Induction of Salmonella stress proteins upon infection of macrophages.

Regulated expression of bacterial genes allows a pathogen to adapt to new environmental conditions within the host. The synthesis of over 30 Salmonella proteins is selectively induced during infection of macrophages. Two proteins induced by Salmonella are the heat shock proteins GroEL and DnaK. Two avirulent, macrophage-sensitive mutants of Salmonella synthesize GroEL and DnaK but fail to synthesize different subsets of proteins normally induced within the macrophage. Enhanced expression of selected Salmonella proteins contributes to bacterial survival within macrophages and may also contribute to the apparent immunodominance of heat shock proteins.

A Salmonella locus that controls resistance to microbicidal proteins from phagocytic cells.

Facultative intracellular pathogens pose an important health problem because they circumvent a primary defense mechanism of the host: killing and degradation by professional phagocytic cells. A gene of the intracellular pathogen Salmonella typhimurium that is required for virulence and intracellular survival was identified and shown to have a role in resistance to defensins and possibly to other microbicidal mechanisms of the phagocyte. This gene may prove to be a regulatory element in the expression of virulence functions.

Epithelial cell surfaces induce Salmonella proteins required for bacterial adherence and invasion.

Salmonella bacteria are capable of entering (invading) and multiplying within eukaryotic cells. Stable adherence to and invasion of epithelial cells by S. choleraesuis and S. typhimurium were found to require de novo synthesis of several new bacterial proteins. This inducible event appears to be a coordinately regulated system dependent on trypsin- and neuraminidase-sensitive structures present on the epithelial cell surface. Mutants of S. choleraesuis and S. typhimurium were unable to synthesize these proteins and did not stably adhere to nor invade eukaryotic cells. Two such S. typhimurium mutants were avirulent in mice, an indication that these proteins are required for Salmonella virulence.

HRR25, a putative protein kinase from budding yeast: association with repair of damaged DNA.

In simple eukaryotes, protein kinases regulate mitotic and meiotic cell cycles, the response to polypeptide pheromones, and the initiation of nuclear DNA synthesis. The protein HRR25 from the budding yeast Saccharomyces cerevisiae was defined by the mutation hrr25-1. This mutation resulted in sensitivity to continuous expression of the HO double-strand endonuclease, to methyl methanesulfonate, and to x-irradiation. Homozygotes of hrr25-1 were unable to sporulate and disruption and deletion of HRR25 interfered with mitotic and meiotic cell division. Sequence analysis revealed two distinctive regions in the protein. The NH2-terminus of HRR25 contains the hallmark features of protein kinases, whereas the COOH-terminus is rich in proline and glutamine. Mutations in HRR25 at conserved residues found in all protein kinases inactivated the gene, and these mutants exhibited the hrr25 null phenotypes. Taken together, the hrr25 mutant phenotypes and the features of the gene product indicate that HRR25 is a distinctive member of the protein kinase superfamily.

In vivo analysis of the Saccharomyces cerevisiae HO nuclease recognition site by site-directed mutagenesis.

HO nuclease introduces a specific double-strand break in the mating-type locus (MAT) of Saccharomyces cerevisiae, initiating mating-type interconversion. To define the sequence recognized by HO nuclease, random mutations were produced in a 30-base-pair region homologous to either MAT alpha or MATa by a chemical synthesis procedure. The mutant sites were introduced into S. cerevisiae on a shuttle vector and tested for the ability to stimulate recombination in an assay that mimics mating-type interconversion. The results suggest that a core of 8 noncontiguous bases near the Y-Z junction of MAT is essential for HO nuclease to bind and cleave its recognition site. Other contacts must be required because substrates that contain several mutations outside an intact core reduce or eliminate cleavage in vivo. The results show that HO site recognition is a complex phenomenon, similar to promoter-polymerase interactions.

Double-strand breaks stimulate alternative mechanisms of recombination repair.

To test the double-strand break repair model, we used HO nuclease to introduce double-strand breaks at several sites along a yeast chromosome containing duplicated DNA. Depending on the configuration of the double-strand break and recombining markers, different spectra of recombinant products were observed. Different repair kinetics and recombinant products were observed when a double-strand break was introduced in unique or duplicated DNA. The results of this study suggest that double-strand breaks in yeast stimulate recombination by several mechanisms, and we propose an alternative mechanism for double-strand break-induced gene conversion that does not depend on direct participation of the broken ends.

Analysis of Tn3 sequences required for transposition and immunity.

Tn3 is a 5-kb transposon (Tn) with 38-bp inverted terminal repeats (ITR). The two 38-bp terminal sequences are required in cis for Tn3 transposition. In this study, the role of the ITR in Tn3 transposition has been further dissected by the use of various mini-Tn3 Tn's. The transposition frequency of these mini-Tn's demonstrate that Tn3 contains no sequence other than the ITR sequences that are necessary for the first step in transposition; the two terminal repeats must be oriented as ITR for transposition to occur; the outside 34 bp of the ITR are required for transposition; and reducing the distance between the terminal sequences does not affect transposition frequency. Moreover, mutant copies of the ITR sequences that cannot function in transposition do not confer transposition immunity.

Identification of a new iron regulated locus of Salmonella typhi.

In order to identify genes belonging to the Fur regulon of Salmonella typhi which are absent from Escherichia coli K-12, a plasmid gene bank consisting of 4000 independent clones was screened for Fur regulated promoters using the Fur titration assay (FURTA). DNA probes generated from FURTA positive plasmids were then used for hybridization with chromosomal DNA from S. typhi, Salmonella typhimurium and E. coli. Using these techniques we identified an iron regulated locus present in S. typhi and S. typhimurium but not in E. coli. Further cloning and nucleotide sequence analysis identified two open reading frames, termed iroBC, organized in a typical operon structure. The genes iroBC were located at 4 and 57 centisomes on the physical maps of Salmonella typhi and S. typhimurium, respectively. This region of the S. typhimurium chromosome contains a large DNA loop which is absent from the corresponding area of the E. coli chromosome. Finally, we developed a new method for generation of single copy transcriptional fusions. A suicide vector was constructed, which allows for the generation of chromosomal fusions to the promoterless E. coli lacZYA genes. By integration of this construct at the iro locus we could establish iron responsive expression of iroBC.

Synthesis and characterization of a recombinant myohemerythrin protein encoded by a synthetic gene.

The antigenic epitopes of the myohemerythrin (MHr) molecule have been studied extensively. The critical amino acid residues responsible for its immune recognition have been identified by using synthetic peptides and the technique of epitope scanning. To assess the true relevance of these techniques for determining the molecular mechanism of antigenic recognition and immunogenicity, the results obtained with isolated peptides should be tested in the context of the folded protein. To this end, we have designed and constructed a synthetic MHr gene, in modular form, which will allow subsequent alterations of nucleotide sequence encoding epitopes of interest. We have produced the recombinant protein at high level, and have shown by several criteria that it possesses the chemical, physical and immunological properties of the native worm protein. Thus, we have developed a valuable system for detailed immunological studies of the structure and chemistry required for antibody binding to protein.

The inhibition of cholesterol esterification by cyclandelate in transformed mouse macrophages.

Cyclandelate (trimethylcyclohexanyl mandelate) inhibited cholesterol esterification in a transformed mouse macrophage cell line (J774) with a concentration of approximately 20 microM being required for half-maximal inhibition. The intact drug was required for its inhibitory action since neither of its hydrolysis products, trimethylcyclohexanol and mandelic acid, caused any inhibition even at high concentrations. The drug entered the cells very rapidly with inhibition being apparent within the shortest time possible to measure esterification (15 min after drug addition). The rate of cholesterol esterification returned to control values when drug-inhibited cells were incubated in drug-free medium indicating a rapid loss of drug from the cells. Loading of cells with cholesterol had no effect on the inhibitory action of cyclandelate, and the inhibition of esterification of cholesterol appeared to be specific, since the syntheses of phospholipid and triacylglycerol (which also involve the action of acyltransferases) were not affected by the drug. Similar inhibitions of cholesterol esterification were seen in four other cell lines, a human osteosarcoma, Chinese hamster ovary cells, a human transformed macrophage cell line (U937) and human umbilical cord vein endothelial cells, as well as in slices of pig aorta, indicating a general action in extra-hepatic tissues where the drug is not hydrolysed.

Inhibition of acyl coenzyme A: cholesterol acyl transferase by trimethylcyclohexanylmandelate (cyclandelate).

Cyclandelate was an effective inhibitor of rat hepatic acycloenzyme A: cholesterol acyltransferase (ACAT) with a concentration of 80 microM being required for half maximal inhibition. A similar effect was seen with human and rabbit liver microsomal enzymes. The drug did not compete with oleoyl CoA or cholesterol and could be removed from enzyme preparations by washing. It was hydrolysed rapidly by rat liver microsomes to products which were non inhibitory. No hydrolysis of the drug was seen with non hepatic microsomes and the concentration of cyclandelate required to cause half maximal inhibition of ACAT in the transformed mouse macrophage J774 microsomal fraction was less than 30 microM. The possible significance of the differential actions of cyclandelate towards hepatic and extra hepatic ACAT in vivo is discussed.

The effects of two acylcoenzyme A: cholesterol acyltransferase (ACAT) inhibitors, cyclandelate and a non-hydrolysable ether analogue, benzyl3,3,5-trimethylcyclohexanol on low density lipoprotein metabolism in macrophages and hepatocytes.

Cyclandelate (3,3,5-trimethylcyclohexanylmandelate) caused a dose-dependent decrease in the metabolism of radioiodinated low density lipoprotein [125I-LDL] by J774 mouse macrophages. This was probably an indirect effect due to the inhibition of cholesterol esterification by the cells rather than a direct one on the interaction of LDL with its receptor, since no inhibition was seen in cells which had been cholesterol-depleted by prior incubation with lipoprotein-depleted serum for 48 hr. Cyclandelate also inhibited immediately de novo synthesis of cholesterol from [1-14C]acetate in J774 cells, suggesting a direct action of the drug on an enzyme of the cholesterol biosynthetic pathway. The drug was an efficient inhibitor of hamster and rat intestinal acylcoenzyme A: cholesterol acyltransferase (ACAT) activity in vitro with an IC50 of 20 microM. Addition of cyclandelate to the diet of meal-fed rats caused a marked inhibition of the rate of appearance of dietary [4-14C]cholesterol in the plasma. A nonhydrolysable ether analogue of cyclandelate, benzyl3,3,5-trimethylcyclohexanol, was prepared to compare hepatic and extrahepatic actions of the two molecules. The analogue inhibited cholesterol esterification in J774 cells, transformed human macrophages U937 and human umbilical vein endothelial cells with an IC50 of 20 microM and had effects similar to those of cyclandelate on 125I-LDL metabolism in J774 cells. Differences between the analogue and cyclandelate were seen in hepatocytes and hepatic microsomal fractions, where preincubation with the analogue inhibited cholesterol esterification in both systems while cyclandelate had no inhibitory action in either. Consequently, preincubation of rat hepatocytes with benzyl3,3,5-trimethylcyclohexanol for 17 hr caused a marked decrease in the binding of 125I-LDL to the cells, whereas binding to cells preincubated with cyclandelate was the same as to control cells.

Fimbrial adhesins of Salmonella typhimurium. Role in bacterial interactions with epithelial cells.

S. typhimurium initiates infection of its mammalian host by attachment to mucosal surfaces in the intestine and subsequent invasion of epithelial cells. To date, three S. typhimurium fimbrial operons, fim, lpf and pef, have been characterized. This analysis suggests that fimbrial adhesins fulfill multiple functions during the initial phase of an infection. In addition to their role in colonization of the small intestine, adhesins contribute to the tissue tropism for Peyer's patches, which is characteristic for Salmonella infections. Furthermore, by mediating the initial contact to epithelial cells, fimbrial adhesins appear to be necessary for invasion and possibly for elicitation of an inflammatory response. Thus, fimbriae are important virulence factors of S. typhimurium and their future analysis promises to yield fascinating new insights into host-parasite interactions of this pathogen.