Recently, mitochondria have been identified as important contributors to the virulence and drug tolerance of human fungal pathogens. In different scenarios, either hypo-or hypervirulence can result from changes in mitochondrial function. Similarly, specific mitochondrial mutations lead to either sensitivity or resistance to antifungal drugs. Here, we provide a synthesis of this emerging field, proposing that mitochondrial function in membrane lipid homeostasis is the common denominator underlying the observed effects of mitochondria in drug tolerance (both sensitivity and resistance). We discuss how the contrasting effects of mitochondrial dysfunction on fungal drug tolerance and virulence could be explained and the potential for targeting mitochondrial factors for future antifungal drug development.Although it has been studied quite extensively in the model yeast Saccharomyces cerevisiae, mitochondrial function has remained understudied in human fungal pathogens. A possible reason is that several pathogenic fungi, such as Candida albicans and Cryptococcus neoformans, are so-called "petite-negative" yeasts, i.e., they cannot survive mitochondrial genome loss, which is a classic and extensively used tool for studying mitochondrial function in S. cerevisiae. Recent work from several laboratories has revealed that mitochondria have a fundamental role as a control point in the cellular networks impacted by antifungal drugs, as well as a prominent role in fungal virulence. These studies suggest that the functions of mitochondria in these pathways are complex. With antifungal drugs that target cell membranes, such as the azoles and the polyenes, both resistance and sensitivity of mitochondrial mutants have been reported (9,10,18,22,45,75,85,86). In contrast, only sensitivity has been observed with agents that target the cell wall, which includes the echinocandin class of antifungal drugs (3,15,17,22,38,90). A similarly complex picture is observed in regard to virulence of mitochondrial mutants, with both hypo-and hypervirulence of Candida spp. mitochondrial mutants observed in animal infection models (1,4,10,19,25,64). These studies pose several questions. What is the biochemical basis for the impact of mitochondria on drug tolerance? When (and why) does a change to mitochondrial function lead to hypo-versus hypervirulence? Would mitochondrial factors be useful targets for antifungal drug development? In this review, we will consider the molecular and cellular mechanisms behind the observed drug sensitivity and virulence phenotypes of mitochondrial mutants, mostly discussing Candida spp., for which the most is known, and mentioning the other pathogens where appropriate. We will also draw on the model fungus S. cerevisiae, because mitochondrial functions have been extensively studied in this yeast and there is reasonable evidence that the mechanisms of mitochondrial involvement in drug resistance are conserved with pathogenic fungi. Finally, we will discuss the potential for targeting mitochondrial factors for the deve...

αβ T-cell receptor (TCR) activation plays a crucial role for T-cell function. However, the TCR itself does not possess signaling domains. Instead, the TCR is noncovalently coupled to a conserved multisubunit signaling apparatus, the CD3 complex, that comprises the CD3eγ, CD3eδ, and CD3ζζ dimers. How antigen ligation by the TCR triggers CD3 activation and what structural role the CD3 extracellular domains (ECDs) play in the assembled TCR-CD3 complex remain unclear. Here, we use two complementary structural approaches to gain insight into the overall organization of the TCR-CD3 complex. Small-angle X-ray scattering of the soluble TCR-CD3eδ complex reveals the CD3eδ ECDs to sit underneath the TCR α-chain. The observed arrangement is consistent with EM images of the entire TCR-CD3 integral membrane complex, in which the CD3eδ and CD3eγ subunits were situated underneath the TCR α-chain and TCR β-chain, respectively. Interestingly, the TCR-CD3 transmembrane complex bound to peptide-MHC is a dimer in which two TCRs project outward from a central core composed of the CD3 ECDs and the TCR and CD3 transmembrane domains. This arrangement suggests a potential ligand-dependent dimerization mechanism for TCR signaling. Collectively, our data advance our understanding of the molecular organization of the TCR-CD3 complex, and provides a conceptual framework for the TCR activation mechanism.T-cell receptor | electron microscopy | small-angle X-ray scattering T cells are key mediators of the adaptive immune response.Each αβ T cell contains a unique αβ T-cell receptor (TCR), which binds antigens (Ags) displayed by major histocompatibility complexes (MHCs) and MHC-like molecules (1). The TCR serves as a remarkably sensitive driver of cellular function: although TCR ligands typically bind quite weakly (1-200 μM), even a handful of TCR ligands are sufficient to fully activate a T cell (2, 3). The TCR does not possess intracellular signaling domains, uncoupling Ag recognition from T-cell signaling. The TCR is instead noncovalently associated with a multisubunit signaling apparatus, consisting of the CD3eγ and CD3eδ heterodimers and the CD3ζζ homodimer, which collectively form the TCR-CD3 complex (4, 5). The CD3γ/δ/e subunits each consist of a single extracellular Ig domain and a single immunoreceptor tyrosine-based activation motif (ITAM), whereas CD3ζ has a short extracellular domain (ECD) and three ITAMs (6-11). The TCR-CD3 complex exists in 1:1:1:1 stoichiometry for the αβTCR: CD3eγ:CD3eδ:CD3ζζ dimers (12). Phosphorylation of the intracellular CD3 ITAMs and recruitment of the adaptor Nck lead to T-cell activation, proliferation, and survival (13,14). Understanding the underlying principles of TCR-CD3 architecture and T-cell signaling is of therapeutic interest. For example, TCR-CD3 is the target of therapeutic antibodies such as the immunosuppressant OKT3 (15), and there is increasing interest in manipulating T cells in an Ag-dependent manner by using naturally occurring and engineered TCRs (16).Assembly of the TCR-CD3 complex is p...

Functional interactions of the translational activator Mss51 with both the mitochondrially encoded COX1 mRNA 5-untranslated region and with newly synthesized unassembled Cox1 protein suggest that it has a key role in coupling Cox1 synthesis with assembly of cytochrome c oxidase. Mss51 is present at levels that are near rate limiting for expression of a reporter gene inserted at COX1 in mitochondrial DNA, and a substantial fraction of Mss51 is associated with Cox1 protein in assembly intermediates. Thus, sequestration of Mss51 in assembly intermediates could limit Cox1 synthesis in wild type, and account for the reduced Cox1 synthesis caused by most yeast mutations that block assembly. Mss51 does not stably interact with newly synthesized Cox1 in a mutant lacking Cox14, suggesting that the failure of nuclear cox14 mutants to decrease Cox1 synthesis, despite their inability to assemble cytochrome c oxidase, is due to a failure to sequester Mss51. The physical interaction between Mss51 and Cox14 is dependent upon Cox1 synthesis, indicating dynamic assembly of early cytochrome c oxidase intermediates nucleated by Cox1. Regulation of COX1 mRNA translation by Mss51 seems to be an example of a homeostatic mechanism in which a positive effector of gene expression interacts with the product it regulates in a posttranslational assembly process. INTRODUCTIONThe largest subunit of mitochondrial cytochrome c oxidase, Cox1, is encoded in the mitochondrial DNA (mtDNA) of all eukaryotic species that have been examined (Gray et al., 2004), and it is synthesized by their organellar genetic systems. Cox1 is highly hydrophobic, spanning the inner mitochondrial membrane 12 times, and it is complexed with several metal ions and two heme A moieties that participate directly in electron transport (Tsukihara et al., 1996). It is assembled into the core of cytochrome c oxidase, largely surrounded by subunits encoded by nuclear genes. The processes by which Cox1 is assembled with the other subunits and cofactors into an active enzyme are highly complex, requiring at least 30 genes in Saccharomyces cerevisiae (Herrmann and Funes, 2005;Khalimonchuk and Rodel, 2005;Cobine et al., 2006;Fontanesi et al., 2006;Barrientos et al., 2009). The assembly pathway is not understood in detail. In mammals, analysis of mutant and drug-treated cell lines indicates that Cox1 is a component of the earliest assembly intermediates (Nijtmans et al., 1998;Williams et al., 2004), and similar analysis in yeast is consistent with this idea (Horan et al., 2005).An important function of this assembly process may be to prevent incompletely assembled components of cytochrome c oxidase from generating damaging reactive oxygen species, before they are contained by the holoenzyme. Indeed, mutations in several yeast genes required for cytochrome c oxidase assembly cause hypersensitivity to hydrogen peroxide (Pungartnik et al., 1999;Williams et al., 2005;Banting and Glerum, 2006), and a key component of the reactive prooxidant species is Cox1 . One feature of the assembly p...

dThe transcriptional response of Acinetobacter baumannii, a major cause of nosocomial infections, to the DNA-damaging agent mitomycin C (MMC) was studied using DNA microarray technology. Most of the 39 genes induced by MMC were related to either prophages or encoded proteins involved in DNA repair. Electrophoretic mobility shift assays demonstrated that the product of the A. baumannii MMC-inducible umuD gene (umuDAb) specifically binds to the palindromic sequence TTGAAAATGTAAC TTTTTCAA present in its promoter region. Mutations in this palindromic region abolished UmuDAb protein binding. A comparison of the promoter regions of all MMC-induced genes identified four additional transcriptional units with similar palindromic sequences recognized and specifically bound by UmuDAb. Therefore, the UmuDAb regulon consists of at least eight genes encoding seven predicted error-prone DNA polymerase V components and DddR, a protein of unknown function. Expression of these genes was not induced in the MMC-treated recA mutant. Furthermore, inactivation of the umuDAb gene resulted in the deregulation of all DNA-damage-induced genes containing the described palindromic DNA motif. Together, these findings suggest that UmuDAb is a direct regulator of the DNA damage response in A. baumannii. In Escherichia coli and many other bacterial species, extensive DNA damage that cannot be repaired by other cellular mechanisms induces a mutagenic repair pathway known as the SOS response (1). As part of the SOS response, the RecA protein binds to the single-stranded region of the damaged DNA, where it is activated and forms a nucleoprotein filament (2). Activated RecA induces the autoprotease activity of LexA, which in the absence of activated RecA represses the expression of SOS genes by binding to specific sequences in their promoters. As cleaved LexA is unable to bind to these regulator sequences, the SOS genes are derepressed (1). The proteins encoded by SOS genes mediate DNA repair and replication in addition to pausing the cell cycle. The activities in the SOS response include those of highly error-prone polymerases (1). Once the damaged DNA is repaired, RecA no longer induces the autocleavage of LexA, which then accumulates and shuts down the SOS response (3).The SOS genetic network is widely present in Eubacteria (4), but in Acinetobacter spp. the DNA damage response is characterized by several atypical features: (i) there is no damage-induced mutagenesis response to DNA damage, with the remarkable exception of the opportunistic pathogens Acinetobacter baumannii, Acinetobacter ursingii, and Acinetobacter beijerinckii (5-7); (ii) after DNA damage, further induction of recA does not require the RecA protein (8); (iii) none of the promoters of the DNAdamage-inducible genes of Acinetobacter spp. contain a known SOS box (7, 9, 10); (iv) a canonical LexA homologue has not been identified (8, 10, 11); and (v) a UmuD homologue in A. baumannii (UmuDAb) has been proposed as a putative indirect regulator of the DNA damage response (6). Given the...