Starvation sensing by mycobacterial RelA/SpoT homologue through constitutive surveillance of translation.

The stringent response, which leads to persistence of nutrient-starved mycobacteria, is induced by activation of the RelA/SpoT homolog (Rsh) upon entry of a deacylated-tRNA in a translating ribosome. However, the mechanism by which Rsh identifies such ribosomes in vivo remains unclear. Here, we show that conditions inducing ribosome hibernation result in loss of intracellular Rsh in a Clp protease-dependent manner. This loss is also observed in nonstarved cells using mutations in Rsh that block its interaction with the ribosome, indicating that Rsh association with the ribosome is important for Rsh stability. The cryo-EM structure of the Rsh-bound 70S ribosome in a translation initiation complex reveals unknown interactions between the ACT domain of Rsh and components of the ribosomal L7/L12 stalk base, suggesting that the aminoacylation status of A-site tRNA is surveilled during the first cycle of elongation. Altogether, we propose a surveillance model of Rsh activation that originates from its constitutive interaction with the ribosomes entering the translation cycle.

What makes Candida auris pan-drug resistant? Integrative insights from genomic, transcriptomic, and phenomic analysis of clinical strains resistant to all four major classes of antifungal drugs.

The global epidemic of drug-resistant Candida auris continues unabated. The initial report on pan-drug resistant (PDR) C. auris strains in a hospitalized patient in New York was unprecedented. PDR C. auris showed both known and unique mutations in the prominent gene targets of azoles, amphotericin B, echinocandins, and flucytosine. However, the factors that allow C. auris to acquire pan-drug resistance are not known. Therefore, we conducted a genomic, transcriptomic, and phenomic analysis to better understand PDR C. auris. Among 1,570 genetic variants in drug-resistant C. auris, 299 were unique to PDR strains. The whole-genome sequencing results suggested perturbations in genes associated with nucleotide biosynthesis, mRNA processing, and nuclear export of mRNA. Whole transcriptome sequencing of PDR C. auris revealed two genes to be significantly differentially expressed-a DNA repair protein and DNA replication-dependent chromatin assembly factor 1. Of 59 novel transcripts, 12 transcripts had no known homology. We observed no fitness defects among multi-drug resistant (MDR) and PDR C. auris strains grown in nutrient-deficient or -enriched media at different temperatures. Phenotypic profiling revealed wider adaptability to nitrogenous nutrients and increased utilization of substrates critical in upper glycolysis and tricarboxylic acid cycle. Structural modeling of a 33-amino acid deletion in the gene for uracil phosphoribosyl transferase suggested an alternate route in C. auris to generate uracil monophosphate that does not accommodate 5-fluorouracil as a substrate. Overall, we find evidence of metabolic adaptations in MDR and PDR C. auris in response to antifungal drug lethality without deleterious fitness costs.

Development and Validation of TaqMan Chemistry Probe-Based Rapid Assay for the Detection of Echinocandin-Resistance in Candida auris.

Candida auris is a multidrug-resistant yeast pathogen causing outbreaks in health care facilities worldwide, and the emergence of echinocandin-resistant C. auris is a concern. Currently used Clinical and Laboratory Standards Institute (CLSI) and commercial antifungal susceptibility tests (AFST) are phenotype-based, slow, and not scalable, limiting their effectiveness in the surveillance of echinocandin-resistant C. auris. The urgent need for accurate and rapid methods of assessment of echinocandin resistance cannot be overstated, as this class of antifungal drugs is preferred for patient management. We report the development and validation of a TaqMan chemistry probe-based fluorescence melt curve analysis (FMCA) following asymmetric polymerase chain reaction (PCR) to assess mutations within the hot spot one (HS1) region of FKS1, the gene responsible for encoding 1,3-ß-d-glucan synthase that is a target for echinocandins. The assay correctly identified F635C, F635Y, F635del, F635S, S639F or S639Y, S639P, and D642H/R645T mutations. Of these mutations, F635S and D642H/R645T were not involved in echinocandin resistance, while the rest were, as confirmed by AFST. Of 31 clinical cases, the predominant mutation conferring echinocandin resistance was S639F/Y (20 cases) followed by S639P (4 cases), F635del (4 cases), F635Y (2 cases), and F635C (1 case). The FMCA assay was highly specific and did not cross-react with closely and distantly related Candida and other yeast and mold species. Structural modeling of the Fks1 protein, its mutants, and docked conformations of three echinocandin drugs suggest a plausible Fks1 binding orientation for echinocandins. These findings lay the groundwork for future evaluations of additional FKS1 mutations and their impact on the development of drug resistance. The TaqMan chemistry probe-based FMCA would allow rapid, high throughput, and accurate detection of FKS1 mutations conferring echinocandin resistance in C. auris.

Structural Articulation of Biochemical Reactions Using Restrained Geometries and Topology Switching.

A strategy named "restrained geometries and topology switching" (RGATS) is presented to obtain detailed trajectories for complex biochemical reactions using molecular mechanics (MM) methods. It enables prediction of realistic dynamical pathways for chemical reactions, especially for accurately characterizing the structural adjustments of highly complex environments to any proximal biochemical reaction. It can be used to generate reactive conformations, model stepwise or concerted reactions in complex environments, and probe the influence of changes in the environment. Its ability to take reactively nonoptimal conformations and generate favorable starting conformations for a biochemical reaction is illustrated for a proton transfer between two model compounds. Its ability to study concerted reactions in explicit solvent is illustrated using proton transfers between an ammonium ion and two conserved histidines in an ammonia transporter channel embedded in a lipid membrane. Its ability to characterize the changes induced by subtle differences in the active site environment is illustrated using nucleotide addition by a DNA polymerase in the presence of two versus three Mg2+ ions. RGATS can be employed within any MM program and requires no additional software implementation. This allows the full assortment of computational methods implemented in all available MM programs to be used to tackle virtually any question about biochemical reactions that is answerable without using a quantum mechanical (QM) model. It can also be applied to generate reasonable starting structures for more detailed and expensive QM or QM/MM methods. In particular, this strategy enables rapid prediction of reactant, intermediary, or product state structures in any macromolecular context, with the only requirement being that the structure in any one of these states is either known or can be accurately modeled.

Cytosine unstacking and strand slippage at an insertion-deletion mutation sequence in an overhang-containing DNA duplex.

Base unstacking in template strands, when accompanied by strand slippage, can result in deletion mutations during strand extension by nucleic acid polymerases. In a GCCC mutation hot-spot sequence, which was previously identified to have a 50% probability of causing such mutations during DNA replication by a Y-family polymerase, a single-base deletion mutation could result from such unstacking of any one of its three template cytosines. In this study, the intrinsic energetic differences in unstacking among these three cytosines in a solvated DNA duplex overhang model were examined using umbrella sampling molecular dynamics simulations. The free energy profiles obtained show that cytosine unstacking grows progressively more unfavorable as one moves inside the duplex from the 5'-end of the overhang template strand. Spontaneous strand slippage occurs in response to such base unstacking in the direction of both the major and minor grooves for all three cytosines. Unrestrained simulations run from three distinct strand-slipped states and one non-strand-slipped state suggest that a more duplexlike environment can help stabilize strand slippage. The possible underlying reasons and biological implications of these observations are discussed in the context of nucleic acid replication active site dynamics.

What makes Candida auris pan-drug resistant? Integrative insights from genomic, transcriptomic, and phenomic analysis of clinical strains resistant to all four major classes of antifungal drugs.

The global epidemic of drug-resistant Candida auris continues unabated. We do not know what caused the unprecedented appearance of pan-drug resistant (PDR) Candida auris strains in a hospitalized patient in New York; the initial report highlighted both known and unique mutations in the prominent gene targets of azoles, amphotericin B, echinocandins, and flucytosine antifungal drugs. However, the factors that allow C. auris to acquire multi-drug resistance and pan-drug resistance are not known. Therefore, we conducted a comprehensive genomic, transcriptomic, and phenomic analysis to better understand PDR C. auris . Among 1,570 genetic variants in drug-resistant C. auris , 299 were unique to PDR strains. The whole genome sequencing results suggested perturbations in genes associated with nucleotide biosynthesis, mRNA processing, and nuclear export of mRNA. Whole transcriptome sequencing of PDR C. auris revealed two genes to be significantly differentially expressed - a DNA repair protein and DNA replication-dependent chromatin assembly factor 1. Of 59 novel transcripts, 12 candidate transcripts had no known homology among expressed transcripts found in other organisms. We observed no fitness defects among multi-drug resistant (MDR) and PDR C. auris strains grown in nutrient-deficient or - enriched media at different temperatures. Phenotypic profiling revealed wider adaptability to nitrogenous nutrients with an uptick in the utilization of substrates critical in upper glycolysis and tricarboxylic acid cycle. Structural modelling of 33-amino acid deletion in the gene for uracil phosphoribosyl transferase suggested an alternate route in C. auris to generate uracil monophosphate that does not accommodate 5-fluorouracil as a substrate. Overall, we find evidence of metabolic adaptations in MDR and PDR C. auris in response to antifungal drug lethality without deleterious fitness costs.

Drug resistance through ribosome splitting and rRNA disordering in mycobacteria.

HflX is known to rescue stalled ribosomes and is implicated in antibiotic resistance in several bacteria. Here we present several high-resolution cryo-EM structures of mycobacterial HflX in complex with the ribosome and its 50S subunit, with and without antibiotics. These structures reveal a distinct mechanism for HflX-mediated ribosome splitting and antibiotic resistance in mycobacteria. In addition to dissociating ribosome into two subunits, mycobacterial HflX mediates persistent disordering of multiple 23S rRNA helices to generate an inactive pool of 50S subunits. Mycobacterial HflX also acts as an anti-association factor by binding to pre-dissociated 50S subunits. A mycobacteria-specific insertion in HflX reaches further into the peptidyl transferase center. The position of this insertion overlaps with ribosome-bound macrolides or lincosamide class of antibiotics. The extended conformation of insertion seen in the absence of these antibiotics retracts and adjusts around the bound antibiotics instead of physically displacing them. It therefore likely imparts antibiotic resistance by sequestration of the antibiotic-bound inactive 50S subunits.

Clinical Course, Antifungal Susceptibility, and Genomic Sequencing of Trichophyton indotineae.

Importance: Trichophyton indotineae is an emerging dermatophyte causing outbreaks of extensive tinea infections often unresponsive to terbinafine. This species has been detected worldwide and in multiple US states, yet detailed US data on infections with T indotineae are sparse and could improve treatment practices and medical understanding of transmission. Objective: To correlate clinical features of T indotineae infections with in vitro antifungal susceptibility testing results, squalene epoxidase gene sequence variations, and isolate relatedness using whole-genome sequencing. Design, Setting, and Participants: This retrospective cohort study of patients with T indotineae infections in New York City spanned May 2022 to May 2023. Patients with confirmed T indotineae infections were recruited from 6 New York City medical centers. Main Outcome and Measure: Improvement or resolution at the last follow-up assessment. Results: Among 11 patients with T indotineae (6 male and 5 female patients; median [range] age, 39 [10-65] years), 2 were pregnant; 1 had lymphoma; and the remainder were immunocompetent. Nine patients reported previous travel to Bangladesh. All had widespread lesions with variable scale and inflammation, topical antifungal monotherapy failure, and diagnostic delays (range, 3-42 months). Terbinafine treatment failed in 7 patients at standard doses (250 mg daily) for prolonged duration; these patients also had isolates with amino acid substitutions at positions 393 (L393S) or 397 (F397L) in squalene epoxidase that correlated with elevated terbinafine minimum inhibitory concentrations of 0.5 µg/mL or higher. Patients who were treated with fluconazole and griseofulvin improved in 2 of 4 and 2 of 5 instances, respectively, without correlation between outcomes and antifungal minimum inhibitory concentrations. Furthermore, 5 of 7 patients treated with itraconazole cleared or had improvement at the last follow-up, and 2 of 7 were lost to follow-up or stopped treatment. Based on whole-genome sequencing analysis, US isolates formed a cluster distinct from Indian isolates. Conclusion and Relevance: The results of this case series suggest that disease severity, diagnostic delays, and lack of response to typically used doses and durations of antifungals for tinea were common in this primarily immunocompetent patient cohort with T indotineae, consistent with published data. Itraconazole was generally effective, and the acquisition of infection was likely in Bangladesh.

The structure of a hibernating ribosome in a Lyme disease pathogen.

The spirochete bacterial pathogen Borrelia (Borreliella) burgdorferi (Bbu) affects more than 10% of the world population and causes Lyme disease in about half a million people in the US annually. Therapy for Lyme disease includes antibiotics that target the Bbu ribosome. Here we present the structure of the Bbu 70S ribosome obtained by single particle cryo-electron microscopy at 2.9 Å resolution, revealing a bound hibernation promotion factor protein and two genetically non-annotated ribosomal proteins bS22 and bL38. The ribosomal protein uL30 in Bbu has an N-terminal α-helical extension, partly resembling the mycobacterial bL37 protein, suggesting evolution of bL37 and a shorter uL30 from a longer uL30 protein. Its analogy to proteins uL30m and mL63 in mammalian mitochondrial ribosomes also suggests a plausible evolutionary pathway for expansion of protein content in mammalian mitochondrial ribosomes. Computational binding free energy predictions for antibiotics reflect subtle distinctions in antibiotic-binding sites in the Bbu ribosome. Discovery of these features in the Bbu ribosome may enable better ribosome-targeted antibiotic design for Lyme disease treatment.

The structure of a hibernating ribosome in a Lyme disease pathogen.

The spirochete bacterial pathogen Borrelia ( Borreliella) burgdorferi ( Bbu ) affects more than 10% of the world population and causes Lyme disease in about half a million people in the US annually. Therapy for Lyme disease includes antibiotics that target the Bbu ribosome. We determined the structure of the Bbu 70S ribosome by single particle cryo-electron microscopy (cryo-EM) at a resolution of 2.9 Å, revealing its distinctive features. In contrast to a previous study suggesting that the single hibernation promoting factor protein present in Bbu (bbHPF) may not bind to its ribosome, our structure reveals a clear density for bbHPF bound to the decoding center of the small ribosomal 30S subunit. The 30S subunit has a non-annotated ribosomal protein, bS22, that has been found only in mycobacteria and Bacteroidetes so far. The protein bL38, recently discovered in Bacteroidetes, is also present in the Bbu large 50S ribosomal subunit. The protein bL37, previously seen only in mycobacterial ribosomes, is replaced by an N-terminal α-helical extension of uL30, suggesting that the two bacterial ribosomal proteins uL30 and bL37 may have evolved from one longer uL30 protein. The longer uL30 protein interacts with both the 23S rRNA and the 5S rRNA, is near the peptidyl transferase center (PTC), and could impart greater stability to this region. Its analogy to proteins uL30m and mL63 in mammalian mitochondrial ribosomes also suggests a plausible evolutionary pathway for expansion of protein content in mammalian mitochondrial ribosomes. Computational binding free energies are predicted for antibiotics, bound to the decoding center or PTC and are in clinical use for Lyme disease, that account for subtle distinctions in antibiotic-binding regions in the Bbu ribosome structure. Besides revealing unanticipated structural and compositional features for the Bbu ribosome, our study thus provides groundwork to enable ribosome-targeted antibiotic design for more effective treatment of Lyme disease.

Temperature- and pressure-dependence of the outer-sphere reorganization free energy for electron transfer reactions: a continuum approach.

The outer-sphere reorganization free energy for electron-transfer reactions in polar solvents and its variations with temperature and pressure are studied in the dielectric continuum framework by extending the recent fluctuating cavity description [J. Chem. Phys. 2005, 123, 014504]. The diabatic free energies are obtained as a function of three variables, i.e., radii of two spherical cavities for the donor and acceptor moieties of an electron-transfer complex and a solvent coordinate that gauges an arbitrary configuration of solvent orientational polarization. Equilibrium cavities relevant to the reactant and product states are determined via the variational principle. This incorporates cavity size readjustment accompanying electron transfer and related electrostrictive effects. Another important consequence of the variational determination of equilibrium cavities is that their size depends on thermodynamic conditions. The application of the theoretical formulation presented here to electron self-exchange shows that in contrast to the prediction of the standard Marcus theory, the solvent reorganization free energy decreases with temperature. This is in excellent accord with a recent experiment on a mixed valence dinuclear iron complex in acetonitrile [J. Phys. Chem. A 1999, 103, 7888]. It is also found that electrostriction makes a significant contribution to outer-sphere reorganization. Model calculations for the dinuclear iron complex system show that about 25-30% of the total solvent reorganization free energy arises from cavity size changes, while solvent repolarization is responsible for the rest.

A comparative study of quantum transport properties of silver and copper nanowires using first principles calculations.

The electronic structure and transport properties of silver (Ag) and copper (Cu) nanowires of diameters up to 1.7 nm are investigated using first principles density functional theory and the Landauer formalism in conjunction with a supercell approach. A direct comparison of the ballistic conductances, quantum capacitances, and kinetic inductances indicates that Ag and Cu nanowires show very similar performances. Compared to the electrostatic capacitance, the quantum capacitance is found to have a negligible effect on the total capacitance of the nanowire interconnect. In contrast, the overall inductance has a dominant contribution from the kinetic inductance over the magnetic inductance.

Free energy, entropy and volume of activation for electron transfer reactions in a polar solvent.

A continuum theory with account of cavity size fluctuations is employed to study free energy, volume and entropy of activation for nonadiabatic electron transfer (ET) reactions in polar solvents. By using a two-sphere cavity description, model calculations are performed for charge separation and recombination processes in acetonitrile under ambient conditions. It is found that the cavity size at the transition state varies with the free energy of reaction as well as with the thermodynamic conditions. In contrast to the Marcus theory predictions, the volume and entropy of activation show a monotonic behavior with the free energy of reaction and a strong correlation with each other. For example, for a given ET process, the volume and entropy of activation have the same sign. Their values for the charge separation and recombination processes are opposite in sign. These findings are in good qualitative agreement with measurements.

On the temperature and pressure dependences of cavities in the dielectric continuum picture.

Cavity size at equilibrium and its variations with thermodynamic conditions are studied in the dielectric continuum framework of solvents. By employing Gibbs' theory of dividing surfaces, the fluctuating cavity description of Kim [H.J. Kim, J. Chem. Phys. 105, 6818 (1996)] is extended to include effects related to the local solvent density inhomogeneity near the cavity. The resulting theory is applied to study cavity size variations with temperature and pressure in dipolar and nondipolar solvents. Model calculations show that the cavity size tends to increase with temperature along an isobar and decrease with pressure along an isotherm.