Diversion of phagosome trafficking by pathogenic Rhodococcus equi depends on mycolic acid chain length.

Rhodococcus equi is a close relative of Mycobacterium spp. and a facultative intracellular pathogen which arrests phagosome maturation in macrophages before the late endocytic stage. We have screened a transposon mutant library of R. equi for mutants with decreased capability to prevent phagolysosome formation. This screen yielded a mutant in the gene for ß-ketoacyl-(acyl carrier protein)-synthase A (KasA), a key enzyme of the long-chain mycolic acid synthesizing FAS-II system. The longest kasA mutant mycolic acid chains were 10 carbon units shorter than those of wild-type bacteria. Coating of non-pathogenic E. coli with purified wild-type trehalose dimycolate reduced phagolysosome formation substantially which was not the case with shorter kasA mutant-derived trehalose dimycolate. The mutant was moderately attenuated in macrophages and in a mouse infection model, but was fully cytotoxic.Whereas loss of KasA is lethal in mycobacteria, R. equi kasA mutant multiplication in broth was normal proving that long-chain mycolic acid compounds are not necessarily required for cellular integrity and viability of the bacteria that typically produce them. This study demonstrates a central role of mycolic acid chain length in diversion of trafficking by R. equi.

Considerable increase in resveratrol production by recombinant industrial yeast strains with use of rich medium.

Resveratrol synthesis from p-coumarate was analyzed in different Saccharomyces cerevisiae strains expressing the 4-coumaroyl-coenzyme A ligase (4CL1) from Arabidopsis thaliana and the stilbene synthase (STS) from Vitis vinifera and compared between yeast cultures growing in rich or synthetic medium. The use of rich medium considerably improved resveratrol production, and resveratrol yields of up to 391 mg/liter could be achieved with an industrial Brazilian sugar cane-fermenting yeast.

A mycolyl transferase mutant of Rhodococcus equi lacking capsule integrity is fully virulent.

Rhodococcus equi is a mucoid Gram-positive facultative intracellular pathogen which can cause severe bronchopneumonia in foals and AIDS patients. A polysaccharide capsule which gives R. equi a mucoid appearance has long been suspected to be a virulence factor. Here, we describe a transposome mutant in the gene fbpA of strain R. equi 103 causing absence of a capsular structure. FbpA is a chromosomal gene homologous to antigen 85 (Ag85) mycolyl chain transferase gene of Mycobacterium tuberculosis. The mutant multiplied normally in isolated macrophages, was able to establish the unusual R. equi-containing vacuole in macrophages, was cytotoxic for macrophages, and was virulent in a mouse model. Colonies had a dry appearance on nutrient agar and defective capsule structure. Surprisingly, fbpA mutants cured of the virulence-associated plasmid were found in a phagosome that was more alkaline than that of the corresponding wild-type bacteria, were more cytotoxic and even multiplied to some extent. This study suggests that the capsule is not an important virulence factor of R. equi and that it may even counteract virulence traits.

Control of Ste6 recycling by ubiquitination in the early endocytic pathway in yeast.

We present evidence that ubiquitination controls sorting of the ABC-transporter Ste6 in the early endocytic pathway. The intracellular distribution of Ste6 variants with reduced ubiquitination was examined. In contrast to wild-type Ste6, which was mainly localized to internal structures, these variants accumulated at the cell surface in a polar manner. When endocytic recycling was blocked by Ypt6 inactivation, the ubiquitination deficient variants were trapped inside the cell. This indicates that the polar distribution is maintained dynamically through endocytic recycling and localized exocytosis ("kinetic polarization"). Ste6 does not appear to recycle through late endosomes, because recycling was not blocked in class E vps (vacuolar protein sorting) mutants (Deltavps4, Deltavps27), which are affected in late endosome function and in the retromer mutant Deltavps35. Instead, recycling was partially affected in the sorting nexin mutant Deltasnx4, which serves as an indication that Ste6 recycles through early endosomes. Enhanced recycling of wild-type Ste6 was observed in class D vps mutants (Deltapep12, Deltavps8, and Deltavps21). The identification of putative recycling signals in Ste6 suggests that recycling is a signal-mediated process. Endocytic recycling and localized exocytosis could be important for Ste6 polarization during the mating process.

Cholesterol oxidase (ChoE) is not important in the virulence of Rhodococcus equi.

To analyze further the role in virulence of the prominent cholesterol oxidase (ChoE) of Rhodococcus equi, an allelic exchange choE mutant from strain 103+ was constructed and assessed for virulence in macrophages, in mice, and in foals. There was no difference between the mutant and parent strain in cytotoxic activity for macrophages or in intra-macrophage multiplication. No evidence of attenuation was obtained in macrophages and in mice, but there was slight attenuation apparent in four intra-bronchially infected foals compared to infection of four foals with the virulent parent strain, based on a delayed rise in temperature of the choE-mutant infected foals. However, bacterial colony counts in the lung 2 weeks after infection were not significantly different, although there was a slight but non-significant (P=0.12) difference in lung:body weight ratio of the choE mutant versus virulent parent infected foals (mean 2.67+/-0.25% compared to 4.58+/-0.96%). We conclude that the cholesterol oxidase is not important for the virulence of R. equi.

Necrotic death of Rhodococcus equi-infected macrophages is regulated by virulence-associated plasmids.

Rhodococcus equi is a gram-positive intracellular pathogen that can cause severe bronchopneumonia in foals and AIDS patients. It has been reported that advanced infection of foals is characterized by tissue necrosis, coinciding with the presence of degenerate bacteria-laden macrophages. Here, we report that the possession of the VapA-expressing plasmid, which has been previously correlated with a high level of virulence for foals and mice, strongly increases cytotoxicity of R. equi for murine macrophage-like (J774E) cells. Isolates containing different, VapB-expressing plasmids are less virulent and also have a lower cytotoxic potential. Isogenic strains lacking either plasmid are avirulent and have a very low cytotoxic potential. We show, using fluorescence-activated cell sorter analysis (annexin V/7-amino-actinomycin D and sub-G1-analysis), Western blotting [poly(ADP-ribose) polymerase processing analysis], and electron microscopy (macrophage and nucleus morphologies) that the deaths of murine macrophages are the result of necrotic rather than apoptotic events. We demonstrate that the bacteria must be alive in order to act cytotoxic. Therefore, one effect of the virulence-associated plasmids during infection with R. equi is the promotion of necrotic damage to the host.