. This and the fact that the K5 polysaccharide is formed in a kpsU mutant (defective in capsule-specific K-CMP-KDO synthetase) showed that CMP-KDO is engaged not in initiation of polymerization but in translocation of the polysaccharide.

The Escherichia coli serA-linked capsule locus and its flanking sequences are polymorphic, genetic evidence for the existence of more than two groups of capsule gene clusters.

Two families of Escherichia coli capsules, termed groups I and II, have been defined previously on the basis of a number of biochemical and genetic criteria. Recently, a third group of capsules, termed I/II has been suggested on the basis of chemical structure and mode of expression. In this paper, we show that group I capsule-producing strains lack the serA-linked group II capsule genes. In addition, group I/II capsule-producing strains lack the group II capsule genes despite the former genes also mapping near to serA. Therefore, the genetic data presented in this paper support the existence of three groups of capsule gene clusters, two of which are linked to serA. Sequences flanking the K4 capsule genes were found in the chromosome of all E. coli strains examined and were sometimes present in multiple copies at different loci, indicating that this chromosomal region is highly polymorphic.

Genetic analysis of the gene cluster encoding nonfimbrial adhesin I from an Escherichia coli uropathogen.

The chromosomally encoded nonfimbrial adhesion I (NFA-I) from Escherichia coli urinary tract isolate 827 (O83:K1:H4) mediates agglutination of human erythrocytes. Subclones were constructed from an NFA-I-expressing recombinant E. coli K-12 clone, derived from a genomic library of E. coli 827. Minicell analysis and nucleotide sequencing revealed that proteins of 30.5, 9, 80, 15, and 19 kDa encoded on a stretch of approximately 6 kb are involved in the expression of NFA-I. NFA-I exhibits a polymeric structure, which disintegrates with elevated temperature into a 19-kDa monomer but with some relatively stable dimers. By using gold-conjugated monoclonal antibodies directed against NFA-I in electron microscopy, the adhesin could be localized on the outer surface of the recombinant E. coli K-12 bacteria. The nucleotide sequence of the nfaA gene encoding the monomeric structural subunit of the adhesin was determined. An open reading frame of 184 amino acids encoding the NfaA precursor, which is processed to the mature protein, was found; it consisted of 156 amino acids with a calculated molecular weight of 16,000. Peptide sequencing of the NFA-I subunit protein confirmed that this open reading frame corresponds to the NfaA coding locus. Furthermore, the nucleotide sequence of the open reading frame termed NfaE, located at the proximal part of the DNA stretch responsible for NFA-I expression, was elaborated. NfaE consists of 247 amino acids, including a presumptive 29-amino-acid signal peptide, leading to a molecular weight of 24,000 for the mature protein. The nfaE sequence shares homology with the 27-kDa CS3 protein, which is involved in the assembly of CS3 fibrillae, and might encode the 30.5-kDa protein, detected in minicells.

Cytotoxic effects on hair cells of guinea pig cochlea produced by pneumolysin, the thiol activated toxin of Streptococcus pneumoniae.

The cytolytic toxin, pneumolysin, from the gram positive bacterium, Streptococcus pneumoniae, when perfused through the scala tympani of the guinea pig cochlea reduced the amplitude of both the compound action potential and the cochlear microphonic potential. When the surface of the organ of Corti was examined by scanning electron microscopy, both inner and outer hair cells and supporting cells were found to be damaged. Inner hair cells and outer hair cells of row 3 were the most susceptible to damage by pneumolysin, followed by row 2 and then by row 1 of the outer hair cells. Damage to hair cells included disruption and splaying of stereocilia, loss of stereocilia and complete dissolution of hair bundles. Apical surfaces of hair cells and supporting cells were torn, pitted and cratered with shrinkage and tearing of cell boundaries. Within the dose range perfused (0.05-1 micrograms/microliters in a 10 microliters aliquot), the magnitude of the physiological and anatomical lesions was concentration dependent. The cytotoxic effects of pneumolysin reported here may be clinically significant factors in deafness caused by meningitis and otitis media in humans.

Molecular analysis of the pathogenicity of Streptococcus pneumoniae: the role of pneumococcal proteins.

For many years the virulence of Streptococcus pneumoniae has largely been attributed to its antiphagocytic polysaccharide capsule. Recent evidence, however, indicates that certain pneumococcal proteins play an important part in the pathogenesis of disease, either as mediators of inflammation or by directly attacking host tissues. Pneumococci carrying defined mutations in the genes encoding any one of at least three pneumococcal proteins (the toxin pneumolysin, the major pneumococcal autolysin, and pneumococcal surface protein A) have significantly reduced virulence. Pneumococcal hydrolytic enzymes, such as neuraminidase, hyaluronidase, and IgA1 protease may also contribute to colonization and/or invasion of the host. Several of these proteins (or their detoxified derivatives) are protective immunogens in animal models and therefore warrant consideration for inclusion in human antipneumococcal vaccine formulations.

Genome diversity at the serA-linked capsule locus in Escherichia coli.

Individual isolates of Escherichia coli synthesize one of more than 70 chemically distinct polysaccharides which form the capsule. In this article we review the genetics of capsule production in E. coli and highlight what this is beginning to reveal in terms of the genetic basis of the structural diversity of polysaccharides. The serA-linked capsule locus can take three different allelic forms. Two of these are associated with capsule genes and are themselves internally variant, whilst the third form has not so far been implicated in capsule biogenesis. Thus the serA-linked region of the E. coli genome is strikingly polymorphic.

Effect of immunization with Freund's adjuvant and pneumolysin on histologic features of pneumococcal infection in the rat lung in vivo.

Immunization with Freund's adjuvant and pneumolysin and stimulation with Freund's adjuvant alone both reduced the severity of the pneumonia caused by injections of bacteria into the apical lobe bronchi of rats. Neither protocol influenced the incidence of pneumococcal bacteremia. Illness sufficiently severe to require sacrifice was delayed from 2.8 days in nonimmunized animals to 5.7 days in those immunized with Freund's adjuvant and pneumolysin (P < 0.05) and 4.5 days in those stimulated with Freund's adjuvant alone (P, not significant).

Homology among Escherichia coli K1 and K92 polysialytransferases.

The neuS-encoded polysialytransferase (polyST) in Escherichia coli K1 catalyzes synthesis of polysialic acid homopolymers composed of unbranched sialyl alpha 2,8 linkages. Subcloning and complementation experiments showed that the K1 neuS was functionally interchangeable with the neuS from E. coli K92 (S. M. Steenbergen, T. J. Wrona, and E. R. Vimr, J. Bacteriol. 174:1099-1108, 1992), which synthesizes polysialic acid capsules with alternating sialyl alpha 2,8-2,9 linkages. To better understand the relationship between these polySTs, the complete K92 neuS sequence was determined. The results demonstrated that K1 and K92 neuS genes are homologous and indicated that the K92 copy may have evolved from its K1 homolog. Both K1 and K92 structural genes comprised 1,227 bp. There were 156 (12.7%) differences between the two sequences; among these mutations, 55 did not affect the derived primary structure of K92 polyST and hence were synonymous with the K1 sequence. Assuming maximum parsimony, another estimated 17 synonymous mutations plus 84 nonsynonymous mutations could account for the 70 amino acid replacements in K92 polyST; 36 of these replacements were judged to be conservative when compared with those of K1 polyST. There were no changes detected in the first 146 5' or last 129 3' bp of either gene, suggesting, in addition to the observed mutational differences, the possibility of a past recombination event between neuS loci of two different kps clusters. The results indicate that relatively few amino acid changes can account for the evolution of a glycosyltransferase with novel linkage specificity.