CRISPRi-mediated characterization of novel anti-tuberculosis targets: Mycobacterial peptidoglycan modifications promote beta-lactam resistance and intracellular survival.

The lack of effective therapeutics against emerging multi-drug resistant strains of Mycobacterium tuberculosis (Mtb) prompts the identification of novel anti-tuberculosis targets. The essential nature of the peptidoglycan (PG) layer of the mycobacterial cell wall, which features several distinctive modifications, such as the N-glycolylation of muramic acid and the amidation of D-iso-glutamate, makes it a target of particular interest. To understand their role in susceptibility to beta-lactams and in the modulation of host-pathogen interactions, the genes encoding the enzymes responsible for these PG modifications (namH and murT/gatD, respectively) were silenced in the model organism Mycobacterium smegmatis using CRISPR interference (CRISPRi). Although beta-lactams are not included in TB-therapy, their combination with beta-lactamase inhibitors is a prospective strategy to treat MDR-TB. To uncover synergistic effects between the action of beta-lactams and the depletion of these PG modifications, knockdown mutants were also constructed in strains lacking the major beta-lactamase of M. smegmatis BlaS, PM965 (M. smegmatis ΔblaS1) and PM979 (M. smegmatis ΔblaS1 ΔnamH). The phenotyping assays affirmed the essentiality of the amidation of D-iso-glutamate to the survival of mycobacteria, as opposed to the N-glycolylation of muramic acid. The qRT-PCR assays confirmed the successful repression of the target genes, along with few polar effects and differential knockdown level depending on PAM strength and target site. Both PG modifications were found to contribute to beta-lactam resistance. While the amidation of D-iso-glutamate impacted cefotaxime and isoniazid resistance, the N-glycolylation of muramic acid substantially promoted resistance to the tested beta-lactams. Their simultaneous depletion provoked synergistic reductions in beta-lactam MICs. Moreover, the depletion of these PG modifications promoted a significantly faster bacilli killing by J774 macrophages. Whole-genome sequencing revealed that these PG modifications are highly conserved in a set of 172 clinical strains of Mtb, demonstrating their potential as therapeutic targets against TB. Our results support the development of new therapeutic agents targeting these distinctive mycobacterial PG modifications.

Dietary muramidase increases ileal amino acid digestibility of wheat and corn-based broiler diets without affecting endogenous amino acid losses.

The objective of these studies was to evaluate the impact of dietary muramidase (MUR) on endogenous amino acids (AA) losses and digestibility of nutrients in wheat and corn-based broiler diets. In experiment 1, the effect of dietary MUR on the flow of endogenous AA (EAA) at the jejunum and terminal ileum of broilers were assessed using either the nitrogen (N) free diet method (NFD) or the highly digestible protein diet method (HDP; 100 g casein/kg diet). Sialic acid and muramic acid concentrations were measured in the jejunal content. In experiment 2, a 2x2x2 factorial arrangement of treatments with 2 base grains (wheat or corn), with low or high metabolizable energy (ME) levels, and without or with MUR supplementation was implemented. All diets contained phytase, xylanase, and cellulase. Apparent ileal digestibility (AID) of dry matter (DM), protein (CP), amino acids (AA), crude fat, and energy, as well as the apparent total tract metabolizability (ATTM) of DM, CP, and gross energy (GE) were determined. The standardized ileal digestibility (SID) of AA was obtained by correcting AID values for basal ileal EAA obtained from chicks fed with NFD or HDP in experiment 1, jejunal EAA flow of all AA was higher (P < 0.001) compared to the ileum, but this effect was method dependent. Jejunal, but not ileal, EAA flow measured with HDP was higher compared to NFD, as well as sialic acid (P < 0.001) and muramic acid (P < 0.004) concentrations. Muramidase inclusion had no effect on basal EAA flow, independently of the segment and the method used. In experiment 2, dietary MUR supplementation increased the AID of CP (P < 0.05), all AA, and tended (P = 0.07) to increase the AID of GE, independently of the cereal type used. However, ATTM of DM and GE, but not CP, increased with MUR inclusion compared with the control treatments, especially in wheat and low ME diets (P < 0.05). In conclusion, MUR supplementation improved AID of CP and AA without affecting EAA losses and increases energy utilization.

Selective Monovalent Galectin-8 Ligands Based on 3-Lactoylgalactoside.

Galectin-8 has gained attention as a potential new pharmacological target for the treatment of various diseases, including cancer, inflammation, and disorders associated with bone mass reduction. To that end, new molecular probes are needed in order to better understand its role and its functions. Herein we aimed to improve the affinity and target selectivity of a recently published galectin-8 ligand, 3-O-[1-carboxyethyl]-ß-d-galactopyranoside, by introducing modifications at positions 1 and 3 of the galactose. Affinity data measured by fluorescence polarization show that the most potent compound reached a KD of 12 µM. Furthermore, reasonable selectivity versus other galectins was achieved, making the highlighted compound a promising lead for the development of new selective and potent ligands for galectin-8 as molecular probes to examine the protein's role in cell-based and in vivo studies.

Design and synthesis of novel antimicrobial peptide scaffolds.

Muramic acid (Mur), a sugar amino acid (SAA), is present in the cell walls of bacteria asN-acetyl muramic acid (MurNAc) where together with ofN-acetylglucosamine (GlcNAc) and peptide makes main building block of peptidoglycan (PGN). It was challenging to incorporate muramic acid as SAA characteristic for bacteria into the peptides and investigate the antimicrobial activity of these scaffolds. Four building units were used in designing the desired peptide: muramic acid, tetrapeptide Leu-Ser-Lys-Leu, Nε-Lys, and Asn. Positions of three components were changeable while the position of Asn was always C-terminal (in linear peptides). The glycopeptide libraries of linear and cyclic peptides were synthesized using solid-phase peptide synthesis (SPPS). The antimicrobial effect of linear and cyclic glycopeptides, as well as the LSKL sequence used as a control, was investigated on several standard laboratory microbial strains. Liner glycopeptide with sequences Leu-Ser-Lys-Leu-Nε-Lys-Mur-Asn was active onStaphylococcus aureus(Gram-positive bacteria). Prepared compounds did not show activity towards applied tumor and normal human cell lines.

1,6-AnhMurNAc derivatives for assay development of amidase AmiD.

Various peptidoglycan fragments were synthesized from two anhydro-muramic acid derivatives protected with a Bn or a PMB group at the 4th position, in homogenate phase or on a solid support. In order to facilitate HPLC detection, a chromophoric group was attached to the peptide chain. The periplasmic amidase sAmiD of Escherichia coli was used to cleave the amide bond between the lactyl group of the MurNAc and the α-amino group of L-Ala where the peptide chain was at least a dipeptide (L-Ala-γ-D-Glu) amidated by benzylamine on the γ-carboxyl group of D-Glu. In the presence of a tripeptide chain (L-Ala-γ-D-Glu-L-Lys) or a tetrapeptide chain (L-Ala-γ-D-Glu-m-A(2)pm-D-Ala) higher hydrolysis rates were observed. We have also demonstrated that the presence of TNB on the ε-amino group of L-Lys only has a small influence on the hydrolysis capacity of sAmiD.

Synthesis and biological evaluation of analogues of bacterial lipid I.

Bacterial Lipid I analogues containing different anomeric groups at the muramic acid moiety were synthesized and screened in MurG enzyme assays run in the presence and absence of cell wall membranes. The results obtained in this study help elucidate the role of the lipid diphosphate in the recognition of Lipid I by MurG.

Effects of muramyl peptides on macrophages, monokines, and sleep.

Muramyl peptides are fragments of peptidoglycan from the cell walls of bacteria. Because of their unique chemistry, the immune system recognizes that muramyl peptides are products of bacteria, and it responds by becoming activated to resist infection. This resistance to infection is nonspecific, and extends to unrelated species of bacteria, fungi, and viruses. A key mechanism of the resistance to infection is activation of macrophages. Macrophage activation results in increased production of microbicidal oxygen radicals like superoxide and peroxide, and in increased secretion of inflammatory cytokines like interleukin-1beta and tumor necrosis factor-alpha. These cytokines, besides activating neutrophils, B lymphocytes, and T lymphocytes, act on the central nervous system to induce physiological responses like fever and sleep. These physiological responses also aid in combating infection. Muramyl peptides also activate macrophages and other cells of the immune system to kill cancer cells. Muramyl peptides and similar agents will become more important as therapeutic agents in the future, due to increasing resistance of microbes to antibiotics, and increasing numbers of patients with immunodeficiencies.

Teichuronic acid operon of Bacillus subtilis 168.

Sequence analysis reveals that the Bacillus subtilis 168 tuaABCDEFGH operon encodes enzymes required for the polymerization of teichuronic acid as well as for the synthesis of one of its precursors, the UDP-glucuronate. Mutants deficient in any of the tua genes, grown in batch cultures under conditions of phosphate limitation, were characterized by reduced amounts of uronate in their cell walls. The teichuronic acid operon belongs to the Pho regulon, as phosphate limitation induces its transcription. Placing the tuaABCDEFGH operon under the control of the inducible Pspac promoter allowed its constitutive expression independently of the phosphate concentration in the medium; the level of uronic acid in cell walls was dependent on the concentration of the inducer. Apparently, owing to an interdependence between teichoic and teichuronic acid incorporation into the cell wall, in examined growth conditions, the balance between the two polymers is maintained in order to insure a constant level of the wall negative charge.

Structures of biologically active muramyl peptides from peptidoglycan of Streptococcus sanguis.

The structures of major muramyl peptides derived from peptidoglycan of the oral pathogen Streptococcus sanguis were determined and the biological activity of the peptides was tested in vitro on human monocytes. The muramyl peptides, produced by muramidase digestion of the purified peptidoglycan, were separated by reversed-phase high-performance liquid chromatography, either in their native form or after reduction with sodium borohydride. Chemical structures of the peptides were elucidated by a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, amino acid analysis, post-source decay analysis and Edman sequencing. The study revealed two distinct monomers: N-acetylglucosaminyl-N-acetylmuramyl-Ala-iGln-Lys(Ala-Ala) (1), where the Ala-Ala is connected to the epsilon-amino group of lysine, and N-acetylglucosaminyl-N-acetylmuramyl-Ala-iGln-Lys(Ala-Ala)-Ala-Ala (2), where an additional dialanyl residue is attached to the lysine alpha-carboxyl group. Two sets of higher oligomers (di-, tri- and tetramers), related structurally to monomers 1 or 2 were also detected. In these oligomers, the monomeric subunits are linked together by Ala-Ala-Ala bridges. The native muramyl peptides primed human monocytes in vitro for the increased production of the microbicidal superoxide radical.

CD11a/CD18 and CD11b/18 modulation by lipoteichoic acid, N-acetyl-muramyl-alpha-alanyl-D-isoglutamine, muramic acid and protein A from Staphylococcus aureus.

This study investigates the effect of some components of the Staphylococcus aureus cell wall [lipoteichoic acid (LTA), N-acetyl-muramyl-alanyl-D-isoglutamine (MD), muramic acid (MA) and protein A (PA)] in modulating expression of cell-surface adhesion molecules CD11a/CD18, CD11b/C18 on monocytes qualitatively and quantitatively. Monocytes incubated with bacterial components presented different CD11b/CD18 expressions which were dose-dependent in contrast to controls. The results obtained demonstrated that lymphocytes incubated with bacterial components also increased the expression of CD11a/CD18. The modifications in activation of CD11a/CD18 and CD11b/CD18 expression are probably correlated with modifications of membrane fluidity measured as polarisation fluorescence (P).

Relationship between NO-synthase activity and TNF-alpha secretion in mouse macrophage lines stimulated by a muramyl peptide entrapped in nanocapsules.

The present study evaluates the ability of a new drug carrier: nanocapsules of poly(D,L-lactide) containing muramyldipeptide-L-alanyl-cholesterol (MTP-Chol NC) to induce activation of mouse macrophage cell lines. MTP-Chol NC stimulated nitric oxide (NO) expression and tumor necrosis factor-alpha (TNF-alpha) production, these are two important mediators of macrophage-mediated cytotoxicity. The encapsulated form was more effective than free muramyldipeptide, at low immunomodulator concentrations. The dose-response curves were completely different for NO and TNF-alpha, implying different regulatory mechanisms. In RAW 264.7 cells, the addition of anti-TNF-alpha antibodies during the activation period did not affect the level of nitrite induced by MTP-Chol Nc and lipopolysaccharide. Therefore, autocrine stimulation by TNF-alpha did not contribute to NO production. On the other hand, the presence of an NO synthase inhibitor led to an increase in TNF-alpha secretion. In J774.A1 cells, which were activated by MTP-Chol NC and interferon-gamma, TNF-alpha production seemed to act as a second messenger. Thus, under certain conditions, NO can play a role in modulating the cytotoxic activities of mouse macrophages.

Selective induction of CD11a,b,c/CD18 and CD54 expression at the cell surface of human leukocytes by muramyl peptides.

Muramyl dipeptide (MDP), murametide, and murabutide which belong to the family of the immunoadjuvant muramyl dipeptides were applied directly to fresh human whole blood and the expression of some surface markers involved in cell adherence in distinct leukocyte populations was investigated. CD11a,b,c/CD18, CD54, CD49d were selected for their involvement in cell adherence, and transferrin receptor (CD71) and low-affinity IgE receptor (CD23) were selected as markers for activated cells. Whereas CD11a was increased only on monocytes, CD11b, CD11c, and CD18 were strongly enhanced on monocytes and polymorphonuclear cells (PMNs) after treatment with MDPs. This increase in membrane expression of integrins, such as CD11b, was not associated with mRNA synthesis, suggesting a mobilization of the CD11b,c/CD18 intracellular pools present in these cells. In contrast, treatment with MDP, murametide, or murabutide enhanced ICAM-1 (CD54) expression on monocyte and PMN cell surface in association with ICAM-1 mRNA synthesis. No variation of CD49d expression was detected on leukocyte surface after incubation with MDPs. Transferrin receptor (CD71) expression and low-affinity receptor for IgE (CD23) expression were increased on monocyte only after incubation with LPS used as positive control. Moreover, no observable change in the selected markers was detected on lymphocyte after MDPs or LPS treatment. These results indicate that MDPs seem to act preferentially on monocytes and PMNs in increasing the level of molecules involved in cellular adhesion process, either in provoking the expression of preformed molecules or in inducing their synthesis. This contributes to understanding the mechanism of the activities of muramyl peptides on specific and nonspecific immunity.

Cytokines in sleep regulation.

The central thesis of this essay is that the cytokine network in brain is a key element in the humoral regulation of sleep responses to infection and in the physiological regulation of sleep. We hypothesize that many cytokines, their cellular receptors, soluble receptors, and endogenous antagonists are involved in physiological sleep regulation. The expressions of some cytokines are greatly amplified by microbial challenge. This excess cytokine production during infection induces sleep responses. The excessive sleep and wakefulness that occur at different times during the course of the infectious process results from dynamic changes in various cytokines that occur during the host's response to infectious challenge. Removal of any one somnogenic cytokine inhibits normal sleep, alters the cytokine network by changing the cytokine mix, but does not completely disrupt sleep due to the redundant nature of the cytokine network. The cytokine network operates in a paracrine/autocrine fashion and is responsive to neuronal use. Finally, cytokines elicit their somnogenic actions via endocrine and neurotransmitter systems as well as having direct effects neurons and glia. Evidence in support of these postulates is reviewed in this essay.

Protein A and other surface components of Staphylococcus aureus stimulate production of IL-1 alpha, IL-4, IL-6, TNF and IFN-gamma.

Studies were carried out on the ability of protein A (PA) and of muramic acid (MA) from S. aureus to induce the release of cytokines both from monocytes and lymphocytes in vitro. Results show that protein A induces the greatest activity, compared to the activity already known for the theicoic acid (TA) and for muramyl dipeptide (MDP). At concentration of 10 micrograms/ml; PA induces roughly +180% release of TNF with respect to controls, while release of IL-1 alpha is about 500% control values, and is higher than those obtained when cells are treated with TA and MDP; IL-6 release is higher than that stimulated by Con A, used as standard challenge. At PA concentrations of 5 micrograms/ml, IL-4 release is about five times higher than that induced by Con A. Release of IFN-gamma showed similar dose-dependent stimulations. Muramic acid (MA) is particularly active in inducing the release of cytokines from target cells, inducing TNF release of about +75% with respect to the controls. This increase is less than that obtained with PA. Also IL-4 and IFN-gamma are released by PA in quantities higher than those induced by TA and MDP. Our results lead us to believe that during infections by Gram-positive bacteria, their surface components are able to induce a series of chain reactions ranging from the inflammatory to the immunologic responses which are also conditioned by release of cytokines.

N-acetylmuramic acid inhibits spore germination and germination enzymes.

This report describes the inhibition of spore germination by N-acetylmuramic acid. At concentrations of 20 mM and higher, the rate of germination of the treated spores was ten-fold lower than that of the control spores. The inhibitor also depressed the activity of two germination-related enzymes; a coat-associated hexosaminidase and a core enzyme. These findings suggests that N-acetylmuramic acid affects spore germination by inhibition of the corticolytic enzymes. The results are discussed with respect to the possible role of these enzymes in germination.

Dependence of proliferation of Bacteroides forsythus on exogenous N-acetylmuramic acid.

Bacteroides forsythus is the first wild-type or mutant bacterium found to require exogenous N-acetylmuramic acid for proliferation and maintenance of cell shape. This implies so far unknown pathways for peptidoglycan synthesis and a strict dependence of B. forsythus on other bacteria in its oral habitat. Addition of N-acetylmuramic acid to conventional bacteriological media allows routine cultivation of this fastidious organism.

Induction of coordinated movement of Myxococcus xanthus cells.

Rhythmically advancing waves of cells, called ripples, arise spontaneously during the aggregation of Myxococcus xanthus into fruiting bodies. Extracts prepared by washing rippling cells contain a substance that will induce quiescent cells to ripple. Three lines of evidence indicate that murein (peptidoglycan) is the ripple-inducing substance in the extracts. First, ripple-inducing activity is associated with the cell envelope of sonically disrupted M. xanthus cells. Second, whole cells, cell extracts, or purified murein from a variety of different bacteria are capable of inducing ripples. In contrast, extracts prepared from Methanobacterium spp. which contain pseudomurein instead of typical bacterial murein fail to induce ripples. Third, four components of M. xanthus murein, N-acetylglucosamine, N-acetylmuramic acid, diaminopimelate, and D-alanine, are able to induce ripples. Ripples produced by aggregating cells have a wavelength of 45 micrometers and a maximum velocity of 2 micrometers/min. Both of the multigene systems that control gliding motility appear to be required for rippling, and all known mutations at the spoC locus eliminate both rippling and sporulation.

Murein components rescue developmental sporulation of Myxococcus xanthus.

Murein (peptidoglycan) components are able to rescue sporulation in certain sporulation-defective mutants of Myxococcus xanthus. N-Acetylglucosamine, N-acetylmuramic acid, diaminopimelic acid, and D-alanine each increase the number of spores produced by SpoC mutants. When all four components are included they have a synergistic effect, raising the number of spores produced by SpoC mutants to the wild-type level. Murein-rescued spores are resistant to heat and sonic oscillation and germinate when plated on a nutrient-rich medium. They appear to be identical to fruiting body spores in their ultrastructure, in their protein composition, and in their resistance to boiling sodium dodecyl sulfate. Murein rescue of sporulation, like fruiting body sporulation, requires high cell density, a low nutrient level, and a solid surface.