RESUMO
A pet cockatoo was the suspected source of Cryptococcus neoformans recovered from an immunocompromised patient with cryptococcosis based on molecular analyses available in 2000. Here, we report whole genome sequence analysis of the clinical and cockatoo strains. Both are closely related MATα strains belonging to the VNII lineage, confirming that the human infection likely originated from pet bird exposure. The two strains differ by 61 single nucleotide polymorphisms, including eight nonsynonymous changes involving seven genes. To ascertain whether changes in these genes are selected for during mammalian infection, we passaged the cockatoo strain in mice. Remarkably, isolates obtained from mouse tissue possess a frameshift mutation in one of the seven genes altered in the human sample (LQVO5_000317), a gene predicted to encode an SWI-SNF chromatin-remodeling complex protein. In addition, both cockatoo and patient strains as well as mouse-passaged isolates obtained from brain tissue had a premature stop codon in a homologue of ZFC3 (LQVO5_004463), a predicted single-zinc finger containing protein, which is associated with larger capsules when deleted and reverted to a full-length protein in the mouse-passaged isolates obtained from lung tissue. The patient strain and mouse-passaged isolates show variability in virulence factors, with differences in capsule size, melanization, rates of nonlytic expulsion from macrophages, and amoeba predation resistance. Our results establish that environmental strains undergo genomic and phenotypic changes during mammalian passage, suggesting that animal virulence can be a mechanism for genetic change and that the genomes of clinical isolates may provide a readout of mutations acquired during infection.
Assuntos
Criptococose , Cryptococcus neoformans , Humanos , Animais , Camundongos , Cryptococcus neoformans/genética , Virulência/genética , Fatores de Virulência/genética , Evolução Biológica , MamíferosRESUMO
Constant domains in antibody molecules at the level of the Fab (CH1 and CL) have long been considered to be simple scaffolding elements that physically separate the paratope-defining variable (V) region from the effector function-mediating constant (C) regions. However, due to recent findings that C domains of different isotypes can modulate the fine specificity encoded in the V region, elucidating the role of C domains in shaping the paratope and influencing specificity is a critical area of interest. To dissect the relative contributions of each C domain to this phenomenon, we generated antibody fragments with different C regions omitted, using a set of antibodies targeting capsular polysaccharides from the fungal pathogen, Cryptococcus neoformans. Antigen specificity mapping and functional activity measurements revealed that V region-only antibody fragments exhibited poly-specificity to antigenic variants and extended to recognition of self-antigens, while measurable hydrolytic activity of the capsule was greatly attenuated. To better understand the mechanistic origins of the remarkable loss of specificity that accompanies the removal of C domains from identical paratopes, we performed molecular dynamics simulations which revealed increased paratope plasticity in the scFv relative to the corresponding Fab. Together, our results provide insight into how the remarkable specificity of immunoglobulins is governed and maintained at the level of the Fab through the enforcement of structural restrictions on the paratope by CH1 domains.
Assuntos
Cryptococcus neoformans , Epitopos , Cryptococcus neoformans/imunologia , Cryptococcus neoformans/química , Epitopos/química , Epitopos/imunologia , Regiões Constantes de Imunoglobulina/química , Regiões Constantes de Imunoglobulina/genética , Simulação de Dinâmica Molecular , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Humanos , Especificidade de Anticorpos , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/genética , Animais , Anticorpos Antifúngicos/imunologia , Anticorpos Antifúngicos/químicaRESUMO
Many species of pathogenic gram-positive bacteria display covalently crosslinked protein polymers (called pili or fimbriae) that mediate microbial adhesion to host tissues. These structures are assembled by pilus-specific sortase enzymes that join the pilin components together via lysine-isopeptide bonds. The archetypal SpaA pilus from Corynebacterium diphtheriae is built by the Cd SrtA pilus-specific sortase, which crosslinks lysine residues within the SpaA and SpaB pilins to build the shaft and base of the pilus, respectively. Here, we show that Cd SrtA crosslinks SpaB to SpaA via a K139(SpaB)-T494(SpaA) lysine-isopeptide bond. Despite sharing only limited sequence homology, an NMR structure of SpaB reveals striking similarities with the N-terminal domain of SpaA (N SpaA) that is also crosslinked by Cd SrtA. In particular, both pilins contain similarly positioned reactive lysine residues and adjacent disordered AB loops that are predicted to be involved in the recently proposed "latch" mechanism of isopeptide bond formation. Competition experiments using an inactive SpaB variant and additional NMR studies suggest that SpaB terminates SpaA polymerization by outcompeting N SpaA for access to a shared thioester enzyme-substrate reaction intermediate.
Assuntos
Aminoaciltransferases , Corynebacterium diphtheriae , Proteínas de Fímbrias/química , Proteínas de Fímbrias/metabolismo , Corynebacterium diphtheriae/metabolismo , Proteínas de Bactérias/metabolismo , Lisina , Cádmio/metabolismo , Aminoaciltransferases/metabolismoRESUMO
Classic antibody functions include opsonization, complement activation, and enhancement of cellular antimicrobial function. Antibodies can also have catalytic activity, although the contribution of catalysis to their biological functions has been more difficult to establish. With the ubiquity of catalytic antibodies against glycans virtually unknown, we sought to advance this knowledge. The use of a glycan microarray allowed epitope mapping of several monoclonal antibodies (mAbs) against the capsule of Cryptococcus neoformans From this, we designed and synthesized two glycan-based FRET probes, which we used to discover antibodies with innate glycosidase activity and analyze their enzyme kinetics, including mAb 2H1, the most efficient identified to date. The validity of the FRET assay was confirmed by demonstrating that the mAbs mediate glycosidase activity on intact cryptococcal capsules, as observed by a reduction in capsule diameter. Furthermore, the mAb 18B7, a glycosidase hydrolase, resulted in the appearance of reducing ends in the capsule as labeled by a hydroxylamine-armed fluorescent (HAAF) probe. Finally, we demonstrate that exposing C. neoformans cells to catalytic antibodies results in changes in complement deposition and increased phagocytosis by macrophages, suggesting that the antiphagocytic properties of the capsule have been impaired. Our results raise questions over the ubiquity of antibodies with catalytic activity against glycans and establish the utility of glycan-based FRET and HAAF probes as tools for investigating this activity.
Assuntos
Anticorpos Catalíticos/imunologia , Anticorpos Antifúngicos/imunologia , Bioensaio , Cryptococcus neoformans/imunologia , Transferência Ressonante de Energia de Fluorescência , Polissacarídeos/química , Proteínas do Sistema Complemento/metabolismo , Mapeamento de Epitopos , Cinética , Macrófagos/imunologia , Modelos Moleculares , Sondas Moleculares/química , Oligossacarídeos/síntese química , Oligossacarídeos/química , Peptídeos/química , Fagocitose , Estrutura Secundária de ProteínaRESUMO
Gram-positive bacteria assemble pili (fimbriae) on their surfaces to adhere to host tissues and to promote polymicrobial interactions. These hair-like structures, although very thin (1 to 5 nm), exhibit impressive tensile strengths because their protein components (pilins) are covalently crosslinked together via lysine-isopeptide bonds by pilus-specific sortase enzymes. While atomic structures of isolated pilins have been determined, how they are joined together by sortases and how these interpilin crosslinks stabilize pilus structure are poorly understood. Using a reconstituted pilus assembly system and hybrid structural biology methods, we elucidated the solution structure and dynamics of the crosslinked interface that is repeated to build the prototypical SpaA pilus from Corynebacterium diphtheriae We show that sortase-catalyzed introduction of a K190-T494 isopeptide bond between adjacent SpaA pilins causes them to form a rigid interface in which the LPLTG sorting signal is inserted into a large binding groove. Cellular and quantitative kinetic measurements of the crosslinking reaction shed light onto the mechanism of pilus biogenesis. We propose that the pilus-specific sortase in C. diphtheriae uses a latch mechanism to select K190 on SpaA for crosslinking in which the sorting signal is partially transferred from the enzyme to a binding groove in SpaA in order to facilitate catalysis. This process is facilitated by a conserved loop in SpaA, which after crosslinking forms a stabilizing latch that covers the K190-T494 isopeptide bond. General features of the structure and sortase-catalyzed assembly mechanism of the SpaA pilus are likely conserved in Gram-positive bacteria.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Corynebacterium diphtheriae/fisiologia , Cisteína Endopeptidases/metabolismo , Fímbrias Bacterianas/fisiologia , Catálise , Proteínas de Fímbrias/metabolismo , Lisina/metabolismo , Ligação ProteicaRESUMO
The polysaccharide capsule of fungal pathogen Cryptococcus neoformans is a critical virulence factor that has historically evaded complete characterization. Cryptococcal polysaccharides are known to either remain attached to the cell as capsular polysaccharides (CPSs) or to be shed into the extracellular space as exopolysaccharides (EPSs). While many studies have examined the properties of EPS, far less is known about CPS. In this work, we detail the development of new physical and enzymatic methods for the isolation of CPS which can be used to explore the architecture of the capsule and isolated capsular material. We show that sonication or Glucanex enzyme cocktail digestion yields soluble CPS preparations, while use of a French pressure cell press or Glucanex digestion followed by cell disruption removed the capsule and produced cell wall-associated polysaccharide aggregates that we call "capsule ghosts", implying an inherent organization that allows the CPS to exist independent of the cell wall surface. Since sonication and Glucanex digestion were noncytotoxic, it was also possible to observe the cryptococcal cells rebuilding their capsule, revealing the presence of reducing end glycans throughout the capsule. Finally, analysis of dimethyl sulfoxide-extracted and sonicated CPS preparations revealed the conservation of previously identified glucuronoxylomannan motifs only in the sonicated CPS. Together, these observations provide new insights into capsule architecture and synthesis, consistent with a model in which the capsule is assembled from the cell wall outward using smaller polymers, which are then compiled into larger ones.
Assuntos
Cryptococcus neoformans , Cápsulas Fúngicas , Polissacarídeos , Parede Celular/química , Parede Celular/metabolismo , Criptococose/microbiologia , Cryptococcus neoformans/metabolismo , Cápsulas Fúngicas/química , Cápsulas Fúngicas/metabolismo , Polissacarídeos/metabolismo , Fatores de Virulência/metabolismoRESUMO
Covalently cross-linked pilus polymers displayed on the cell surface of Gram-positive bacteria are assembled by class C sortase enzymes. These pilus-specific transpeptidases located on the bacterial membrane catalyze a two-step protein ligation reaction, first cleaving the LPXTG motif of one pilin protomer to form an acyl-enzyme intermediate and then joining the terminal Thr to the nucleophilic Lys residue residing within the pilin motif of another pilin protomer. To date, the determinants of class C enzymes that uniquely enable them to construct pili remain unknown. Here, informed by high-resolution crystal structures of corynebacterial pilus-specific sortase (SrtA) and utilizing a structural variant of the enzyme (SrtA2M), whose catalytic pocket has been unmasked by activating mutations, we successfully reconstituted in vitro polymerization of the cognate major pilin (SpaA). Mass spectrometry, electron microscopy, and biochemical experiments authenticated that SrtA2M synthesizes pilus fibers with correct Lys-Thr isopeptide bonds linking individual pilins via a thioacyl intermediate. Structural modeling of the SpaA-SrtA-SpaA polymerization intermediate depicts SrtA2M sandwiched between the N- and C-terminal domains of SpaA harboring the reactive pilin and LPXTG motifs, respectively. Remarkably, the model uncovered a conserved TP(Y/L)XIN(S/T)H signature sequence following the catalytic Cys, in which the alanine substitutions abrogated cross-linking activity but not cleavage of LPXTG. These insights and our evidence that SrtA2M can terminate pilus polymerization by joining the terminal pilin SpaB to SpaA and catalyze ligation of isolated SpaA domains in vitro provide a facile and versatile platform for protein engineering and bio-conjugation that has major implications for biotechnology.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Corynebacterium/metabolismo , Cisteína Endopeptidases/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Catálise , Parede Celular/metabolismo , Cristalografia por Raios X/métodos , Peptidil Transferases/metabolismo , PolimerizaçãoRESUMO
Site-specifically modified protein bioconjugates have important applications in biology, chemistry, and medicine. Functionalizing specific protein side chains with enzymes using mild reaction conditions is of significant interest, but remains challenging. Recently, the lysine-isopeptide bond forming activity of the sortase enzyme that builds surface pili in Corynebacterium diphtheriae (CdSrtA) has been reconstituted in vitro. A mutationally activated form of CdSrtA was shown to be a promising bioconjugating enzyme that can attach Leu-Pro-Leu-Thr-Gly peptide fluorophores to a specific lysine residue within the N-terminal domain of the SpaA protein (NSpaA), enabling the labeling of target proteins that are fused to NSpaA. Here we present a detailed analysis of the CdSrtA catalyzed protein labeling reaction. We show that the first step in catalysis is rate limiting, which is the formation of the CdSrtA-peptide thioacyl intermediate that subsequently reacts with a lysine ε-amine in NSpaA. This intermediate is surprisingly stable, limiting spurious proteolysis of the peptide substrate. We report the discovery of a new enzyme variant (CdSrtAΔ) that has significantly improved transpeptidation activity, because it completely lacks an inhibitory polypeptide appendage ("lid") that normally masks the active site. We show that the presence of the lid primarily impairs formation of the thioacyl intermediate and not the recognition of the NSpaA substrate. Quantitative measurements reveal that CdSrtAΔ generates its cross-linked product with a catalytic turnover number of 1.4 ± 0.004 h-1 and that it has apparent KM values of 0.16 ± 0.04 and 1.6 ± 0.3 mM for its NSpaA and peptide substrates, respectively. CdSrtAΔ is 7-fold more active than previously studied variants, labeling >90% of NSpaA with peptide within 6 h. The results of this study further improve the utility of CdSrtA as a protein labeling tool and provide insight into the enzyme catalyzed reaction that underpins protein labeling and pilus biogenesis.
Assuntos
Corynebacterium diphtheriae/enzimologia , Cisteína Endopeptidases/química , Lisina/química , Peptídeos/química , Biocatálise , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Cinética , Mutação , Domínios ProteicosRESUMO
Proteins that are site-specifically modified with peptides and chemicals can be used as novel therapeutics, imaging tools, diagnostic reagents and materials. However, there are few enzyme-catalyzed methods currently available to selectively conjugate peptides to internal sites within proteins. Here we show that a pilus-specific sortase enzyme from Corynebacterium diphtheriae (CdSrtA) can be used to attach a peptide to a protein via a specific lysine-isopeptide bond. Using rational mutagenesis we created CdSrtA3M, a highly activated cysteine transpeptidase that catalyzes in vitro isopeptide bond formation. CdSrtA3M mediates bioconjugation to a specific lysine residue within a fused domain derived from the corynebacterial SpaA protein. Peptide modification yields greater than >95% can be achieved. We demonstrate that CdSrtA3M can be used in concert with the Staphylococcus aureus SrtA enzyme, enabling dual, orthogonal protein labeling via lysine-isopeptide and backbone-peptide bonds.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Corynebacterium diphtheriae/enzimologia , Cisteína Endopeptidases/metabolismo , Corantes Fluorescentes/metabolismo , Lisina/metabolismo , Peptídeos/metabolismo , Proteínas de Bactérias/química , Corynebacterium diphtheriae/metabolismo , Proteínas de Fímbrias/metabolismo , Corantes Fluorescentes/química , Lisina/química , Modelos Moleculares , Peptídeos/química , Polimerização , Coloração e Rotulagem , Staphylococcus aureus/enzimologiaRESUMO
PURPOSE: The goal of this study was to describe the outcomes associated with daptomycin treatment of documented gram-positive infections in patients with neutropenia. METHODS: All patients with neutropenia (≤500 cells/m(3)) and at least one documented gram-positive culture from 2006-2009 were identified from a retrospective, multicenter, and observational registry (Cubicin(®) Outcome Registry and Experience (CORE(®))). Investigators assessed patient outcome (cured, improved, failed, nonevaluable) at the end of daptomycin therapy. All patients were included in the safety analysis. RESULTS: The efficacy population had 186 patients; 159 (85 %) patients had either cure (n = 108, 58 %) or improved (n = 51, 27 %) as an outcome. Success rates (cure plus improved) by the lowest WBC during daptomycin were 98/116 (84 %) for ≤100 cells/m(3) and 61/70 (87 %) for 101-499 cells/m(3), P = 0.6. Most patients had cancer; 135/186 (73 %) had hematological malignancy; 26/186 (14 %) had solid tumors, and 9 (5 %) had both. One hundred fifty-six (84 %) patients received other antibiotics before daptomycin treatment; 82 % vancomycin, of which 31 % failed vancomycin. The most common infections were bacteremia (78 %), skin and skin structure infections (8 %), and urinary tract infections/pyelonephritis (6 %). The most common pathogens were vancomycin-resistant Enterococcus faecium (47 %), methicillin-resistant Staphylococcus aureus (20 %), and coagulase-negative staphylococci (19 %). The median (min, max) initial daptomycin dose was 6 mg/kg (3.6, 8.3). The median (min, max) daptomycin duration of therapy was 14 days (1, 86). Possibly related adverse events occurred in 12/209 patients (6 %), and 13 patients (6 %) discontinued daptomycin due to adverse event. CONCLUSIONS: The results suggest that daptomycin appeared useful and well tolerated in neutropenic patients, and the degree of neutropenia did not affect daptomycin success rates. Comparative clinical trials are needed to confirm these findings.
Assuntos
Antibacterianos/uso terapêutico , Daptomicina/uso terapêutico , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Neutropenia/microbiologia , Adulto , Idoso , Antibacterianos/efeitos adversos , Bacteriemia/sangue , Bacteriemia/tratamento farmacológico , Daptomicina/efeitos adversos , Feminino , Infecções por Bactérias Gram-Positivas/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/sangue , Neoplasias/tratamento farmacológico , Neoplasias/microbiologia , Neutropenia/induzido quimicamente , Neutropenia/etiologia , Estudos Retrospectivos , Resultado do Tratamento , Vancomicina/uso terapêuticoRESUMO
One of the standard assays for the fungal pathogen Cryptococcus neoformans is the glucuronoxylomannan (GXM) ELISA. This assay utilizes monoclonal antibodies targeted against the critical virulence factor, the polysaccharide (PS) capsule. GXM ELISA is one of the most used assays in the field used for diagnosis of cryptococcal infection, quantification of PS content, and determination of binding specificity for antibodies. Here we present three variations of the GXM ELISA used by our group-indirect, capture, and competition ELISAs. We have also provided some history, perspective, and notes on these methods, which we hope will help the reader choose, and implement, the best assay for their research.While it has long been referred to as the GXM ELISA, we also suggest a name update to better reflect our updated understanding of the polysaccharide antigens targeted by this assay. The Cryptococcal PS ELISA is a more accurate description of this set of methodologies and the antigens they measure. Finally, we discuss the limitations of this assay and put forth future plans for expanding the antigens assayed by ELISA.
Assuntos
Criptococose , Cryptococcus neoformans , Ensaio de Imunoadsorção Enzimática , Polissacarídeos , Ensaio de Imunoadsorção Enzimática/métodos , Cryptococcus neoformans/imunologia , Criptococose/diagnóstico , Criptococose/microbiologia , Criptococose/imunologia , Polissacarídeos/análise , Polissacarídeos/imunologia , Humanos , Antígenos de Fungos/imunologia , Antígenos de Fungos/análise , Polissacarídeos Fúngicos/imunologia , Polissacarídeos Fúngicos/análise , Anticorpos Monoclonais/imunologia , Anticorpos Antifúngicos/imunologiaRESUMO
Antibodies play a vital role in the immune response to infectious diseases and can be administered passively to protect patients. In the case of Cryptococcus neoformans, a WHO critical priority fungal pathogen, infection results in antibodies targeting capsular glucuronoxylomannan (GXM). These antibodies yield protective, non-protective, and disease-enhancing outcomes when administered passively. However, it was unknown how these distinct antibodies recognized their antigens at the molecular level, leading to the hypothesis that they may target different GXM epitopes. To test this hypothesis, we constructed a microarray containing 26 glycans representative of those found in highly virulent cryptococcal strains and utilized it to study 16 well-characterized monoclonal antibodies. Notably, we found that protective and non-protective antibodies shared conserved reactivity to the M2 motif of GXM, irrespective of the strain used in infection or GXM-isolated to produce a conjugate vaccine. Here, only two antibodies, 12A1 and 18B7, exhibited diverse trivalent GXM motif reactivity. IgG antibodies associated with protective responses showed cross-reactivity to at least two GXM motifs. This molecular understanding of antibody binding epitopes was used to map the antigenic diversity of two Cryptococcus neoformans strains, which revealed the exceptional complexity of fungal capsular polysaccharides. A multi-GXM motif vaccine holds the potential to effectively address this antigenic diversity. Collectively, these findings underscore the context-dependent nature of antibody function and challenge the classification of anti-GXM epitopes as either "protective" or "non-protective".
Assuntos
Criptococose , Cryptococcus neoformans , Humanos , Anticorpos Antifúngicos/metabolismo , Cryptococcus neoformans/metabolismo , Epitopos , Anticorpos Monoclonais , PolissacarídeosRESUMO
Many species of pathogenic gram-positive bacteria display covalently crosslinked protein polymers (called pili or fimbriae) that mediate microbial adhesion to host tissues. These structures are assembled by pilus-specific sortase enzymes that join the pilin components together via lysine-isopeptide bonds. The archetypal SpaA pilus from Corynebacterium diphtheriae is built by the Cd SrtA pilus-specific sortase, which crosslinks lysine residues within the SpaA and SpaB pilins to build the shaft and base of the pilus, respectively. Here, we show that Cd SrtA crosslinks SpaB to SpaA via a K139(SpaB)-T494(SpaA) lysine-isopeptide bond. Despite sharing only limited sequence homology, an NMR structure of SpaB reveals striking similarities with the N-terminal domain of SpaA ( N SpaA) that is also crosslinked by Cd SrtA. In particular, both pilins contain similarly positioned reactive lysine residues and adjacent disordered AB loops that are predicted to be involved in the recently proposed "latch" mechanism of isopeptide bond formation. Competition experiments using an inactive SpaB variant and additional NMR studies suggest that SpaB terminates SpaA polymerization by outcompeting N SpaA for access to a shared thioester enzyme-substrate reaction intermediate.
RESUMO
Understanding the mechanisms of antibody-mediated neutralization of SARS-CoV-2 is critical in combating the COVID-19 pandemic. Based on previous reports of antibody catalysis, we investigated the proteolysis of spike (S) by antibodies in COVID-19 convalescent plasma (CCP) and its contribution to viral neutralization. Quenched fluorescent peptides were designed based on S epitopes to sensitively detect antibody-mediated proteolysis. We observed epitope cleavage by CCP from different donors which persisted when plasma was heat-treated or when IgG was isolated from plasma. Further, purified CCP antibodies proteolyzed recombinant S domains, as well as authentic viral S. Cleavage of S variants suggests CCP antibody-mediated proteolysis is a durable phenomenon despite antigenic drift. We differentiated viral neutralization occurring via direct interference with receptor binding from that occurring by antibody-mediated proteolysis, demonstrating that antibody catalysis enhanced neutralization. These results suggest that antibody-catalyzed damage of S is an immunologically relevant function of neutralizing antibodies against SARS-CoV-2.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Proteólise , Pandemias , COVID-19/terapia , Soroterapia para COVID-19 , Glicoproteína da Espícula de Coronavírus , Peptídeo Hidrolases , Anticorpos Neutralizantes , Epitopos , Anticorpos AntiviraisRESUMO
BACKGROUND: Daptomycin is a rapidly bactericidal agent with broad coverage against Gram-positive organisms, including Staphylococcus aureus, the most frequent cause of osteomyelitis. The objective of this study was to describe the clinical outcome of patients with non-hardware associated osteomyelitis, and the safety profile of daptomycin in the treatment of these infections. METHODS: All patients with osteomyelitis, excluding concurrent orthopedic foreign body infections, treated with daptomycin and identified between 2007-2008 in a retrospective, multicenter, observational registry, were included. Investigators assessed patient outcome (cured, improved, failed, non-evaluable) at the end of daptomycin therapy. Patients with a successful outcome at the end of daptomycin therapy were reassessed in 2009. All patients were included in the safety analysis; evaluable patients were included in the efficacy analysis. Data was assessed using descriptive statistics. A Kaplan Meier analysis was used to assess time to clinical failure. RESULTS: Two-hundred and nine osteomyelitis patients successfully completed daptomycin therapy in 2007-2008, 71 of which (34%) had a follow-up visit in 2009 and had an evaluable clinical outcome. The median (min, max) daptomycin dose and duration were 6 mg/kg (4, 10) and 42 days (1, 88), respectively. Of the 52 patients with a documented pathogen, S. aureus was the most common (42%); primarily methicillin-resistant S. aureus. All patients were included in the safety analysis; evaluable patients were included in the efficacy analysis. Clinical resolution was reported in 94% (CI - 86.2%, 98.44%) of patients. A Kaplan Meier analysis of time to clinical failure showed that approximately 85% (CI - 64%, 95%) of patients had a continued successful outcome at the time of re-evaluation. Eighteen patients (25%) in the safety population experienced an adverse event; 13 patients (18%) had an adverse event that was possibly-related to daptomycin treatment. CONCLUSIONS: Daptomycin appears to be an effective therapeutic choice with an acceptable safety profile in the management of osteomyelitis that does not involve hardware.
Assuntos
Antibacterianos/uso terapêutico , Daptomicina/uso terapêutico , Osteomielite/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/efeitos adversos , Daptomicina/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Resultado do TratamentoRESUMO
BACKGROUND: Vancomycin is often the drug of choice in critically ill patients with gram-positive infections, although circumstances often prevent its use. In these situations, clinicians are frequently left with limited data regarding alternative agents. OBJECTIVE: To describe patients with reported sepsis receiving daptomycin in a critical care unit. METHODS: This multicenter, noncomparative, noninterventional study identified patients in critical care units, using the Cubicin Outcomes Registry and Experience (CORE) 2005-2009 registry. A descriptive account of patient characteristics, infectious etiology, outcomes at the end of daptomycin therapy, and 30-day mortality is reported. Nonevaluable patients were excluded from the efficacy analysis but included in the safety analysis. RESULTS: We identified 128 patients, 98 (77%) of whom were evaluable for efficacy. Patient characteristics for the efficacy population were 55 (56%) males, 30 (31%) aged 66 years or older, 38 (39%) had creatinine clearance less than 30 mL/min, and 27 (28%) were on dialysis. Common underlying diseases included acute or chronic renal failure 44 (45%), hypertension 40 (41%), and diabetes 27 (28%). Seventy-two (73%) patients were bacteremic. The most common pathogens found were methicillin-resistant Staphylococcus aureus (32%), vancomycin-resistant Enterococcus faecium (21%), and coagulase-negative staphylococci (20%). Prior to daptomycin, antibiotics were used in 84 (86%) patients, most commonly vancomycin (65/84; 77%). The median (range) initial daptomycin dose was 6 mg/kg (3-10) and duration of 10 days (1-58). Overall success rate was 70% (31% cured; 39% improved). Twelve adverse events possibly related to daptomycin were reported in 9 of 128 (7%) patients in the safety population; 4 of these in 4 (3%) patients were serious. The mortality rate within 30 days of completing daptomycin was 42 of 128 (33%) patients. CONCLUSIONS: These data provide preliminary results on the use of daptomycin in critically ill patients with complicated conditions. Controlled studies are needed to best evaluate daptomycin use in these patients.
Assuntos
Antibacterianos/uso terapêutico , Daptomicina/uso terapêutico , Sepse/tratamento farmacológico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/administração & dosagem , Antibacterianos/efeitos adversos , Criança , Cuidados Críticos , Daptomicina/administração & dosagem , Daptomicina/efeitos adversos , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sistema de Registros/estatística & dados numéricos , Estudos Retrospectivos , Sepse/microbiologia , Sepse/mortalidade , Resultado do Tratamento , Vancomicina/uso terapêutico , Adulto JovemRESUMO
Microbial polysaccharide characterization requires purification that often involves detergent precipitation and lyophilization. Here we examined physicochemical changes following lyophilization of Cryptococcus neoformans exopolysaccharide (EPS). Solution 1H Nuclear Magnetic Resonance (NMR) reveals significant anomeric signal attenuation following lyophilization of native EPS while 1H solid-state Nuclear Magnetic Resonance (ssNMR) shows few changes, suggesting diminished molecular motion and consequent broadening of 1H NMR polysaccharide resonances. 13C ssNMR, dynamic light scattering, and transmission electron microscopy show that, while native EPS has rigid molecular characteristics and contains small, loosely packed polysaccharide assemblies, lyophilized and resuspended EPS is disordered and contains larger dense aggregates, suggesting that structural water molecules in the interior of the polysaccharide assemblies are removed during extensive lyophilization. Importantly, mAbs to C. neoformans polysaccharide bind native EPS more strongly than lyophilized EPS. Together, these observations argue for caution when interpreting the biological and immunological attributes of polysaccharides that have been lyophilized to dryness.
Assuntos
Cryptococcus neoformans , Polissacarídeos , Cryptococcus neoformans/metabolismo , Liofilização , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Transmissão , Polissacarídeos/metabolismo , Polissacarídeos Bacterianos/químicaRESUMO
Clinical data for daptomycin in the treatment of neutropenic cancer patients with documented gram-positive infections are limited. For this study, neutropenic patients were identified from an ongoing retrospective registry (Cubicin Outcome Registry and Experience [CORE]; 2006 program year). Clinical outcomes included cure, improved, failed, and nonevaluable response, and were assessed at the end of daptomycin therapy. Patients who had a nonevaluable clinical response were only included in the safety analysis. Eighty-four patients were identified, of which 72 (86%) were clinically evaluable. Thirty-four (47%) evaluable patients had severe neutropenia (less than 100 cells/mm(3)). Hematologic malignancies were most common (82%). Bacteremia was the most common infection (76%), and vancomycin-resistant enterococci (50%), coagulase-negative staphylococci (24%), and Staphylococcus aureus (11%) were the most common pathogens isolated. Sixty-three patients (88%) received prior antibiotics, including vancomycin (83%), cefepime (17%), and linezolid (16%). The overall success rate (cure + improved) was 90%. Success rates stratified by degree of neutropenia were 85% for patients with less than 100 cells/mm(3), 93% for those with 100-499 cells/mm(3), and 100% for those with 500-1,000 cells/mm(3). The median final daptomycin dose was 6 mg/kg (range, 3-8) and the median duration of therapy was 13 days (range, 1-86). Of the 84 patients analyzed for safety, 24 (29%) developed 44 adverse events; only 5 (6%) patients had adverse events possibly related to daptomycin. The results suggest that daptomycin may be useful for specific cases involving neutropenic patients, and comparative clinical trials are feasible.
Assuntos
Antibacterianos/uso terapêutico , Daptomicina/uso terapêutico , Neoplasias/complicações , Neutropenia/tratamento farmacológico , Adulto , Idoso , Daptomicina/efeitos adversos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sistema de Registros , Estudos Retrospectivos , Resultado do TratamentoRESUMO
The functions of enzymes can be strongly affected by their higher-order spatial arrangements. In this study we combine multiple new technologies-designer protein cages and sortase-based enzymatic attachments between proteins-as a novel platform for organizing multiple enzymes (of one or more types) in specified configurations. As a scaffold we employ a previously characterized 24-subunit designed protein cage whose termini are outwardly exposed for attachment. As a first-use case, we test the attachment of two cellulase enzymes known to act synergistically in cellulose degradation. We show that, after endowing the termini of the cage subunits with a short "sort-tag" sequence (LPXTG) and the opposing termini of the cellulase enzymes with a short polyglycine sequence tag, addition of sortase covalently attaches the enzymes to the cage with good reactivity and high copy number. The doubly modified cages show enhanced activity in a cellulose degradation assay compared to enzymes in solution, and compared to a combination of singly modified cages. These new engineering strategies could be broadly useful in the development of enzymatic material and synthetic biology applications.
Assuntos
Celulase/metabolismo , Nanocápsulas/química , Engenharia de Proteínas , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Celulase/genética , Celulose/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Especificidade por SubstratoRESUMO
The pharmacokinetics and safety of extended-interval dosing of prophylactic liposomal amphotericin B (L-AMB) in peripheral stem cell transplant recipients were evaluated. The patients received L-AMB daily at 1 mg/kg of body weight or weekly at 7.5 mg/kg or received L-AMB as a single dose (15 mg/kg). The buccal mucosal tissue concentrations of L-AMB were measured. Of the 24 patients enrolled, 5 withdrew after the initial dose due to an infusion-related reaction (n = 2) or significant increases in the serum creatinine (Scr) levels (n = 3). Weekly L-AMB dosing (7.5 mg/kg) produced mean plasma concentrations of >0.300 microg/ml for the first 7 days and >0.220 microg/ml for 7 days after the second dose. A single L-AMB dose (15 mg/kg) produced mean plasma concentrations of >0.491 microg/ml for at least 7 seven days. These concentrations are within the range of the MICs reported in the literature for susceptible strains of Candida and are at the lower limits of the MICs for Aspergillus spp. Extended-interval dosing produced buccal mucosal tissue concentrations well in excess of the MICs reported in the literature for susceptible strains of Candida and Aspergillus spp. Infusion-related reactions occurred in 24% of the patients. Baseline and end-of-study Scr, electrolyte (K+, Mg2+, PO4), and serum transaminase levels were similar across the dosage groups. Five (31%) patients met the nephrotoxicity definition prior to completion of the study. Patients in the weekly or single-dose groups experienced nephrotoxicity significantly faster than the patients in the daily dosing cohort. A weekly L-AMB dose (7.5 mg/kg) or a single L-AMB dose (15 mg/kg) produced sufficient concentrations in plasma and highly vascular tissue to warrant further studies of the safety, efficacy, and practicality of the weekly prophylactic administration of L-AMB.