Structural and Functional Studies on IroB: A Pathogen-Associated C-glycosyltransferase

Bacterial iron acquisition by the means of enterobactin (ENT) is constrained in mammalian hosts due to ENT-binding proteins such as siderocalin and serum albumin. To evade sequestration by these proteins, ENT can be modified by the C glycosyltransferase IroB, which is located in the iroA locus of Salmonella and certain extraintestinal E. coli strains such as uropathogenic E. coli CFT073. The glycosylation of ENT has been reported to be a bacterial evasion mechanism to restore the iron scavenging ability of ENT in the presence of mammalian ENT-binding proteins by the installation of a steric impediment. The C glycosyltransferase IroB catalyses the transfer of a glucose moiety to the DHB subunit of ENT under formation of a C-C bond between the anomeric C1 of the glucose moiety and the C5 of the 2,3-DHB subunit of ENT. The formation of mono-, di- and triglycosylated Ent (MGE/DGE/TGE) products where observed in vitro. The formation of a C-C bond is remarkable because of its chemical stability and resilience against enzymatic degradation. In this M.Sc. thesis, we initially identified the iroB gene product in the iroA harbouring E. coli strain Nissle 1917 on transcriptional and translational level and expressed and purified IroB recombinant. Then, we investigated the mechanism of the C-C bond formation catalysed by IroB in vitro. Based on the hypothesis that deprotonation of the catechol 2 hydroxyl renders the catechol C5 para to the 2-hydroxyl nucleophilic, the C-C bond would then be formed in a general SN2 reaction between the attacking nucleophile and the anomeric carbon of glucose, which is further facilitated by the excellent phosphate leaving group of the UDP-glucose donor. By the means of homology modelling and superposition strategies, we were able to identify the binding sites of the glycosyl donor UDP-glucose and the glycosyl acceptor ENT and to locate residues that could potentially act as base catalysts to increase the phenolate anionic character of the 2,3-DHB subunit during catalysis. We established an activity assay for IroB, separated products arising from IroB activity by reversed phase chromatography and compared so the activity of wild-type IroB and several variants. Additionally, all variants were characterized biophysically, mainly to confirm that the structural integrity was not impaired by mutations. Ultimately, our results enable us to propose a mechanism for C-glycosylation of IroB that is consistent with other glycosyltransferases found in nature

The C-glycosyltransferase IroB from Pathogenic Escherichia coli: Identification of Residues Required for Efficient Catalysis

The E. coli C-glycosyltransferase IroB catalyzes formation of a C-C bond between enterobactin and the glucose moiety of UDP-glucose, resulting in the production of mono-, di- and tri-glucosylated enterobactin (MGE, DGE, TGE). To identify catalytic residues, we generated a homology model of IroB from aligned structures of two similar C-glycosyltransferases as templates. Superposition of our homology model onto the structure of a TDP-bound orthologue revealed residue W264 as a possible stabilizer of UDP-glucose. D304 in our model was located near the predicted site of the glucose moiety of UDP-glucose. A loop containing possible catalytic residues (H65, H66, E67) was found at the predicted enterobactin-binding site. We generated IroB variants at positions 65-67, 264, and 304 and investigated variant protein conformations and enzymatic activities. Variants were found to have Tm values similar to wild-type IroB. Fluorescence emission spectra of H65A/H66A, E67A, and D304N were superimposable with wild-type IroB. However, the emission spectrum of W264L was blue-shifted, suggesting solvent exposure of W264. While H65A/H66A retained activity (92% conversion of enterobactin, with MGE as a major product), all other IroB variants were impaired in their abilities to glucosylate enterobactin: E67A catalyzed partial (29%) conversion of enterobactin to MGE; W264L converted 55% of enterobactin to MGE; D304N was completely inactive. Activity-impaired variants were found to bind enterobactin with affinities within 2.5-fold of wild-type IroB. Given our outcomes, we propose that IroB W264 and D304 are required for binding and orienting UDP-glucose, while E67, possibly supported by H65/H66, participates in enterobactin/MGE/DGE deprotonation

Optimization of cell-free protein synthesis by proteomics and metabolic engineering of Escherichia coli A19

Cell-free protein synthesis (CFPS) has emerged as a standard protein production system over the last two decades. Due to its open nature and various methods of directly influencing protein expression, it has replaced or complemented in vivo expression systems, especially for the expression of toxins, membrane proteins and other difficult-to-express proteins. Despite the widespread use of CFPS, the main component of the system, an extract derived from the centrifugation of a bacterial lysate (S30 extract), still has not been defined thoroughly. S30 extract preparation often causes changes in protein composition, altering the original proteome of exponentially growing Escherichia coli (E. coli). To optimize CFPS in a rational manner, S30 extracts from the E. coli K12-derivative A19 were analyzed using a GeLC-MS approach. The S30 core proteome, consisting of 821 proteins detected in several replicates, was functionally integrated and categorized using GO terms, revealing the presence of complete pathways that can be explored for energy regeneration or precursor generation. To evaluate the effects of alternative growth conditions, S30 extracts derived from cells grown at SOS response-inducing conditions were analyzed by quantitative GeLC-MS using isotope-coded protein labeling (ICPL). These modified S30-S extracts contained 3-10-fold increased folding factors and were shown to improve the solubility and folding of difficult proteins. Therefore, the manipulation of the S30 extract proteome by modifying the cultivation conditions is an effective approach for the expression of challenging proteins. A second approach to improve CFPS productivity was the engineering of specific metabolic pathways through genetic modifications. Using the previously generated proteome as a guideline, 13 genes coding for various enzymes affecting protein, amino acid and mRNA stability were either tagged or knocked out in E. coli strains A19 and D10. After verifying the modifications by PCR and sequencing, the viability and fitness of the strains were examined. Additionally, the transcriptional profile of a heavily modified strain was compared with the original A19 strain, revealing highly coregulated transcriptome in response to the genetic modification. The amino acid concentrations of 19 amino acids were traced during a CFPS reaction, demonstrating that amino acids can be stabilized by genetic modifications. The engineered strains showed an increase in yield for some target proteins, highlighting the relevance of metabolic engineering when optimizing CFPS. Finally, one of the metabolically engineered strains was used as an extract source and combined with purified chaperones (DsbC, Skp and FkpA) to produce different antibody fragments. DsbC was the most important chaperone for Fab folding, whereas Skp and FkpA were beneficial to produce scFab

Association of surgical treatment, systemic therapy, and survival in patients with abdominal visceral melanoma metastases, 1965-2014: relevance of surgical cure in the era of modern systemic therapy

Systemic therapy for metastatic melanoma has evolved rapidly during the last decade, and patient treatment has become more complex.To evaluate the survival benefit achieved through surgical resection of melanoma metastatic to the abdominal viscera in patients treated in the modern treatment environment.This retrospective review of the institutional melanoma database from the John Wayne Cancer Institute at Providence St Johns Health Center, a tertiary-level melanoma referral center, included 1623 patients with melanoma diagnosed as having potentially resectable abdominal metastases before (1969-2003) and after (2004-2014) advances in systemic therapy.Overall survival (OS).Of the 1623 patients identified in the database with abdominal melanoma metastases, 1097 were men (67.6%), and the mean (SD) age was 54.6 (14.6) years. Of the patients with metastatic melanoma, 1623 (320 [19.7%] in the 2004-2014 period) had abdominal metastases, including 336 (20.7%) with metastases in the gastrointestinal tract, 697 (42.9%) in the liver, 138 (8.5%) in the adrenal glands, 38 (2.3%) in the pancreas, 109 (6.7%) in the spleen, and 305 (18.8%) with multiple sites. Median OS was superior in surgical (n = 392; 18.0 months) vs nonsurgical (n = 1231; 7.0 months) patients (P

The E. coli S30 lysate proteome: A prototype for cell-free protein production

Protein production using processed cell lysates is a core technology in synthetic biology and these systems are excellent to produce difficult toxins or membrane proteins. However, the composition of the central lysate of cell-free systems is still a black box. Escherichia coli lysates are most productive for cell-free expression, yielding several mgs of protein per ml of reaction. Their preparation implies proteome fractionation, resulting in strongly biased and yet unknown lysate compositions. Many metabolic pathways are expected to be truncated or completely removed. The lack of knowledge of basic cell-free lysate proteomes is a major bottleneck for directed lysate engineering approaches as well as for assay design using non-purified reaction mixtures. This study is starting to close this gap by providing a blueprint of the S30 lysate proteome derived from the commonly used E. coli strain A19. S30 lysates are frequently used for cell-free protein production and represent the basis of most commercial E. coli cell-free expression systems. A fraction of 821 proteins was identified as the core proteome in S30 lysates, representing approximately a quarter of the known E. coli proteome. Its classification into functional groups relevant for transcription/translation, folding, stability and metabolic processes will build the framework for tailored cell-free reactions. As an example, we show that SOS response induction during cultivation results in tuned S30 lysate with better folding capacity, and improved solubility and activity of synthesized proteins. The presented data and protocols can serve as platform for the generation of customized cell-free systems and product analysis.Andalusian Government P11-CVI-7216 BIO198Spanish Ministry of Science and Innovation BFU2015-71017-

Regional Node Basin Recurrence in Melanoma Patients:More Common After Node Dissection for Macroscopic Rather than Clinically Occult Nodal Disease

Background Recommended treatment for patients with sentinel lymph node (SLN)-positive melanoma has recently changed. Randomized trials demonstrated equivalent survival with close observation versus completion lymph node dissection (CLND), but increased regional node recurrence. We evaluated factors related to in-basin nodal recurrence after lymphadenectomy (LND) for SLN-positive or macroscopic nodal metastases. Methods An institutional database and the first Multicenter Selective Lymphadenectomy Trial (MSLT-I) were analyzed independently. Exclusions were multiple primaries, multi-basin involvement, or in-transit metastases. Patient demographics, primary tumor thickness and ulceration, lymph nodes retrieved, and use of adjuvant radiotherapy were analyzed. Multivariate analyses were performed to determine factors predicting in-basin nodal recurrence (significance