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In this work, an overview of the biosimilars market, pipeline and industry targets is discussed. Biosimilars typically have a shorter timeline for approval (8 years) compared to 12 years for innovator drugs and the development cost can be 10–20% of the innovator drug. The biosimilar pipeline is reviewed as well as the quality management system (QMS) that is needed to generate traceable, trackable data sets. One difference between developing a biosimilar compared to an originator is that a broader analytical foundation is required for biosimilars and advances made in developing analytical similarity to characterize these products are discussed. An example is presented on the decisions and considerations explored in the development of a biosimilar and includes identification of the best process parameters and methods based on cost, time, and titer. Finally factors to consider in the manufacture of a biosimilar and approaches used to achieve the target-directed development of a biosimilar are discussed.

Heme is a cofactor that is essential for cellular respiration and for the function of many enzymes. If heme levels become too low within the cell, S. aureus switches from producing energy via respiration to producing energy by fermentation. S. aureus encodes two heme oxygenases, IsdI and IsdG, which cleave the porphyrin heme ring releasing iron for use as a nutrient source. Both isdI and isdG are only expressed under low iron conditions and are regulated by the canonical Ferric Uptake Regulator (Fur). Here we demonstrate that unregulated expression of isdI and isdG within S. aureus, leads to reduced growth under low iron conditions. Additionally, the constitutive expression of these enzymes leads to decreased heme abundance in S. aureus, an increase in the fermentation product lactate, and increased resistance to gentamicin. This work demonstrates that S. aureus has developed tuning mechanisms, such as Fur regulation, to ensure that the cell has sufficient quantities of heme for efficient ATP production through aerobic respiration.

During infection, bacteria must steal metals, including iron, from the host tissue. Therefore, pathogenic bacteria have evolved metal acquisition systems to overcome the elaborate processes mammals use to withhold metal from pathogens. Staphylococcus aureus uses IsdB, a hemoglobin receptor, to thieve iron-containing heme from hemoglobin within human blood. We find evidence that primate hemoglobin has undergone rapid evolution at protein surfaces contacted by IsdB. Additionally, variation in the hemoglobin sequences among primates, or variation in IsdB of related staphylococci, reduces bacterial hemoglobin capture. Together, these data suggest that S. aureus has evolved to recognize human hemoglobin in the face of rapid evolution at the IsdB binding interface, consistent with repeated evolutionary conflicts in the battle for iron during host-pathogen interactions., Metals are a limiting resource for pathogenic bacteria and must be scavenged from host proteins. Hemoglobin provides the most abundant source of iron in the human body and is required by several pathogens to cause invasive disease. However, the consequences of hemoglobin evolution for bacterial nutrient acquisition remain unclear. Here we show that the α- and β-globin genes exhibit strikingly parallel signatures of adaptive evolution across simian primates. Rapidly evolving sites in hemoglobin correspond to binding interfaces of IsdB, a bacterial hemoglobin receptor harbored by pathogenic Staphylococcus aureus. Using an evolution-guided experimental approach, we demonstrate that the divergence between primates and staphylococcal isolates governs hemoglobin recognition and bacterial growth. The reintroduction of putative adaptive mutations in α- or β-globin proteins was sufficient to impair S. aureus binding, providing a mechanism for the evolution of disease resistance. These findings suggest that bacterial hemoprotein capture has driven repeated evolutionary conflicts with hemoglobin during primate descent.

Metals are a limiting resource for pathogenic bacteria and must be scavenged from host proteins. Hemoglobin provides the most abundant source of iron in the human body and is required by several pathogens to cause invasive disease. However, the consequences of hemoglobin evolution for bacterial nutrient acquisition remain unclear. Here we show that the α- and β-globin genes exhibit strikingly parallel signatures of adaptive evolution across simian primates. Rapidly evolving sites in hemoglobin correspond to binding interfaces of IsdB, a bacterial hemoglobin receptor encoded by pathogenicStaphylococcus aureus. Using an evolution-guided experimental approach, we demonstrate that divergence between primates and staphylococcal isolates governs hemoglobin recognition and bacterial growth. Reintroducing putative adaptive mutations in α- or β-globin proteins is sufficient to impairS. aureusbinding, providing a mechanism for the evolution of disease resistance. These findings suggest that bacterial hemoprotein capture has driven repeated evolutionary conflicts with hemoglobin during primate descent.

Bacteria alter their cell surface in response to changing environments, including those encountered upon invasion of a host during infection. One alteration that occurs in several Gram-positive pathogens is the presentation of cell wall-anchored components of the iron-regulated surface determinant (Isd) system, which extracts heme from host hemoglobin to fulfill the bacterial requirement for iron. Staphylococcus lugdunensis , an opportunistic pathogen that causes infective endocarditis, encodes an Isd system. Unique among the known Isd systems, S. lugdunensis contains a gene encoding a putative autolysin located adjacent to the Isd operon. To elucidate the function of this putative autolysin, here named IsdP, we investigated its contribution to Isd protein localization and hemoglobin-dependent iron acquisition. S. lugdunensis IsdP was found to be iron regulated and cotranscribed with the Isd operon. IsdP is a specialized peptidoglycan hydrolase that cleaves the stem peptide and pentaglycine crossbridge of the cell wall and alters processing and anchoring of a major Isd system component, IsdC. Perturbation of IsdC localization due to isdP inactivation results in a hemoglobin utilization growth defect. These studies establish IsdP as an autolysin that functions in heme acquisition and describe a role for IsdP in cell wall reorganization to accommodate nutrient uptake systems during infection.

The majority of cholesterol-dependent cytolysins (CDCs) utilize cholesterol as a membrane receptor, whereas a small number are restricted to the GPI-anchored protein CD59 for initial membrane recognition. Two cholesterol-binding CDCs, perfringolysin O (PFO) and streptolysin O (SLO), were found to exhibit strikingly different binding properties to cholesterol-rich natural and synthetic membranes. The structural basis for this difference was mapped to one of the loops (L3) in the membrane binding interface that help anchor the toxin monomers to the membrane after receptor (cholesterol) binding by the membrane insertion of its amino acid side chains. A single point mutation in this loop conferred the binding properties of SLO to PFO and vice versa. Our studies strongly suggest that changing the side chain structure of this loop alters its equilibrium between membrane-inserted and uninserted states, thereby affecting the overall binding affinity and total bound toxin. Previous studies have shown that the lipid environment of cholesterol has a dramatic effect on binding and activity. Combining this data with the results of our current studies on L3 suggests that the structure of this loop has evolved in the different CDCs to preferentially direct binding to cholesterol in different lipid environments. Finally, the efficiency of β-barrel pore formation was inversely correlated with the increased binding and affinity of the PFO L3 mutant, suggesting that selection of a compatible lipid environment impacts the efficiency of membrane insertion of the β-barrel pore.

Protein turnover is a key process for bacterial survival mediated by intracellular proteases. Proteolytic degradation reduces the levels of unfolded and misfolded peptides that accumulate in the cell during stress conditions. Three intracellular proteases, ClpP, HslV, and FtsH, have been identified in the Gram-positive bacteriumStaphylococcus aureus, a pathogen responsible for significant morbidity and mortality worldwide. Consistent with their crucial role in protein turnover, ClpP, HslV, and FtsH affect a number of cellular processes, including metabolism, stress responses, and virulence. The ClpP protease is believed to be the principal degradation machinery inS. aureus. This study sought to identify the effect of the Clp protease on theiron-regulatedsurfacedeterminant (Isd) system, which extracts heme-iron from host hemoglobin during infection and is critical toS. aureuspathogenesis. Inactivation of components of the Clp protease alters abundance of several Isd proteins, including the hemoglobin receptor IsdB. Furthermore, the observed changes in IsdB abundance are the result of transcriptional regulation, since transcription ofisdBis decreased byclpPorclpXinactivation. In contrast, inactivation ofclpCenhancesisdBtranscription and protein abundance. Loss ofclpPorclpXimpairs host hemoglobin binding and utilization and results in severe virulence defects in a systemic mouse model of infection. These findings suggest that the Clp proteolytic system is important for regulating nutrient iron acquisition inS. aureus. The Clp protease and Isd complex are widely conserved in bacteria; therefore, these data reveal a novel Clp-dependent regulation pathway that may be present in other bacterial pathogens.

During infection, vertebrates limit access to manganese and zinc, starving invading pathogens, such as Staphylococcus aureus , of these essential metals in a process termed “nutritional immunity.” The manganese and zinc binding protein calprotectin is a key component of the nutrient-withholding response, and mice lacking this protein do not sequester manganese from S. aureus liver abscesses. One potential mechanism utilized by S. aureus to minimize host-imposed manganese and zinc starvation is the expression of the metal transporters MntABC and MntH. We performed transcriptional analyses of both mntA and mntH , which revealed increased expression of both systems in response to calprotectin treatment. MntABC and MntH compete with calprotectin for manganese, which enables S. aureus growth and retention of manganese-dependent superoxide dismutase activity. Loss of MntABC and MntH results in reduced staphylococcal burdens in the livers of wild-type but not calprotectin-deficient mice, suggesting that these systems promote manganese acquisition during infection. During the course of these studies, we observed that metal content and the importance of calprotectin varies between murine organs, and infection leads to profound changes in the anatomical distribution of manganese and zinc. In total, these studies provide insight into the mechanisms utilized by bacteria to evade host-imposed nutrient metal starvation and the critical importance of restricting manganese availability during infection.

CD59 is a glycosylphosphatidylinositol-anchored protein that inhibits the assembly of the terminal complement membrane attack complex (MAC) pore, whereas Streptococcus intermedius intermedilysin (ILY), a pore forming cholesterol-dependent cytolysin (CDC), specifically binds to human CD59 (hCD59) to initiate the formation of its pore. The identification of the residues of ILY and hCD59 that form their binding interface revealed a remarkably deep correspondence between the hCD59 binding site for ILY and that for the MAC proteins C8α and C9. ILY disengages from hCD59 during the prepore to pore transition, suggesting that loss of this interaction is necessary to accommodate specific structural changes associated with this transition. Consistent with this scenario, mutants of hCD59 or ILY that increased the affinity of this interaction decreased the cytolytic activity by slowing the transition of the prepore to pore but not the assembly of the prepore oligomer. A signature motif was also identified in the hCD59 binding CDCs that revealed a new hCD59-binding member of the CDC family. Although the binding site on hCD59 for ILY, C8α, and C9 exhibits significant homology, no similarity exists in their binding sites for hCD59. Hence, ILY and the MAC proteins interact with common amino acids of hCD59 but lack detectable conservation in their binding sites for hCD59.

The recognition and binding of cholesterol is an important feature of many eukaryotic, viral, and prokaryotic proteins, but the molecular details of such interactions are understood only for a few proteins. The pore-forming cholesterol-dependent cytolysins (CDCs) contribute to the pathogenic mechanisms of a large number of Gram-positive bacteria. Cholesterol dependence of the CDC mechanism is a hallmark of these toxins, yet the identity of the CDC cholesterol recognition motif has remained elusive. A detailed analysis of membrane interactive structures at the tip of perfringolysin O (PFO) domain 4 reveals that a threonine-leucine pair mediates CDC recognition of and binding to membrane cholesterol. This motif is conserved in all known CDCs and conservative changes in its sequence or order are not well tolerated. Thus, the Thr-Leu pair constitutes a common structural basis for mediating CDC-cholesterol recognition and binding, and defines a unique paradigm for membrane cholesterol recognition by surface-binding proteins.

Pore-forming proteins are weapons often used by bacterial pathogens to breach the membrane barrier of target cells. Despite their critical role in infection important structural aspects of the mechanism of how these proteins assemble into pores remain unknown. Streptococcus pneumoniae is the world’s leading cause of pneumonia, meningitis, bacteremia and otitis media. Pneumolysin (PLY) is a major virulence factor of S. pneumoniae and a target for both small molecule drug development and vaccines. PLY is a member of the cholesterol-dependent cytolysins (CDCs), a family of pore-forming toxins that form gigantic pores in cell membranes. Here we present the structure of PLY determined by X-ray crystallography and, in solution, by small-angle X-ray scattering. The crystal structure reveals PLY assembles as a linear oligomer that provides key structural insights into the poorly understood early monomer-monomer interactions of CDCs at the membrane surface.

Staphylococcus aureus is a frequent human pathogen that is capable of causing a wide range of life-threatening infections. A promising antibacterial target is the Clp proteolytic system, which performs the vital function of maintaining protein turnover within the cell. This system primarily impacts the bacterial response to various stresses by degrading specific proteins but can also regulate a number of physiological processes through protein degradation. A critical stress to which S. aureus must adapt during infection of a vertebrate host is nutrient iron limitation. We have previously shown that the Clp system impacts expression of genes required for heme-iron acquisition during iron limitation and is required for staphylococcal infection. Based on these data, we sought to further define the Clp-dependent impact on S. aureus during iron limitation by characterizing the proteomic profiles of mutants inactivated for components of the Clp protease, including ClpP, ClpC and ClpX, in high- and low-iron conditions. Our results reveal numerous proteins altered in abundance in the clp mutants and provide new insights into the staphylococcal proteolytic network during nutrient iron limitation.

The assembly of the cholesterol-dependent cytolysin (CDC) oligomeric pore complex requires a complex choreography of secondary and tertiary structural changes in domain 3 (D3) of the CDC monomer structure. A point mutation was identified in the archetype CDC, perfringolysin O, that blocks detectable D3 structural changes and traps the membrane-bound monomers in an early and reversible stage of oligomer assembly. Using this and other mutants we show that specific D3 structural changes are propagated from one membrane-bound monomer to another. Propagation of these structural changes results in the exposure of a β-strand in D3 that allows it to pair and form edge-on interactions with a second β-strand of a free membrane-bound monomer. Pairing of these strands establishes the final geometry of the pore complex and is necessary to drive the formation of the β-barrel pore. These studies provide new insights into how structural information is propagated between membrane-bound monomers of a self-assembling system and the interactions that establish the geometry of the final pore complex.

The cholesterol-dependent cytolysins (CDCs) constitute a family of pore-forming toxins that contribute to the pathogenesis of a large number of Gram-positive bacterial pathogens.The most highly conserved region in the primary structure of the CDCs is the signature undecapeptide sequence (ECTGLAWEWWR). The CDC pore forming mechanism is highly sensitive to changes in its structure, yet its contribution to the molecular mechanism of the CDCs has remained enigmatic. Using a combination of fluorescence spectroscopic methods we provide evidence that shows the undecapeptide motif of the archetype CDC, perfringolysin O (PFO), is a key structural element in the allosteric coupling of the cholesterol-mediated membrane binding in domain 4 (D4) to distal structural changes in domain 3 (D3) that are required for the formation of the oligomeric pore complex. Loss of the undecapeptide function prevents all measurable D3 structural transitions, the intermolecular interaction of membrane bound monomers and the assembly of the oligomeric pore complex. We further show that this pathway does not exist in intermedilysin (ILY), a CDC that exhibits a divergent undecapeptide and that has evolved to use human CD59 rather than cholesterol as its receptor. These studies show for the first time that the undecapeptide of the cholesterol-binding CDCs forms a critical element of the allosteric pathway that controls the assembly of the pore complex., Author Summary The CDCs are a large family of pathogenesis-associated pore-forming toxins that are expressed by many Gram-positive pathogens. The conserved undecapeptide motif of the CDCs has been regarded as the signature peptide sequence for these toxins, yet its function has remained obscure. The studies herein show that the undecapeptide forms a critical structural element in the allosteric pathway that couples membrane binding to cholesterol to the initiation of distal structural changes, which are required for the assembly of the pore forming complex. These studies provide the first insight into the function of this highly conserved sequence and show that through evolution this pathway is missing in the CD59-binding CDCs.

Bacillus cereus causes the most virulent and refractory form of endophthalmitis. The authors analyzed the effectiveness of early treatment with vancomycin or gatifloxacin, with or without dexamethasone, for experimental B. cereus endophthalmitis.Rabbit eyes were injected intravitreally with 100 colony-forming units of B. cereus. At 2, 4, or 6 hours after infection, eyes were injected intravitreally with 0.1 mL gatifloxacin (0.3%), vancomycin (1.0%), either antibiotic plus dexamethasone, dexamethasone alone (1.0%), or PBS. Eyes were analyzed by electroretinography, bacterial quantitation, and antibiotic penetration analysis. Drug toxicity toward Müller cells, retinal pigment epithelium, and cones was also analyzed.Eyes treated at 2 hours with vancomycin or gatifloxacin, with or without dexamethasone, maintained higher ERG amplitudes than the dexamethasone alone and PBS control groups. Eyes treated with antibiotic plus dexamethasone at 6 hours had reduced retinal function compared to antibiotic treatment alone. With the exception of vancomycin with or without dexamethasone at 6 hours, all antibiotic treatments sterilized eyes. Only gatifloxacin reached aqueous concentrations greater than the minimal inhibitory concentration for B. cereus when measured at 8 hours. Neither gatifloxacin nor vancomycin was toxic to retinal cells in vitro.Early intravitreal injection of vancomycin or gatifloxacin improved the therapeutic outcome of B. cereus endophthalmitis. The addition of dexamethasone to antibiotic treatment did not provide a therapeutic benefit over antibiotics alone and appeared to reduce the antibiotic efficacy of vancomycin 6 hours after infection. In this model, delay in treatment past 6 hours significantly reduced the potential for salvaging useful vision.