The plasma membrane H⁺-ATPase Pma1 and the vacuolar V-ATPase act in close harmony to tightly control pH homeostasis, which is essential for a vast number of physiological processes. As these main two regulators of pH are responsive to the nutritional status of the cell, it seems evident that pH homeostasis acts in conjunction with nutrient-induced signalling pathways. Indeed, both PKA and the TORC1-Sch9 axis influence the proton pumping activity of the V-ATPase and possibly also of Pma1. In addition, it recently became clear that the proton acts as a second messenger to signal glucose availability via the V-ATPase to PKA and TORC1-Sch9. Given the prominent role of nutrient signalling in longevity, it is not surprising that pH homeostasis has been linked to ageing and longevity as well. A first indication is provided by acetic acid, whose uptake by the cell induces toxicity and affects longevity. Secondly, vacuolar acidity has been linked to autophagic processes, including mitophagy. In agreement with this, a decline in vacuolar acidity was shown to induce mitochondrial dysfunction and shorten lifespan. In addition, the asymmetric inheritance of Pma1 has been associated with replicative ageing and this again links to repercussions on vacuolar pH. Taken together, accumulating evidence indicates that pH homeostasis plays a prominent role in the determination of ageing and longevity, thereby providing new perspectives and avenues to explore the underlying molecular mechanisms.

The various symptomatic outcomes of cutaneous leishmaniasis relates to the type and potency of its underlying inflammatory responses. Presence of the cytoplasmic Leishmania RNA virus-1 (LRV1) within Leishmania guyanensis, worsens lesional inflammation and parasite burden, as the viral dsRNA genome acts as a potent innate immunogen stimulating Toll-Like-Receptor-3 (TLR3). Here we investigated other innate pattern recognition receptors capable of reacting to dsRNA and potentially contributing to LRV1-mediated inflammatory pathology. We included the cytoplasmic dsRNA sensors, namely, the RIG-like receptors (RLRs) and the inflammasome-dependent and -independent Nod-like-receptors (NLRs). Our study found no role for RLRs or inflammasome-dependent NLRs in the pathology of L. guyanensis infection irrespective of its LRV1-status. Further, neither LRV1-bearing L. guyanensis (LgyLRV1+) nor LRV1-negative L. guyanensis (LgyLRV1-) activated the inflammasome in vitro. Interestingly, similarly to L. donovani, L. guyanensis infection induced the up-regulation of the A20 protein, known to be involved in the evasion of inflammasome activation. Moreover, we observed that LgyLRV1+ promoted the transcription of inflammasome-independent NLRC2 (also called NOD2) and NLRC5. However, only NLRC2 showed some contribution to LRV1-dependent pathology. These data confirmed that the endosomal TLR3 pathway is the dominant route of LRV1-dependent signalling, thus excluding the cytosolic and inflammasome pathways. We postulate that avoidance of the inflammasome pathways is likely an important mechanism of virulence in Leishmania infection irrespective of the LRV1-status.

The mitochondrial genome is responsible for the production of a handful of polypeptides that are core subunits of the membrane-bound oxidative phosphorylation system. Until now the mechanistic studies of mitochondrial protein synthesis inside cells have been conducted with inhibition of cytoplasmic protein synthesis to reduce the background of nuclear gene expression with the undesired consequence of major disturbances of cellular signaling cascades. Here we have generated a system that allows direct monitoring of mitochondrial translation in unperturbed cells. A recoded gene for superfolder GFP was inserted into the yeast (Saccharomyces cerevisiae) mitochondrial genome and enabled the detection of translation through fluorescence microscopy and flow cytometry in functional mitochondria. This novel tool allows the investigation of the function and regulation of mitochondrial translation during stress signaling, aging and mitochondrial biogenesis.

Ubiquitination is a posttranslational protein modification that regulates most aspects of cellular life. The sheer number of ubiquitination enzymes that are present in a mammalian cell, over 700 in total, has thus far hampered the analysis of distinct protein ubiquitination cascades in a cellular context. To overcome this complexity we have developed a versatile vector system that allows the reconstitution of specific ubiquitination cascades in the model eukaryote Saccharomyces cerevisae (baker’s yeast). The vector system consists of 32 modular yeast shuttle plasmids allowing inducible or constitutive expression of up to four proteins of interest in a single cell. To demonstrate the validity of the system, we show that co-expression in yeast of the mammalian HECT type E3 ubiquitin ligase E6AP (E6-Associated Protein) and a model substrate faithfully recapitulates E6AP-dependent substrate ubiquitination and degradation. In addition, we show that the endogenous sumoylation pathway of S. cerevisiae can specifically sumoylate mouse PML (Promyelocytic leukemia protein). In conclusion, the yeast vector system described in this paper provides a versatile tool to study complex post-translational modifications in a cellular setting.

The bacterial cell wall preserves cell integrity in response to external insults and the internal turgor pressure. The major component of the cell wall is the peptidoglycan (PG); a giant macromolecule formed by glycan chains cross-linked by short peptides. The PG is synthesized by a stepwise process that includes cytosolic and periplasmic reactions. The building subunits -muropeptides- are incorporated into the growing macromolecule by transglycolyslation (TG) and transpeptidation (TP) reactions, which constitute the last biosynthetic steps. TP reactions, involving cleavage of the terminal D‑Ala-D-Ala bond in the stem peptide, are carried out by enzymes known generically as penicillin-binding proteins (PBPs) due to their capacity to bind β‑lactam antibiotics, which are D‑Ala-D-Ala structural analogues. On an average, bacterial genomes harbour a minimum of 10 PBP-encoding genes, most of them non-essential. This dispensability has led to the widely accepted concept of functional redundancy for many PBPs. An exemption is the PBP dedicated to build the septal PG required to separate daughter cells during cell division. To date, this division‑specific PBP was reported as unique in all known bacteria and, as a consequence, “essential”. Our recent results obtained in the intracellular bacterial pathogen Salmonella enterica serovar Typhimurium challenges this view since this bacterium has two PBPs that can independently build the division septum. One of these two division PG enzymes is orthologue of the division-specific PBP3 of Escherichia coli. The second enzyme, named PBP3_SAL, is absent in non-pathogenic bacteria and, at least in S. Typhimurium, displays PG biosynthetic activity restricted to acidic conditions. Our work also revealed that it is possible to generate a S. Typhimurium mutant defective in PBP3, which cannot divide at neutral pH.