ABSTRACT
Eukaryotic translation initiation factor (eIF) 3 is a multi-subunit protein complex that binds both ribosomes and messenger RNAs (mRNAs) in order to drive a diverse set of mechanistic steps during translation. Despite its importance, a unifying framework explaining how eIF3 performs these numerous activities is lacking. Using single-molecule light scattering microscopy, we demonstrate that Saccharomyces cerevisiae eIF3 is an equilibrium mixture of the full complex, subcomplexes, and subunits. By extending our microscopy approach to an in vitro reconstituted eIF3 and complementing it with biochemical assays, we define the subspecies comprising this equilibrium and show that, rather than being driven by the full complex, mRNA binding by eIF3 is instead driven by the eIF3a subunit within eIF3a-containing subcomplexes. Our findings provide a mechanistic model for the role of eIF3 in the mRNA recruitment step of translation initiation and establish a mechanistic framework for explaining and investigating the other activities of eIF3.
ABSTRACT
Cryptococcus neoformans ( Cn ) is an opportunistic fungal microorganism that causes life-threatening meningoencephalitis. During the infection, the microbial population is heterogeneously composed of cells with varying generational ages, with older cells accumulating during chronic infections. This is attributed to their enhanced resistance to phagocytic killing and tolerance of antifungals like fluconazole (FLC). In this study, we investigated the role of ergosterol synthesis, ATP-binding cassette (ABC) transporters, and mitochondrial metabolism in the regulation of age-dependent FLC tolerance. We find that old Cn cells increase the production of ergosterol and exhibit upregulation of ABC transporters. Old cells also show transcriptional and phenotypic characteristics consistent with increased metabolic activity, leading to increased ATP production. This is accompanied by increased production of reactive oxygen species (ROS), which results in mitochondrial fragmentation. This study demonstrates that the metabolic changes occurring in the mitochondria of old cells drive the increase in ergosterol synthesis and the upregulation of ABC transporters, leading to FLC tolerance. IMPORTANCE: Infections caused by Cryptococcus neoformans cause more than 180,000 deaths annually. Estimated one-year mortality for patients receiving care ranges from 20% in developed countries to 70% in developing countries, suggesting that current treatments are inadequate. Some fungal cells can persist and replicate despite the usage of current antifungal regimens, leading to death or treatment failure. In replicative aging, older cells display a resilient phenotype, characterized by their enhanced tolerance against antifungals and resistance to killing by host cells. This study shows that age-dependent increase in mitochondrial reactive oxygen species drive changes in ABC transporters and ergosterol synthesis, ultimately leading to the heightened tolerance against fluconazole in old C. neoformans cells. Understanding the underlying molecular mechanisms of this age-associated antifungal tolerance will enable more targeted antifungal therapies for cryptococcal infections.
ABSTRACT
Cryptococcus neoformans is a facultative intracellular fungal pathogen. Ten-generation-old (10GEN) C. neoformans cells are more resistant to phagocytosis and killing by macrophages than younger daughter cells. However, mechanisms that mediate this resistance and intracellular parasitism are poorly understood. Here, we identified important factors for the intracellular survival of 10GEN C. neoformans, such as urease activity, capsule synthesis, and DNA content using flow cytometry and fluorescent microscopy techniques. The real-time visualization of time-lapse imaging was applied to determine the phagosomal acidity, membrane permeability, and vomocytosis (non-lytic exocytosis) rate in J774 macrophages that phagocytosed C. neoformans of different generational ages. Our results showed that old C. neoformans exhibited higher urease activity and enhanced Golgi activity. In addition, old C. neoformans were more likely to be arrested in the G2 phase, resulting in the occasional formation of aberrant trimera-like cells. To finish, the advanced generational age of the yeast cells slightly reduced vomocytosis events within host cells, which might be associated with increased phagolysosome pH and membrane permeability. Altogether, our results suggest that old C. neoformans prevail within acidic phagolysosomes and can manipulate the phagosome pH. These strategies may be used by old C. neoformans to resist phagosomal killing and drive cryptococcosis pathogenesis. The comprehension of these essential host-pathogen interactions could further shed light on mechanisms that bring new insights for novel antifungal therapeutic design.
ABSTRACT
Noncoding small RNAs (sRNAs) are crucial for the posttranscriptional regulation of gene expression in all organisms and are known to be involved in the regulation of bacterial virulence. In the human pathogen Bordetella pertussis, which causes whooping cough, virulence is controlled primarily by the master two-component system BvgA (response regulator)/BvgS (sensor kinase). In this system, BvgA is phosphorylated (Bvg+ mode) or nonphosphorylated (Bvg- mode), with global transcriptional differences between the two. B. pertussis also carries the bacterial sRNA chaperone Hfq, which has previously been shown to be required for virulence. Here, we conducted transcriptomic analyses to identify possible B. pertussis sRNAs and to determine their BvgAS dependence using transcriptome sequencing (RNA-seq) and the prokaryotic sRNA prediction program ANNOgesic. We identified 143 possible candidates (25 Bvg+ mode specific and 53 Bvg- mode specific), of which 90 were previously unreported. Northern blot analyses confirmed all of the 10 ANNOgesic candidates that we tested. Homology searches demonstrated that 9 of the confirmed sRNAs are highly conserved among B. pertussis, Bordetella parapertussis, and Bordetella bronchiseptica, with one that also has homologues in other species of the Alcaligenaceae family. Using coimmunoprecipitation with a B. pertussis FLAG-tagged Hfq, we demonstrated that 3 of the sRNAs interact directly with Hfq, which is the first identification of sRNA binding to B. pertussis Hfq. Our study demonstrates that ANNOgesic is a highly useful tool for the identification of sRNAs in this system and that its combination with molecular techniques is a successful way to identify various BvgAS-dependent and Hfq-binding sRNAs. IMPORTANCE Noncoding small RNAs (sRNAs) are crucial for posttranscriptional regulation of gene expression in all organisms and are known to be involved in the regulation of bacterial virulence. We have investigated the presence of sRNAs in the obligate human pathogen B. pertussis, using transcriptome sequencing (RNA-seq) and the recently developed prokaryotic sRNA search program ANNOgesic. This analysis has identified 143 sRNA candidates (90 previously unreported). We have classified their dependence on the B. pertussis two-component system required for virulence, namely, BvgAS, based on their expression in the presence/absence of the phosphorylated response regulator BvgA, confirmed several by Northern analyses, and demonstrated that 3 bind directly to B. pertussis Hfq, the RNA chaperone involved in mediating sRNA effects. Our study demonstrates the utility of combining RNA-seq, ANNOgesic, and molecular techniques to identify various BvgAS-dependent and Hfq-binding sRNAs, which may unveil the roles of sRNAs in pertussis pathogenesis.
