Identification of two penicillin-binding multienzyme complexes in Haemophilus influenzae.

Dansyl-labeled penicillin, reversed-phase chromatography, and peptide mapping have been used to detect, separate, and study penicillin-binding proteins (PBPs) and PBP multienzyme complexes of H. influenzae. The cross-linking of proteins in the multienzyme complex was accomplished with the aid of cyanogen, a salt-bridge specific cross-linking agent. The chromatographic profile of the PBPs clearly showed a dramatic change in the number and identity of peaks after treatment of the bacterial cells with cyanogen. The disappearance of all seven peaks corresponding to the PBPs was accompanied by the emergence of two new peaks with molecular weights between 400 kDa and 600 kDa. The results hint at the existence of two penicillin-binding multienzyme complexes, each containing subunits that interact via salt-bridges. Chromatographic active site peptide mapping of PBPs and PBP complexes was used to determine the identity of PBPs involved in each complex. It is postulated that one multienzyme complex containing PBP 2 may be involved in cell elongation while the other complex containing PBP 3 may be responsible for cell division.     

Tetracyclin-stimulated expression of ampicillin resistance in Haemophilus influenzae.

Tetracycline at a low concentration stimulated the expression of ampicillin resistance in certain strains of Haemophilus influenzae.     

Characteristics of major outer membrane proteins of Haemophilus influenzae.

Several properties of Haemophilus influenzae outer membrane proteins were analyzed to define related proteins in various isolates. H. influenzae type b 760705 had six major outer membrane proteins with the following characteristics. Protein a (Mr, 47,000) demonstrated heat modifiability in sodium dodecyl sulfate; its apparent molecular weight was 34,000 at temperatures below 60 degrees C. This protein was extracted from cell envelopes by using Triton X-100-10 mM MgCl2; in cell envelope preparations, the protein was degraded by trypsin. Proteins b (Mr, 41,000) and c (Mr, 40,000) were insensitive to trypsin degradation, were not heat modifiable in sodium dodecyl sulfate, and were peptidoglycan associated in 0.5% Triton X-100-0.2% sodium dodecyl sulfate. The amount of protein b was reduced in ultrasonically obtained cell envelopes. Protein d (Mr, 37,000) was heat modifiable in sodium dodecyl sulfate with an Mr of 28,000 at temperatures below 100 degrees C and was degraded by trypsin, leaving a membrane-bound fragment of Mr, 27,000. Both the intact and degraded proteins were immunologically cross-reactive with the heat-modifiable OmpA protein of Escherichia coli K-12. Protein d was absent in LiCl-EDTA extracts of cells. Protein e (Mr, 30,000), invariably present in all H. influenzae strains tested, was insensitive to trypsin and absent in LiCl-EDTA extracts of cells. Protein k (Mr, 58,000) was extracted from cell envelopes with 2% Triton X-100-10 mM MgCl2 and, in cell envelopes, appeared to be sensitive to trypsin degradation. Proteins with similar properties to those of proteins a to k were found in 10 other H. influenzae b strains, reference strains with serotype a, c, d, e, and f capsules, and 18 of 20 nonencapsulated strains. Their relative molecular weights, however, varied.     

Identification of two iron-repressed periplasmic proteins in Haemophilus influenzae.

Protein expression by Haemophilus influenzae under iron-limiting growth conditions was examined. The five type b strains and four nontypeable strains studied all expressed a new protein of about 40 kDa when deprived of iron during growth. Most strains also expressed a protein of about 31 kDa under the same growth conditions. Both the 40- and 31-kDa proteins were not expressed by cells grown in iron-replete medium. The 40- and 31-kDa proteins were not expressed in iron-deficient medium to which an excess of ferric nitrate had been added, and therefore it was concluded that their expression was iron regulated. These iron-repressed proteins were localized to the periplasmic space. The amino-terminal sequences of both proteins were determined. The N-terminal sequence of the 40-kDa protein had 81% similarity to the N terminus of Fbp, the major iron-binding protein of Neisseria gonorrhoeae and N. meningitidis. The 31-kDa protein sequence showed no homology with any known protein sequence. As no plasmids were found in the strains, it was concluded that these proteins were chromosomally encoded.     

Haemophilus influenzae biochemotyping

Biochemotyping was performed in 129 strains of Haemophilus influenzae. 95% of strains could be assigned to one of the five biochemotypes proposed by Kilian. Most of the serotype B strains isolated in meningitis belonged to biochemotype I. The biochemical differentiation of Haemophilus influenzae is regarded as a reliable technique deserving further application.     

Immunogenic outer-membrane proteins of Haemophilus influenzae type b in infection.

Outer-membrane proteins (OMPs) from Haemophilus influenzae type b (strain Eagan), grown both in vitro (broth) and in vivo (rat intra-peritoneal), were separated by SDS-PAGE. The major OMPs were present in both growth conditions although the amounts of OMP a and OMP d were reduced in rat-grown organisms. There were strong additional bands in in-vivo-grown organisms at 51 and 92 kDa. Antiserum was raised in rabbits against in-vivo-grown bacteria, and absorbed with lysates of in-vitro-grown bacteria. This serum was used in Western blot analysis of OMPs from in-vitro- and in-vivo-grown cells to identify immunogenic proteins present in infection. These infection-associated OMPs had apparent molecular masses of 43 kDa, 48 kDa, 81 kDa and greater than 200 kDa. Bands of reactivity, of the same molecular mass as some of these, were found on immunoblots when rat and human convalescent sera were used as the source of primary antibody. In particular, a band of 81 kDa was recognized by pooled rat and three human convalescent sera.     

Circularized copies of amplifiable resistance genes from Haemophilus influenzae plasmids.

Tandem repeat amplification of resistance determinants in Haemophilus influenzae plasmids is associated with the occurrence of separate circular DNA molecules. They were demonstrated to represent mono- and multimeric forms of the amplifiable segments of the plasmids which comprise the respective resistance transposons and an additional region designated as an amplification sequence. The latter region mediates the recombinational events involved in amplification. The DNA circles apparently lack the ability to replicate autonomously but most probably provide an effective means for the translocation of resistance genes from one plasmid to another.     

Lactoperoxidase and Iodo-Gen-catalyzed iodination labels inner and outer membrane proteins of Haemophilus influenzae

Both inner and outer membrane proteins of Haemophilus influenzae type b were labeled by iodination procedures believed to be specific for exposed surface proteins only. It is suggested that this is due to specific properties of the outer membrane of H. influenzae and that use of these procedures with other gram-negative bacteria be evaluated carefully.     

Biological and biophysical properties of Haemophilus influenzae transforming DNA encapsided by viral proteins

Summary The expression of the transforming ability of Haemophilus influenzae DNA was investigated after its encapsidation by the coat protein of two different plant viruses, Brome Mosaic Virus (BMV) and Alfalfa Mosaic Virus (AMV). The influence of the encapsidation on the various steps of the transformation process was studied, as well as the protection of the DNA molecule inside of the DNA-protein complex particles against nucleolytic attack.The kinetics of uptake and penetration of free and encapsided DNA and their respective competitive abilities were compared in order to explain the differences which appeared between the rates of transformation by free and encapsided high molecular weight DNAs.Finally, some conclusions are drawn concerning the uncoating process of these nucleoprotein complex particles and the strength of the DNA-protein and protein-protein interactions existing in these particles.Abbreviations M.W. molecular weight - T.A. transforming ability     

β-Lactam resistance in Streptococcus pneumoniae: penicillin-binding proteins and non-penicillin-binding proteins

The beta-lactams are by far the most widely used and efficacious of all antibiotics. Over the past few decades, however, widespread resistance has evolved among most common pathogens. Streptococcus pneumoniae has become a paradigm for understanding the evolution of resistance mechanisms, the simplest of which, by far, is the production of beta-lactamases. As these enzymes are frequently plasmid encoded, resistance can readily be transmitted between bacteria. Despite the fact that pneumococci are naturally transformable organisms, no beta-lactamase-producing strain has yet been described. A much more complex resistance mechanism has evolved in S. pneumoniae that is mediated by a sophisticated restructuring of the targets of the beta-lactams, the penicillin-binding proteins (PBPs); however, this may not be the whole story. Recently, a third level of resistance mechanisms has been identified in laboratory mutants, wherein non-PBP genes are mutated and resistance development is accompanied by deficiency in genetic transformation. Two such non-PBP genes have been described: a putative glycosyltransferase, CpoA, and a histidine protein kinase, CiaH. We propose that these non-PBP genes are involved in the biosynthesis of cell wall components at a step prior to the biosynthetic functions of PBPs, and that the mutations selected during beta-lactam treatment counteract the effects caused by the inhibition of penicillin-binding proteins.