The Francisella tularensis live vaccine strain (LVS), in contrast to its iglC mutant, replicates in the cytoplasm of macrophages. We studied the outcome of infection of the murine macrophagelike cell line J774A.1 with LVS and with iglC, iglD, and mglA mutants, the latter of which is deficient in a global regulator. Compared to LVS, all of the mutants showed impaired intracellular replication up to 72 h, and the number of the mglA mutant bacteria even decreased. Colocalization with LAMP-1 was significantly increased for all mutants compared to LVS, indicating an impaired ability to escape into the cytoplasm. A lysosomal acidity-dependent dye accumulated in approximately 40% of the vacuoles containing mutant bacteria but not at all in vacuoles containing LVS. Preactivation of the macrophages with gamma interferon inhibited the intracellular growth of all strains and significantly increased acidification of phagosomes containing the mutants, but it only slightly increased the LAMP-1 colocalization. The intracellular replication and phagosomal escape of the iglC and iglD mutants were restored by complementation in trans. In conclusion, the IglC, IglD, and MglA proteins each directly or indirectly critically contribute to the virulence of F. tularensis LVS, including its intracellular replication, cytoplasmic escape, and inhibition of acidification of the phagosomes.

In Saccharomyces cerevisiae, acetate is formed by acetaldehyde dehydrogenase (ACDH), a key enzyme of the pyruvate dehydrogenase (PDH) bypass, which fulfils the essential task of generating acetyl-CoA in the cytosol. The role of the five members of the ACDH family (ALD genes) was investigated during anaerobic growth on glucose. Single and multiple aldΔ mutants were generated in the wine-yeast-derived V5 and laboratory CEN.PK strains and analysed under standard (YPD 5 % glucose) and wine (MS 20 % glucose) fermentation conditions. The deletion of ALD6 and ALD5 decreased acetate formation in both strains, demonstrating for the first time that the mitochondrial Ald5p isoform is involved in the biosynthesis of acetate during anaerobic growth on glucose. Acetate production of the ald4Δ mutant was slightly decreased in the CEN.PK strain during growth on YPD only. In contrast, the deletion of ALD2 or ALD3 had no effect on acetate production. The absence of Ald6p was compensated by the mitochondrial isoforms and this involves the transcriptional activation of ALD4. Consistent with this, growth retardation was observed in ald6Δald4Δ, and this effect was amplified by the additional deletion of ALD5. A aldΔ null mutant, devoid of ACDH activity, was viable and produced similar levels of acetate to the ald6Δald4Δald5Δ strain, excluding a role of Ald2p and Ald3p. Thus, acetate is mainly produced by the cytosolic PDH bypass via Ald6p and by a mitochondrial route involving Ald5p. An unknown alternative pathway can compensate for the loss of Ald6p, Ald4p and Ald5p.

Lack of neonatal angiotensin II type-1 receptor stimulation produces irreversible abnormalities of renal function and morphology, which can be prevented by simultaneous administration of insulin-like growth factor-I (IGF-I). Given the fact that growth hormone (GH) is the strongest secretagogue for IGF-I, we wanted to explore whether GH could reproduce the effect of IGF-I. Rats were treated from 3 to 13 days of age with the angiotensin-converting enzyme inhibitor enalapril (10 mg/kg/day) and GH (4 mg/kg/day), alone or in combination. Renal gene expression of IGF-I and IGF-binding proteins (IGFBP) was determined during and after treatment. Renal function and morphology were investigated at adult age. In contrast to the beneficial effect of IGF-I, GH treatment in combination with enalapril further deteriorated both renal function and morphology as compared with enalapril treatment alone, demonstrating: reduced glomerular filtration rate, increased tubular dilation and further expansion of the outer medulla. Enalapril decreased medullary expression of IGF-I and increased renal expression of IGFBP-1, changes that were not affected by concomitant GH treatment. These findings indicate that GH and IGF-I have different roles in the renin-angiotensin system-mediated kidney development.