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.

Spliceosomal Prp8 intein at the crossroads of protein and RNA splicing.

The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNAs. At its functional core lies the essential pre-mRNA processing factor 8 (Prp8) protein. Across diverse eukaryotes, this protein cofactor of RNA catalysis harbors a self-splicing element called an intein. Inteins in Prp8 are extremely pervasive and are found at 7 different sites in various species. Here, we focus on the Prp8 intein from Cryptococcus neoformans (Cne), a human fungal pathogen. We solved the crystal structure of this intein, revealing structural homology among protein splicing sequences in eukaryotes, including the Hedgehog C terminus. Working with the Cne Prp8 intein in a reporter assay, we find that the biologically relevant divalent metals copper and zinc inhibit intein splicing, albeit by 2 different mechanisms. Copper likely stimulates reversible modifications on a catalytically important cysteine, whereas zinc binds at the terminal asparagine and the same critical cysteine. Importantly, we also show that copper treatment inhibits Prp8 protein splicing in Cne. Lastly, an intein-containing Prp8 precursor model is presented, suggesting that metal-induced protein splicing inhibition would disturb function of both Prp8 and the spliceosome. These results indicate that Prp8 protein splicing can be modulated, with potential functional implications for the spliceosome.

Ribosome hibernation: a new molecular framework for targeting nonreplicating persisters of mycobacteria.

Treatment of tuberculosis requires a multi-drug regimen administered for at least 6 months. The long-term chemotherapy is attributed in part to a minor subpopulation of nonreplicating Mycobacterium tuberculosis cells that exhibit phenotypic tolerance to antibiotics. The origins of these cells in infected hosts remain unclear. Here we discuss some recent evidence supporting the hypothesis that hibernation of ribosomes in M. tuberculosis, induced by zinc starvation, could be one of the primary mechanisms driving the development of nonreplicating persisters in hosts. We further analyse inconsistencies in previously reported studies to clarify the molecular principles underlying mycobacterial ribosome hibernation.

The Mechanism of Cholesterol Modification of Hedgehog Ligand.

Hedgehog (Hh) proteins are important components of signal transduction pathways involved in animal development, and their defects are implicated in carcinogenesis. Their N-terminal domain (HhN) acts as a signaling ligand, and their C-terminal domain (HhC) performs an autocatalytic function of cleaving itself away, while adding a cholesterol moiety to HhN. HhC has two sub-domains: a hedgehog/intein (hint) domain that primarily performs the autocatalytic activity, and a sterol-recognition region (SRR) that binds to cholesterol and properly positions it with respect to HhN. The three-dimensional details of this autocatalytic mechanism remain unknown, as does the structure of the precursor Hh protein. In this study, a complete cholesterol-bound precursor form of the drosophila Hh precursor is modeled using known crystal structures of HhN and the hint domain, and a hypothesized similarity of SRR to an unrelated but similar-sized cholesterol binding protein. The restrained geometries and topology switching (RGATS) strategy is then used to predict atomic-detail pathways for the full autocatalytic reaction starting from the precursor and ending in a cholesterol-linked HhN domain and a cleaved HhC domain. The RGATS explicit solvent simulations indicate the roles of individual HhC residues in facilitating the reaction, which can be confirmed through mutational experiments. These simulations also provide plausible structural models for the N/S acyl transfer intermediate and the product states of this reaction. This study thus provides a good framework for future computational and experimental studies to develop a full structural and dynamic understanding of Hh autoprocessing. © 2019 Wiley Periodicals, Inc.

Chemical activation of adenylyl cyclase Rv1625c inhibits growth of Mycobacterium tuberculosis on cholesterol and modulates intramacrophage signaling.

Mycobacterium tuberculosis (Mtb) uses a complex 3', 5'-cyclic AMP (cAMP) signaling network to sense and respond to changing environments encountered during infection, so perturbation of cAMP signaling might be leveraged to disrupt Mtb pathogenesis. However, understanding of cAMP signaling pathways is hindered by the presence of at least 15 distinct adenylyl cyclases (ACs). Recently, the small molecule V-58 was shown to inhibit Mtb replication within macrophages and stimulate cAMP production in Mtb. Here we determined that V-58 rapidly and directly activates Mtb AC Rv1625c to produce high levels of cAMP regardless of the bacterial environment or growth medium. Metabolic inhibition by V-58 was carbon source dependent in Mtb and did not occur in Mycobacterium smegmatis, suggesting that V-58-mediated growth inhibition is due to interference with specific Mtb metabolic pathways rather than a generalized cAMP toxicity. Chemical stimulation of cAMP production by Mtb within macrophages also caused down regulation of TNF-α production by the macrophages, indicating a complex role for cAMP in Mtb pathogenesis. Together these studies describe a novel approach for targeted stimulation of cAMP production in Mtb, and provide new insights into the myriad roles of cAMP signaling in Mtb, particularly during Mtb's interactions with macrophages.

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.

Geometric Patterns for Neighboring Bases Near the Stacked State in Nucleic Acid Strands.

Structural variation in base stacking has been analyzed frequently in isolated double helical contexts for nucleic acids, but not as often in nonhelical geometries or in complex biomolecular environments. In this study, conformations of two neighboring bases near their stacked state in any environment are comprehensively characterized for single-strand dinucleotide (SSD) nucleic acid crystal structure conformations. An ensemble clustering method is used to identify a reduced set of representative stacking geometries based on pairwise distances between select atoms in consecutive bases, with multiple separable conformational clusters obtained for categories divided by nucleic acid type (DNA/RNA), SSD sequence, stacking face orientation, and the presence or absence of a protein environment. For both DNA and RNA, SSD conformations are observed that are either close to the A-form, or close to the B-form, or intermediate between the two forms, or further away from either form, illustrating the local structural heterogeneity near the stacked state. Among this large variety of distinct conformations, several common stacking patterns are observed between DNA and RNA, and between nucleic acids in isolation or in complex with proteins, suggesting that these might be stable stacking orientations. Noncanonical face/face orientations of the two bases are also observed for neighboring bases in the same strand, but their frequency is much lower, with multiple SSD sequences across categories showing no occurrences of such unusual stacked conformations. The resulting reduced set of stacking geometries is directly useful for stacking-energy comparisons between empirical force fields, prediction of plausible localized variations in single-strand structures near their canonical states, and identification of analogous stacking patterns in newly solved nucleic acid containing structures.

Post-translational environmental switch of RadA activity by extein-intein interactions in protein splicing.

Post-translational control based on an environmentally sensitive intervening intein sequence is described. Inteins are invasive genetic elements that self-splice at the protein level from the flanking host protein, the exteins. Here we show in Escherichia coli and in vitro that splicing of the RadA intein located in the ATPase domain of the hyperthermophilic archaeon Pyrococcus horikoshii is strongly regulated by the native exteins, which lock the intein in an inactive state. High temperature or solution conditions can unlock the intein for full activity, as can remote extein point mutations. Notably, this splicing trap occurs through interactions between distant residues in the native exteins and the intein, in three-dimensional space. The exteins might thereby serve as an environmental sensor, releasing the intein for full activity only at optimal growth conditions for the native organism, while sparing ATP consumption under conditions of cold-shock. This partnership between the intein and its exteins, which implies coevolution of the parasitic intein and its host protein may provide a novel means of post-translational control.

RNA approaches the B-form in stacked single strand dinucleotide contexts.

Duplex RNA adopts an A-form structure, while duplex DNA interconverts between the A- and B-forms depending on the environment. The C2'-endo sugar pucker seen in B-form DNA can occur infrequently in ribose sugars as well, but RNA is not understood to assume B-form conformations. Through analysis of over 45,000 stacked single strand dinucleotide (SSD) crystal structure conformations, this study demonstrates that RNA is capable of adopting a wide conformational range between the canonical A- and B-forms at the localized SSD level, including many B-form-like conformations. It does so through C2'-endo ribose conformations in one or both nucleotides, and B-form-like neighboring base stacking patterns. As chemical reactions on nucleic acids involve localized changes in chemical bonds, the understanding of how enzymes distinguish between DNA and RNA nucleotides is altered by the energetic accessibility of these rare B-form-like RNA SSD conformations. The existence of these conformations also has direct implications in parametrization of molecular mechanics energy functions used extensively to model nucleic acid behavior., 2016. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 65-82, 2016.

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.

Molecular evidence for ß-tubulin neofunctionalization in Retaria (Foraminifera and radiolarians).

Foraminifera and radiolarians are closely related amoeboid protists (i.e., retarians) often characterized by their shells and pseudopodia. Previous studies hypothesized that the unusual "Type 2" ß-tubulin (ß2) is critically involved in forming helical filaments (HFs), a unique microtubule (MT) assembly/disassembly intermediate found in foraminiferan reticulopodia. Such noncanonical ß-tubulin sequences have also been found in two radiolarian species and appear to be closely related to the foraminiferan ß2. In this study, we report 119 new ß-tubulin transcript sequences from six foraminiferans, four radiolarians, and a related non-retarian species. We found that foraminiferan and radiolarian ß2-tubulins share some of the unusual substitutions in the structurally essential and usually conserved domains. In the ß-tubulin phylogeny, retarian ß2-tubulin forms a monophyletic clade, well separated from the canonical ß-tubulin (ß1) ubiquitous in eukaryotes. Furthermore, we found that foraminiferan and radiolarian ß2-tubulin lineages were under positive selection, and used homology models for foraminiferan α- and ß-tubulin hexamers to understand the structural effect of the positively selected substitutions. We suggest that the positively selected substitutions play key roles in the transition of MT to HF by altering the lateral and longitudinal interactions between α- and ß-tubulin heterodimers. Our results indicate that the unusual ß2-tubulin is a molecular synapomorphy of retarians, and the ß-tubulin gene duplication occurred before the divergence of Foraminifera and radiolarians. The duplicates have likely been subjected to neofunctionalization responsible for the unique MT to HF assembly/disassembly dynamics, and/or other unknown physiological processes in retarian protists.

Insertion domain within mammalian mitochondrial translation initiation factor 2 serves the role of eubacterial initiation factor 1.

Mitochondria have their own translational machineries for the synthesis of thirteen polypeptide chains that are components of the complexes that participate in the process of oxidative phosphorylation (or ATP generation). Translation initiation in mammalian mitochondria requires two initiation factors, IF2(mt) and IF3(mt), instead of the three that are present in eubacteria. The mammalian IF2(mt) possesses a unique 37 amino acid insertion domain, which is known to be important for the formation of the translation initiation complex. We have obtained a three-dimensional cryoelectron microscopic map of the mammalian IF2(mt) in complex with initiator fMet-tRNA(iMet) and the eubacterial ribosome. We find that the 37 amino acid insertion domain interacts with the same binding site on the ribosome that would be occupied by the eubacterial initiation factor IF1, which is absent in mitochondria. Our finding suggests that the insertion domain of IF2(mt) mimics the function of eubacterial IF1, by blocking the ribosomal aminoacyl-tRNA binding site (A site) at the initiation step.

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.

Partial base flipping is sufficient for strand slippage near DNA duplex termini.

Strand slippage is a structural mechanism by which insertion-deletion (indel) mutations are introduced during replication by polymerases. Three-dimensional atomic-resolution structural pathways are still not known for the decades-old template slippage description. The dynamic nature of the process and the higher energy intermediates involved increase the difficulty of studying these processes experimentally. In the present study, restrained and unrestrained molecular dynamics simulations, carried out using multiple nucleic acid force fields, are used to demonstrate that partial base-flipping can be sufficient for strand slippage at DNA duplex termini. Such strand slippage can occur in either strand, i.e. near either the 3' or the 5' terminus of a DNA strand, which suggests that similar structural flipping mechanisms can cause both primer and template slippage. In the repetitive mutation hot-spot sequence studied, non-canonical base-pairing with exposed DNA groove atoms of a neighboring G:C base-pair stabilizes a partially flipped state of the cytosine. For its base-pair partner guanine, a similar partially flipped metastable intermediate was not detected, and the propensity for sustained slippage was also found to be lower. This illustrates that a relatively small metastable DNA structural distortion in polymerase active sites could allow single base insertion or deletion mutations to occur, and stringent DNA groove molecular recognition may be required to maintain intrinsic DNA polymerase fidelity. The implications of a close relationship between base-pair dissociation, base unstacking, and strand slippage are discussed in the context of sequence dependence of indel mutations.

The small mycobacterial ribosomal protein, bS22, modulates aminoglycoside accessibility to its 16S rRNA helix-44 binding site.

Treatment of tuberculosis continues to be challenging due to the widespread latent form of the disease and the emergence of antibiotic-resistant strains of the pathogen, Mycobacterium tuberculosis. Bacterial ribosomes are a common and effective target for antibiotics. Several second line anti-tuberculosis drugs, e.g. kanamycin, amikacin, and capreomycin, target ribosomal RNA to inhibit protein synthesis. However, M. tuberculosis can acquire resistance to these drugs, emphasizing the need to identify new drug targets. Previous cryo-EM structures of the M. tuberculosis and M. smegmatis ribosomes identified two novel ribosomal proteins, bS22 and bL37, in the vicinity of two crucial drug-binding sites: the mRNA-decoding center on the small (30S), and the peptidyl-transferase center on the large (50S) ribosomal subunits, respectively. The functional significance of these two small proteins is unknown. In this study, we observe that an M. smegmatis strain lacking the bs22 gene shows enhanced susceptibility to kanamycin compared to the wild-type strain. Cryo-EM structures of the ribosomes lacking bS22 in the presence and absence of kanamycin suggest a direct role of bS22 in modulating the 16S rRNA kanamycin-binding site. Our structures suggest that amino-acid residue Lys-16 of bS22 interacts directly with the phosphate backbone of helix 44 of 16S rRNA to influence the micro-configuration of the kanamycin-binding pocket. Our analysis shows that similar interactions occur between eukaryotic homologues of bS22, and their corresponding rRNAs, pointing to a common mechanism of aminoglycoside resistance in higher organisms.

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.