Catalytically Active Cas9 Mediates Transcriptional Interference to Facilitate Bacterial Virulence.

In addition to defense against foreign DNA, the CRISPR-Cas9 system of Francisella novicida represses expression of an endogenous immunostimulatory lipoprotein. We investigated the specificity and molecular mechanism of this regulation, demonstrating that Cas9 controls a highly specific regulon of four genes that must be repressed for bacterial virulence. Regulation occurs through a protospacer adjacent motif (PAM)-dependent interaction of Cas9 with its endogenous DNA targets, dependent on a non-canonical small RNA (scaRNA) and tracrRNA. The limited complementarity between scaRNA and the endogenous DNA targets precludes cleavage, highlighting the evolution of scaRNA to repress transcription without lethally targeting the chromosome. We show that scaRNA can be reprogrammed to repress other genes, and with engineered, extended complementarity to an exogenous target, the repurposed scaRNA:tracrRNA-FnoCas9 machinery can also direct DNA cleavage. Natural Cas9 transcriptional interference likely represents a broad paradigm of regulatory functionality, which is potentially critical to the physiology of numerous Cas9-encoding pathogenic and commensal organisms.

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.

Bacterial lyso-form lipoproteins are synthesized via an intramolecular acyl chain migration.

All bacterial lipoproteins share a variably acylated N-terminal cysteine residue. Gram-negative bacterial lipoproteins are triacylated with a thioether-linked diacylglycerol moiety and an N-acyl chain. The latter is transferred from a membrane phospholipid donor to the α-amino terminus by the enzyme lipoprotein N-acyltransferase (Lnt), using an active-site cysteine thioester covalent intermediate. Many Gram-positive Firmicutes also have N-acylated lipoproteins, but the enzymes catalyzing N-acylation remain uncharacterized. The integral membrane protein Lit (lipoprotein intramolecular transacylase) from the opportunistic nosocomial pathogen Enterococcus faecalis synthesizes a specific lysoform lipoprotein (N-acyl S-monoacylglycerol) chemotype by an unknown mechanism that helps this bacterium evade immune recognition by the Toll-like receptor 2 family complex. Here, we used a deuterium-labeled lipoprotein substrate with reconstituted Lit to investigate intramolecular acyl chain transfer. We observed that Lit transfers the sn-2 ester-linked lipid from the diacylglycerol moiety to the α-amino terminus without forming a covalent thioester intermediate. Utilizing Mut-Seq to analyze an alanine scan library of Lit alleles, we identified two stretches of functionally important amino acid residues containing two conserved histidines. Topology maps based on reporter fusion assays and cysteine accessibility placed both histidines in the extracellular half of the cytoplasmic membrane. We propose a general acid base-promoted catalytic mechanism, invoking direct nucleophilic attack by the substrate α-amino group on the sn-2 ester to form a cyclic tetrahedral intermediate that then collapses to produce lyso-lipoprotein. Lit is a unique example of an intramolecular transacylase differentiated from that catalyzed by Lnt, and provides insight into the heterogeneity of bacterial lipoprotein biosynthetic systems.

Crustacean Hemolymph Lipoproteins.

Lipoproteins mediate the transport of apolar lipids in the hydrophilic environment of physiological fluids such as the vertebrate blood and the arthropod hemolymph. In this overview, we will focus on the hemolymph lipoproteins in Crustacea that have received most attention during the last years: the high density lipoprotein/ß-glucan binding proteins (HDL-BGBPs), the vitellogenins (VGs), the clotting proteins (CPs) and the more recently discovered large discoidal lipoproteins (dLPs). VGs are female specific lipoproteins which supply both proteins and lipids as storage material for the oocyte for later use by the developing embryo. Unusual within the invertebrates, the crustacean yolk proteins-formerly designated VGs-are more related to the ApoB type lipoproteins of vertebrates and are now termed apolipocrustaceins. The CPs on the other hand, which are present in both sexes, are related to the (sex specific) VGs of insects and vertebrates. CPs serve in hemostasis and wound closure but also as storage proteins in the oocyte. The HDL-BGBPs are the main lipid transporters, but are also involved in immune defense. Most crustacean lipoproteins belong to the family of the large lipid transfer proteins (LLTPs) such as the intracellular microsomal triglyceride transfer protein, the VGs, CPs and the dLPs. In contrast, the HDL-BGBPs do not belong to the LLTPs and their relationship with other lipoproteins is unknown. However, they originate from a common precursor with the dLPs, whose functions are as yet unknown. The majority of lipoprotein studies have focused on decapod crustaceans, especially shrimps, due to their economic importance. However, we will present evidence that the HDL-BGBPs are restricted to the decapod crustaceans which raises the question as to the main lipid transporting proteins of the other crustacean groups. The diversity of crustaceans lipoproteins thus appears to be more complex than reflected by the present state of knowledge.

Translation initiation in bacterial polysomes through ribosome loading on a standby site on a highly translated mRNA.

During translation, consecutive ribosomes load on an mRNA and form a polysome. The first ribosome binds to a single-stranded mRNA region and moves toward the start codon, unwinding potential mRNA structures on the way. In contrast, the following ribosomes can dock at the start codon only when the first ribosome has vacated the initiation site. Here we show that loading of the second ribosome on a natural 38-nt-long 5' untranslated region of lpp mRNA, which codes for the outer membrane lipoprotein from Escherichia coli, takes place before the leading ribosome has moved away from the start codon. The rapid formation of this standby complex depends on the presence of ribosomal proteins S1/S2 in the leading ribosome. The early recruitment of the second ribosome to the standby site before translation by the leading ribosome and the tight coupling between translation elongation by the first ribosome and the accommodation of the second ribosome can contribute to high translational efficiency of the lpp mRNA.

Construction of a eukaryotic expression system with stable and secretory expression of mycobacterium tuberculosis 38 kDa protein.

The 38 kDa protein is a major antigen of mycobacterium tuberculosis and has been widely used in TB serodiagnosis, due to its highly sensitivity and specificity. Here we attempt to establish a production platform of recombinant 38 kDa protein in mammalian cells and to evaluate the potential value of 38 kDa protein in TB serodiagnosis. The 38 kDa gene is synthesized and cloned into a lentiviral expressing vector. Recombinant lentiviral vector LV-CMV-38 kDa-eGFP was packaged, titered, and then transduced into HEK 293 T cells. Recombinant cell lines were selected by limiting dilution. Supernatants were collected and purified by HisTrapTM HP column. Western blot showed a molecular weight of approximate 38 kDa in cell supernatants as expected. ELISA assay confirmed the immunological specificity of the obtained protein in the presence of MTB-infected human serum samples. In all, we have obtained a stable cell line with long-term and robust expression of secretory MTB 38 kDa protein, which may provide a promising candidate antigen for the development of TB serological diagnosis.

Long-term efficacy of olipudase alfa in adults with acid sphingomyelinase deficiency (ASMD): Further clearance of hepatic sphingomyelin is associated with additional improvements in pro- and anti-atherogenic lipid profiles after 42 months of treatment.

The liver is a major site of lipoprotein synthesis and metabolism. Liver manifestations of chronic visceral ASMD include hepatomegaly, fibrosis, elevated liver enzymes and a pro-atherogenic lipid profile. Measurements of sphingomyelin (SM) levels in liver biopsies and lyso-SM in plasma were used as pharmacodynamic biomarkers. Five adult patients with chronic visceral ASMD were enrolled in a 26-week phase 1b trial of enzyme replacement therapy (ERT) with olipudase alfa (NCT01722526) followed by an ongoing long-term extension study (NCT02004704). We compare the changes in hepatic SM levels, plasma lyso-SM, and lipoprotein profiles after 42 months of treatment. Progressive clearance of histologic SM storage was observed throughout the trial, along with similar reductions in plasma lyso-SM. Improvements in liver enzymes were observed at 6 months and remained stable at 42 months. Progressive reductions from baseline in pro-atherogenic lipid profiles (total cholesterol, LDL-C, VLDL-C, triglycerides) were observed at month 6 and 42. Conversely, there were progressive increases in anti-atherogenic markers, HDL-C and apolipoprotein A-I, with HDL-C increases up to 200% over baseline levels after 42 months of treatment. These data demonstrate that hepatic clearance of SM during olipudase alfa treatment over 42 months is associated with overall improvements in the lipid profiles of ASMD patients. The clinical relevance of these findings needs to be determined in the future, but we speculate that these improvements may reduce the risk for liver cirrhosis and cardiovascular disease. Trial registration: Clintrials.gov trial registration # NCT01722526.

NAPE-PLD controls OEA synthesis and fat absorption by regulating lipoprotein synthesis in an in vitro model of intestinal epithelial cells.

Oleoylethanolamide (OEA), a fatty acid ethanolamide (FAE), is a lipid mediator that controls food intake and lipid metabolism. Accumulating data imply the importance of intestinal OEA in controlling satiety in addition to gastrointestinal peptide hormones. Although the biochemical pathway of FAE production has been illustrated, the enzymes responsible for the cleavage of OEA from its precursor N-acyl-phosphatidylethanolamine (NAPE) must be identified among reported candidates in the gut. In this study, we assessed the involvement of NAPE-specific phospholipase D (NAPE-PLD), which can directly release FAEs from NAPE, in intestinal OEA synthesis and lipid metabolism. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPER-associated protein 9 (Cas9)-mediated deletion of the NAPE-PLD gene in intestinal epithelial-like Caco-2 cells reduced OEA levels, regardless of their differentiation states. Transcriptome analysis revealed that deletion of NAPE-PLD activates a transcriptional program for nutrient transportation, including lipids and lipoproteins, and inactivates cell-cycle or mitosis-related genes in Caco-2 cells. In addition, the basolateral secretion of lipoproteins was increased in NAPE-PLD-deleted cells although lipoprotein size was not affected. By contrast, cellular lipid levels were reduced in NAPE-PLD-deleted cells. Overall, these results indicate that NAPE-PLD plays important roles in OEA synthesis and fat absorption by regulating lipoprotein production in the intestinal epithelial cells.-Igarashi, M., Watanabe, K., Tsuduki, T., Kimura, I., Kubota, N. NAPE-PLD controls OEA synthesis and fat absorption by regulating lipoprotein synthesis in an in vitro model of intestinal epithelial cells.

Characterization of protein interaction surface on fatty acyl selectivity of starter condensation domain in lipopeptide biosynthesis.

Lipopeptides are important non-ribosomal peptide synthetases (NRPSs) products with broad therapeutic potential in biotechnology and biopharmaceutical applications. Fatty acyl modifications in N-terminal of lipopeptides have attracted wide interest in the engineering processes of altered fatty acyl selectivity. In this study, we focused on the starter condensation domain of antibiotic A54145 (lptC1) and its indiscriminate selectivity of fatty acyl substrates, which results in multi-component products. Using in silico analysis, five site-directed mutations at protein-protein interface were identified with altered activity and selectivity towards wild type lptC1. The variants Y149W and A330T exhibited changed substrate selectivity to prefer longer branched chain fatty acyl substrate, while T16A and A350D showed improved selectivity for shorter linear chain fatty acyl substrates. Subsequently, molecular dynamics (MD) simulations were performed to analyze the impact of these residues on the changes of catalytic activity and conformation. Through in silico analysis, the altered binding free energy were coincident with the corresponding activity performance of the variants, and surface forces indicated that other factors or processes may influence the activity and selectivity. Moreover, the MD results revealed even altered active center conformations, implying the importance of these interface residues affected on distant active center thus reflected to catalysis activity. Based on the biochemistry and computational results, our work provides detailed insights from molecular and dynamics aspects into the role of C1's interface residues during complex NRPS biosynthesis machinery, prompting further rational engineering for lipopeptide catalysis.

Lipoproteins: Structure, Function, Biosynthesis.

The Lpp lipoprotein of Escherichia coli is the first identified protein with a covalently linked lipid. It is chemically bound by its C-terminus to murein (peptidoglycan) and inserts by the lipid at the N-terminus into the outer membrane. As the most abundant protein in E. coli (106 molecules per cell) it plays an important role for the integrity of the cell envelope. Lpp represents the type protein of a large variety of lipoproteins found in Gram-negative and Gram-positive bacteria and in archaea that have in common the lipid structure for anchoring the proteins to membranes but otherwise strongly vary in sequence, structure, and function. Predicted lipoproteins in known prokaryotic genomes comprise 2.7% of all proteins. Lipoproteins are modified by a unique phospholipid pathway and transferred from the cytoplasmic membrane into the outer membrane by a special system. They are involved in protein incorporation into the outer membrane, protein secretion across the cytoplasmic membrane, periplasm and outer membrane, signal transduction, conjugation, cell wall metabolism, antibiotic resistance, biofilm formation, and adhesion to host tissues. They are only found in bacteria and function as signal molecules for the innate immune system of vertebrates, where they cause inflammation and elicit innate and adaptive immune response through Toll-like receptors. This review discusses various aspects of Lpp and other lipoproteins of Gram-negative and Gram-positive bacteria and archaea.

Pyridoxal phosphate-dependent reactions in the biosynthesis of natural products.

Covering: up to mid-2018 Pyridoxal 5'-phosphate (PLP) is a versatile organic cofactor used to catalyze diverse reactions on amino acid, oxoacid, and amine substrates. Here we review the reactions catalyzed by PLP-dependent enzymes, highlighting enzymes reported in the natural product biosynthetic literature. We describe enzymes that catalyze transaminations, Claisen-like condensations, and ß- and γ-eliminations and substitutions, along with epimerizations, decarboxylations, and transaldolations. Finally, we describe a newly reported group of O2-, PLP-dependent enzymes. Altogether, natural product biosynthesis showcases the incredible versatility of PLP-dependent transformations for building chemical complexity.

ABCG5/G8: a structural view to pathophysiology of the hepatobiliary cholesterol secretion.

The ABCG5/G8 heterodimer is the primary neutral sterol transporter in hepatobiliary and transintestinal cholesterol excretion. Inactivating mutations on either the ABCG5 or ABCG8 subunit cause Sitosterolemia, a rare genetic disorder. In 2016, a crystal structure of human ABCG5/G8 in an apo state showed the first structural information on ATP-binding cassette (ABC) sterol transporters and revealed several structural features that were observed for the first time. Over the past decade, several missense variants of ABCG5/G8 have been associated with non-Sitosterolemia lipid phenotypes. In this review, we summarize recent pathophysiological and structural findings of ABCG5/G8, interpret the structure-function relationship in disease-causing variants and describe the available evidence that allows us to build a mechanistic view of ABCG5/G8-mediated sterol transport.

Variable Lipoprotein Hemagglutinin A Gene (vlhA) Expression in Variant Mycoplasma gallisepticum Strains In Vivo.

Mycoplasma gallisepticum, the primary etiologic agent of chronic respiratory disease, is a significant poultry pathogen, causing severe inflammation and leading to economic losses worldwide. Immunodominant proteins encoded by the variable lipoprotein and hemagglutinin (vlhA) gene family are thought to be important for M. gallisepticum-host interaction, pathogenesis, and immune evasion, but their exact role remains unknown. Previous work has demonstrated that vlhA phase variation is dynamic throughout the earliest stages of infection, with vlhA 3.03 being the predominant vlhA expressed during the initial infection, and that the pattern of dominant vlhA expression may be nonrandom and regulated by previously unrecognized mechanisms. To further investigate this gene family, we assessed the vlhA profile of two well-characterized vaccine strains, GT5 and Mg7, a vlhA 3.03 mutant strain, and an M. gallisepticum population expressing an alternative immunodominant vlhA Here, we report that two M. gallisepticum vaccine strains show different vlhA profiles over the first 2 days of infection compared to that of wild-type Rlow, while the population expressing an alternative immunodominant vlhA gene reverted to a profile indistinguishable from that of wild-type Rlow Additionally, we observed a slight shift in the vlhA gene expression profile but no reduction in virulence in a vlhA 3.03 mutant. Taken together, these data further support the hypothesis that M. gallisepticum vlhA genes change in a nonstochastic temporal progression of expression and that vlhA 3.03, while preferred, is not required for virulence. Collectively, these data may be important in elucidating mechanisms of colonization and overall pathogenesis of M. gallisepticum.

Transcriptional development of phospholipid and lipoprotein metabolism in different intestinal regions of Atlantic salmon (Salmo salar) fry.

BACKGROUND: It has been suggested that the high phospholipid (PL) requirement in Atlantic salmon (Salmo salar) fry is due to insufficient intestinal de-novo synthesis causing low lipoprotein (LP) production and reduced transport capacity of dietary lipids. However, in-depth ontogenetic analysis of intestinal PL and LP synthesis with the development of salmon has yet to be performed. Therefore, in this paper we used RNA-Seq technology to investigate the expression of genes involved in PL synthesis and LP formation throughout early developmental stages and associate insufficient expression of synthesis pathways in salmon fry with its higher dietary PL requirement. There was a special focus on the understanding homologous genes, especially those from salmonid-specific fourth vertebrate whole-genome duplication (Ss4R), and their contribution to salmonid specific features of regulation of PL metabolic pathways. Salmon fry were sampled at 0.16 g (1 day before first-feeding), 2.5 and 10 g stages of development and transcriptomic analysis was applied separately on stomach, pyloric caeca and hindgut of the fish. RESULTS: In general, we found up-regulated pathways involved in synthesis of phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), and LP in pyloric caeca of salmon between 0.16 and 10 g. Thirteen differentially expressed genes (q < 0.05) in these pathways were highly up-regulated in 2.5 g salmon compared to 0.16 g, while only five more differentially expressed (q < 0.05) genes were found when the fish grew up to 10 g. Different homologous genes were found dominating in stomach, pyloric caeca and hindgut. However, the expression of dominating genes in pathways of PL and LP synthesis were much higher in pyloric caeca than stomach and hindgut. Salmon-specific homologous genes (Ss4R) had similar expression during development, while other homologs had more diverged expression. CONCLUSIONS: The up-regulation of the de-novo PtdCho and PtdEtn pathways confirm that salmon have decreasing requirement for dietary PL as the fish develops. The similar expressions between Ss4R homologous genes suggest that the functional divergence of these genes was incomplete compared to homologs derived from other genome duplication. The results of the present study have provided new information on the molecular mechanisms of phospholipid synthesis and lipoprotein formation in fish.

Novel role of Vpmas as major adhesins of Mycoplasma agalactiae mediating differential cell adhesion and invasion of Vpma expression variants.

Mycoplasma agalactiae exhibits antigenic variation by switching the expression of multiple surface lipoproteins called Vpmas. Although implicated to have a significant influence on the pathogenicity, their exact role in pathogen-host interactions has not been investigated so far. Initial attachment to host cells is regarded as one of the most important steps for colonization but this pathogen lacks the typical mycoplasma attachment organelle. The aim of this study was to determine the role of Vpmas in adhesion of M. agalactiae to host cells. 'Phase-Locked' Mutants (PLMs) steadily expressing single well-characterized Vpma lipoproteins served as ideal tools to evaluate the role of each of the six Vpmas in cytadhesion, which was otherwise not possible due to the high-frequency switching of Vpmas in the wildtype strain PG2. Using in vitro adhesion assays with HeLa and sheep mammary epithelial (MECs) and stromal (MSCs) cells, we could demonstrate differences in the adhesion capabilities of each of the six PLMs compared to the wildtype strain. The PLMV mutant expressing VpmaV exhibited the highest adhesion rate, whereas PLMU, which expresses VpmaU showed the lowest adhesion values explaining the reduced in vivo fitness of PLMU in sheep during experimental intramammary and conjunctival infections. Furthermore, adhesion inhibition assays using Vpma-specific polyclonal antisera were performed to confirm the role of Vpmas in M. agalactiae cytadhesion. This led to a significant decrease (p<0.05) in the adhesion percentage of each PLM. Immunofluorescence staining of TX-114 phase proteins extracted from each PLM showed binding of the respective Vpma to HeLa cells and MECs proving the direct role of Vpmas in cytadhesion. Furthermore, as adhesion is a prerequisite for cell invasion, the ability of the six PLMs to invade HeLa cells was also evaluated using the gentamicin protection assay. The results showed a strong correlation between the adhesion rates and invasion frequencies of the individual PLMs. This is the first report that describes a novel function of Vpma proteins in cell adhesion and invasion. Besides the variability of these proteins causing surface antigenic variation, the newly identified phenotypes are likely to play critical roles in the pathogenicity potential of this ruminant pathogen.

A Novel Nonantibiotic, lgt-Based Selection System for Stable Maintenance of Expression Vectors in Escherichia coli and Vibrio cholerae.

Antibiotic selection for the maintenance of expression plasmids is discouraged in the production of recombinant proteins for pharmaceutical or other human uses due to the risks of antibiotic residue contamination of the final products and the release of DNA encoding antibiotic resistance into the environment. We describe the construction of expression plasmids that are instead maintained by complementation of the lgt gene encoding a (pro)lipoprotein glyceryl transferase essential for the biosynthesis of bacterial lipoprotein. Mutations in lgt are lethal in Escherichia coli and other Gram-negative organisms. The lgt gene was deleted from E. coli and complemented by the Vibrio cholerae-derived gene provided in trans on a temperature-sensitive plasmid, allowing cells to grow at 30°C but not at 37°C. A temperature-insensitive expression vector carrying the V. cholerae-derived lgt gene was constructed, whereby transformants were selected by growth at 39°C. The vector was successfully used to express two recombinant proteins, one soluble and one forming insoluble inclusion bodies. Reciprocal construction was done by deleting the lgt gene from V. cholerae and complementing the lesion with the corresponding gene from E. coli The resulting strain was used to produce the secreted recombinant cholera toxin B subunit (CTB) protein, a component of licensed as well as newly developed oral cholera vaccines. Overall, the lgt system described here confers extreme stability on expression plasmids, and this strategy can be easily transferred to other Gram-negative species using the E. coli-derived lgt gene for complementation.IMPORTANCE Many recombinant proteins are produced in bacteria from genes carried on autonomously replicating DNA elements called plasmids. These plasmids are usually inherently unstable and rapidly lost. This can be prevented by using genes encoding antibiotic resistance. Plasmids are thus maintained by allowing only plasmid-containing cells to survive when the bacteria are grown in medium supplemented with antibiotics. In the described antibiotic-free system for the production of recombinant proteins, an essential gene is deleted from the bacterial chromosome and instead provided on a plasmid. The loss of the plasmid becomes lethal for the bacteria. Such plasmids can be used for the expression of recombinant proteins. This broadly applicable system removes the need for antibiotics in recombinant protein production, thereby contributing to reducing the spread of genes encoding antibiotic resistance, reducing the release of antibiotics into the environment, and freeing the final products (often used in pharmaceuticals) from contamination with potentially harmful antibiotic residues.

Lipoprotein LprI of Mycobacterium tuberculosis Acts as a Lysozyme Inhibitor.

Mycobacterium tuberculosis executes numerous defense strategies for the successful establishment of infection under a diverse array of challenges inside the host. One such strategy that has been delineated in this study is the abrogation of lytic activity of lysozyme by a novel glycosylated and surface-localized lipoprotein, LprI, which is exclusively present in M. tuberculosis complex. The lprI gene co-transcribes with the glbN gene (encoding hemoglobin (HbN)) and both are synchronously up-regulated in M. tuberculosis during macrophage infection. Recombinant LprI, expressed in Escherichia coli, exhibited strong binding (Kd ≤ 2 nm) with lysozyme and abrogated its lytic activity completely, thereby conferring protection to fluorescein-labeled Micrococcus lysodeikticus from lysozyme-mediated hydrolysis. Expression of the lprI gene in Mycobacterium smegmatis (8-10-fold) protected its growth from lysozyme inhibition in vitro and enhanced its phagocytosis and survival during intracellular infection of peritoneal and monocyte-derived macrophages, known to secrete lysozyme, and in the presence of exogenously added lysozyme in secondary cell lines where lysozyme levels are low. In contrast, the presence of HbN enhanced phagocytosis and intracellular survival of M. smegmatis only in the absence of lysozyme but not under lysozyme stress. Interestingly, co-expression of the glbN-lprI gene pair elevated the invasion and survival of M. smegmatis 2-3-fold in secondary cell lines in the presence of lysozyme in comparison with isogenic cells expressing these genes individually. Thus, specific advantage against macrophage-generated lysozyme, conferred by the combination of LprI-HbN during invasion of M. tuberculosis, may have vital implications on the pathogenesis of tuberculosis.

EPAS1/HIF-2 alpha-mediated downregulation of tissue factor pathway inhibitor leads to a pro-thrombotic potential in endothelial cells.

Neovascularization and hemorrhaging are evident in advanced atherosclerotic plaques due to hypoxic conditions, and mediate the accumulation of metabolic substrates, inflammatory cells, lipids, and other blood born factors inside the plaque. Tissue factor (TF) pathway inhibitor (TFPI) is mainly expressed by endothelial cells and is the endogenous inhibitor of the coagulation activator TF, which together with the downstream product thrombin can drive plaque progression and atherogenesis. We aimed to investigate the effect of hypoxic conditions on endothelial cell expression and activity of TFPI and TF that are important in coagulation initiation. Hypoxia was induced in primary human umbilical vein endothelial cells using chemicals or 1% oxygen tension, and mRNA and protein expressions were measured using qRT-PCR, ELISA, and Western blot analysis. Microscopy of fluorescence-labeled cells was used to visualize cell-associated TFPI. Cell-surface factor Xa (FXa) activity was measured using a two-stage chromogenic substrate method. We found that hypoxia reduced the TFPI mRNA and protein levels and increased the TF mRNA expression in a dose-dependent manner. The effect on TFPI was apparent on all the protein pools of TFPI, i.e., secreted TFPI, cell-surface associated TFPI, and intracellular TFPI, and seemed to be dependent upon hypoxia inducible factor-2α (HIF-2α). An increase in FXa activity was also observed on the endothelial cell surface, reflecting an increase in pro-thrombotic potential of the cells. Our findings indicate that hypoxic conditions may enhance the pro-coagulant activity of endothelial cells, which may promote atherogenesis in addition to clinical events and thus the severity of atherosclerotic disorders.

Bacterial lipoproteins; biogenesis, sorting and quality control.

Bacterial lipoproteins are a subset of membrane proteins localized on either leaflet of the lipid bilayer. These proteins are anchored to membranes through their N-terminal lipid moiety attached to a conserved Cys. Since the protein moiety of most lipoproteins is hydrophilic, they are expected to play various roles in a hydrophilic environment outside the cytoplasmic membrane. Gram-negative bacteria such as Escherichia coli possess an outer membrane, to which most lipoproteins are sorted. The Lol pathway plays a central role in the sorting of lipoproteins to the outer membrane after lipoprotein precursors are processed to mature forms in the cytoplasmic membrane. Most lipoproteins are anchored to the inner leaflet of the outer membrane with their protein moiety in the periplasm. However, recent studies indicated that some lipoproteins further undergo topology change in the outer membrane, and play critical roles in the biogenesis and quality control of the outer membrane. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.

Lichenysin production is improved in codY null Bacillus licheniformis by addition of precursor amino acids.

Lichenysin is categorized into the family of lipopeptide biosurfactants and has a variety of applications in the petroleum industry, bioremediation, pharmaceuticals, and the food industry. Currently, large-scale production is limited due to the low yield. This study found that lichenysin production was repressed by supplementation of extracellular amino acids. The global transcriptional factor CodY was hypothesized to prevent lichenysin biosynthesis under an amino acid-rich condition in Bacillus licheniformis. Thus, the codY null strain was constructed, and lichenysin production was increased by 31.0% to 2356 mg/L with the addition of precursor amino acids, and the lichenysin production efficiency was improved by 42.8% to 98.2 mg/L• h. Correspondingly, the transcription levels of the lichenysin synthetase gene lchAA, and its corresponding regulator genes comA, degQ, and degU, were upregulated. Also, the codY deletion enhanced biosynthesis of lichenysin precursor amino acids (Gln, Ile, Leu, and Val) and reduced the formation of byproducts, acetate, acetoin, and 2,3-butanediol. This study firstly reported that lichenysin biosynthesis was negatively regulated by CodY and lichenysin production could be further improved with the precursor amino acid amendment in the codY null strain.