SUMMARY Salmonella enterica serotype Typhimurium thrives in the lumen of the acutely inflamed intestine, which suggests that this pathogen is resistant to antimicrobials encountered in this environment. However, the identity of these antimicrobials and the corresponding bacterial resistance genes remains elusive. Here we show that enteric infection with S. Typhimurium evoked marked interleukin (IL)–22/IL-17 mediated induction in intestinal epithelial cells of lipocalin-2, an antimicrobial protein that prevents bacterial iron acquisition. Lipocalin-2 accumulated in the intestinal lumen of rhesus macaques during S. Typhimurium infection. Resistance to lipocalin-2, mediated by the iroBCDE iroN locus, conferred a competitive advantage upon the S. Typhimurium wild-type in colonizing the inflamed intestine of wild-type, but not of lipocalin-2 deficient mice. These data support that resistance to lipocalin-2 defines a specific adaptation to growth in the inflamed intestine.

Cysteine can be specifically functionalized by a myriad of acid-base conjugation strategies for applications ranging from probing protein function to antibody-drug conjugates and proteomics. In contrast, selective ligation to the other sulfur-containing amino acid, methionine, has been precluded by its intrinsically weaker nucleophilicity. Here, we report a strategy for chemoselective methionine bioconjugation through redox reactivity, using oxaziridine-based reagents to achieve highly selective, rapid, and robust methionine labeling under a range of biocompatible reaction conditions. We highlight the broad utility of this conjugation method to enable precise addition of payloads to proteins, synthesis of antibody-drug conjugates, and identification of hyperreactive methionine residues in whole proteomes.

Antibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC50<20 nm), high stability (Tm∼80 °C), good expression in E. coli (∼5 mg/L), and ability to bind antigen in complex cell lysates. We evaluated a subset of Fabs generated to homologous SCAN domains for binding specificities. These Fragment antigen-binding domains were monospecific to their target SCAN antigen except in rare cases where they cross-reacted with a few highly related antigens. Remarkably, immunofluorescence experiments in six cell lines for 270 of the TF antigens, each having multiple antibodies, show that ∼70% stain predominantly in the cytosol and ∼20% stain in the nucleus which reinforces the dominant role that translocation plays in TF biology. These cloned antibody reagents are being made available to the academic community through our web site recombinant-antibodies.org to allow a more system-wide analysis of TF and chromatin biology. We believe these platforms, infrastructure, and automated approaches will facilitate the next generation of renewable antibody reagents to the human proteome in the coming decade.

The substrate activity screening method, a substrate-based fragment identification and optimization method for the development of enzyme inhibitors, was previously applied to cathepsin S to obtain low nanomolar 1,4-disubstituted-1,2,3-triazole-based aldehyde inhibitors (Wood, W. J. L.; Patterson, A. W.; Tsuruoka, H.; Jain, R. K.; Ellman, J. A. J. Am. Chem. Soc. 2005, 127, 15521-15527). Replacement of the metabolically labile aldehyde pharmacophore with the nitrile pharmacophore provided inhibitors with moderate potency for cathepsin S. The inhibitors showed good selectivity over cathepsins B and L but no selectivity over cathepsin K. X-ray structures of two crystal forms (1.5 and 1.9 A) of a complex between cathepsin S and a triazole inhibitor incorporating a chloromethyl ketone pharmacophore guided the design of triazole substrates with increased cleavage efficiency and selectivity for cathepsin S over cathepsins B, L, and K. Conversion of select substrates to nitrile inhibitors yielded a low molecular weight (414 Da) and potent (15 nM) cathepsin S inhibitor that showed >1000-fold selectivity over cathepsins B, L, and K.

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Background and Aims-We used the rhesus macaque model to study the effects of the cag pathogenicity island (cag PAI) on the H. pylori host-pathogen interaction.

Antibody phage display libraries combined with high-throughput selections have recently demonstrated tremendous promise to create the next generation of renewable, recombinant antibodies to study proteins and their many post-translational modification states; however many challenges still remain, such as optimized antibody scaffolds. Recently, a single-chain Fab (scFab) format, in which the carboxy-terminus of the light chain is linked to the amino-terminus of the heavy chain, was described to potentially combine the high display levels of a single-chain Fv with the high stability of purified Fabs. However, this format required removal of the interchain disulfide bond to achieve modest display levels and subsequent bacterial expression resulted in high levels of aggregated scFab, hindering further use of scFabs. Here, we developed an improved scFab format that retains the interchain disulfide bond by increasing the linker length between the light and heavy chains to improve display and bacterial expression levels to 1–3 mg per liter. Furthermore, rerouting of the scFab to the co-translational signal recognition particle (SRP) pathway combined with reengineering of the signal peptide sequence results in display levels 24-fold above the original scFab format and 3-fold above parent Fab levels. This optimized scFab scaffold can be easily reformatted in a single step for expression in a bacterial or mammalian host to produce stable (81°C Tm), predominantly monomeric (>90%) antibodies at a high yield. Ultimately, this new scFab format will advance high-throughput antibody generation platforms to discover the next generation of research and therapeutic antibodies.

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