Angiotensin mediates renal fibrosis in the nephropathy of glycogen storage disease type Ia.

Patients with glycogen storage disease type Ia (GSD-Ia) develop renal disease of unknown etiology despite intensive dietary therapies. This renal disease shares many clinical and pathological similarities to diabetic nephropathy. We studied the expression of angiotensinogen, angiotensin type 1 receptor, transforming growth factor-beta1, and connective tissue growth factor in mice with GSD-Ia and found them to be elevated compared to controls. While increased renal expression of angiotensinogen was evident in 2-week-old mice with GSD-Ia, the renal expression of transforming growth factor-beta and connective tissue growth factor did not increase for another week; consistent with upregulation of these factors by angiotensin II. The expression of fibronectin and collagens I, III, and IV was also elevated in the kidneys of mice with GSD-Ia, compared to controls. Renal fibrosis was characterized by a marked increase in the synthesis and deposition of extracellular matrix proteins in the renal cortex and histological abnormalities including tubular basement membrane thickening, tubular atrophy, tubular dilation, and multifocal interstitial fibrosis. Our results suggest that activation of the angiotensin system has an important role in the pathophysiology of renal disease in patients with GSD-Ia.

Characterization of a putative RND-type efflux system in Agrobacterium tumefaciens.

Sequencing of a 7277 bp fragment adjacent to the chvH locus of Agrobacterium tumefaciens revealed four open reading frames (ORFs), designated ameR, ameA, ameB and ameC, respectively. These ORFs exhibit amino acid similarities to components of Resistance-Nodulation-Cell Division (RND) type efflux systems. AmeA and AmeB show high homology to membrane fusion proteins (MFP) and RND-type transporters, whereas AmeC shows similarity to NodT and other members of outer membrane factor families. Mutations of the ameA and ameB genes did not affect the susceptibility profile of the wild-type strain to several detergents and antibiotics. In contrast, mutations of the ameC gene dramatically affected the susceptibility of the strain to these same inhibitory compounds. RT-PCR analysis demonstrated that the ameABC genes form an operon. In addition, ameC gene has its own promoter gene located in the intergenic region between ameB and ameC. Mapping upstream of ameA is ameR, which encodes a protein that shows similarity especially at its N-terminal end to the TetR family of bacterial transcriptional regulators. AmeR negatively regulates expression of the ameABC operon. A mutation in ameR increased the resistance of the cells to several antimicrobial agents. This regulatory locus appears to be in the same operon as a gene located upstream which codes for a product that has high similarity to numerous 4-(N-succinocarboxamide)-5-aminoimidazole ribonucleotide (SAICAR) synthetases. The possible role of the putative efflux pump coded by the ame genes is discussed.

The chvH locus of Agrobacterium encodes a homologue of an elongation factor involved in protein synthesis.

The virulence of Agrobacterium tumefaciens depends on both chromosome- and Ti plasmid-encoded gene products. In this study, we characterize a chromosomal locus, chvH, previously identified by TnphoA mutagenesis and shown to be required for tumor formation. Through DNA sequencing and comparison of the sequence with identified sequences in the database, we show that this locus encodes a protein similar in sequence to elongation factor P, a protein thought to be involved in peptide bond synthesis in Escherichia coli. The analysis of vir-lacZ and vir-phoA translational fusions as well as Western immunoblotting revealed that the expression of Vir proteins such as VirE2 was significantly reduced in the chvH mutant compared with the wild-type strain. The E. coli efp gene complemented detergent sensitivity, virulence, and expression of VirE2 in the chvH mutant, suggesting that chvH and efp are functionally homologous. As expected, ChvH exerts its activity at the posttranscriptional level. Southern analysis suggests that the gene encoding this elongation factor is present as a single copy in A. tumefaciens. We constructed a chvH deletion mutant in which a 445-bp fragment within its coding sequence was deleted and replaced with an omega fragment. On complex medium, this mutant grew more slowly than the wild-type strain, indicating that elongation factor P is important but not essential for the growth of Agrobacterium.

VirE1 is a specific molecular chaperone for the exported single-stranded-DNA-binding protein VirE2 in Agrobacterium.

Agrobacterium tumefaciens induces tumours on plants by transferring a nucleoprotein complex, the T-complex, from the bacterium to the plant cell. The T-complex consists of a single-stranded DNA (ssDNA) segment, the T-DNA, and VirD2, an endonuclease covalently attached to the 5' end of the T-DNA. A type IV secretion system encoded by the virB operon and virD4 is required for the entry of the T-complex and VirE2, a ssDNA-binding protein, into plant cells. The VirE1 protein is specifically required for the export of the VirE2 protein, as demonstrated by extracellular complementation and tumour formation. In this report, using a yeast two-hybrid system, we demonstrated that the VirE1 and VirE2 proteins interact and confirmed this interaction by in vitro binding assays. Although VirE2 is a ssDNA-binding protein, addition of ssDNA into the binding buffer did not interfere with the interaction of VirE1 and VirE2. VirE2 also interacts with itself, but the interaction between VirE1 and VirE2 is stronger than the VirE2 self-interaction, as measured in a lacZ reporter gene assay. In addition, the interaction of VirE2 with itself is inhibited by VirE1, indicating that VirE2 binds VirE1 preferentially. Analysis of various virE2 deletions indicated that the VirE1 interaction domain of VirE2 overlaps the VirE2 self-interaction domain. Incubation of extracts from Escherichia coli overexpressing His-VirE1 with the extracts of E. coli overexpressing His-VirE2 increased the yield of His-VirE2 in the soluble fraction. In a similar purified protein solubility assay, His-VirE1 increased the amount of His-VirE2 partitioning into the soluble fraction. In Agrobacterium, VirE2 was undetectable in the soluble protein fraction unless VirE1 was co-expressed. When urea was added to solubilize any large protein aggregates, a low level of VirE2 was detected. These results indicate that VirE1 prevents VirE2 from aggregating, enhances the stability of VirE2 and, perhaps, maintains VirE2 in an export-competent state. Analysis of the deduced amino acid sequence of the VirE1 protein revealed that the VirE1 protein shares a number of properties with molecular chaperones that are involved in the transport of specific proteins into animal and plant cells using type III secretion systems. We suggest that VirE1 functions as a specific molecular chaperone for VirE2, the first such chaperone linked to the presumed type IV secretion system.

The phenolic recognition profiles of the Agrobacterium tumefaciens VirA protein are broadened by a high level of the sugar binding protein ChvE.

The formation of crown gall tumors by Agrobacterium tumefaciens requires that the virulence (vir) genes be induced by chemical signals which consist of specific phenolic compounds and monosaccharides, synthesized at plant wound sites. Signal transduction in the activation of these genes is mediated by the VirA-VirG two-component regulatory system, together with ChvE, a glucose-galactose binding protein which interacts with VirA. We have previously presented genetic evidence that virA senses phenolic compounds directly (Y.-W. Lee, S. Jin, W.-S. Sim, and E. W. Nester, Proc. Natl. Acad. Sci. USA 92:12245-12249, 1995). The vir genes of strain KU12 can be induced by 4-hydroxyacetophenone, p-coumaric acid, and phenol, whereas these same phenolic compounds are weak inducers of the vir genes of strain A6. In this report, we show that a specific inducing sugar can broaden the specificity of the phenolic compound which VirA senses. 4-Hydroxyacetophenone and other related phenolic compounds function as inducing phenolic compounds with the virA gene of A6 if arabinose replaces glucose as the inducing sugar. We further demonstrate that this broadened specificity for phenolic inducers results from the increased level of ChvE through induction by arabinose via the regulatory protein GbpR. If high levels of ChvE are present, then poorly inducing phenolic compounds can induce the vir genes to high levels in combination with glucose. Comparing the induction response of the wild type and that of a VirA mutant with a mutation in its receiver domain revealed that the activity of the receiver domain is controlled by the periplasmic domain. We discuss these observations in terms of how VirA senses and transduces signals elicited by the two classes of plant signal molecules.