Mikael Rhen

Molecular analysis of spv virulence genes of the salmonella virulence plasmids

Genes on an 8 kb region common to the virulence plasmids of several serovars of Salmonella are sufficient to replace the entire plasmid in enabling systemic infection in animal models. This virulence region encompasses five genes which previously have been designated with different names from each investigating laboratory. A common nomenclature has been devised for the five genes, i.e. spv for salmonella plasmid virulence. The first gene, spvR, encodes a positive activator for the following four genes, spvABCD. DNA sequence analysis of the spv genes from Salmonella typhimurium. Salmonella dublin, and Salmonella choleraesuis demonstrated extremely high conservation of the DNA and amino acid sequences. The spv genes are induced at stationary phase and in carbon‐poor media, and optimal expression is dependent on the katF locus. The cirulence functions of the spv genes are not known, but these genes may increase the growth rate of salmonellae in host cells and affect the interaction of salmonellae with the host immune system.

Transcriptional regulation of Salmonella enterica virulence plasmid genes in cultured macrophages

The plasmid‐carried spv genes promote virulence of salmonellae in mice by allowing bacterial growth in the reticuloendothelial tissue. When the bacteria are cultivated under normal laboratory conditions the spv genes appear dormant. This communication explores the transcriptional regulation of spv genes within murine macrophage‐like J774‐A.1 cells utilizing a new reporter system. Transcriptional fusions were constructed between promoter elements of the Salmonella enterica var. Typhimurium spv genes and the KS71A fimbrial gene cluster. The expression of KS71A fimbriae In fusion‐carrying Escherichia coli strains was found to be under the control of the transcriptional activator gene spvR. In strains overproducing SpvR, KS71A fimbriae were assembled on the bacterial cell surface and could be detected by bacterial agglutination or immunofluorescence of intact bacteria; the reporter activity was quantified by estimating the percentage of fluorescent bacteria and by immunoblotting of cell lysates. The activity of the reporters, when transformed into the parent Typhimurium TML R66, was low and revealed less than 0.3% fimbriated cells under in vitro culture conditions. A 15–30‐fold increase in fimbriation was observed when the bacteria were cultivated within J774‐A.1 cells. No such increase occurred when the reporter fusions were transformed into TML R66 cured of the virulence plasmid. Insertional inactivation of the spvR gene of the virulence plasmid in Typhimurium TML R66 also abolished induction, whereas corresponding inactivation of spvA or spvB did not reduce induction. No increase in reporter activity was obtained in Typhimurium of line Q1, which is naturally avirulent for mice, although the strain was provided with virulence plasmid pEX102 of line TML R66. We conclude that the intracellular environment of J774‐A.1 cells induces the spv genes and that this induction requires gene functions of both the bacterial chromosome and the virulence plasmid.

Location of adhesion sites for P-fimbriated and for 075X-positive Escherichia coli in the human kidney☆

High-affinity binding sites for P-fimbriated and for 075X-positive Escherichia coli were located in the human kidney. Frozen sections of normal human kidney were double-stained first with fluorochrome-labelled bacteria and then with fluorochrome-labelled nephron site-specific lectins or antibodies. The P-fimbriate recombinant E. coli strain used showed specific adherence to glomerular structures, to the lumen of proximal and sital tubules and to vascular endothelium but did not adhere to collecting ducts or to peritubular sites. Two E. coli strains having the 075X adhesin showed specific adherence to renal interstitium, to glomerular elements and to Bowmans capsule. The method described allows the detailed determination of tissue-substructure specificity of bacterial adhesion. Our results demonstrate tissue tropism in the adhesion of E. coli to human kidneys and suggest a pathogenetic role for X adhesins.

Molecular organization of genes constituting the virulence determinant on the Salmonella typhimurium 96 kilobase pair plasmid

The ability of intracellular growth is plasmid‐dependent in Salmonella typhimurium. Only a small portion of this 96 kilobase pair plasmid appears essential for intracellular growth. The genetic organization of this region (the essential virulence determinant) was resolved. Fragments of the virulence determinant were cloned from the 96‐kb plasmid pEX102 and transformed into minicell‐producing E. coli. Plasmid‐directed protein synthesis was investigated in metabolically labeled minicells. This analysis indicated the presence of at least four genes, mkaA, mkaB, mkaC and mkaD, within the virulence determinant encoding proteins of 70, 31, 30 and 29 kDa, respectively. The genes were positioned on the restriction map of the 96‐kb virulence plasmid and the map locations confirmed by nucleotide sequence analysis of two new virulence genes (mkaB and mkaC)

The virulence plasmid does not contribute to growth of Salmonella in cultured murine macrophages

The virulence plasmid, characteristic of many serovars of Salmonella sp., and specifically its spv genes, promote intracellular growth of the bacteria in the liver and spleen and are essential for the virulence of these Salmonella serovars in the mouse. In an attempt to establish an in vitro model for studying its function, we evaluated its effect on the intracellular growth of the bacteria in macrophages in culture. We used a number of different macrophage-like cell lines (J774-A.1, IC-21 and PU5-1.8), as well as peritoneal or splenic macrophages from genetically Salmonella-sensitive (Itys, BALB/c) or resistant (Ityr, C3H/HeN) mice, and at different states of activation, stimulated in vivo or in vitro with lipopolysaccharide and/or recombinant gamma interferon. These were found to differ in their ability to suppress or sustain intracellular growth of several Salmonella serovars, but in all cases the growth was independent of the spv genes.

Plasmid-mediated serum resistance in Salmonella enterica

The ability of Salmonella enterica to survive an incubation of 2 h in fresh, pooled guinea-pig serum was determined comparing strain sets of serovariants Dublin and Typhimurium, harbouring or lacking the virulence plasmid. All strains showed marked serum resistance, which was slightly decreased among cured strains of Typhimurium. However, when introduced into a rough Escherichia coli strain, all Typhimurium plasmids substantially increased the resistance of the host strain to guinea-pig serum, whereas the Dublin plasmid did not. The traT gene, previously shown to affect serum resistance, was identified on all Typhimurium plasmids, but not on the Dublin plasmid. Mutational inactivation of the traT gene on the Typhimurium plasmids eliminated the serum resistance mediated by the plasmids.

Organization of genes expressing the blood-group-M-specific hemagglutinin of Escherichia coli: identification and nucleotide sequence of the M-agglutinin subunit gene

The organization of genes encoding the blood group M-specific hemagglutinin (M-agglutinin) of Escherichia coli strain IH11165 was studied with a cloned 6.5-kb DNA segment. This DNA segment contains at least five genes which code for the polypeptides of 12.5, 30, 80, 18.5 and 21 kDa. The 30-, 80- and 21-kDa polypeptides are synthesized as precursors that are approximately 2 kDa larger. The 21-kDa polypeptide was identified as the M-agglutinin subunit by its reactivity with anti-M-agglutinin serum. Nucleotide sequence analysis of the corresponding gene showed that the M-agglutinin precursor had a 24-amino acid (aa) signal sequence, while the mature protein is 146 aa residues long. Although the organization of the M-agglutinin gene cluster resembles those of other E. coli adhesins, there is no significant sequence homology between the M-agglutinin subunit and the subunits of the other potentially related proteins in E. coli.

Evidence for functional polymorphism of the spvR gene regulating virulence gene expression in Salmonella

The expression of Salmonella enterica spv virulence genes was studied in serovariants Dublin and Typhimurium using Western blotting (immunoblotting), spv-lacZ operon fusions and Northern blotting. The SpvA protein was detected in immunoblots from stationary phase cultures of Dublin but not from the corresponding cultures of Typhimurium. Transcriptional measurements, using a spvA-lacZ operon fusion, indicated 8–10 times higher SpvA transcription in Dublin. In an isogenic Escherichia coli chromosomal background, virulence plasmids from various Dublin strains systematically had a significantly higher induction level of the spvA-lacZ operon fusion than virulence plasmids from Typhimurium strains. The cloned spvR transcriptional activator gene of Dublin strain 2229 was found to activate both spvR-lacZ and spvA-lacZ operon fusions, as well as to raise spv mRNA levels in E. coli TG1. In contrast, the corresponding cloned gene of Typhimurium strain SL2965 possessed a lower induction potential and required higher spvR gene dosage for activation. A comparison of the nucleotide sequences of spvR genes from two Dublin and four Typhimurium strains revealed conserved, serovariant-associated basepair substitutions. Our results indicate that the spv virulence gene cluster possesses different functional alleles of the regulator gene spvR. This finding has important consequences for comparative studies of regulation and virulence in different serovariants of Salmonella.

Complementation and regulatory interaction between two cloned fimbrial gene clusters of Escherichia coli strain KS71

SummaryComplementation experiments with cloned DNA fragments encoding either the KS71A, the KS71B or the KS71C fimbriae of the pyelonephritogenic Escherichia coli strain KS71 were used to localise the P-fimbrillin genes and to demonstrate regulatory interactions between the cloned genes. The structural genes of the KS71A and KS71B fimbriae were located within a common 1.1 kilobase pair ClaI-SmaI fragment, and it was shown that the gene clusters for these fimbriae could complement each other in trans. The gene cluster encoding the KS71C fimbriae did not complement for the other KS71 fimbriae. A DNA fragment, located near the KS71A fimbrillin gene, was found to enhance the production of the KS71B fimbriae in trans.

The Salmonella Typhimurium Virulence Plasmid

Salmonellosis is a good example of an infectious disease in which intracellular growth is an essential step; Salmonellae undergo a major multiplication step within host tissue macrophages. Many different serovars of Salmonella cause extraintestinal infections in man and mouse, yet the course of the infection is similar, if not identical.