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Purpose: To investigate the light transmission (LT) of UV-A and green light through infected corneas saturated with riboflavin or rose bengal in an ex vivo porcine model for infectious keratitis., Setting: University of Zurich and EMPA., Design: Laboratory study., Methods: Ex vivo porcine eyes (n=162) were divided into three groups: control eyes, eyes infected with Staphylococcus aureus, and eyes infected with Pseudomonas aeruginosa. Corneas remained either uninfected, or were infected with S. aureus, and P. aeruginosa, respectively, and were either left untreated, or were instilled with 0.1% riboflavin or 0.1% rose bengal. Corneal buttons were prepared, and corneal LT was measured at 365 nm and 522 nm using a spectrophotometer. LTs were calculated and compared. Transmission electron microscopy (TEM) was used to visualize structural damage and bacteria within infected corneas., Results: Riboflavin-saturated corneas infected by S. aureus or P. aeruginosa (LT = 0.77% [0.41-1.87] and 0.81% [0.23, 1.46]) exhibited 3.18-fold and 3.02-fold lower LTs than uninfected corneas (LT = 2.45% [2.15, 5.89]) (both p-values < 0.001). No LT difference was found between rose bengal-saturated corneas infected by S. aureus or P. aeruginosa and uninfected corneas (all LTs = 0.01% [0.01-0.01]; both p-values = 0.08). TEM showed bacteria on corneal stroma borders and occasionally inside the stroma., Conclusion: Our results indicate that the amount of light arriving at the corneal endothelium is substantially reduced in infected corneas. The total fluence of clinical PACK-CXL protocols can be safely increased substantially while maintaining a low risk of corneal endothelial damage., (Copyright © 2024 Published by Wolters Kluwer on behalf of ASCRS and ESCRS.)

Background: Patients with hemorrhagic stroke and an external ventricular drain in situ are at risk for ventriculostomy-related-infections (VRI). Because of the contamination of the cerebrospinal fluid (CSF) with blood and the high frequency of false negative CSF culture, the diagnosis of VRI remains challenging. This study investigated the introduction of CSF broad range eubacterial polymerase chain reaction (ePCR) and its effect on frequency and duration of antibiotic therapy for VRI, neurocritical care unit (NCCU) length of stay, related costs, and outcome., Methods: Between 2020 and 2022, we prospectively included 193 patients admitted to the NCCU of the University Hospital of Zürich with hemorrhagic stroke and an external ventricular drain for more than 48 h. Patient characteristics, serum inflammatory markers, white blood cell count in CSF, use and duration of antibiotic treatment for VRI, microbiological findings (CSF cultures and ePCR tests), and NCCU length of stay were compared in patients with no infection, noncerebral infection, suspected VRI, and confirmed VRI. Data of patients with suspected VRI of this cohort were compared with a retrospective cohort of patients with suspected VRI treated at our NCCU before the introduction of CSF ePCR testing (2013-2019)., Results: Out of 193 patients, 12 (6%) were diagnosed with a confirmed VRI, 66 (34%) with suspected VRI, 90 (47%) with a noncerebral infection, and 25 (13%) had no infection at all. Compared with the retrospective cohort of patients, the use of CSF ePCR resulted in a reduction of patients treated for suspected VRI for the whole duration of 14 days (from 51 to 11%). Furthermore, compared with the retrospective group of patients with suspected VRI (n = 67), after the introduction of CSF ePCR, patients with suspected VRI had shorter antibiotic treatment duration of almost 10 days and, hence, lower related costs with comparable outcome at 3 months., Conclusions: The use of CSF ePCR to identify VRI resulted in shorter antibiotic treatment duration without changing the outcome, as compared with a retrospective cohort of patients with suspected VRI., (© 2023. The Author(s).)

Purpose: To investigate and compare the efficacy of high-fluence accelerated photoactivated chromophore for keratitis-corneal cross-linking (PACK-CXL) using either riboflavin/ultraviolet (UV)-A light or rose bengal/green light to treat Staphylococcus aureus or Pseudomonas aeruginosa infections in an ex vivo porcine cornea model., Methods: One hundred and seventeen ex vivo porcine corneas were injected with clinical isolates of S. aureus or P. aeruginosa, divided into eight groups, and cultured for 24 hours. Then, either riboflavin with UV-A light irradiation (30 mW/cm2; 8 minutes, 20 seconds; 15 J/cm2) or rose bengal with green light irradiation (15 mW/cm2, 16 minutes, 40 seconds; 15 J/cm2) was applied; unirradiated infected groups served as controls. All corneas were incubated for another 24 hours. Next, corneal buttons were obtained and vortexed to release the bacterial cells. The irradiated and unirradiated solutions were then plated and incubated on agar plates. The amount of colony-forming units was quantified and the bacterial killing ratios (BKRs) resulting from different PACK-CXL protocols relative to non-treated controls were calculated., Results: Riboflavin/UV-A light PACK-CXL resulted in median BKRs of 52.8% and 45.8% in S. aureus and P. aeruginosa, respectively, whereas rose bengal/green light PACK-CXL resulted in significantly greater BKRs of 76.7% and 81.0%, respectively (both P < 0.01)., Conclusions: Both accelerated PACK-CXL protocols significantly decreased S. aureus and P. aeruginosa bacterial loads. Comparing the riboflavin/UV-A light and rose bengal/green light PACK-CXL approaches in the same experimental setup may help develop strain-specific and depth-dependent PACK-CXL approaches that could be used alongside the current standard of care., Translational Relevance: Our study used an animal model to gain insight into the efficacy of high-fluence accelerated PACK-CXL using either riboflavin/UV-A light or rose bengal/green light to treat Staphylococcus aureus or Pseudomonas aeruginosa infections.

The rapid detection and species-level differentiation of bacterial pathogens facilitates antibiotic stewardship and improves disease management. Here, we develop a rapid bacteriophage-based diagnostic assay to detect the most prevalent pathogens causing urinary tract infections: Escherichia coli, Enterococcus spp., and Klebsiella spp. For each uropathogen, two virulent phages were genetically engineered to express a nanoluciferase reporter gene upon host infection. Using 206 patient urine samples, reporter phage-induced bioluminescence was quantified to identify bacteriuria and the assay was benchmarked against conventional urinalysis. Overall, E. coli, Enterococcus spp., and Klebsiella spp. were each detected with high sensitivity (68%, 78%, 87%), specificity (99%, 99%, 99%), and accuracy (90%, 94%, 98%) at a resolution of ≥10 3  CFU/ml within 5 h. We further demonstrate how bioluminescence in urine can be used to predict phage antibacterial activity, demonstrating the future potential of reporter phages as companion diagnostics that guide patient-phage matching prior to therapeutic phage application., (© 2023. The Author(s).)

Introduction: Streptococcus agalactiae, also known as group B streptococci (GBS), is associated with invasive infections in neonates. Identification of GBS vaginal colonization in pregnant women before delivery is essential for treatment with antibiotics to prevent intrapartum vertical transmission to the newborn. This study was designed to evaluate applicability of two rapid real-time PCRs in comparison to standard culture identification., Material and Methods: We compared the Xpert GBS assay, hereafter referred to as Xpert, and GenomEra GBS PCR, hereafter referred to as GenomEra. The standard culture identification consisted of two different agar plates as well as an enrichment broth., Results: We analyzed vaginal samples of 260 pregnant women; 42 samples were tested GBS-positive by using standard culture as a gold standard, 30 by Xpert, and 37 by GenomEra. Xpert and GenomEra assays performed with sensitivities of 71.4% and 88.1% as well as specificities of 98.6% and 99.1%, respectively. Twelve vaginal samples were false-negative by Xpert and five samples by GenomEra. Interestingly, three negative Xpert results of standard culture-positive samples exhibited high Ct-values indicating the presence of GBS. If higher Ct-values are taken into consideration, the sensitivity of Xpert increases up to 78.6%. Moreover, only three Xpert PCRs had to be repeated, whereas two Genomera were invalid even after repetition and further 15 GenomEra PCRs were repeated because of borderline results or inhibition of the PCR test., Conclusions: In this study, GenomEra assay performed with a higher sensitivity than the Xpert PCR. On the other hand, the Xpert assay needs less hands-on-time for a sample preparation and requires approximately four-fold less repetitions as compared to the GenomEra assay. This robust performance of the Xpert assay make it applicable as a rapid intrapartum point-of-care test, although a higher sensitivity would be desirable. Therefore, culture in the 35-37 week of gestation remains the gold standard to detect vaginal colonization., (© 2023 The Authors. Acta Obstetricia et Gynecologica Scandinavica published by John Wiley & Sons Ltd on behalf of Nordic Federation of Societies of Obstetrics and Gynecology (NFOG).)

Purpose: To determine whether high-fluence photoactivated chromophore for keratitis cross-linking (PACK-CXL) can be accelerated., Methods: Solutions of Staphylococcus aureus and Pseudomonas aeruginosa with 0.1% riboflavin were prepared and exposed to 365 nm ultraviolet (UV)-A irradiation of intensities and fluences from 9 to 30 mW/cm2 and from 5.4 to 15.0 J/cm2, respectively, representing nine different accelerated PACK-CXL protocols. Irradiated solutions and unirradiated controls were diluted, plated, and inoculated on agar plates so that the bacterial killing ratios (BKR) could be calculated. Additionally, strains of Achromobacter xylosoxidans, Staphylococcus epidermidis, and Stenotrophomonas maltophilia were exposed to a single accelerated PACK-CXL protocol (intensity: 30 mW/cm2, total fluence: 15.0 J/cm2)., Results: With total fluences of 5.4, 10.0, and 15.0 J/cm2, the range of mean BKR for S. aureus was 45.78% to 50.91%, 84.13% to 88.16%, and 97.50% to 99.90%, respectively; the mean BKR for P. aeruginosa was 69.09% to 70.86%, 75.37% to 77.93%, and 82.27% to 91.44%, respectively. The mean BKR was 41.97% for A. xylosoxidans, 65.38% for S. epidermidis, and 78.04% for S. maltophilia for the accelerated PACK-CXL protocol (30 mW/cm2, 15 J/cm2)., Conclusions: The BKR of high-fluence PACK-CXL protocols can be accelerated while maintaining a high, but species-dependent, BKR. The Bunsen to Roscoe law is respected in fluences up to 10 J/cm2 in S. aureus and P. aeruginosa, whereas fluences above 10 J/cm2 show strain dependence., Translational Relevance: The high-fluence PACK-CXL protocols can be accelerated in clinical practice while maintaining high levels of BKR.

The disaccharide trehalose is essential for viability of Mycobacterium tuberculosis, which synthesizes trehalose de novo but can also utilize exogenous trehalose. The mycobacterial cell wall encompasses two permeability barriers, the cytoplasmic membrane and the outer mycolic acid-containing mycomembrane. The ABC transporter LpqY-SugA-SugB-SugC has previously been demonstrated to mediate the specific uptake of trehalose across the cytoplasmic membrane. However, it is still unclear how the transport of trehalose molecules across the mycomembrane is mediated. In this study, we harnessed the antimycobacterial activity of the analogue 6-azido trehalose to select for spontaneous resistant M. tuberculosis mutants in a merodiploid strain harbouring two LpqY-SugA-SugB-SugC copies. Mutations mediating resistance to 6-azido trehalose mapped to the proline-proline-glutamate (PPE) family member PPE51 (Rv3136), which has recently been shown to be an integral mycomembrane protein involved in uptake of low-molecular weight compounds. A site-specific ppe51 gene deletion mutant of M. tuberculosis was unable to grow on trehalose as the sole carbon source. Furthermore, bioorthogonal labelling of the M. tuberculosis Δppe51 mutant incubated with 6-azido trehalose corroborated the impaired internalization. Taken together, the results indicate that the transport of trehalose and trehalose analogues across the mycomembrane of M. tuberculosis is exclusively mediated by PPE51., (© 2022. The Author(s).)

Macrophages use diverse strategies to restrict intracellular pathogens, including either depriving the bacteria of (micro)nutrients such as transition metals or intoxicating them via metal accumulation. Little is known about the chemical warfare between Mycobacterium marinum , a close relative of Mycobacterium tuberculosis (Mtb), and its hosts. We use the professional phagocyte Dictyostelium discoideum to investigate the role of Zn 2+ during M. marinum infection. We show that M. marinum senses toxic levels of Zn 2+ and responds by upregulating one of its isoforms of the Zn 2+ efflux transporter CtpC. Deletion of ctpC (MMAR&#95;1271) leads to growth inhibition in broth supplemented with Zn 2+ as well as reduced intracellular growth. Both phenotypes were fully rescued by constitutive ectopic expression of the Mtb CtpC orthologue demonstrating that MMAR&#95;1271 is the functional CtpC Zn 2+ efflux transporter in M. marinum Infection leads to the accumulation of Zn 2+ inside the Mycobacterium -containing vacuole (MCV), achieved by the induction and recruitment of the D. discoideum Zn 2+ efflux pumps ZntA and ZntB. In cells lacking ZntA, there is further attenuation of M. marinum growth, presumably due to a compensatory efflux of Zn 2+ into the MCV, carried out by ZntB, the main Zn 2+ transporter in endosomes and phagosomes. Counterintuitively, bacterial growth is also impaired in zntB KO cells, in which MCVs appear to accumulate less Zn 2+ than in wild-type cells, suggesting restriction by other Zn 2+ -mediated mechanisms. Absence of CtpC further epistatically attenuates the intracellular proliferation of M. marinum in zntA and zntB KO cells, confirming that mycobacteria face noxious levels of Zn 2+ IMPORTANCE Microelements are essential for the function of the innate immune system. A deficiency in zinc or copper results in an increased susceptibility to bacterial infections. Zn 2+ serves as an important catalytic and structural cofactor for a variety of enzymes including transcription factors and enzymes involved in cell signaling. But Zn 2+ is toxic at high concentrations and represents a cell-autonomous immunity strategy that ensures killing of intracellular bacteria in a process called zinc poisoning. The cytosolic and lumenal Zn 2+ concentrations result from the balance of import into the cytosol via ZIP influx transporters and efflux via ZnT transporters. Here, we show that Zn 2+ poisoning is involved in restricting Mycobacterium marinum infections. Our study extends observations during Mycobacterium tuberculosis infection and explores for the first time how the interplay of ZnT transporters affects mycobacterial infection by impacting Zn 2+ homeostasis., (Copyright © 2021 Hanna et al.)

Mycobacterium marinum is a model organism for pathogenic Mycobacterium species, including Mycobacterium tuberculosis, the causative agent of tuberculosis. These pathogens enter phagocytes and replicate within the Mycobacterium-containing vacuole, possibly followed by vacuole exit and growth in the host cell cytosol. Mycobacteria release siderophores called mycobactins to scavenge iron, an essential yet poorly soluble and available micronutrient. To investigate the role of M. marinum mycobactins, we purified by organic solvent extraction and identified by mass spectrometry the lipid-bound mycobactin (MBT) and the water-soluble variant carboxymycobactin (cMBT). Moreover, we generated by specialised phage transduction a defined M. marinum ΔmbtB deletion mutant predicted to be defective for mycobactin production. The M. marinum ΔmbtB mutant strain showed a severe growth defect in broth and phagocytes, which was partially complemented by supplying the mbtB gene on a plasmid. Furthermore, purified Fe-MBT or Fe-cMBT improved the growth of wild type as well as ΔmbtB mutant bacteria on minimal plates, but only Fe-cMBT promoted the growth of wild-type M. marinum during phagocyte infection. Finally, the intracellular growth of M. marinum ΔmbtB in Acanthamoeba castellanii amoebae was restored by coinfection with wild-type bacteria. Our study identifies and characterises the M. marinum MBT and cMBT siderophores and reveals the requirement of mycobactins for extra- and intracellular growth of the pathogen., (© 2020 John Wiley & Sons Ltd.)

The causative agent of tuberculosis, Mycobacterium tuberculosis, and its close relative Mycobacterium marinum manipulate phagocytic host cells, thereby creating a replication-permissive compartment termed the Mycobacterium-containing vacuole (MCV). The phosphoinositide (PI) lipid pattern is a crucial determinant of MCV formation and is targeted by mycobacterial PI phosphatases. In this study, we establish an efficient phage transduction protocol to construct defined M. marinum deletion mutants lacking one or three phosphatases, PtpA, PtpB, and/or SapM. These strains were defective for intracellular replication in macrophages and amoebae, and the growth defect was complemented by the corresponding plasmid-borne genes. Fluorescence microscopy of M. marinum-infected Dictyostelium discoideum revealed that MCVs harbouring mycobacteria lacking PtpA, SapM, or all three phosphatases accumulate significantly more phosphatidylinositol-3-phosphate (PtdIns3P) compared with MCVs containing the parental strain. Moreover, PtpA reduced MCV acidification by blocking the recruitment of the V-ATPase, and all three phosphatases promoted bacterial escape from the pathogen vacuole to the cytoplasm. In summary, the secreted M. marinum phosphatases PtpA, PtpB, and SapM determine the MCV PI pattern, compartment acidification, and phagosomal escape., (© 2019 John Wiley & Sons Ltd.)

Molecular research on mycobacteria relies on a multitude of tools for the genetic manipulation of these clinically important bacteria. However, a uniform set of vectors allowing for standardized cloning procedures is not available. Here, we developed a versatile series of mycobacterial vectors for gene deletion, complementation and protein production and purification. The vectors are compatible with fragment exchange (FX) cloning, a recently developed high-throughput cloning principle taking advantage of the type IIS restriction enzyme SapI and its capacity to generate sticky trinucleotide ends outside of its recognition sequence. FX cloning allows for the efficient cloning into an entry vector and the facile transfer of the sequenced insert into a variety of destination vectors. We generated a set of mycobacterial expression vectors spanning a wide range of expression strengths, tagging variants and selection markers to rapidly screen for the optimal expression construct in order to purify membrane proteins from the model organism Mycobacterium smegmatis. Further, we generated a series of suicide vectors containing two counterselection markers and used them to delete twenty genes encoding for potential drug efflux pumps in M. smegmatis. The vectors will further facilitate genetic and biochemical research on various mycobacterial species.

Trehalose biosynthesis is considered an attractive target for the development of antimicrobials against fungal, helminthic and bacterial pathogens including Mycobacterium tuberculosis. The most common biosynthetic route involves trehalose-6-phosphate (T6P) synthase OtsA and T6P phosphatase OtsB that generate trehalose from ADP/UDP-glucose and glucose-6-phosphate. In order to assess the drug target potential of T6P phosphatase, we generated a conditional mutant of M. tuberculosis allowing the regulated gene silencing of the T6P phosphatase gene otsB2. We found that otsB2 is essential for growth of M. tuberculosis in vitro as well as for the acute infection phase in mice following aerosol infection. By contrast, otsB2 is not essential for the chronic infection phase in mice, highlighting the substantial remodelling of trehalose metabolism during infection by M. tuberculosis. Blocking OtsB2 resulted in the accumulation of its substrate T6P, which appears to be toxic, leading to the self-poisoning of cells. Accordingly, blocking T6P production in a ΔotsA mutant abrogated otsB2 essentiality. T6P accumulation elicited a global upregulation of more than 800 genes, which might result from an increase in RNA stability implied by the enhanced neutralization of toxins exhibiting ribonuclease activity. Surprisingly, overlap with the stress response caused by the accumulation of another toxic sugar phosphate molecule, maltose-1-phosphate, was minimal. A genome-wide screen for synthetic lethal interactions with otsA identified numerous genes, revealing additional potential drug targets synergistic with OtsB2 suitable for combination therapies that would minimize the emergence of resistance to OtsB2 inhibitors., Competing Interests: The authors have declared that no competing interests exist.

Mycobacterium tuberculosis synthesizes intra- and extracellular α-glucans that were believed to originate from separate pathways. The extracellular glucose polymer is the main constituent of the mycobacterial capsule that is thought to be involved in immune evasion and virulence. However, the role of the α-glucan capsule in pathogenesis has remained enigmatic due to an incomplete understanding of α-glucan biosynthetic pathways preventing the generation of capsule-deficient mutants. Three separate and potentially redundant pathways had been implicated in α-glucan biosynthesis in mycobacteria: the GlgC-GlgA, the Rv3032 and the TreS-Pep2-GlgE pathways. We now show that α-glucan in mycobacteria is exclusively assembled intracellularly utilizing the building block α-maltose-1-phosphate as the substrate for the maltosyltransferase GlgE, with subsequent branching of the polymer by the branching enzyme GlgB. Some α-glucan is exported to form the α-glucan capsule. There is an unexpected convergence of the TreS-Pep2 and GlgC-GlgA pathways that both generate α-maltose-1-phosphate. While the TreS-Pep2 route from trehalose was already known, we have now established that GlgA forms this phosphosugar from ADP-glucose and glucose 1-phosphate 1000-fold more efficiently than its hitherto described glycogen synthase activity. The two routes are connected by the common precursor ADP-glucose, allowing compensatory flux from one route to the other. Having elucidated this unexpected configuration of the metabolic pathways underlying α-glucan biosynthesis in mycobacteria, an M. tuberculosis double mutant devoid of α-glucan could be constructed, showing a direct link between the GlgE pathway, α-glucan biosynthesis and virulence in a mouse infection model.

Actinomycetes, such as mycobacteria and streptomycetes, synthesize α-glucan with α-1,4 linkages and α-1,6 branching to help evade immune responses and to store carbon. α-Glucan is thought to resemble glycogen except for having shorter constituent linear chains. However, the fine structure of α-glucan and how it can be defined by the maltosyl transferase GlgE and branching enzyme GlgB were not known. Using a combination of enzymolysis and mass spectrometry, we compared the properties of α-glucan isolated from actinomycetes with polymer synthesized in vitro by GlgE and GlgB. We now propose the following assembly mechanism. Polymer synthesis starts with GlgE and its donor substrate, α-maltose 1-phosphate, yielding a linear oligomer with a degree of polymerization (∼16) sufficient for GlgB to introduce a branch. Branching involves strictly intrachain transfer to generate a C chain (the only constituent chain to retain its reducing end), which now bears an A chain (a nonreducing end terminal branch that does not itself bear a branch). GlgE preferentially extends A chains allowing GlgB to act iteratively to generate new A chains emanating from B chains (nonterminal branches that themselves bear a branch). Although extension and branching occur primarily with A chains, the other chain types are sometimes extended and branched such that some B chains (and possibly C chains) bear more than one branch. This occurs less frequently in α-glucans than in classical glycogens. The very similar properties of cytosolic and capsular α-glucans from Mycobacterium tuberculosis imply GlgE and GlgB are sufficient to synthesize them both.

Chemical investigation of the Indonesian sponge Callyspongia aerizusa afforded five new cyclic peptides, callyaerins I-M (1-5), along with the known callyaerins A-G (6-12). The structures of the new compounds were unambiguously elucidated on the basis of one- and two-dimensional NMR spectroscopy and mass spectrometry. In addition, the structures of callyaerins D (9), F (11), and G (12), previously available in only small amounts, have been reinvestigated and revised. All compounds were tested in vitro against Mycobacterium tuberculosis, as well as against THP-1 (human acute monocytic leukemia) and MRC-5 (human fetal lung fibroblast) cell lines, in order to assess their general cytotoxicity. Callyaerins A (6) and B (7) showed potent anti-TB activity with MIC₉₀ values of 2 and 5 μM, respectively. Callyaerin C (8) was found to be less active, with an MIC₉₀ value of 40 μM. Callyaerin A (6), which showed the strongest anti-TB activity, was not cytotoxic to THP-1 or MRC-5 cells (IC₅₀ > 10 μM), which highlights the potential of these compounds as promising anti-TB agents.

Trehalose [alpha-D-glucopyranosyl-(1→1)-alpha-D-glucopyranoside] is a highly abundant disaccharide in mycobacteria that fulfills many biological roles and has a plethora of possible metabolic fates. Trehalose is synthesized in mycobacteria de novo either from glycolytic intermediates or from alpha-glucans via two alternative routes, the OtsA-OtsB and the TreY-TreZ pathways, respectively. Intracellular trehalose can serve as an endogenous remobilizable carbon storage compound and as a biocompatible stress protectant. Furthermore, trehalose functions as the sugar core of many glycolipids with important structural or immunomodulatory functions such as the cord factor trehalose dimycolate, sulfolipids, and polyacyltrehalose. Moreover, trehalose plays a central role in the formation of the mycolic acid cell wall layer because it serves as a carrier molecule that shuttles mycolic acids in the form of the glycolipid trehalose monomycolate between the cytoplasm and the periplasm. In this process, a specific importer recycles the free trehalose that is extracellularly released as a by-product during mycolate processing via the antigen 85 complex, which might represent a specific adaptation to the intracellular lifestyle of Mycobacterium tuberculosis with limited carbohydrate availability. Finally, trehalose is converted to glycogen-like branched alpha-glucans by a four-step metabolic pathway involving the essential maltosyltransferase GlgE, which may be further processed to derivatives with intracellular or extracellular destinations such as polymethylated lipopolysaccharides or capsular alpha-glucans, respectively. In this article we summarize the current knowledge of the genetic basis of trehalose biosynthesis and metabolism in mycobacteria, the biological functions of trehalose-based molecules, and their roles in virulence of the human pathogen M. tuberculosis.

Trehalose synthase (TreS) was thought to catalyze flux from maltose to trehalose, a precursor of essential trehalose mycolates in mycobacterial cell walls. However, we now show, using a genetic approach, that TreS is not required for trehalose biosynthesis in Mycobacterium smegmatis, whereas two alternative trehalose-biosynthetic pathways (OtsAB and TreYZ) are crucial. Consistent with this direction of flux, trehalose levels in Mycobacterium tuberculosis decreased when TreS was overexpressed. In addition, TreS was shown to interconvert the α anomer of maltose and trehalose using (1)H and (19)F-nuclear magnetic resonance spectroscopies using its normal substrates and deoxyfluoromaltose analogs, with the nonenzymatic mutarotation of α/β-maltose being slow. Therefore, flux through TreS in mycobacteria flows from trehalose to α-maltose, which is the appropriate anomer for maltose kinase of the GlgE α-glucan pathway, which in turn contributes to intracellular and/or capsular polysaccharide biosynthesis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In vertebrates, mannose 6-phosphate receptors [MPR300 (Mr 300 kDa) and MPR46 (Mr 46 kDa)] are highly conserved transmembrane glycoproteins that mediate transport of lysosomal enzymes to lysosomes. Our studies have revealed the appearance of these putative receptors in invertebrates such as the molluscs and deuterostomes. Starfish tissue extracts contain several lysosomal enzyme activities and here we describe the affinity purification of α-fucosidase. The purified enzyme is a glycoprotein that exhibited a molecular mass of ∼56 kDa in SDS-PAGE under reducing conditions. It has also cross-reacted with an antiserum to the mollusc enzyme suggesting antigenic similarities among the two invertebrate enzymes. LC-MS/MS analysis of the proteolytic peptides of the purified enzyme in combination with de novo sequencing allowed us to do partial amino acid sequence determination of the enzyme. These data suggest that this invertebrate enzyme is homologous to the known mammalian enzyme. The purified enzyme exhibited a mannose 6-phosphate dependent interaction with the immobilized starfish MPR300 protein. Our results demonstrate that the lysosomal enzyme targeting pathway is conserved even among the invertebrates., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Siglec-2, also known as CD22, is involved in the regulation and survival of B-cells and has been successfully targeted in cell depletion therapies with antibody-based approaches. Sialic acid derivatives, already known to bind with high affinity to myelin-associated glycoprotein (MAG, Siglec-4), were screened for their binding affinity for CD22 by surface plasmon resonance. The best compound identified was further modified with various hydrophobic substituents at the 2-, 5-, and 9-positions of the sialic acid scaffold, leading to nanomolar derivatives, of which ligand 17 b shows the most promising pharmacodynamic and pharmacokinetic profiles. Isothermal titration calorimetry measurements demonstrate that the binding is enthalpy driven. Interestingly, the thermodynamic fingerprints reveal an excellent correlation between gains in enthalpy and compensation by increased entropy costs. Moreover, 17 b exhibits a residence time in the range of a few seconds, clearly prolonged relative to residence times typically observed for carbohydrate-lectin interactions. Finally, initial tests regarding drug-like properties of 17 b demonstrate the required high plasma protein binding yet a lack of oral availability, although its distribution coefficient (log D) is in the required range., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Animal African trypanosomiasis, sleeping sickness in humans and Nagana in cattle, is a resurgent disease in Africa caused by Trypanosoma parasites. Trans-sialidases expressed by trypanosomes play an important role in the infection cycle of insects and mammals. Whereas trans-sialidases of other trypanosomes like the American T. cruzi are well investigated, relatively little research has been done on these enzymes of T. congolense., Results: Based on a partial sequence and an open reading frame in the WTSI database, DNA sequences encoding for eleven T. congolense trans-sialidase 1 variants with 96.3% overall amino acid identity were amplified. Trans-sialidase 1 variants were expressed as recombinant proteins, isolated and assayed for trans-sialylation activity. The purified proteins produced α2,3-sialyllactose from lactose by desialylating fetuin, clearly demonstrating their trans-sialidase activity. Using an HPLC-based assay, substrate specificities and kinetic parameters of two variants were characterized in detail indicating differences in substrate specificities for lactose, fetuin and synthetic substrates. Both enzymes were able to sialylate asialofetuin to an extent, which was sufficient to reconstitute binding sites for Siglec-4. A mass spectrometric analysis of the sialylation pattern of glycopeptides from fetuin revealed clear but generally similar changes in the sialylation pattern of the N-glycans on fetuin catalyzed by the trans-sialidases investigated., Conclusions: The identification and characterization of a trans-sialidase gene family of the African parasite T. congolense has opened new perspectives for investigating the biological role of these enzymes in Nagana and sleeping sickness. Based on this study it will be interesting to address the expression pattern of these genes and their activities in the different stages of the parasite in its infection cycle. Furthermore, these trans-sialidases have the biotechnological potential to be used for enzymatic modification of sialylated glycoconjugates.

A lipid bilayer deposited on an electrode surface can serve as a benchmark system to investigate lipid-protein interactions in the presence of physiological electric fields. Recoverin and myelin-associated glycoprotein (MAG) are used to study the impact of strong and weak protein-lipid interactions on the structure of model lipid bilayers, respectively. The structural changes in lipid bilayers are followed using electrochemical polarization modulation infrared reflection-absorption spectroscopy (PM IRRAS). Recoverin contains a myristoyl group that anchors in the hydrophobic part of a cell membrane. Insertion of the protein into the 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC)-cholesterol lipid bilayer leads to an increase in the capacitance of the lipid film adsorbed on a gold electrode surface. The stability and kinetics of the electric-field-driven adsorption-desorption process are not affected by the interaction with protein. Upon interaction with recoverin, the hydrophobic hydrocarbon chains become less ordered. The polar head groups are separated from each other, which allows for recoverin association in the membrane. MAG is known to interact with glycolipids present on the surface of a cell membrane. Upon probing the interaction of the DMPC-cholesterol-glycolipid bilayer with MAG a slight decrease in the capacity of the adsorbed lipid film is observed. The stability of the lipid bilayer increases towards negative potentials. At the molecular scale this interaction results in minor changes in the structure of the lipid bilayer. MAG causes small ordering in the hydrocarbon chains region and an increase in the hydration of the polar head groups. Combining an electrochemical approach with a structure-sensitive technique, such as PM IRRAS, is a powerful tool to follow small but significant changes in the structure of a supramolecular assembly., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

High concentrations of 2-deoxy-D-ribose (2dRib) have been reported to cause oxidative stress and to disturb the glutathione (GSH) metabolism of various cell types. Exposure of astrocyte-rich primary cultures to millimolar concentrations of 2dRib or its stereoisomer 2-deoxy-L-ribose, but not the incubation with ribose, 2-deoxyglucose, glucose, fructose or saccharose, lowered the cellular GSH content in a time and concentration dependent manner. After exposure for 4 h to 30 mM 2dRib the cells contained 2dRib in a concentration of about 24 mM. Under these conditions 2dRib did not compromise cell viability and the ability of the cells to synthesise GSH, nor were the cellular ratio of glutathione disulfide (GSSG) to GSH and the extracellular concentrations of GSH or GSSG increased. These data demonstrate that 2dRib deprives viable cultured astrocytes of GSH and suggest that a cellular reaction of GSH with 2dRib or its metabolites is involved in the deprivation of astrocytic GSH.

Broad modifications of various positions of the minimal natural epitope recognized by the myelin-associated glycoprotein (MAG), a blocker of regeneration of neurite injuries, produced sialosides with nanomolar affinities. However, important pharmacokinetic issues, for example, the metabolic stability of these sialosides, remain to be addressed. For this reason, the novel non-carbohydrate mimic 3 was designed and synthesized from (-)-quinic acid. For the design of 3, previously identified beneficial modifications of side chains of Neu5Ac were combined with the replacement of the ring oxygen by a methylene group and the substitution of the C(4)-OH by an acetamide. Although docking experiments to a homology model of MAG revealed that mimic 3 forms all but one of the essential hydrogen bonds identified for the earlier reported lead 2, its affinity was substantially reduced. Extensive molecular-dynamics simulation disclosed that the missing hydrogen bond of the former C(8)-OH leads to a change of the orientation of the side chain. As a consequence, an important hydrophobic contact is compromised leading to a loss of affinity., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Paraplegia is caused by injuries of the central nervous system (CNS) and especially young people suffer from these severe consequences as, for example, the loss of motor functions. The lack of repair of the injured nerve strands originates from the inhibitory environment for axon regeneration in the CNS. Specific inhibitory proteins block the regrowth of nerve roots. One of these neurite outgrowth inhibitors is the myelin-associated glycoprotein (MAG), which is a member of the Siglec family (sialic acid-binding immunoglobulin-like lectin). In previous studies, we identified potent small molecule MAG antagonists. In this communication, we report new neuraminic acid derivatives modified in the 4- and 5-position, and the influence of various structural modifications on their kinetic and thermodynamic binding properties., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The injured adult mammalian central nervous system is an inhibitory environment for axon regeneration due to specific inhibitors, among them the myelin-associated glycoprotein (MAG), a member of the Siglec family (sialic-acid binding immunoglobulin-like lectin). In earlier studies, we identified the lead structure 5, which shows a 250-fold improved in vitro affinity for MAG compared to the tetrasaccharide binding epitope of GQ1balpha (1), the best physiological MAG ligand described so far. By modifying the 2- and 5-position, the affinity of 5 could be further improved to the nanomolar range (-->19a). Docking studies to a homology model of MAG allowed the rationalization of the experimental binding properties. Finally, pharmacokinetic parameters (stability in the cerebrospinal fluid, logD and permeation through the BBB) indicate the drug-like properties of the high-affinity antagonist 19a.