No abstract

To investigate the relevance of zinc in host-pathogen interactions, we have constructed Salmonella enterica mutant strains in which the znuA gene, which encodes the periplasmic component of the ZnuABC high-affinity Zn 2؉ transporter, was deleted. This mutation does not alter the ability of Salmonella to grow in rich media but drastically reduces its ability to multiply in media deprived of zinc. In agreement with this phenotype, ZnuA accumulates only in bacteria cultivated in environments poor in zinc. In spite of the nearly millimolar intracellular concentration of zinc, we have found that znuA is highly expressed in intracellular salmonellae recovered either from cultivated cells or from the spleens of infected mice. We have also observed that znuA mutants are impaired in their ability to grow in Caco-2 epithelial cells and that bacteria starved for zinc display decreased ability to multiply in phagocytes. A dramatic reduction in the pathogenicity of the znuA mutants was observed in Salmonella-susceptible (BALB/c) or Salmonella-resistant (DBA-2) mice infected intraperitoneally or orally. This study shows that the amount of free metals available for bacterial growth within the infected animal is limited, despite the apparent elevated concentration of free metals within cells and in plasma and suggests that Salmonella exploits the ZnuABC zinc transporter to maximize zinc availability in such conditions. These results shed new light on the complex functions of zinc in vertebrate and bacterial physiology and pave the way for a better comprehension of pathogenic mechanisms in Salmonella infections.The ability of bacteria to colonize specific environments relies on their ability to obtain adequate supplies of the nutrients that are indispensable for their growth. Of particular relevance for human and animal health is to understand how bacterial pathogens face the problem of nutrient limitation in the infected host, an environment where several essential elements are not freely available for infectious microorganisms (44). Well-studied examples are the strategies adopted by pathogens to obtain iron within their host. In fact, iron availability in eukaryotes is strictly controlled by metal-binding proteins (i.e., ferritin, transferrin, and lactoferrin) which prevent its reactivity and limit the uptake ability by invasive microorganisms (40,42,43). Moreover, growth of intracellular bacteria is also controlled by specific pumps which remove iron from the bacterium-containing phagosomes (19, 48). As iron plays crucial catalytic roles in a large number of bacterial proteins, an adequate supply of this transition metal is necessary for bacterial survival and multiplication. Therefore, different pathogenic bacteria have evolved sophisticated strategies to acquire and utilize host iron, including the production of molecules (siderophores, hemophores, and membrane-associated pumps) characterized by an extraordinarily elevated iron affinity (40,42,43). The outcome of the competition for iron between the host cell and the micro...

Cells induced to apoptosis extrude glutathione in the reduced form concomitantly with (U937 cells) or before (HepG2 cells) the development of apoptosis, much earlier than plasma membrane leakage. Two specific inhibitors of carrier-mediated GSH extrusion, methionine or cystathionine, are able to decrease apoptotic GSH efflux across the intact plasma membrane, demonstrating that in these cell systems GSH extrusion occurs via a specific mechanism. While decreasing GSH efflux, cystathionine or methionine also decrease the extent of apoptosis. They fail to exert anti-apoptotic activity in cells previously deprived of GSH, indicating that the target of the protection is indeed GSH efflux. The cells rescued by methionine or cystathionine remained viable after removal of the apoptogenic inducers and were even able to replicate. This shows that a real rescue to perfect viability and not just a delay of apoptosis is achieved by forcing GSH to stay within the cells during apoptogenic treatment. All this evidence indicates that extrusion of reduced glutathione precedes and is responsible for the irreversible morphofunctional changes of apoptosis, probably by altering the intracellular redox state without intervention of reactive oxygen species, thus giving a rationale for the development of redox-dependent apoptosis under anaerobic conditions.

The transcriptional co-activator PGC-1␣ and the NAD ؉ -dependent deacetylase SIRT1 are considered important inducers of mitochondrial biogenesis because in the nucleus they regulate transcription of nucleus-encoded mitochondrial genes. We demonstrate that PGC-1␣ and SIRT1 are also present inside mitochondria and are in close proximity to mtDNA. The mitochondrial proteome includes ϳ1500 proteins among which only 13 are expressed by the mitochondrial genome (1). Accordingly, abundance, morphology, and functional properties of mitochondria are finely controlled at the nuclear genome, where an interconnected network of transcription factors regulates the expression of mitochondrial proteins, including those that control replication and transcription of the mitochondrial genome. The same transcriptional network senses alterations of energetic homeostasis of the cells and is able to adapt mitochondrial function and biogenesis in response to nutrient availability and energy demand (2, 3).In the last few years, the NAD ϩ -dependent protein deacetylase sirtuin 1 (SIRT1) is emerging as a crucial regulator of mitochondrial biogenesis (4). Mammalian SIRT1 is homologous to the yeast silent information regulator 2 (Sir2) and belongs to class III histone deacetylates (HDACIII) that include other sirtuins (SIRT2-7) with specific subcellular localization and protein substrates. The dependence of SIRT1 on NAD ϩ links its enzymatic activity directly to the energy status of the cell, thus being activated in conditions of nutrient deprivation such as fasting and caloric restriction (5). Besides its role in modification of chromatin and silencing of transcription, by heterochromatin formation through histones modification, SIRT1 targets a wide range of transcriptional factors, including protein 53 (p53) and forkhead box O (FoxO) (6, 7). However, by regulating peroxisome proliferator-activated receptor ␥ co-activator 1␣ (PGC-1␣), through a functional protein-protein interaction, SIRT1 influences the activity of one of the most versatile metabolic transcriptional co-activators of genes involved in energy metabolism (5,8,9). In particular, PGC-1␣ represents an upstream inducer of genes of mitochondrial metabolism by positively affecting the activity of some hormone nuclear receptors (peroxisome proliferator-activated receptor ␥ and estrogen-related receptor ␣) and nuclear transcription factors (NRF-1,2) (10). NRF-1 is a downstream effector of SIRT1/PGC-1␣ and activates the expression of OxPhos components, mitochondrial transporters, and ribosomal proteins. Additionally, NRF-1 regulates the activation of the Tfam, Tfb1m, and Tfb2m promoters and indirectly affects the expression of Cox genes, Glut4 and PGC-1␣ itself (1). Importantly, the coordination of the two genomes seems to be exclusively achieved by the nucleus-encoded proteins TFAM, 2 TFB1M, and TFB2M, among which the mitochondrial transcription factor A seems to play a central role being essential for transcription, replication, and maintenance of mtDNA (11,12). mtDNA is packaged i...