RESUMEN
Preclinical and clinical studies have suggested that cancer treatment with antitumor antibodies induces a specific adaptive T cell response. A central role in this process has been attributed to CD4+ T cells, but the relevant T cell epitopes, mostly derived from non-mutated self-antigens, are largely unknown. In this study, we have characterized human CD20-derived epitopes restricted by HLA-DR1, HLA-DR3, HLA-DR4, and HLA-DR7, and investigated whether T cell responses directed against CD20-derived peptides can be elicited in human HLA-DR-transgenic mice and human samples. Based on in vitro binding assays to recombinant human MHC II molecules and on in vivo immunization assays in H-2 KO/HLA-A2+-DR1+ transgenic mice, we have identified 21 MHC II-restricted long peptides derived from intracellular, membrane, or extracellular domains of the human non-mutated CD20 protein that trigger in vitro IFN-γ production by PBMCs and splenocytes from healthy individuals and by PBMCs from follicular lymphoma patients. These CD20-derived MHC II-restricted peptides could serve as a therapeutic tool for improving and/or monitoring anti-CD20 T cell activity in patients treated with rituximab or other anti-CD20 antibodies.
Asunto(s)
Antígenos CD20/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfoma/tratamiento farmacológico , Animales , Femenino , Cadenas HLA-DRB1/inmunología , Humanos , Interferón gamma/biosíntesis , Linfoma/inmunología , Ratones , Rituximab/uso terapéuticoRESUMEN
Bypass of the d,d-transpeptidase activity of penicillin-binding proteins by an l,d-transpeptidase (Ldtfm) results in resistance to ampicillin and glycopeptides in Enterococcus faecium M9, a mutant obtained by nine consecutive selection steps. Resistance requires activation of a cryptic locus for production of the essential tetrapeptide-containing substrate of Ldtfm and impaired activity of protein phosphatase StpA. Here, whole-genome sequencing revealed a high mutation rate for the entire selection procedure (79 mutations in 900 generations). Acquisition of a mutation in the mismatch repair gene mutL had little impact on the frequency of rifampin-resistant mutants although the mutation spectrum of M9 was typical of impaired MutL with high transversion to transition (40/11) and substitution to deletion (51/28) ratios. M9 did not mainly accumulate neutral mutations since base substitutions occurred more frequently in coding sequences than expected (χ(2) = 5.0; P < 0.05) and silent mutations were underrepresented (χ(2) = 5.72; P < 0.02). None of the mutations directly affected recognition of the tetrapeptide substrate of Ldtfm by peptidoglycan synthesis enzymes. Instead, mutations appear to remodel regulatory circuits involving two-component regulatory systems and sugar metabolism. The high number of mutations required for activation of the l,d-transpeptidase pathway may strongly limit emergence of cross-resistance to ampicillin and glycopeptides by this mechanism.
Asunto(s)
Enterococcus faecium/efectos de los fármacos , Glicopéptidos/farmacología , beta-Lactamas/farmacología , Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Enterococcus faecium/genética , MutaciónRESUMEN
Colicin M (ColM) is the only enzymatic colicin reported to date that inhibits cell wall peptidoglycan biosynthesis. It catalyzes the specific degradation of the lipid intermediates involved in this pathway, thereby provoking lysis of susceptible Escherichia coli cells. A gene encoding a homologue of ColM was detected within the exoU-containing genomic island A carried by certain pathogenic Pseudomonas aeruginosa strains. This bacteriocin (pyocin) that we have named PaeM was crystallized, and its structure with and without an Mg(2+) ion bound was solved. In parallel, site-directed mutagenesis of conserved PaeM residues from the C-terminal domain was performed, confirming their essentiality for the protein activity both in vitro (lipid II-degrading activity) and in vivo (cytotoxicity against a susceptible P. aeruginosa strain). Although PaeM is structurally similar to ColM, the conformation of their active sites differs radically; in PaeM, residues essential for enzymatic activity and cytotoxicity converge toward a same pocket, whereas in ColM they are spread along a particularly elongated active site. We have also isolated a minimal domain corresponding to the C-terminal half of the PaeM protein and exhibiting a 70-fold higher enzymatic activity as compared with the full-length protein. This isolated domain of the PaeM bacteriocin was further shown to kill E. coli cells when addressed to the periplasm of these bacteria.
Asunto(s)
Bacteriocinas/química , Colicinas/química , Hidrolasas Diéster Fosfóricas/química , Pseudomonas aeruginosa/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Bacteriocinas/metabolismo , Bacteriocinas/farmacología , Dominio Catalítico , Colicinas/metabolismo , Colicinas/farmacología , Secuencia Conservada , Cristalografía por Rayos X , Escherichia coli/efectos de los fármacos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/farmacología , Estructura Secundaria de Proteína , Homología Estructural de Proteína , Especificidad por SustratoRESUMEN
One billion people worldwide get flu every year, including patients with non-small cell lung cancer (NSCLC). However, the impact of acute influenza A virus (IAV) infection on the composition of the tumor microenvironment (TME) and the clinical outcome of patients with NSCLC is largely unknown. We set out to understand how IAV load impacts cancer growth and modifies cellular and molecular players in the TME. Herein, we report that IAV can infect both tumor and immune cells, resulting in a long-term protumoral effect in tumor-bearing mice. Mechanistically, IAV impaired tumor-specific T-cell responses, led to the exhaustion of memory CD8+ T cells and induced PD-L1 expression on tumor cells. IAV infection modulated the transcriptomic profile of the TME, fine-tuning it toward immunosuppression, carcinogenesis, and lipid and drug metabolism. Consistent with these data, the transcriptional module induced by IAV infection in tumor cells in tumor-bearing mice was also found in human patients with lung adenocarcinoma and correlated with poor overall survival. In conclusion, we found that IAV infection worsened lung tumor progression by reprogramming the TME toward a more aggressive state.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Virus de la Influenza A , Gripe Humana , Neoplasias Pulmonares , Infecciones por Orthomyxoviridae , Humanos , Animales , Ratones , Neoplasias Pulmonares/patología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Microambiente Tumoral , Linfocitos T CD8-positivos , Pulmón , Infecciones por Orthomyxoviridae/patologíaRESUMEN
Bypass of the penicillin-binding proteins by an L,D-transpeptidase (Ldt(fm)) confers cross-resistance to beta-lactam and glycopeptide antibiotics in mutants of Enterococcus faecium selected in vitro. Ldt(fm) is produced by the parental strain D344S although it insignificantly contributes to peptidoglycan cross-linking as pentapeptide stems cannot be used as acyl donors by this enzyme. Here we show that production of the tetrapeptide substrate of Ldt(fm) is controlled by a two-component regulatory system (DdcRS) and a metallo-D,D-carboxypeptidase (DdcY). The locus was silent in D344S and its activation was due to amino acid substitutions in DdcS or DdcR that led to production of DdcY and hydrolysis of the C-terminal D-Ala residue of the cytoplasmic peptidoglycan precursor UDP-MurNAc-pentapeptide. The T(161)A and T(161)M substitutions affected a position of DdcS known to be essential for the phosphatase activity of related sensor kinases. Complete elimination of UDP-MurNAc-pentapeptide, which was required specifically for resistance to glycopeptides, involved substitutions in DdcY that increased the catalytic efficiency of the enzyme (E(127)K) and affected its interaction with the cell envelope (I(14)N). The ddc locus displays striking similarities with portions of the van vancomycin resistance gene clusters, suggesting possible routes of emergence of cross-resistance to glycopeptides and beta-lactams in natural conditions.
Asunto(s)
Enterococcus faecium/metabolismo , Peptidoglicano/metabolismo , Proteínas/metabolismo , Antibacterianos/farmacología , Secuencia de Bases , Pared Celular/metabolismo , Farmacorresistencia Bacteriana , Enterococcus faecium/efectos de los fármacos , Enterococcus faecium/enzimología , Glicopéptidos/farmacología , Datos de Secuencia Molecular , Peptidoglicano/biosíntesis , Peptidoglicano/química , Proteínas/genética , Especificidad por Sustrato , Uridina Difosfato Ácido N-Acetilmurámico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurámico/metabolismo , beta-Lactamas/farmacologíaRESUMEN
SARS-CoV-2 infection leads to a highly variable clinical evolution, ranging from asymptomatic to severe disease with acute respiratory distress syndrome, requiring intensive care units (ICU) admission. The optimal management of hospitalized patients has become a worldwide concern and identification of immune biomarkers predictive of the clinical outcome for hospitalized patients remains a major challenge. Immunophenotyping and transcriptomic analysis of hospitalized COVID-19 patients at admission allow identifying the two categories of patients. Inflammation, high neutrophil activation, dysfunctional monocytic response and a strongly impaired adaptive immune response was observed in patients who will experience the more severe form of the disease. This observation was validated in an independent cohort of patients. Using in silico analysis on drug signature database, we identify differential therapeutics that specifically correspond to each group of patients. From this signature, we propose a score-the SARS-Score-composed of easily quantifiable biomarkers, to classify hospitalized patients upon arrival to adapt treatment according to their immune profile.
Asunto(s)
COVID-19/inmunología , SARS-CoV-2/fisiología , Inmunidad Adaptativa/genética , Adulto , Anciano , Antivirales/uso terapéutico , Biomarcadores , COVID-19/terapia , Estudios de Cohortes , Femenino , Hospitalización , Humanos , Inflamación/genética , Masculino , Persona de Mediana Edad , Medicina de Precisión , Estudios Prospectivos , Índice de Severidad de la Enfermedad , TranscriptomaRESUMEN
To investigate the mechanisms underlying the SARS-CoV-2 infection severity observed in patients with obesity, we performed a prospective study of 51 patients evaluating the impact of multiple immune parameters during 2 weeks after admission, on vital organs' functions according to body mass index (BMI) categories. High-dimensional flow cytometric characterization of immune cell subsets was performed at admission, 30 systemic cytokines/chemokines levels were sequentially measured, thirteen endothelial markers were determined at admission and at the zenith of the cytokines. Computed tomography scans on admission were quantified for lung damage and hepatic steatosis (n = 23). Abnormal BMI (> 25) observed in 72.6% of patients, was associated with a higher rate of intensive care unit hospitalization (p = 0.044). SARS-CoV-2 RNAaemia, peripheral immune cell subsets and cytokines/chemokines were similar among BMI groups. A significant association between inflammatory cytokines and liver, renal, and endothelial dysfunctions was observed only in patients with obesity (BMI > 30). In contrast, early signs of lung damage (ground-glass opacity) correlated with Th1/M1/inflammatory cytokines only in normal weight patients. Later lesions of pulmonary consolidation correlated with BMI but were independent of cytokine levels. Our study reveals distinct physiopathological mechanisms associated with SARS-CoV-2 infection in patients with obesity that may have important clinical implications.
Asunto(s)
COVID-19/patología , Citocinas/metabolismo , Hígado/fisiopatología , Pulmón/fisiopatología , Obesidad/patología , Anciano , Biomarcadores/metabolismo , Índice de Masa Corporal , COVID-19/complicaciones , COVID-19/virología , Quimiocinas/sangre , Quimiocinas/metabolismo , Citocinas/sangre , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Femenino , Hospitalización/estadística & datos numéricos , Humanos , Unidades de Cuidados Intensivos , Hígado/diagnóstico por imagen , Pulmón/diagnóstico por imagen , Masculino , Persona de Mediana Edad , Obesidad/complicaciones , Estudios Prospectivos , ARN Viral/sangre , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Índice de Severidad de la EnfermedadRESUMEN
Anti-CD20 treatment represents a therapeutic benefit for patients with B-cell lymphomas, although more efficient therapies are needed for refractory or relapsing patients. Among them, the combination of anti-CD20 and IL-2 that induces T cell response has been hampered by the expansion of FoxP3+ Tregs that strongly express the high affinity IL-2 receptor (IL-2R αßγ). We explore here the anti-tumor effect of an anti-CD20 antibody combined with a mutated IL-2 (no-alpha mutein) which has a disrupted affinity for the IL-2R αßγ. We demonstrate that anti-CD20/no-alpha mutein combination significantly augments the survival rate of mice challenged with huCD20+ cells as compared to animals treated with anti-CD20 ± IL-2. Moreover, the combination with no-alpha mutein but not IL-2 provokes an increase of granzyme B and perforin in splenic NK and CD8+ T cells, a reduction of Tregs and an increase in activated macrophages. The former combination also induces a T helper profile different from that obtained with IL-2, with an earlier polarization to Th1 and no increase in Th17. The therapeutic effect of anti-CD20/no-alpha mutein was accompanied by an expansion of peripheral central (TCM) and effector (TEM) memory CD8+ T cell compartments. Last, as opposed to IL-2, no-alpha mutein administered at the beginning of anti-CD20 treatment did not dampen the long-term protection of surviving mice after tumor rechallenge. Thus, this study shows that the combination of anti-tumor antibodies and no-alpha mutein is a promising approach to improve the therapeutic effect of these antibodies by potentiating NK/macrophage-mediated innate immunity and the adaptive T-cell response.
Asunto(s)
Linfocitos T CD8-positivos , Interleucina-2 , Animales , Femenino , Humanos , Ratones , Rituximab/farmacologíaRESUMEN
BACKGROUND: Natural killer (NK) cells play a crucial role in tumor immunosurveillance through their cytotoxic effector functions and their capacity to interact with other immune cells to build a coordinated antitumor immune response. Emerging data reveal NK cell dysfunction within the tumor microenvironment (TME) through checkpoint inhibitory molecules associated with a regulatory phenotype. OBJECTIVE: We aimed at analyzing the gene expression profile of intratumoral NK cells compared with non-tumorous NK cells, and to characterize their inhibitory function in the TME. METHODS: NK cells were sorted from human lung tumor tissue and compared with non- tumoral distant lungs. RESULTS: In the current study, we identify a unique gene signature of NK cell dysfunction in human non-small cell lung carcinoma (NSCLC). First, transcriptomic analysis reveals significant changes related to migratory pattern with a downregulation of sphingosine-1-phosphate receptor 1 (S1PR1) and CX3C chemokine receptor 1 (CX3CR1) and overexpression of C-X-C chemokine receptor type 5 (CXCR5) and C-X-C chemokine receptor type 6 (CXCR6). Second, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and killer cell lectin like receptor (KLRC1) inhibitory molecules were increased in intratumoral NK cells, and CTLA-4 blockade could partially restore MHC class II level on dendritic cell (DC) that was impaired during the DCs/NK cell cross talk. Finally, NK cell density impacts the positive prognostic value of CD8+ T cells in NSCLC. CONCLUSIONS: These findings demonstrate novel molecular cues associated with NK cell inhibitory functions in NSCLC.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Inmunoterapia/métodos , Células Asesinas Naturales/inmunología , Transcriptoma/genética , Humanos , Microambiente TumoralRESUMEN
Peptidoglycan is polymerized by monofunctional d,d-transpeptidases belonging to class B penicillin-binding proteins (PBPs) and monofunctional glycosyltransferases and by bifunctional enzymes that combine both activities (class A PBPs). Three genes encoding putative class A PBPs (pbpF, pbpZ, and ponA) were deleted from the chromosome of Enterococcus faecium D344R in all possible combinations in order to identify the glycosyltransferases that cooperate with low-affinity class B Pbp5 for synthesis of peptidoglycan in the presence of beta-lactam antibiotics. The viability of the triple mutant indicated that glycan strands can be polymerized independently from class A PBPs by an unknown glycosyltranferase. The susceptibility of the DeltapbpF DeltaponA mutant and triple mutants to extended spectrum cephalosporins (ceftriaxone and cefepime) identified either PbpF or PonA as essential partners of Pbp5 for peptidoglycan polymerization in the presence of the drugs. Mass spectrometry analysis of peptidoglycan structure showed that loss of PonA and PbpF activity led to a minor decrease in the extent of peptidoglycan cross-linking by the remaining PBPs without any detectable compensatory increase in the participation of the L,D-transpeptidase in peptidoglycan synthesis. Optical density measurements and electron microscopy analyses showed that the DeltapbpF DeltaponA mutant underwent increased stationary-phase autolysis compared to the parental strain. Unexpectedly, deletion of the class A pbp genes revealed dissociation between the expression of resistance to cephalosporins and penicillins, although the production of Pbp5 was required for resistance to both classes of drugs. Thus, susceptibility of Pbp5-mediated peptidoglycan cross-linking to different beta-lactam antibiotics differed as a function of its partner glycosyltransferase.
Asunto(s)
Proteínas Bacterianas/metabolismo , Enterococcus faecium/metabolismo , Proteínas de Unión a las Penicilinas/metabolismo , Resistencia betalactámica/genética , Proteínas Bacterianas/genética , Southern Blotting , Ceftriaxona/farmacología , Cefalosporinas/farmacología , Electroporación , Enterococcus faecium/efectos de los fármacos , Enterococcus faecium/genética , Enterococcus faecium/ultraestructura , Espectrometría de Masas , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Transmisión , Mutación , Proteínas de Unión a las Penicilinas/genética , Penicilinas/farmacología , Peptidoglicano/química , Peptidoglicano/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
The FemX(Wv) aminoacyl transferase of Weissella viridescens initiates the synthesis of the side chain of peptidoglycan precursors by transferring l-Ala from Ala-tRNA(Ala) to UDP-MurNAc-pentadepsipeptide. FemX(Wv) is an attractive target for the development of novel antibiotics, since the side chain is essential for the last cross-linking step of peptidoglycan synthesis. Here, we show that FemX(Wv) is highly specific for incorporation of l-Ala in vivo based on extensive analysis of the structure of peptidoglycan. Comparison of various natural and in vitro-transcribed tRNAs indicated that the specificity of FemX(Wv) depends mainly upon the sequence of the tRNA although additional specificity determinants may include post-transcriptional modifications and recognition of the esterified amino acid. Site-directed mutagenesis identified cytosines in the G1-C72 and G2-C71 base pairs of the acceptor stem as critical for FemX(Wv) activity in agreement with modeling of tRNA(Ala) in the catalytic cavity of the enzyme. In contrast, semi-synthesis of Ala-tRNA(Ala) harboring nucleotide substitutions in the G3-U70 wobble base pair showed that this main identity determinant of alanyl-tRNA synthetase is non-essential for FemX(Wv). The different modes of recognition of the acceptor stem indicate that specific inhibition of FemX(Wv) could be achieved by targeting the distal portion of tRNA(Ala) for the design of substrate analogues.
Asunto(s)
Bacterias/metabolismo , Pared Celular/metabolismo , ARN de Transferencia de Alanina/química , Bacterias/química , Bacterias/enzimología , Secuencia de Bases , Pared Celular/química , Pared Celular/enzimología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Péptido Sintasas/metabolismo , Peptidoglicano/química , Peptidoglicano/metabolismo , ARN de Transferencia de Alanina/genética , ARN de Transferencia de Alanina/metabolismoRESUMEN
Clinical responses to anti-tumor monoclonal antibody (mAb) treatment have been regarded for many years only as a consequence of the ability of mAbs to destroy tumor cells by innate immune effector mechanisms. More recently, it has also been shown that anti-tumor antibodies can induce a long-lasting anti-tumor adaptive immunity, likely responsible for durable clinical responses, a phenomenon that has been termed the vaccinal effect of antibodies. However, some of these anti-tumor antibodies are directed against molecules expressed both by tumor cells and normal immune cells, in particular lymphocytes, and, hence, can also strongly affect the host adaptive immunity. In addition to a delayed recovery of target cells, lymphocyte depleting-mAb treatments can have dramatic consequences on the adaptive immune cell network, its rebound, and its functional capacities. Thus, in this review, we will not only discuss the mAb-induced vaccinal effect that has emerged from experimental preclinical studies and clinical trials but also the multifaceted impact of lymphocytes-depleting therapeutic antibodies on the host adaptive immunity. We will also discuss some of the molecular and cellular mechanisms of action whereby therapeutic mAbs induce a long-term protective anti-tumor effect and the relationship between the mAb-induced vaccinal effect and the immune response against self-antigens.
RESUMEN
The last step of peptidoglycan polymerization involves two families of unrelated transpeptidases that are the essential targets of ß-lactam antibiotics. D,D-transpeptidases of the penicillin-binding protein (PBP) family are active-site serine enzymes that use pentapeptide precursors and are the main or exclusive cross-linking enzymes in nearly all bacteria. However, peptidoglycan cross-linking is performed mainly by active-site cysteine L,D-transpeptidases that use tetrapeptides in Mycobacterium tuberculosis, Clostridium difficile, and ß-lactam-resistant mutants of Enterococcus faecium. We have investigated reprogramming of the E. faecium peptidoglycan assembly pathway by a switch from pentapeptide to tetrapeptide precursors and bypass of PBPs by L,D-transpeptidase Ldtfm. Mutational alterations of two signal transduction systems were necessary and sufficient for activation of the L,D-transpeptidation pathway, which is essentially cryptic in wild-type strains. The first one is a classical two-component regulatory system, DdcRS, that controls the activity of Ldtfm at the substrate level. As previously described, loss of DdcS phosphatase activity leads to production of the D,D-carboxypeptidase DdcY and conversion of the pentapeptide into the tetrapeptide substrate of Ldtfm. Here we show that full bypass of PBPs by Ldtfm also requires increased Ser/Thr protein phosphorylation resulting from impaired activity of phosphoprotein phosphatase StpA. This enzyme negatively controlled the level of protein phosphorylation both by direct dephosphorylation of target proteins and by dephosphorylation of its cognate kinase Stk. In combination with production of DdcY, increased protein phosphorylation by this eukaryotic-enzyme-like Ser/Thr protein kinase was sufficient for activation of the L,D-transpeptidation pathway in the absence of mutational alteration of peptidoglycan synthesis enzymes. Importance: The mechanism of acquisition of high-level ampicillin resistance involving bypass of the penicillin-binding proteins (PBPs) by L,D-transpeptidase Ldtfm was incompletely understood, as production of tetrapeptide precursors following transcriptional activation of the ddc locus by the DdcRS two-component regulatory system was necessary but not sufficient for full activation of the L,D-transpeptidation pathway. Here, we identified the release of a negative control of Ser/Thr protein phosphorylation mediated by phosphatase StpA as the additional factor essential for ampicillin resistance. Thus, bypass of PBPs by Ldtfm requires the modification of signal transduction regulatory systems without any gain of function by mutational alteration of peptidoglycan biosynthetic enzymes. In contrast, previously characterized mechanisms of antibiotic resistance involve horizontal gene transfer and mutational alteration of drug targets. Activation of the L,D-transpeptidation pathway reported in this study is an unprecedented mechanism of emergence of a new metabolic pathway since it involved the recruitment of preexisting functions following modifications of regulatory circuits.
Asunto(s)
Proteínas Bacterianas/metabolismo , Enterococcus faecium/enzimología , Enterococcus faecium/metabolismo , Peptidoglicano Glicosiltransferasa/metabolismo , Peptidoglicano/metabolismo , Fosfoproteínas Fosfatasas/metabolismoRESUMEN
Glycopeptides and beta-lactams are the major antibiotics available for the treatment of infections due to Gram-positive bacteria. Emergence of cross-resistance to these drugs by a single mechanism has been considered as unlikely because they inhibit peptidoglycan polymerization by different mechanisms. The glycopeptides bind to the peptidyl-D-Ala(4)-D-Ala(5) extremity of peptidoglycan precursors and block by steric hindrance the essential glycosyltransferase and D,D-transpeptidase activities of the penicillin-binding proteins (PBPs). The beta-lactams are structural analogues of D-Ala(4)-D-Ala(5) and act as suicide substrates of the D,D-transpeptidase module of the PBPs. Here we have shown that bypass of the PBPs by the recently described beta-lactam-insensitive L,D-transpeptidase from Enterococcus faecium (Ldt(fm)) can lead to high level resistance to glycopeptides and beta-lactams. Cross-resistance was selected by glycopeptides alone or serially by beta-lactams and glycopeptides. In the corresponding mutants, UDP-MurNAc-pentapeptide was extensively converted to UDP-MurNAc-tetrapeptide following hydrolysis of D-Ala(5), thereby providing the substrate of Ldt(fm). Complete elimination of D-Ala(5), a residue essential for glycopeptide binding, was possible because Ldt(fm) uses the energy of the L-Lys(3)-D-Ala(4) peptide bond for cross-link formation in contrast to PBPs, which use the energy of the D-Ala(4)-D-Ala(5) bond. This novel mechanism of glycopeptide resistance was unrelated to the previously identified replacement of D-Ala(5) by D-Ser or D-lactate.
Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Enterococcus faecium/metabolismo , Glicopéptidos/farmacología , Alanina/metabolismo , Sustitución de Aminoácidos , Reactivos de Enlaces Cruzados/farmacología , Citoplasma/metabolismo , Enterococcus faecium/genética , Hidrólisis , Pruebas de Sensibilidad Microbiana , Modelos Biológicos , Muramoilpentapéptido Carboxipeptidasa/metabolismo , Fragmentos de Péptidos/química , Peptidoglicano/biosíntesis , Peptidoglicano/química , Peptidoglicano/metabolismo , Especificidad por Sustrato , beta-Lactamas/metabolismoRESUMEN
Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classes A and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. In Enterococcus faecalis, class B PBP5 mediates intrinsic resistance to the cephalosporin class of beta-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes of E. faecalis JH2-2 (ponA, pbpF, and pbpZ). Among mutants with single or double deletions, only JH2-2 DeltaponA DeltapbpF was susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferase of Staphylococcus aureus. Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of beta-lactams formed a subset of the class A PBPs of E. faecalis, and heterospecific complementation was observed with an ortholog from E. faecium. Site-directed mutagenesis of pbpF confirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As the E. faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Enterococcus faecalis/efectos de los fármacos , Hexosiltransferasas/metabolismo , Muramoilpentapéptido Carboxipeptidasa/metabolismo , Peptidil Transferasas/metabolismo , Resistencia betalactámica , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Proteínas Portadoras/clasificación , Proteínas Portadoras/genética , Enterococcus faecalis/genética , Enterococcus faecalis/crecimiento & desarrollo , Inhibidores Enzimáticos/farmacología , Eliminación de Gen , Glicosiltransferasas/antagonistas & inhibidores , Hexosiltransferasas/clasificación , Hexosiltransferasas/genética , Pruebas de Sensibilidad Microbiana , Muramoilpentapéptido Carboxipeptidasa/clasificación , Muramoilpentapéptido Carboxipeptidasa/genética , Oligosacáridos/farmacología , Proteínas de Unión a las Penicilinas , Peptidoglicano/química , Peptidoglicano/metabolismo , Peptidil Transferasas/clasificación , Peptidil Transferasas/genéticaRESUMEN
The enzymatic synthesis of the complete l-alanyl(1)-l-alanine(2) side chain of the peptidoglycan precursors of Enterococcus faecalis was obtained in vitro using purified enzymes. The pathway involved alanyl-tRNA synthetase and two ligases, BppA1 and BppA2, that specifically transfer alanine from Ala-tRNA to the first and second positions of the side chain, respectively. The structure of the UDP-N-acetylmuramoyl-l-Ala-gamma-d-Glu-l-Lys(N(epsilon)-l-Ala(1)-l-Ala(2))-d-Ala-d-Ala product of BppA1 and BppA2 was confirmed by mass spectrometry (MS) and MS/MS analyses. The peptidoglycan structure of the wild-type E. faecalis strain JH2-2 was determined by tandem reverse-phase high-pressure liquid chromatography-MS revealing that most muropeptides contained two l-alanyl residues in the cross-bridges and in the free N-terminal ends. Deletion of the bppA2 gene was associated with production of muropeptides containing a single alanyl residue at these positions. The relative abundance of monomers, dimers, trimers, and tetramers in the peptidoglycan of the bppA2 mutant indicated that precursors containing an incomplete side chain were efficiently used by the dd-transpeptidases in the cross-linking reaction. However, the bppA2 deletion impaired expression of intrinsic beta-lactam resistance suggesting that the low affinity penicillin-binding protein 5 did not function optimally with precursors substituted by a single alanine.
Asunto(s)
Alanina/química , Dipéptidos/síntesis química , Enterococcus faecalis/metabolismo , Peptidoglicano/químicaRESUMEN
The peptidoglycan cross-bridges of Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium consist of the sequences Gly(5), l-Ala(2), and d-Asx, respectively. Expression of the fmhB, femA, and femB genes of S. aureus in E. faecalis led to the production of peptidoglycan precursors substituted by mosaic side chains that were efficiently used by the penicillin-binding proteins for cross-bridge formation. The Fem transferases were specific for incorporation of glycyl residues at defined positions of the side chains in the absence of any additional S. aureus factors such as tRNAs used for amino acid activation. The PBPs of E. faecalis displayed a broad substrate specificity because mosaic side chains containing from 1 to 5 residues and Gly instead of l-Ala at the N-terminal position were used for peptidoglycan cross-linking. Low affinity PBP2a of S. aureus conferred beta-lactam resistance in E. faecalis and E. faecium, thereby indicating that there was no barrier to heterospecific expression of resistance caused by variations in the structure of peptidoglycan precursors. Thus, conservation of the structure of the peptidoglycan cross-bridges in members of the same species reflects the high specificity of the enzymes for side chain synthesis, although this is not essential for the activity of the PBPs.