Anti-Prion Systems in Yeast and Inositol Polyphosphates.

The amyloid-based yeast prions are folded in-register parallel ß-sheet polymers. Each prion can exist in a wide array of variants, with different biological properties resulting from different self-propagating amyloid conformations. Yeast has several anti-prion systems, acting in normal cells (without protein overexpression or deficiency). Some anti-prion proteins partially block prion formation (Ssb1,2p, ribosome-associated Hsp70s); others cure a large portion of prion variants that arise [Btn2p, Cur1p, Hsp104 (a disaggregase), Siw14p, and Upf1,2,3p, nonsense-mediated decay proteins], and others prevent prion-induced pathology (Sis1p, essential cytoplasmic Hsp40). Study of the anti-prion activity of Siw14p, a pyrophosphatase specific for 5-diphosphoinositol pentakisphosphate (5PP-IP5), led to the discovery that inositol polyphosphates, signal transduction molecules, are involved in [PSI+] prion propagation. Either inositol hexakisphosphate or 5PP-IP4 (or 5PP-IP5) can supply a function that is needed by nearly all [PSI+] variants. Because yeast prions are informative models for mammalian prion diseases and other amyloidoses, detailed examination of the anti-prion systems, some of which have close mammalian homologues, will be important for the development of therapeutic measures.

Prion propagation and inositol polyphosphates.

The [PSI+] prion is a folded in-register parallel ß-sheet amyloid (filamentous polymer) of Sup35p, a subunit of the translation termination factor. Our searches for anti-prion systems led to our finding that certain soluble inositol polyphosphates (IPs) are important for the propagation of the [PSI+] prion. The IPs affect a wide range of processes, including mRNA export, telomere length, phosphate and polyphosphate metabolism, energy regulation, transcription and translation. We found that 5-diphosphoinositol tetra(or penta)kisphosphate or inositol hexakisphosphate could support [PSI+] prion propagation, and 1-diphosphoinositol pentakisphosphate appears to inhibit the process.

Characterization of Function of the GlgA2 Glycogen/Starch Synthase in Cyanobacterium sp. Clg1 Highlights Convergent Evolution of Glycogen Metabolism into Starch Granule Aggregation.

At variance with the starch-accumulating plants and most of the glycogen-accumulating cyanobacteria, Cyanobacterium sp. CLg1 synthesizes both glycogen and starch. We now report the selection of a starchless mutant of this cyanobacterium that retains wild-type amounts of glycogen. Unlike other mutants of this type found in plants and cyanobacteria, this mutant proved to be selectively defective for one of the two types of glycogen/starch synthase: GlgA2. This enzyme is phylogenetically related to the previously reported SSIII/SSIV starch synthase that is thought to be involved in starch granule seeding in plants. This suggests that, in addition to the selective polysaccharide debranching demonstrated to be responsible for starch rather than glycogen synthesis, the nature and properties of the elongation enzyme define a novel determinant of starch versus glycogen accumulation. We show that the phylogenies of GlgA2 and of 16S ribosomal RNA display significant congruence. This suggests that this enzyme evolved together with cyanobacteria when they diversified over 2 billion years ago. However, cyanobacteria can be ruled out as direct progenitors of the SSIII/SSIV ancestral gene found in Archaeplastida. Hence, both cyanobacteria and plants recruited similar enzymes independently to perform analogous tasks, further emphasizing the importance of convergent evolution in the appearance of starch from a preexisting glycogen metabolism network.

Toward an understanding of the function of Chlamydiales in plastid endosymbiosis.

Plastid endosymbiosis defines a process through which a fully evolved cyanobacterial ancestor has transmitted to a eukaryotic phagotroph the hundreds of genes required to perform oxygenic photosynthesis, together with the membrane structures, and cellular compartment associated with this process. In this review, we will summarize the evidence pointing to an active role of Chlamydiales in metabolic integration of free living cyanobacteria, within the cytosol of the last common plant ancestor.

Convergent evolution of polysaccharide debranching defines a common mechanism for starch accumulation in cyanobacteria and plants.

Starch, unlike hydrosoluble glycogen particles, aggregates into insoluble, semicrystalline granules. In photosynthetic eukaryotes, the transition to starch accumulation occurred after plastid endosymbiosis from a preexisting cytosolic host glycogen metabolism network. This involved the recruitment of a debranching enzyme of chlamydial pathogen origin. The latter is thought to be responsible for removing misplaced branches that would otherwise yield a water-soluble polysaccharide. We now report the implication of starch debranching enzyme in the aggregation of semicrystalline granules of single-cell cyanobacteria that accumulate both glycogen and starch-like polymers. We show that an enzyme of analogous nature to the plant debranching enzyme but of a different bacterial origin was recruited for the same purpose in these organisms. Remarkably, both the plant and cyanobacterial enzymes have evolved through convergent evolution, showing novel yet identical substrate specificities from a preexisting enzyme that originally displayed the much narrower substrate preferences required for glycogen catabolism.

Conservation of the glycogen metabolism pathway underlines a pivotal function of storage polysaccharides in Chlamydiae.

The order Chlamydiales includes obligate intracellular pathogens capable of infecting mammals, fishes and amoeba. Unlike other intracellular bacteria for which intracellular adaptation led to the loss of glycogen metabolism pathway, all chlamydial families maintained the nucleotide-sugar dependent glycogen metabolism pathway i.e. the GlgC-pathway with the notable exception of both Criblamydiaceae and Waddliaceae families. Through detailed genome analysis and biochemical investigations, we have shown that genome rearrangement events have resulted in a defective GlgC-pathway and more importantly we have evidenced a distinct trehalose-dependent GlgE-pathway in both Criblamydiaceae and Waddliaceae families. Altogether, this study strongly indicates that the glycogen metabolism is retained in all Chlamydiales without exception, highlighting the pivotal function of storage polysaccharides, which has been underestimated to date. We propose that glycogen degradation is a mandatory process for fueling essential metabolic pathways that ensure the survival and virulence of extracellular forms i.e. elementary bodies of Chlamydiales.

Yeast Prions Compared to Functional Prions and Amyloids.

Saccharomyces cerevisiae is an occasional host to an array of prions, most based on self-propagating, self-templating amyloid filaments of a normally soluble protein. [URE3] is a prion of Ure2p, a regulator of nitrogen catabolism, while [PSI+] is a prion of Sup35p, a subunit of the translation termination factor Sup35p. In contrast to the functional prions, [Het-s] of Podospora anserina and [BETA] of yeast, the amyloid-based yeast prions are rare in wild strains, arise sporadically, have an array of prion variants for a single prion protein sequence, have a folded in-register parallel ß-sheet amyloid architecture, are detrimental to their hosts, arouse a stress response in the host, and are subject to curing by various host anti-prion systems. These characteristics allow a logical basis for distinction between functional amyloids/prions and prion diseases. These infectious yeast amyloidoses are outstanding models for the many common human amyloid-based diseases that are increasingly found to have some infectious characteristics.

Was the Chlamydial Adaptative Strategy to Tryptophan Starvation an Early Determinant of Plastid Endosymbiosis?

Chlamydiales were recently proposed to have sheltered the future cyanobacterial ancestor of plastids in a common inclusion. The intracellular pathogens are thought to have donated those critical transporters that triggered the efflux of photosynthetic carbon and the consequent onset of symbiosis. Chlamydiales are also suspected to have encoded glycogen metabolism TTS (Type Three Secretion) effectors responsible for photosynthetic carbon assimilation in the eukaryotic cytosol. We now review the reasons underlying other chlamydial lateral gene transfers evidenced in the descendants of plastid endosymbiosis. In particular we show that half of the genes encoding enzymes of tryptophan synthesis in Archaeplastida are of chlamydial origin. Tryptophan concentration is an essential cue triggering two alternative modes of replication in Chlamydiales. In addition, sophisticated tryptophan starvation mechanisms are known to act as antibacterial defenses in animal hosts. We propose that Chlamydiales have donated their tryptophan operon to the emerging plastid to ensure increased synthesis of tryptophan by the plastid ancestor. This would have allowed massive expression of the tryptophan rich chlamydial transporters responsible for symbiosis. It would also have allowed possible export of this valuable amino-acid in the inclusion of the tryptophan hungry pathogens. Free-living single cell cyanobacteria are devoid of proteins able to transport this amino-acid. We therefore investigated the phylogeny of the Tyr/Trp transporters homologous to E. coli TyrP/Mre and found yet another LGT from Chlamydiales to Archaeplastida thereby considerably strengthening our proposal.

Sequestration of host metabolism by an intracellular pathogen.

For intracellular pathogens, residence in a vacuole provides a shelter against cytosolic host defense to the cost of limited access to nutrients. The human pathogen Chlamydia trachomatis grows in a glycogen-rich vacuole. How this large polymer accumulates there is unknown. We reveal that host glycogen stores shift to the vacuole through two pathways: bulk uptake from the cytoplasmic pool, and de novo synthesis. We provide evidence that bacterial glycogen metabolism enzymes are secreted into the vacuole lumen through type 3 secretion. Our data bring strong support to the following scenario: bacteria co-opt the host transporter SLC35D2 to import UDP-glucose into the vacuole, where it serves as substrate for de novo glycogen synthesis, through a remarkable adaptation of the bacterial glycogen synthase. Based on these findings we propose that parasitophorous vacuoles not only offer protection but also provide a microorganism-controlled metabolically active compartment essential for redirecting host resources to the pathogens.