Deletion of a previously uncharacterized lipoprotein lirL confers resistance to an inhibitor of type II signal peptidase in Acinetobacter baumannii.

Acinetobacter baumannii is a clinically important, predominantly health care-associated gram-negative bacterium with high rates of emerging resistance worldwide. Given the urgent need for novel antibacterial therapies against A. baumannii, we focused on inhibiting lipoprotein biosynthesis, a pathway that is essential for envelope biogenesis in gram-negative bacteria. The natural product globomycin, which inhibits the essential type II signal peptidase prolipoprotein signal peptidase (LspA), is ineffective against wild-type A. baumannii clinical isolates due to its poor penetration through the outer membrane. Here, we describe a globomycin analog, G5132, that is more potent against wild-type and clinical A. baumannii isolates. Mutations leading to G5132 resistance in A. baumannii map to the signal peptide of a single hypothetical gene, which we confirm encodes an alanine-rich lipoprotein and have renamed lirL (prolipoprotein signal peptidase inhibitor resistance lipoprotein). LirL is a highly abundant lipoprotein primarily localized to the inner membrane. Deletion of lirL leads to G5132 resistance, inefficient cell division, increased sensitivity to serum, and attenuated virulence. Signal peptide mutations that confer resistance to G5132 lead to the accumulation of diacylglyceryl-modified LirL prolipoprotein in untreated cells without significant loss in cell viability, suggesting that these mutations overcome a block in lipoprotein biosynthetic flux by decreasing LirL prolipoprotein substrate sensitivity to processing by LspA. This study characterizes a lipoprotein that plays a critical role in resistance to LspA inhibitors and validates lipoprotein biosynthesis as a antibacterial target in A. baumannii.

EHMT2 methyltransferase governs cell identity in the lung and is required for KRAS G12D tumor development and propagation.

Lung development, integrity and repair rely on precise Wnt signaling, which is corrupted in diverse diseases, including cancer. Here, we discover that EHMT2 methyltransferase regulates Wnt signaling in the lung by controlling the transcriptional activity of chromatin-bound ß-catenin, through a non-histone substrate in mouse lung. Inhibition of EHMT2 induces transcriptional, morphologic, and molecular changes consistent with alveolar type 2 (AT2) lineage commitment. Mechanistically, EHMT2 activity functions to support regenerative properties of KrasG12D tumors and normal AT2 cells-the predominant cell of origin of this cancer. Consequently, EHMT2 inhibition prevents KrasG12D lung adenocarcinoma (LUAD) tumor formation and propagation and disrupts normal AT2 cell differentiation. Consistent with these findings, low gene EHMT2 expression in human LUAD correlates with enhanced AT2 gene expression and improved prognosis. These data reveal EHMT2 as a critical regulator of Wnt signaling, implicating Ehmt2 as a potential target in lung cancer and other AT2-mediated lung pathologies.

Multiplexed proteomics of autophagy-deficient murine macrophages reveals enhanced antimicrobial immunity via the oxidative stress response.

Defective autophagy is strongly associated with chronic inflammation. Loss-of-function of the core autophagy gene Atg16l1 increases risk for Crohn's disease in part by enhancing innate immunity through myeloid cells such as macrophages. However, autophagy is also recognized as a mechanism for clearance of certain intracellular pathogens. These divergent observations prompted a re-evaluation of ATG16L1 in innate antimicrobial immunity. In this study, we found that loss of Atg16l1 in myeloid cells enhanced the killing of virulent Shigella flexneri (S.flexneri), a clinically relevant enteric bacterium that resides within the cytosol by escaping from membrane-bound compartments. Quantitative multiplexed proteomics of murine bone marrow-derived macrophages revealed that ATG16L1 deficiency significantly upregulated proteins involved in the glutathione-mediated antioxidant response to compensate for elevated oxidative stress, which simultaneously promoted S.flexneri killing. Consistent with this, myeloid-specific deletion of Atg16l1 in mice accelerated bacterial clearance in vitro and in vivo. Pharmacological induction of oxidative stress through suppression of cysteine import enhanced microbial clearance by macrophages. Conversely, antioxidant treatment of macrophages permitted S.flexneri proliferation. These findings demonstrate that control of oxidative stress by ATG16L1 and autophagy regulates antimicrobial immunity against intracellular pathogens.

Unstable Mechanisms of Resistance to Inhibitors of Escherichia coli Lipoprotein Signal Peptidase.

Clinical development of antibiotics with novel mechanisms of action to kill pathogenic bacteria is challenging, in part, due to the inevitable emergence of resistance. A phenomenon of potential clinical importance that is broadly overlooked in preclinical development is heteroresistance, an often-unstable phenotype in which subpopulations of bacterial cells show decreased antibiotic susceptibility relative to the dominant population. Here, we describe a new globomycin analog, G0790, with potent activity against the Escherichia coli type II signal peptidase LspA and uncover two novel resistance mechanisms to G0790 in the clinical uropathogenic E. coli strain CFT073. Building on the previous finding that complete deletion of Lpp, the major Gram-negative outer membrane lipoprotein, leads to globomycin resistance, we also find that an unexpectedly modest decrease in Lpp levels mediated by insertion-based disruption of regulatory elements is sufficient to confer G0790 resistance and increase sensitivity to serum killing. In addition, we describe a heteroresistance phenotype mediated by genomic amplifications of lspA that result in increased LspA levels sufficient to overcome inhibition by G0790 in culture. These genomic amplifications are highly unstable and are lost after as few as two subcultures in the absence of G0790, which places amplification-containing resistant strains at high risk of being misclassified as susceptible by routine antimicrobial susceptibility testing. In summary, our study uncovers two vastly different mechanisms of resistance to LspA inhibitors in E. coli and emphasizes the importance of considering the potential impact of unstable and heterogenous phenotypes when developing antibiotics for clinical use.IMPORTANCE Despite increasing evidence suggesting that antibiotic heteroresistance can lead to treatment failure, the significance of this phenomena in the clinic is not well understood, because many clinical antibiotic susceptibility testing approaches lack the resolution needed to reliably classify heteroresistant strains. Here we present G0790, a new globomycin analog and potent inhibitor of the Escherichia coli type II signal peptidase LspA. We demonstrate that in addition to previously known mechanisms of resistance to LspA inhibitors, unstable genomic amplifications containing lspA can lead to modest yet biologically significant increases in LspA protein levels that confer a heteroresistance phenotype.

Preclinical Safety Assessment of a Highly Selective and Potent Dual Small-Molecule Inhibitor of CBP/P300 in Rats and Dogs.

Cyclic adenosine monophosphate-response element (CREB)-binding protein (CBP) and EP300E1A-binding protein (p300) are members of the bromodomain and extraterminal motif (BET) family. These highly homologous proteins have a key role in modulating transcription, including altering the status of chromatin or through interactions with or posttranslational modifications of transcription factors. As CBP and p300 have known roles for stimulating c-Myc oncogenic activity, a small-molecule inhibitor, GNE-781, was developed to selectively and potently inhibit the CBP/p300 bromodomains (BRDs). Genetic models have been challenging to develop due to embryonic lethality arising from germline homozygous mutations in either CBP or P300. Hence, the purpose of this study was to characterize the role of dual inhibition of these proteins in adult rats and dogs. Repeat dose toxicity studies were conducted, and toxicologic and pathologic end points were assessed. GNE-781 was generally tolerated; however, marked effects on thrombopoiesis occurred in both species. Evidence of inhibition of erythroid, granulocytic, and lymphoid cell differentiation was also present, as well as deleterious changes in gastrointestinal and reproductive tissues. These findings are consistent with many preclinical (and clinical) effects reported with BET inhibitors targeting BRD proteins; thus, the current study findings indicate a likely important role for CBP/p300 in stem cell differentiation.

Ubiquilin1 promotes antigen-receptor mediated proliferation by eliminating mislocalized mitochondrial proteins.

Ubiquilins (Ubqlns) are a family of ubiquitin receptors that promote the delivery of hydrophobic and aggregated ubiquitinated proteins to the proteasome for degradation. We carried out a proteomic analysis of a B cell lymphoma-derived cell line, BJAB, that requires UBQLN1 for survival to identify UBQLN1 client proteins. When UBQLN1 expression was acutely inhibited, 120 mitochondrial proteins were enriched in the cytoplasm, suggesting that the accumulation of mitochondrial client proteins in the absence of UBQLN1 is cytostatic. Using a Ubqln1-/- mouse strain, we found that B cell receptor (BCR) ligation of Ubqln1-/- B cells led to a defect in cell cycle entry. As in BJAB cells, mitochondrial proteins accumulated in BCR-stimulated cells, leading to protein synthesis inhibition and cell cycle block. Thus, UBQLN1 plays an important role in clearing mislocalized mitochondrial proteins upon cell stimulation, and its absence leads to suppression of protein synthesis and cell cycle arrest.

Structural insights into lipoprotein N-acylation by Escherichia coli apolipoprotein N-acyltransferase.

Gram-negative bacteria express a diverse array of lipoproteins that are essential for various aspects of cell growth and virulence, including nutrient uptake, signal transduction, adhesion, conjugation, sporulation, and outer membrane protein folding. Lipoprotein maturation requires the sequential activity of three enzymes that are embedded in the cytoplasmic membrane. First, phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) recognizes a conserved lipobox motif within the prolipoprotein signal sequence and catalyzes the addition of diacylglycerol to an invariant cysteine. The signal sequence is then cleaved by signal peptidase II (LspA) to give an N-terminal S-diacylglyceryl cysteine. Finally, apolipoprotein N-acyltransferase (Lnt) catalyzes the transfer of the sn-1-acyl chain of phosphatidylethanolamine to this N-terminal cysteine, generating a mature, triacylated lipoprotein. Although structural studies of Lgt and LspA have yielded significant mechanistic insights into this essential biosynthetic pathway, the structure of Lnt has remained elusive. Here, we present crystal structures of wild-type and an active-site mutant of Escherichia coli Lnt. The structures reveal a monomeric eight-transmembrane helix fold that supports a periplasmic carbon-nitrogen hydrolase domain containing a Cys-Glu-Lys catalytic triad. Two lipids are bound at the active site in the structures, and we propose a putative phosphate recognition site where a chloride ion is coordinated near the active site. Based on these structures and complementary cell-based, biochemical, and molecular dynamics approaches, we propose a mechanism for substrate engagement and catalysis by E. coli Lnt.

Dendritic cells require NIK for CD40-dependent cross-priming of CD8+ T cells.

Dendritic cells (DCs) link innate and adaptive immunity and use a host of innate immune and inflammatory receptors to respond to pathogens and inflammatory stimuli. Although DC maturation via canonical NF-κB signaling is critical for many of these functions, the role of noncanonical NF-κB signaling via the serine/threonine kinase NIK (NF-κB-inducing kinase) remains unclear. Because NIK-deficient mice lack secondary lymphoid organs, we generated transgenic mice with targeted NIK deletion in CD11c(+) cells. Although these mice exhibited normal lymphoid organs, they were defective in cross-priming naive CD8(+) T cells following vaccination, even in the presence of anti-CD40 or polyinosinic:polycytidylic acid to induce DC maturation. This impairment reflected two intrinsic defects observed in splenic CD8(+) DCs in vitro, namely antigen cross-presentation to CD8(+) T cells and secretion of IL-12p40, a cytokine known to promote cross-priming in vivo. In contrast, antigen presentation to CD4(+) T cells was not affected. These findings reveal that NIK, and thus probably the noncanonical NF-κB pathway, is critical to allow DCs to acquire the capacity to cross-present antigen and prime CD8 T cells after exposure to licensing stimuli, such as an agonistic anti-CD40 antibody or Toll-like receptor 3 ligand.

A Crohn's disease variant in Atg16l1 enhances its degradation by caspase 3.

Crohn's disease is a debilitating inflammatory bowel disease (IBD) that can involve the entire digestive tract. A single-nucleotide polymorphism (SNP) encoding a missense variant in the autophagy gene ATG16L1 (rs2241880, Thr300Ala) is strongly associated with the incidence of Crohn's disease. Numerous studies have demonstrated the effect of ATG16L1 deletion or deficiency; however, the molecular consequences of the Thr300Ala (T300A) variant remains unknown. Here we show that amino acids 296-299 constitute a caspase cleavage motif in ATG16L1 and that the T300A variant (T316A in mice) significantly increases ATG16L1 sensitization to caspase-3-mediated processing. We observed that death-receptor activation or starvation-induced metabolic stress in human and murine macrophages increased degradation of the T300A or T316A variants of ATG16L1, respectively, resulting in diminished autophagy. Knock-in mice harbouring the T316A variant showed defective clearance of the ileal pathogen Yersinia enterocolitica and an elevated inflammatory cytokine response. In turn, deletion of the caspase-3-encoding gene, Casp3, or elimination of the caspase cleavage site by site-directed mutagenesis rescued starvation-induced autophagy and pathogen clearance, respectively. These findings demonstrate that caspase 3 activation in the presence of a common risk allele leads to accelerated degradation of ATG16L1, placing cellular stress, apoptotic stimuli and impaired autophagy in a unified pathway that predisposes to Crohn's disease.

Spatial control of gene expression within a scaffold by localized inducer release.

Gene expression can be controlled in genetically modified cells by employing an inducer/promoter system where presence of the inducer molecule regulates the timing and level of gene expression. By applying the principles of controlled release, it should be possible to control gene expression on a biomaterial surface by the presence or absence of inducer release from the underlying material matrix, thus avoiding alternative techniques that rely upon uptake of relatively labile DNA from material surfaces. To evaluate this concept, a modified ecdysone-responsive gene expression system was transfected into B16 murine cells and the ability of an inducer ligand, which was released from elastomeric poly(ester urethane) urea (PEUU), to initiate gene expression was studied. The synthetic inducer ligand was first loaded into PEUU to demonstrate extended release of the bioactive molecule at various loading densities over a one year period in vitro. Patterning films of PEUU variably-loaded with inducer resulted in spatially controlled cell expression of the gene product (green fluorescent protein, GFP). In porous scaffolds made from PEUU by salt leaching, where the central region was exclusively loaded with inducer, cells expressed GFP predominately in the loaded central regions whereas expression was minimal in outer regions where ligand was omitted. This scaffold system may ultimately provide a means to precisely control progenitor cell commitment in a spatially-defined manner in vivo for soft tissue repair and regeneration.

Osteopontin protects the islets and beta-cells from interleukin-1 beta-mediated cytotoxicity through negative feedback regulation of nitric oxide.

Osteopontin (OPN), a phosphorylated glycoprotein that binds to an integrin-binding motif, has been shown to regulate nitric oxide (NO) production via inhibition of induced NO synthase (iNOS) synthesis. In the transplanted islets, iNOS and toxic amounts of NO are produced as a result of islets infiltration with inflammatory cells and production of proinflammatory cytokines. Here, we demonstrate that addition of OPN before IL-1beta in freshly isolated rat islets improved their glucose stimulated insulin secretion dose-dependently and inhibited IL-1beta-induced NO production in an arginine-glycine-aspartate-dependent manner. Transient transfection of OPN gene in RINm5F beta-cells fully prevented the toxic effect of IL-1beta at concentrations that reduced the viability by 50% over 3 d. OPN prevention of IL-1beta-induced toxicity was accompanied by inhibited transcription of iNOS by 80%, resulting in 50% decreased formation of the toxic NO. In OPN-transfected cells, the IL-1beta-induced nuclear factor-kappaB activity was significantly reduced. Islets exposed to IL-1beta revealed a naturally occurring early up-regulated OPN transcription. OPN promoter activity was increased in the presence of IL-1beta, IL-1beta-induced NO, and an inducer of NO synthesis. These data suggest the presence of a cross talk between the IL-1beta and OPN pathways and a unique trans-regulatory mechanism in which IL-1beta-induced NO synthesis feedback regulates itself through up-regulation of OPN gene transcription. Our data also suggest that influencing OPN expression represents an approach for affecting cytokine-induced signal transduction to prevent or reduce activation of the cascade of downstream devastating effects after islet transplantation.