When mitophagy dictates the outcome of cellular infection: the case of Brucella abortus.

Mitochondria are at the basis of various cellular functions ranging from metabolism and redox homeostasis to inflammation and cell death regulation. Mitochondria therefore constitute an attractive target for invading pathogens to fulfil their infectious cycle. This involves the modulation to their advantage of mitochondrial metabolism and dynamics, including the controlled degradation of mitochondria through mitophagy. Mitophagy might for instance be beneficial for bacterial survival as it can clear bactericidal mitochondrial ROS produced by damaged organelle fragments from the intracellular niche. In the case of the bacterial pathogen Brucella abortus, mitophagy induction has another role in the intracellular lifecycle of the bacteria. Indeed, in our study, we showed that B. abortus triggers an iron-dependent BNIP3L-mediated mitophagy response required for proper bacterial egress and infection of neighboring cells. These results highlight the diversity of mitophagy processes that might be crucial for several stages of cellular infection.

To eat or not to eat mitochondria? How do host cells cope with mitophagy upon bacterial infection?

Mitochondria fulfil a plethora of cellular functions ranging from energy production to regulation of inflammation and cell death control. The fundamental role of mitochondria makes them a target of choice for invading pathogens, with either an intracellular or extracellular lifestyle. Indeed, the modulation of mitochondrial functions by several bacterial pathogens has been shown to be beneficial for bacterial survival inside their host. However, so far, relatively little is known about the importance of mitochondrial recycling and degradation pathways through mitophagy in the outcome (success or failure) of bacterial infection. On the one hand, mitophagy could be considered as a defensive response triggered by the host upon infection to maintain mitochondrial homeostasis. However, on the other hand, the pathogen itself may initiate the host mitophagy to escape from mitochondrial-mediated inflammation or antibacterial oxidative stress. In this review, we will discuss the diversity of various mechanisms of mitophagy in a general context, as well as what is currently known about the different bacterial pathogens that have developed strategies to manipulate the host mitophagy.

Host cell egress of Brucella abortus requires BNIP3L-mediated mitophagy.

The facultative intracellular pathogen Brucella abortus interacts with several organelles of the host cell to reach its replicative niche inside the endoplasmic reticulum. However, little is known about the interplay between the intracellular bacteria and the host cell mitochondria. Here, we showed that B. abortus triggers substantive mitochondrial network fragmentation, accompanied by mitophagy and the formation of mitochondrial Brucella-containing vacuoles during the late steps of cellular infection. Brucella-induced expression of the mitophagy receptor BNIP3L is essential for these events and relies on the iron-dependent stabilisation of the hypoxia-inducible factor 1α. Functionally, BNIP3L-mediated mitophagy appears to be advantageous for bacterial exit from the host cell as BNIP3L depletion drastically reduces the number of reinfection events. Altogether, these findings highlight the intricate link between Brucella trafficking and the mitochondria during host cell infection.

Grape ASR Regulates Glucose Transport, Metabolism and Signaling.

To decipher the mediator role of the grape Abscisic acid, Stress, Ripening (ASR) protein, VvMSA, in the pathways of glucose signaling through the regulation of its target, the promoter of hexose transporter VvHT1, we overexpressed and repressed VvMSA in embryogenic and non-embryogenic grapevine cells. The embryogenic cells with organized cell proliferation were chosen as an appropriate model for high sensitivity to the glucose signal, due to their very low intracellular glucose content and low glycolysis flux. In contrast, the non-embryogenic cells displaying anarchic cell proliferation, supported by high glycolysis flux and a partial switch to fermentation, appeared particularly sensitive to inhibitors of glucose metabolism. By using different glucose analogs to discriminate between distinct pathways of glucose signal transduction, we revealed VvMSA positioning as a transcriptional regulator of the glucose transporter gene VvHT1 in glycolysis-dependent glucose signaling. The effects of both the overexpression and repression of VvMSA on glucose transport and metabolism via glycolysis were analyzed, and the results demonstrated its role as a mediator in the interplay of glucose metabolism, transport and signaling. The overexpression of VvMSA in the Arabidopsis mutant abi8 provided evidence for its partial functional complementation by improving glucose absorption activity.

Grape ASR-Silencing Sways Nuclear Proteome, Histone Marks and Interplay of Intrinsically Disordered Proteins.

In order to unravel the functions of ASR (Abscisic acid, Stress, Ripening-induced) proteins in the nucleus, we created a new model of genetically transformed grape embryogenic cells by RNAi-knockdown of grape ASR (VvMSA). Nuclear proteomes of wild-type and VvMSA-RNAi grape cell lines were analyzed by quantitative isobaric tagging (iTRAQ 8-plex). The most significantly up- or down-regulated nuclear proteins were involved in epigenetic regulation, DNA replication/repair, transcription, mRNA splicing/stability/editing, rRNA processing/biogenesis, metabolism, cell division/differentiation and stress responses. The spectacular up-regulation in VvMSA-silenced cells was that of the stress response protein VvLEA D-29 (Late Embryogenesis Abundant). Both VvMSA and VvLEA D-29 genes displayed strong and contrasted responsiveness to auxin depletion, repression of VvMSA and induction of VvLEA D-29. In silico analysis of VvMSA and VvLEA D-29 proteins highlighted their intrinsically disordered nature and possible compensatory relationship. Semi-quantitative evaluation by medium-throughput immunoblotting of eighteen post-translational modifications of histones H3 and H4 in VvMSA-knockdown cells showed significant enrichment/depletion of the histone marks H3K4me1, H3K4me3, H3K9me1, H3K9me2, H3K36me2, H3K36me3 and H4K16ac. We demonstrate that grape ASR repression differentially affects members of complex nucleoprotein structures and may not only act as molecular chaperone/transcription factor, but also participates in plant responses to developmental and environmental cues through epigenetic mechanisms.

Intra-Sample Heterogeneity of Potato Starch Reveals Fluctuation of Starch-Binding Proteins According to Granule Morphology.

Starch granule morphology is highly variable depending on the botanical origin. Moreover, all investigated plant species display intra-tissular variability of granule size. In potato tubers, the size distribution of starch granules follows a unimodal pattern with diameters ranging from 5 to 100 µm. Several evidences indicate that granule morphology in plants is related to the complex starch metabolic pathway. However, the intra-sample variability of starch-binding metabolic proteins remains unknown. Here, we report on the molecular characterization of size-fractionated potato starch granules with average diameters of 14.2 ± 3.7 µm, 24.5 ± 6.5 µm, 47.7 ± 12.8 µm, and 61.8 ± 17.4 µm. In addition to changes in the phosphate contents as well as small differences in the amylopectin structure, we found that the starch-binding protein stoichiometry varies significantly according to granule size. Label-free quantitative proteomics of each granule fraction revealed that individual proteins can be grouped according to four distinct abundance patterns. This study corroborates that the starch proteome may influence starch granule growth and architecture and opens up new perspectives in understanding the dynamics of starch biosynthesis.

Proteome Analysis of Potato Starch Reveals the Presence of New Starch Metabolic Proteins as Well as Multiple Protease Inhibitors.

Starch bound proteins mainly include enzymes from the starch biosynthesis pathway. Recently, new functions in starch molecular assembly or active protein targeting were also proposed for starch associated proteins. The potato genome sequence reveals 77 loci encoding starch metabolizing enzymes with the identification of previously unknown putative isoforms. Here we show by bottom-up proteomics that most of the starch biosynthetic enzymes in potato remain associated with starch even after washing with SDS or protease treatment of the granule surface. Moreover, our study confirmed the presence of PTST1 (Protein Targeting to Starch), ESV1 (Early StarVation1) and LESV (Like ESV), that have recently been identified in Arabidopsis. In addition, we report on the presence of a new isoform of starch synthase, SS6, containing both K-X-G-G-L catalytic motifs. Furthermore, multiple protease inhibitors were also identified that are cleared away from starch by SDS and thermolysin treatments. Our results indicate that SS6 may play a yet uncharacterized function in starch biosynthesis and open new perspectives both in understanding storage starch metabolism as well as breeding improved potato lines.

Comparative metabolomics and glycolysis enzyme profiling of embryogenic and nonembryogenic grape cells.

A novel biological model was created for the comparison of grapevine embryogenic cells (EC) and nonembryogenic cells (NEC) sharing a common genetic background but distinct phenotypes, when cultured on their respective most appropriate media. Cytological characterization, 1H-NMR analysis of intracellular metabolites, and glycolytic enzyme activities provided evidence for the marked metabolic differences between EC and NEC. The EC were characterized by a moderate and organized cell proliferation, coupled with a low flux through glycolysis, high capacity of phosphoenolpyruvate carboxylase and glucokinase, and high oxygen consumption. The NEC displayed strong anarchic growth, and their high rate of glycolysis due to the low energetic efficiency of the fermentative metabolism is confirmed by increased enolase capacity and low oxygen consumption.

Rapid and sensitive quantification of C3- and C6-phosphoesters in starch by fluorescence-assisted capillary electrophoresis.

Phosphate groups are naturally present in starch at C3- or C6-position of the glucose residues and impact the structure of starch granules. Their precise quantification is necessary for understanding starch physicochemical properties and metabolism. Nevertheless, reliable quantification of Glc-3-P remains laborious and time consuming. Here we describe a capillary electrophoresis method for simultaneous measurement of both Glc-6-P and Glc-3-P after acid hydrolysis of starch. The sensitivity threshold was estimated at the fg scale, which is compatible with the analysis of less than a µg of sample. The method was validated by analyzing antisense potato lines deficient in SBEs, GWD or GBSS. We show that Glc-3-P content is altered in the latter and that these variations do not correlate with modifications in Glc-6-P content. We anticipate the method reported here to be an efficient tool for high throughput study of starch phosphorylation at both C3- and C6-position.

Expression of Arabidopsis sugar transport protein STP13 differentially affects glucose transport activity and basal resistance to Botrytis cinerea.

Botrytis cinerea is the causing agent of the grey mold disease in more than 200 crop species. While signaling pathways leading to the basal resistance against this fungus are well described, the role of the import of sugars into host cells remains to be investigated. In Arabidopsis thaliana, apoplastic hexose retrieval is mediated by the activity of sugar transport proteins (STPs). Expression analysis of the 14 STP genes revealed that only STP13 was induced in leaves challenged with B. cinerea. STP13-modified plants were produced and assayed for their resistance to B. cinerea and glucose transport activity. We report that STP13-deficient plants exhibited an enhanced susceptibility and a reduced rate of glucose uptake. Conversely, plants with a high constitutive level of STP13 protein displayed an improved capacity to absorb glucose and an enhanced resistance phenotype. The correlation between STP13 transcripts, protein accumulation, glucose uptake rate and resistance level indicates that STP13 contributes to the basal resistance to B. cinerea by limiting symptom development and points out the importance of the host intracellular sugar uptake in this process. We postulate that STP13 would participate in the active resorption of hexoses to support the increased energy demand to trigger plant defense reactions and to deprive the fungus by changing sugar fluxes toward host cells.