Cloning of the gene encoding the major outer membrane protein of Haemophilus influenzae type b.

The major outer membrane protein (P2) of Haemophilus influenzae type b (Hib) with an apparent molecular weight of 37,000 to 40,000 has been previously shown to function as a porin and also as a target for antibodies protective against experimental Hib disease. The gene encoding the Hib P2 protein was cloned by using a shuttle vector capable of replication in both Escherichia coli and H. influenzae. The amino acid sequence of the amino terminus of the Hib P2 protein was determined and used to design an oligonucleotide probe corresponding to the first 20 amino acids of this protein. This oligonucleotide probe was used to identify Hib chromosomal DNA fragments containing the Hib P2 gene. These DNA fragments were ligated into the plasmid vector pGJB103 and then used to transform a rec-1 mutant of H. influenzae Rd. Recombinant clones expressing the Hib P2 protein were identified in a colony blot-radioimmunoassay by using a monoclonal antibody specific for a surface epitope of the Hib P2 protein. The gene encoding this Hib protein was present on a 10-kilobase Hib DNA insert in the recombinant plasmid. Transformation experiments involving the recombinant plasmid suggested that unregulated synthesis of Hib P2 is a lethal event in E. coli. The recombinant Hib P2 protein was exposed on the surface of the recombinant H. influenzae strain. This recombinant strain was used to develop a system for detecting polyclonal serum antibodies directed against surface determinants of the Hib P2 protein. The availability of the gene encoding the Hib P2 protein should facilitate investigation of both the immunogenicity and the structure-function relationship(s) of this major outer membrane protein.

Cloning and expression in Escherichia coli of the gene encoding the heat-modifiable major outer membrane protein of Haemophilus influenzae type b.

One major outer membrane protein (P1) of Haemophilus influenzae type b (Hib), with an apparent molecular weight of 34,000 (34K) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), has been shown to be heat modifiable. After heating at 100 degrees C for 5 min in 2% SDS, the P1 protein exhibits an apparent molecular weight of 49,000 (49K) in SDS-PAGE. Monoclonal antibodies (MAbs) reactive with P1 bound to the surface of Hib, and one of these MAbs had a protective effect against the development of Hib bacteremia in an animal model for invasive Hib disease. A 6-kilobase Hib DNA insert containing the gene encoding this P1 protein was cloned into Escherichia coli by using the gamma gt11 expression vector. Recombinant phage expressing P1 were identified by screening phage plaques with a MAb directed against the P1 protein. Expression of the P1 protein by an E. coli lysogen carrying the recombinant phage was independent of both vegetative phage growth and induction of lacZ gene-directed transcription of the Hib DNA insert. The Hib DNA insert encoding the P1 protein was subcloned into the plasmid vector pBR322, and a transformant containing the recombinant plasmid pFRG100 was identified with the P1 protein-directed MAb in a colony blot-radioimmunoassay. Western blot (immunoblot) analysis determined that the recombinant P1 protein possessed heat-modifiability characteristics identical to those of the native Hib protein. The P1 protein was expressed on the surface of both the E. coli lysogen containing the recombinant phage and the E. coli transformant containing pFRG100. Western blot analysis of acute- and convalescent-phase sera from infants with Hib meningitis showed that antibodies in the convalescent-phase sera recognized the P1 protein expressed by the E. coli transformant containing pFRG100. The availability of this cloned Hib DNA insert encoding the Hib P1 protein and the expression of this protein on the surface of recombinant E. coli should facilitate the investigation of P1 for both its vaccinogenic potential and its functional role in the outer membrane of Hib.

Ribosomal protein phosphorylation induced during Q fever or by lipopolysaccharide: in vitro translation is stimulated by infected liver ribosomes.

Q fever, as well as the lipopolysaccharide prepared from the rickettsial agent Coxiella burnetii, stimulates the phosphorylation of guinea pig liver ribosomal protein S6. In vitro mRNA and ribosome-dependent rabbit reticulocyte lysate translation systems reconstituted with ribosomes and mRNAs from infected animal livers were more active than those with mRNAs and ribosomes from uninfected animals. Treatment of ribosomes with a ribosomal supernatant phosphatase reduced the in vitro translation activities; the largest decreases occurred in systems with ribosomes and mRNAs from infected liver. These experiments provide a basis for explaining the increased hepatic protein synthesis during Q fever and demonstrate, perhaps for the first time, the phosphorylation of ribosomal protein in response to lipopolysaccharide. The implications of these observations are discussed in the context of previous studies on stimulated transcription and translation during Q fever.

Phosphorylation-dephosphorylation of nuclear proteins during infection (Q fever).

The proposal that gene expression may be regulated by phosphorylation of nonhistone chromatin proteins was tested by studying increased transcription resulting from Q fever. Certain liver nuclear phosphoprotein kinase and phosphatase activities were altered after guinea pigs were infected with Coxiella burnetii. Nonhistone chromatin proteins had increased phosphoprotein kinase activity and were differentially phosphorylated. The addition of spermine equally stimulated nuclear phosphoprotein kinases of uninfected and infected livers. Increased nuclear phosphatase activity accompanied infection. It was concluded that protein phosphorylations are altered by infection and are central events in regulating RNA and protein synthesis. A hypothesis is presented which attempts to correlate the findings in previous reports and those in the present paper regarding biochemical sequelae of Q fever. It is suggested that certain features of regulation described here also may be operative in some other infections or diseases.

Temporal studies of factors associated with changes in transcription during Q fever.

Temporal studies were made of factors associated with increased RNA synthesis in guinea pig liver during Q fever. DNA-dependent RNA polymerase activities increased immediately after infection. The major distribution of RNA polymerase classes shifted from class II to class I during infection. Ornithine decarboxylase activity was induced or stimulated soon after infection and remained elevated throughout the four-day period studied. S-Adenosylmethionine decarboxylase activity increased on the first day after infection and subsequently declined. Concomitantly elevated concentrations of the polyamines putrescine, spermidine and spermine reached a maximum on the first day after infection and then decreased. A model is presented to integrate these and other results to explain how RNA synthesis may be regulated during infection.