Ribosome-binding and anti-microbial studies of the mycinamicins, 16-membered macrolide antibiotics from Micromonospora griseorubida.

Macrolides have been effective clinical antibiotics for over 70 years. They inhibit protein biosynthesis in bacterial pathogens by narrowing the nascent protein exit tunnel in the ribosome. The macrolide class of natural products consist of a macrolactone ring linked to one or more sugar molecules. Most of the macrolides used currently are semi-synthetic erythromycin derivatives, composed of a 14- or 15-membered macrolactone ring. Rapidly emerging resistance in bacterial pathogens is among the most urgent global health challenges, which render many antibiotics ineffective, including next-generation macrolides. To address this threat and advance a longer-term plan for developing new antibiotics, we demonstrate how 16-membered macrolides overcome erythromycin resistance in clinically isolated Staphylococcus aureus strains. By determining the structures of complexes of the large ribosomal subunit of Deinococcus radiodurans (D50S) with these 16-membered selected macrolides, and performing anti-microbial studies, we identified resistance mechanisms they may overcome. This new information provides important insights toward the rational design of therapeutics that are effective against drug resistant human pathogens.

Plate-like Guanine Biocrystals Form via Templated Nucleation of Crystal Leaflets on Preassembled Scaffolds.

Controlling the morphology of crystalline materials is challenging, as crystals have a strong tendency toward thermodynamically stable structures. Yet, organisms form crystals with distinct morphologies, such as the plate-like guanine crystals produced by many terrestrial and aquatic species for light manipulation. Regulation of crystal morphogenesis was hypothesized to entail physical growth restriction by the surrounding membrane, combined with fine-tuned interactions between organic molecules and the growing crystal. Using cryo-electron tomography of developing zebrafish larvae, we found that guanine crystals form via templated nucleation of thin leaflets on preassembled scaffolds made of 20-nm-thick amyloid fibers. These leaflets then merge and coalesce into a single plate-like crystal. Our findings shed light on the biological regulation of crystal morphogenesis, which determines their optical properties.

Structure of Pseudomonas aeruginosa ribosomes from an aminoglycoside-resistant clinical isolate.

Resistance to antibiotics has become a major threat to modern medicine. The ribosome plays a fundamental role in cell vitality by the translation of the genetic code into proteins; hence, it is a major target for clinically useful antibiotics. We report here the cryo-electron microscopy structures of the ribosome of a pathogenic aminoglycoside (AG)-resistant Pseudomonas aeruginosa strain, as well as of a nonresistance strain isolated from a cystic fibrosis patient. The structural studies disclosed defective ribosome complex formation due to a conformational change of rRNA helix H69, an essential intersubunit bridge, and a secondary binding site of the AGs. In addition, a stable conformation of nucleotides A1486 and A1487, pointing into helix h44, is created compared to a non-AG-bound ribosome. We suggest that altering the conformations of ribosomal protein uL6 and rRNA helix H69, which interact with initiation-factor IF2, interferes with proper protein synthesis initiation.

Structural insights of lincosamides targeting the ribosome of Staphylococcus aureus.

Antimicrobial resistance within a wide range of pathogenic bacteria is an increasingly serious threat to global public health. Among these pathogenic bacteria are the highly resistant, versatile and possibly aggressive bacteria, Staphylococcus aureus. Lincosamide antibiotics were proved to be effective against this pathogen. This small, albeit important group of antibiotics is mostly active against Gram-positive bacteria, but also used against selected Gram-negative anaerobes and protozoa. S. aureus resistance to lincosamides can be acquired by modifications and/or mutations in the rRNA and rProteins. Here, we present the crystal structures of the large ribosomal subunit of S. aureus in complex with the lincosamides lincomycin and RB02, a novel semisynthetic derivative and discuss the biochemical aspects of the in vitro potency of various lincosamides. These results allow better understanding of the drugs selectivity as well as the importance of the various chemical moieties of the drug for binding and inhibition.

Avilamycin and evernimicin induce structural changes in rProteins uL16 and CTC that enhance the inhibition of A-site tRNA binding.

Two structurally unique ribosomal antibiotics belonging to the orthosomycin family, avilamycin and evernimicin, possess activity against Enterococci, Staphylococci, and Streptococci, and other Gram-positive bacteria. Here, we describe the high-resolution crystal structures of the eubacterial large ribosomal subunit in complex with them. Their extended binding sites span the A-tRNA entrance corridor, thus inhibiting protein biosynthesis by blocking the binding site of the A-tRNA elbow, a mechanism not shared with other known antibiotics. Along with using the ribosomal components that bind and discriminate the A-tRNA-namely, ribosomal RNA (rRNA) helices H89, H91, and ribosomal proteins (rProtein) uL16-these structures revealed novel interactions with domain 2 of the CTC protein, a feature typical to various Gram-positive bacteria. Furthermore, analysis of these structures explained how single nucleotide mutations and methylations in helices H89 and H91 confer resistance to orthosomycins and revealed the sequence variations in 23S rRNA nucleotides alongside the difference in the lengths of the eukaryotic and prokaryotic α1 helix of protein uL16 that play a key role in the selectivity of those drugs. The accurate interpretation of the crystal structures that could be performed beyond that recently reported in cryo-EM models provide structural insights that may be useful for the design of novel pathogen-specific antibiotics, and for improving the potency of orthosomycins. Because both drugs are extensively metabolized in vivo, their environmental toxicity is very low, thus placing them at the frontline of drugs with reduced ecological hazards.

Structural insights into species-specific features of the ribosome from the pathogen Staphylococcus aureus.

The emergence of bacterial multidrug resistance to antibiotics threatens to cause regression to the preantibiotic era. Here we present the crystal structure of the large ribosomal subunit from Staphylococcus aureus, a versatile Gram-positive aggressive pathogen, and its complexes with the known antibiotics linezolid and telithromycin, as well as with a new, highly potent pleuromutilin derivative, BC-3205. These crystal structures shed light on specific structural motifs of the S. aureus ribosome and the binding modes of the aforementioned antibiotics. Moreover, by analyzing the ribosome structure and comparing it with those of nonpathogenic bacterial models, we identified some unique internal and peripheral structural motifs that may be potential candidates for improving known antibiotics and for use in the design of selective antibiotic drugs against S. aureus.

Transport-Limited Growth of Coccolith Crystals.

Biogenic crystals present a variety of complex morphologies that form with exquisite fidelity. In the case of the intricate morphologies of coccoliths, calcite crystals produced by marine algae, only a single set of crystallographic facets is utilized. It is unclear which growth process can merge this simple crystallographic habit with the species-specific architectures. Here, a suite of state-of-the-art electron microscopies is used to follow both the growth trajectories of the crystals ex situ, and the cellular environment in situ, in the species Emiliania huxleyi. It is shown that crystal growth alternates between a space filling and a skeletonized growth mode, where the crystals elongate via their stable crystallographic facets, but the final morphology is a manifestation of growth arrest. This process is reminiscent of the balance between reaction-limited and transport-limited growth regimes underlying snowflake formation. It is suggested that localized ion transport regulates the kinetic instabilities that are required for transport-limited growth, leading to reproducible morphologies.

The variability in the structural and functional properties of coccolith base plates.

Biomineralization processes exert varying levels of control over crystallization, ranging from poorly ordered polycrystalline arrays to intricately shaped single crystals. Coccoliths, calcified scales formed by unicellular algae, are a model for a highly controlled crystallization process. The coccolith crystals nucleate next to an organic oval structure that was termed the base plate, leading to the assumption that it is responsible for the oriented nucleation of the crystals via stereochemical interactions. In recent years, several works focusing on a well-characterized model species demonstrated a fundamental role for indirect interactions that facilitate coccolith crystallization. Here, we developed the tools to extract the base plates from five different species, giving the opportunity to systematically explore the relations between base plate and coccolith properties. We used multiple imaging techniques to evaluate the structural and chemical features of the base plates under native hydrated conditions. The results show a wide range of properties, overlaid on a common rudimentary scaffold that lacks any detectable structural or chemical motifs that can explain direct nucleation control. This work emphasizes that it is the combination between the base plate and the chemical environment inside the cell that cooperatively facilitate the exquisite control over the crystallization process. STATEMENT OF SIGNIFICANCE: Biological organic scaffolds can serve as functional surfaces that guide the formation of inorganic materials. However, in many cases the specific interactions that facilitate such tight regulation are complex and not fully understood. In this work, we elucidate the architecture of such amodel biological template, an organic scale that directs the assembly of exquisite crystalline arrays of marine microalgae. By using cryo electron microscopy, we reveal the native state organization of these scales from several species. The observed similarities and differences allow us to propose that the chemical microenvironment, rather than stereochemical matching, is the pivotal regulator of the process.

Structural Studies Reveal the Role of Helix 68 in the Elongation Step of Protein Biosynthesis.

The ribosome, a multicomponent assembly consisting of RNA and proteins, is a pivotal macromolecular machine that translates the genetic code into proteins. The large ribosomal subunit rRNA helix 68 (H68) is a key element in the protein synthesis process, as it coordinates the coupled movements of the actors involved in translocation, including the tRNAs and L1 stalk. Examination of cryo-electron microscopy (cryo-EM) structures of ribosomes incubated for various time durations at physiological temperatures led to the identification of functionally relevant H68 movements. These movements assist the transition of the L1 stalk between its open and closed states. H68 spatial flexibility and its significance to the protein synthesis process were confirmed through its effective targeting with antisense PNA oligomers. Our results suggest that H68 is actively involved in ribosome movements that are central to the elongation process. IMPORTANCE The mechanism that regulates the translocation step in ribosomes during protein synthesis is not fully understood. In this work, cryo-EM techniques used to image ribosomes from Staphylococcus aureus after incubation at physiological temperature allowed the identification of a conformation of the helix 68 that has never been observed so far. We then propose a mechanism in which such helix, switching between two different conformations, actively coordinates the translocation step, shedding light on the dynamics of ribosomal components. In addition, the relevance of helix 68 to ribosome function and its potential as an antibiotic target was proved by inhibiting Staphylococcus aureus ribosomes activity in vitro using oligomers with sequence complementarity.

Periplasmic Bacterial Biomineralization of Copper Sulfide Nanoparticles.

Metal sulfides are a common group of extracellular bacterial biominerals. However, only a few cases of intracellular biomineralization are reported in this group, mostly limited to greigite (Fe3 S4 ) in magnetotactic bacteria. Here, a previously unknown periplasmic biomineralization of copper sulfide produced by the magnetotactic bacterium Desulfamplus magnetovallimortis strain BW-1, a species known to mineralize greigite (Fe3 S4 ) and magnetite (Fe3 O4 ) in the cytoplasm is reported. BW-1 produces hundreds of spherical nanoparticles, composed of 1-2 nm substructures of a poorly crystalline hexagonal copper sulfide structure that remains in a thermodynamically unstable state. The particles appear to be surrounded by an organic matrix as found from staining and electron microscopy inspection. Differential proteomics suggests that periplasmic proteins, such as a DegP-like protein and a heavy metal-binding protein, could be involved in this biomineralization process. The unexpected periplasmic formation of copper sulfide nanoparticles in BW-1 reveals previously unknown possibilities for intracellular biomineralization that involves intriguing biological control and holds promise for biological metal recovery in times of copper shortage.

Exit tunnel modulation as resistance mechanism of S. aureus erythromycin resistant mutant.

The clinical use of the antibiotic erythromycin (ery) is hampered owing to the spread of resistance genes that are mostly mutating rRNA around the ery binding site at the entrance to the protein exit tunnel. Additional effective resistance mechanisms include deletion or insertion mutations in ribosomal protein uL22, which lead to alterations of the exit tunnel shape, located 16 Å away from the drug's binding site. We determined the cryo-EM structures of the Staphylococcus aureus 70S ribosome, and its ery bound complex with a two amino acid deletion mutation in its ß hairpin loop, which grants the bacteria resistance to ery. The structures reveal that, although the binding of ery is stable, the movement of the flexible shorter uL22 loop towards the tunnel wall creates a wider path for nascent proteins, thus enabling bypass of the barrier formed by the drug. Moreover, upon drug binding, the tunnel widens further.

Structural Basis for Linezolid Binding Site Rearrangement in the Staphylococcus aureus Ribosome.

An unorthodox, surprising mechanism of resistance to the antibiotic linezolid was revealed by cryo-electron microscopy (cryo-EM) in the 70S ribosomes from a clinical isolate of Staphylococcus aureus This high-resolution structural information demonstrated that a single amino acid deletion in ribosomal protein uL3 confers linezolid resistance despite being located 24 Å away from the linezolid binding pocket in the peptidyl-transferase center. The mutation induces a cascade of allosteric structural rearrangements of the rRNA that ultimately results in the alteration of the antibiotic binding site.IMPORTANCE The growing burden on human health caused by various antibiotic resistance mutations now includes prevalent Staphylococcus aureus resistance to last-line antimicrobial drugs such as linezolid and daptomycin. Structure-informed drug modification represents a frontier with respect to designing advanced clinical therapies, but success in this strategy requires rapid, facile means to shed light on the structural basis for drug resistance (D. Brown, Nat Rev Drug Discov 14:821-832, 2015, https://doi.org/10.1038/nrd4675). Here, detailed structural information demonstrates that a common mechanism is at play in linezolid resistance and provides a step toward the redesign of oxazolidinone antibiotics, a strategy that could thwart known mechanisms of linezolid resistance.

A novel pleuromutilin antibacterial compound, its binding mode and selectivity mechanism.

The increasing appearance of pathogenic bacteria with antibiotic resistance is a global threat. Consequently, clinically available potent antibiotics that are active against multidrug resistant pathogens are becoming exceedingly scarce. Ribosomes are a main target for antibiotics, and hence are an objective for novel drug development. Lefamulin, a semi-synthetic pleuromutilin compound highly active against multi-resistant pathogens, is a promising antibiotic currently in phase III trials for the treatment of community-acquired bacterial pneumonia in adults. The crystal structure of the Staphylococcus aureus large ribosomal subunit in complex with lefamulin reveals its protein synthesis inhibition mechanism and the rationale for its potency. In addition, analysis of the bacterial and eukaryotes ribosome structures around the pleuromutilin binding pocket has elucidated the key for the drug's selectivity.

Ribosomal Antibiotics: Contemporary Challenges.

Most ribosomal antibiotics obstruct distinct ribosomal functions. In selected cases, in addition to paralyzing vital ribosomal tasks, some ribosomal antibiotics are involved in cellular regulation. Owing to the global rapid increase in the appearance of multi-drug resistance in pathogenic bacterial strains, and to the extremely slow progress in developing new antibiotics worldwide, it seems that, in addition to the traditional attempts at improving current antibiotics and the intensive screening for additional natural compounds, this field should undergo substantial conceptual revision. Here, we highlight several contemporary issues, including challenging the common preference of broad-range antibiotics; the marginal attention to alterations in the microbiome population resulting from antibiotics usage, and the insufficient awareness of ecological and environmental aspects of antibiotics usage. We also highlight recent advances in the identification of species-specific structural motifs that may be exploited for the design and the creation of novel, environmental friendly, degradable, antibiotic types, with a better distinction between pathogens and useful bacterial species in the microbiome. Thus, these studies are leading towards the design of "pathogen-specific antibiotics," in contrast to the current preference of broad range antibiotics, partially because it requires significant efforts in speeding up the discovery of the unique species motifs as well as the clinical pathogen identification.