A lectin S-domain receptor kinase mediates lipopolysaccharide sensing in Arabidopsis thaliana.

The sensing of microbe-associated molecular patterns (MAMPs) triggers innate immunity in animals and plants. Lipopolysaccharide (LPS) from Gram-negative bacteria is a potent MAMP for mammals, with the lipid A moiety activating proinflammatory responses via Toll-like receptor 4 (TLR4). Here we found that the plant Arabidopsis thaliana specifically sensed LPS of Pseudomonas and Xanthomonas. We isolated LPS-insensitive mutants defective in the bulb-type lectin S-domain-1 receptor-like kinase LORE (SD1-29), which were hypersusceptible to infection with Pseudomonas syringae. Targeted chemical degradation of LPS from Pseudomonas species suggested that LORE detected mainly the lipid A moiety of LPS. LORE conferred sensitivity to LPS onto tobacco after transient expression, which demonstrated a key function in LPS sensing and indicated the possibility of engineering resistance to bacteria in crop species.

Design of Multidrug-Resistant Tuberculosis Treatment Regimens Based on DNA Sequencing.

BACKGROUND: Comprehensive and reliable drug susceptibility testing (DST) is urgently needed to provide adequate treatment regimens for patients with multidrug-resistant/rifampicin-resistant tuberculosis (MDR/RR-TB). We determined whether next-generation sequencing (NGS) analysis of Mycobacterium tuberculosis complex isolates and genes implicated in drug resistance can guide the design of effective MDR/RR-TB treatment regimens. METHODS: NGS-based genomic DST predictions of M. tuberculosis complex isolates from MDR/RR-TB patients admitted to a TB reference center in Germany between 1 January 2015 and 30 April 2019 were compared with phenotypic DST results of mycobacteria growth indicator tubes (MGIT). Standardized treatment algorithms were applied to design individualized therapies based on either genomic or phenotypic DST results, and discrepancies were further evaluated by determination of minimal inhibitory drug concentrations (MICs) using Sensititre MYCOTBI and UKMYC microtiter plates. RESULTS: In 70 patients with MDR/RR-TB, agreement among 1048 pairwise comparisons of genomic and phenotypic DST was 86.3%; 76 (7.2%) results were discordant, and 68 (6.5%) could not be evaluated due to the presence of polymorphisms with yet unknown implications for drug resistance. Importantly, 549 of 561 (97.9%) predictions of drug susceptibility were phenotypically confirmed in MGIT, and 27 of 64 (42.2%) false-positive results were linked to previously described mutations mediating a low or moderate MIC increase. Virtually all drugs (99.0%) used in combination therapies that were inferred from genomic DST were confirmed to be susceptible by phenotypic DST. CONCLUSIONS: NGS-based genomic DST can reliably guide the design of effective MDR/RR-TB treatment regimens.

Prediction of anti-tuberculosis treatment duration based on a 22-gene transcriptomic model.

BACKGROUND: The World Health Organization recommends standardised treatment durations for patients with tuberculosis (TB). We identified and validated a host-RNA signature as a biomarker for individualised therapy durations for patients with drug-susceptible (DS)- and multidrug-resistant (MDR)-TB. METHODS: Adult patients with pulmonary TB were prospectively enrolled into five independent cohorts in Germany and Romania. Clinical and microbiological data and whole blood for RNA transcriptomic analysis were collected at pre-defined time points throughout therapy. Treatment outcomes were ascertained by TBnet criteria (6-month culture status/1-year follow-up). A whole-blood RNA therapy-end model was developed in a multistep process involving a machine-learning algorithm to identify hypothetical individual end-of-treatment time points. RESULTS: 50 patients with DS-TB and 30 patients with MDR-TB were recruited in the German identification cohorts (DS-GIC and MDR-GIC, respectively); 28 patients with DS-TB and 32 patients with MDR-TB in the German validation cohorts (DS-GVC and MDR-GVC, respectively); and 52 patients with MDR-TB in the Romanian validation cohort (MDR-RVC). A 22-gene RNA model (TB22) that defined cure-associated end-of-therapy time points was derived from the DS- and MDR-GIC data. The TB22 model was superior to other published signatures to accurately predict clinical outcomes for patients in the DS-GVC (area under the curve 0.94, 95% CI 0.9-0.98) and suggests that cure may be achieved with shorter treatment durations for TB patients in the MDR-GIC (mean reduction 218.0 days, 34.2%; p<0.001), the MDR-GVC (mean reduction 211.0 days, 32.9%; p<0.001) and the MDR-RVC (mean reduction of 161.0 days, 23.4%; p=0.001). CONCLUSION: Biomarker-guided management may substantially shorten the duration of therapy for many patients with MDR-TB.

Personalized Medicine for Chronic Respiratory Infectious Diseases: Tuberculosis, Nontuberculous Mycobacterial Pulmonary Diseases, and Chronic Pulmonary Aspergillosis.

Chronic respiratory infectious diseases are causing high rates of morbidity and mortality worldwide. Tuberculosis, a major cause of chronic pulmonary infection, is currently responsible for approximately 1.5 million deaths per year. Although important advances in the fight against tuberculosis have been made, the progress towards eradication of this disease is being challenged by the dramatic increase in multidrug-resistant bacilli. Nontuberculous mycobacteria causing pulmonary disease and chronic pulmonary aspergillosis are emerging infectious diseases. In contrast to other infectious diseases, chronic respiratory infections share the trait of having highly variable treatment outcomes despite longstanding antimicrobial therapy. Recent scientific progress indicates that medicine is presently at a transition stage from programmatic to personalized management. We explain current state-of-the-art management concepts of chronic pulmonary infectious diseases as well as the underlying methods for therapeutic decisions and their implications for personalized medicine. Furthermore, we describe promising biomarkers and techniques with the potential to serve future individual treatment concepts in this field of difficult-to-treat patients. These include candidate markers to improve individual risk assessment for disease development, the design of tailor-made drug therapy regimens, and individualized biomarker-guided therapy duration to achieve relapse-free cure. In addition, the use of therapeutic drug monitoring to reach optimal drug dosing with the smallest rate of adverse events as well as candidate agents for future host-directed therapies are described. Taken together, personalized medicine will provide opportunities to substantially improve the management and treatment outcome of difficult-to-treat patients with chronic respiratory infections.

Secondary cell wall polymers of Enterococcus faecalis are critical for resistance to complement activation via mannose-binding lectin.

The complement system is part of our first line of defense against invading pathogens. The strategies used by Enterococcus faecalis to evade recognition by human complement are incompletely understood. In this study, we identified an insertional mutant of the wall teichoic acid (WTA) synthesis gene tagB in E. faecalis V583 that exhibited an increased susceptibility to complement-mediated killing by neutrophils. Further analysis revealed that increased killing of the mutant was due to a higher rate of phagocytosis by neutrophils, which correlated with higher C3b deposition on the bacterial surface. Our studies indicated that complement activation via the lectin pathway was much stronger on the tagB mutant compared with wild type. In concordance, we found an increased binding of the key lectin pathway components mannose-binding lectin and mannose-binding lectin-associated serine protease-2 (MASP-2) on the mutant. To understand the mechanism of lectin pathway inhibition by E. faecalis, we purified and characterized cell wall carbohydrates of E. faecalis wild type and V583ΔtagB. NMR analysis revealed that the mutant strain lacked two WTAs with a repeating unit of →6)[α-l-Rhap-(1→3)]ß-D-GalpNAc-(1→5)-Rbo-1-P and →6) ß-D-Glcp-(1→3) [α-D-Glcp-(1→4)]-ß-D-GalpNAc-(1→5)-Rbo-1-P→, respectively (Rbo, ribitol). In addition, compositional changes in the enterococcal rhamnopolysaccharide were noticed. Our study indicates that in E. faecalis, modification of peptidoglycan by secondary cell wall polymers is critical to evade recognition by the complement system.

A Single-Run HPLC-MS Multiplex Assay for Therapeutic Drug Monitoring of Relevant First- and Second-Line Antibiotics in the Treatment of Drug-Resistant Tuberculosis.

The treatment of drug-resistant Mycobacterium tuberculosis relies on complex antibiotic therapy. Inadequate antibiotic exposure can lead to treatment failure, acquired drug resistance, and an increased risk of adverse events. Therapeutic drug monitoring (TDM) can be used to optimize the antibiotic exposure. Therefore, we aimed to develop a single-run multiplex assay using high-performance liquid chromatography-mass spectrometry (HPLC-MS) for TDM of patients with multidrug-resistant, pre-extensively drug-resistant and extensively drug-resistant tuberculosis. A target profile for sufficient performance, based on the intended clinical application, was established and the assay was developed accordingly. Antibiotics were analyzed on a zwitterionic hydrophilic interaction liquid chromatography column and a triple quadrupole mass spectrometer using stable isotope-labeled internal standards. The assay was sufficiently sensitive to monitor drug concentrations over five half-lives for rifampicin, rifabutin, levofloxacin, moxifloxacin, bedaquiline, linezolid, clofazimine, terizidone/cycloserine, ethambutol, delamanid, pyrazinamide, meropenem, prothionamide, and para-amino salicylic acid (PAS). Accuracy and precision were sufficient to support clinical decision making (≤±15% in clinical samples and ±20-25% in spiked samples, with 80% of future measured concentrations predicted to fall within ±40% of nominal concentrations). The method was applied in the TDM of two patients with complex drug-resistant tuberculosis. All relevant antibiotics from their regimens could be quantified and high-dose therapy was initiated, followed by microbiological conversion. In conclusion, we developed a multiplex assay that enables TDM of the relevant first- and second-line anti-tuberculosis medicines in a single run and was able to show its applicability in TDM of two drug-resistant tuberculosis patients.

Tuberculostearic Acid-Containing Phosphatidylinositols as Markers of Bacterial Burden in Tuberculosis.

One-fourth of the global human population is estimated to be infected with strains of the Mycobacterium tuberculosis complex (MTBC), the causative agent of tuberculosis (TB). Using lipidomic approaches, we show that tuberculostearic acid (TSA)-containing phosphatidylinositols (PIs) are molecular markers for infection with clinically relevant MTBC strains and signify bacterial burden. For the most abundant lipid marker, detection limits of ∼102 colony forming units (CFUs) and ∼103 CFUs for bacterial and cell culture systems were determined, respectively. We developed a targeted lipid assay, which can be performed within a day including sample preparation─roughly 30-fold faster than in conventional methods based on bacterial culture. This indirect and culture-free detection approach allowed us to determine pathogen loads in infected murine macrophages, human neutrophils, and murine lung tissue. These marker lipids inferred from mycobacterial PIs were found in higher levels in peripheral blood mononuclear cells of TB patients compared to healthy individuals. Moreover, in a small cohort of drug-susceptible TB patients, elevated levels of these molecular markers were detected at the start of therapy and declined upon successful anti-TB treatment. Thus, the concentration of TSA-containing PIs can be used as a correlate for the mycobacterial burden in experimental models and in vitro systems and may prospectively also provide a clinically relevant tool to monitor TB severity.

Glycosylation of wall teichoic acid in Staphylococcus aureus by TarM.

Wall teichoic acid (WTA) glycopolymers are major constituents of cell envelopes in Staphylococcus aureus and related gram-positive bacteria with important roles in cell wall maintenance, susceptibility to antimicrobial molecules, biofilm formation, and host interaction. Most S. aureus strains express polyribitol phosphate WTA substituted with D-alanine and N-acetylglucosamine (GlcNAc). WTA sugar modifications are highly variable and have been implicated in bacteriophage susceptibility and immunogenicity, but the pathway and enzymes of staphylococcal WTA glycosylation have remained unknown. Revisiting the structure of S. aureus RN4220 WTA by NMR analysis revealed the presence of canonical polyribitol phosphate WTA bearing only alpha-linked GlcNAc substituents. A RN4220 transposon mutant resistant to WTA-dependent phages was identified and shown to produce altered WTA, which exhibited faster electrophoretic migration and lacked completely the WTA alpha-GlcNAc residues. Disruption of a gene of unknown function, renamed tarM, was responsible for this phenotype. Recombinant TarM was capable of glycosylating WTA in vitro in a UDP-GlcNAc-dependent manner, thereby confirming its WTA GlcNAc-transferase activity. Deletion of the last seven amino acids from the C terminus abolished the activity of TarM. tarM-related genes were found in the genomes of several WTA-producing bacteria, suggesting that TarM-mediated WTA glycosylation is a general pathway in gram-positive bacteria. Our study represents a basis for dissecting the biosynthesis and function of glycosylated WTA in S. aureus and other bacteria.

Deletion of the glycosyltransferase bgsB of Enterococcus faecalis leads to a complete loss of glycolipids from the cell membrane and to impaired biofilm formation.

BACKGROUND: Deletion of the glycosyltransferase bgsA in Enterococcus faecalis leads to loss of diglucosyldiacylglycerol from the cell membrane and accumulation of its precursor monoglucosyldiacylglycerol, associated with impaired biofilm formation and reduced virulence in vivo. Here we analyzed the function of a putative glucosyltransferase EF2890 designated biofilm-associated glycolipid synthesis B (bgsB) immediately downstream of bgsA. RESULTS: A deletion mutant was constructed by targeted mutagenesis in E. faecalis strain 12030. Analysis of cell membrane extracts revealed a complete loss of glycolipids from the cell membrane. Cell walls of 12030ΔbgsB contained approximately fourfold more LTA, and 1H-nuclear magnetic resonance (NMR) spectroscopy suggested that the higher content of cellular LTA was due to increased length of the glycerol-phosphate polymer of LTA. 12030ΔbgsB was not altered in growth, cell morphology, or autolysis. However, attachment to Caco-2 cells was reduced to 50% of wild-type levels, and biofilm formation on polystyrene was highly impaired. Despite normal resistance to cationic antimicrobial peptides, complement and antibody-mediated opsonophagocytic killing in vitro, 12030ΔbgsB was cleared more rapidly from the bloodstream of mice than wild-type bacteria. Overall, the phenotype resembles the respective deletion mutant in the bgsA gene. Our findings suggest that loss of diglucosyldiacylglycerol or the altered structure of LTA in both mutants account for phenotypic changes observed. CONCLUSIONS: In summary, BgsB is a glucosyltransferase that synthesizes monoglucosyldiacylglycerol. Its inactivation profoundly affects cell membrane composition and has secondary effects on LTA biosynthesis. Both cell-membrane amphiphiles are critical for biofilm formation and virulence of E. faecalis.

Staphylococcus epidermidis clones express Staphylococcus aureus-type wall teichoic acid to shift from a commensal to pathogen lifestyle.

Most clonal lineages of Staphylococcus epidermidis are commensals present on human skin and in the nose. However, some globally spreading healthcare-associated and methicillin-resistant S. epidermidis (HA-MRSE) clones are major causes of difficult-to-treat implant or bloodstream infections. The molecular determinants that alter the lifestyle of S. epidermidis have remained elusive, and their identification might provide therapeutic targets. We reasoned that changes in surface-exposed wall teichoic acid (WTA) polymers of S. epidermidis, which potentially shape host interactions, may be linked to differences between colonization and infection abilities of different clones. We used a combined epidemiological and functional approach to show that while commensal clones express poly-glycerolphosphate WTA, S. epidermidis multilocus sequence type 23, which emerged in the past 15 years and is one of the main infection-causing HA-MRSE clones, contains an accessory genetic element, tarIJLM, that leads to the production of a second, Staphylococcus aureus-type WTA (poly-ribitolphosphate (RboP)). Production of RboP-WTA by S. epidermidis impaired in vivo colonization but augmented endothelial attachment and host mortality in a mouse sepsis model. tarIJLM was absent from commensal human sequence types but was found in several other HA-MRSE clones. Moreover, RboP-WTA enabled S. epidermidis to exchange DNA with S. aureus via siphovirus bacteriophages, thereby creating a possible route for the inter-species exchange of methicillin resistance, virulence and colonization factors. We conclude that tarIJLM alters the lifestyle of S. epidermidis from commensal to pathogenic and propose that RboP-WTA might be a robust target for preventive and therapeutic interventions against MRSE infections.

Glycolipids are involved in biofilm accumulation and prolonged bacteraemia in Enterococcus faecalis.

Biofilm production is thought to be an important step in many enterococcal infections. In several Gram-positive bacteria, membrane glycolipids have been implicated in biofilm formation. We constructed a non-polar deletion mutant of a putative glucosyltransferase designated biofilm-associated glycolipid synthesis A (bgsA) in Enterococcus faecalis 12030. Analysis of major extracted glycolipids by nuclear magnetic resonance spectroscopy revealed that the cell membrane of 12030 Delta bgsA was devoid of diglucosyl-diacylglycerol (DGlcDAG), while monoglucosyl-diacylglycerol was overrepresented. The cell walls of 12030 Delta bgsA contained longer lipoteichoic acid molecules and were less hydrophobic than wild-type bacteria. Inactivation of bgsA in E. faecalis 12030 and E. faecalis V583 led to an almost complete arrest of biofilm formation on plastic surfaces. Overexpression of bgsA, on the other hand, resulted in increased biofilm production. While initial adherence was not affected, bgsA-deficient bacteria did not accumulate in the growing biofilm. Also, adherence of E. faecalis Delta bgsA to Caco-2 cells was impaired. In a mouse bacteraemia model, E. faecalis 12030 Delta bgsA was cleared more rapidly from the bloodstream than the wild-type strain. In summary, BgsA is a glycosyltransferase synthetizing DGlcDAG, a glycolipid and lipoteichoic acid precursor involved in biofilm accumulation, adherence to host cells, and virulence in vivo.

Perspective for Precision Medicine for Tuberculosis.

Tuberculosis is a bacterial infectious disease that is mainly transmitted from human to human via infectious aerosols. Currently, tuberculosis is the leading cause of death by an infectious disease world-wide. In the past decade, the number of patients affected by tuberculosis has increased by ~20 percent and the emergence of drug-resistant strains of Mycobacterium tuberculosis challenges the goal of elimination of tuberculosis in the near future. For the last 50 years, management of patients with tuberculosis has followed a standardized management approach. This standardization neglects the variation in human susceptibility to infection, immune response, the pharmacokinetics of drugs, and the individual duration of treatment needed to achieve relapse-free cure. Here we propose a package of precision medicine-guided therapies that has the prospect to drive clinical management decisions, based on both host immunity and M. tuberculosis strains genetics. Recently, important scientific discoveries and technological advances have been achieved that provide a perspective for individualized rather than standardized management of patients with tuberculosis. For the individual selection of best medicines and host-directed therapies, personalized drug dosing, and treatment durations, physicians treating patients with tuberculosis will be able to rely on these advances in systems biology and to apply them at the bedside.

Mycobacterium Growth Inhibition Assay of Human Alveolar Macrophages as a Correlate of Immune Protection Following Mycobacterium bovis Bacille Calmette-Guérin Vaccination.

BACKGROUND: In order to eliminate tuberculosis (TB), an effective vaccine is urgently needed to prevent infection with Mycobacterium tuberculosis. A key obstacle for the development of novel TB vaccines is the lack of surrogate markers for immune protection against M. tuberculosis. METHODS: We investigated growth rates of M. tuberculosis in the mycobacterial growth inhibition assay (MGIA) as a marker for mycobacterial growth control of human bronchoalveolar lavage (BALC) and peripheral blood mononuclear cells (PBMC) before and after vaccination with Mycobacterium bovis Bacille Calmette-Guérin (BCG) of healthy adult volunteers. RESULTS: Vaccination induced a positive response (p < 0.001) to purified protein derivate (PPD) in 58.8% of the individuals in an interferon-γ release assay-ELISpot. Intraindividual evaluation of the MGIA growth rates before and after M. bovis BCG-vaccination revealed no significant difference in time to culture positivity before and after vaccination in BALC (p = 0.604) and PBMC (p = 0.199). The magnitude of the PPD-response induced by M. bovis BCG-vaccination did not correlate with growth control in BALC and PBMC (correlation = 0.468, 95% CI: -0.016 to 0.775). CONCLUSION: In conclusion, M. bovis BCG-vaccination-induced mycobacterial-specific cytokine immune response does not result in functional immune control against M. tuberculosis in the MGIA.

Structure of a highly phosphorylated lipopolysaccharide core in the Delta algC mutants derived from Pseudomonas aeruginosa wild-type strains PAO1 (serogroup O5) and PAC1R (serogroup O3).

Lipopolysaccharides (LPS) were isolated from rough-type mutant strains of Pseudomonas aeruginosa (Delta algC) derived from wild-type strains PAO1 (serogroup O5) and PAC1R (serogroup O3). Structural studies of the LPS core region with a special focus on the phosphorylation pattern were performed by 2D NMR spectroscopy, including a 1H,(31)P HMQC-TOCSY experiment, MALDI-TOF MS, and Fourier-transform ion cyclotron resonance ESIMS using the capillary skimmer dissociation technique. Both LPS were found to contain two residues each of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and L-glycero-D-manno-heptose (Hep), one residue of N-(L-alanyl)-D-galactosamine and one O-carbamoyl group (Cm) on the distal Hep residue. The following structures of a tetrasaccharide trisphosphate from strain PAC1R Delta algC and that carrying an additional ethanolamine phosphate group (PEtN) from strain PAO1 Delta algC were elucidated: [carbohydrate structre: see text] where R=P in PAC1R Delta algC and PPEtN in PAO1 Delta algC. To our knowledge, in this work the presence of ethanolamine diphosphate is unambiguously confirmed and its position established for the first time in the LPS core of a rough-type strain of P. aeruginosa. In addition, the structure of the complete LPS core of wild-type strain P. aeruginosa PAO1 was reinvestigated and the position of the phosphorylation sites was revised.

Biosynthesis of the unique wall teichoic acid of Staphylococcus aureus lineage ST395.

ABSTRACT The major clonal lineages of the human pathogen Staphylococcus aureus produce cell wall-anchored anionic poly-ribitol-phosphate (RboP) wall teichoic acids (WTA) substituted with d-Alanine and N-acetyl-d-glucosamine. The phylogenetically isolated S. aureus ST395 lineage has recently been found to produce a unique poly-glycerol-phosphate (GroP) WTA glycosylated with N-acetyl-d-galactosamine (GalNAc). ST395 clones bear putative WTA biosynthesis genes on a novel genetic element probably acquired from coagulase-negative staphylococci (CoNS). We elucidated the ST395 WTA biosynthesis pathway and identified three novel WTA biosynthetic genes, including those encoding an α-O-GalNAc transferase TagN, a nucleotide sugar epimerase TagV probably required for generation of the activated sugar donor substrate for TagN, and an unusually short GroP WTA polymerase TagF. By using a panel of mutants derived from ST395, the GalNAc residues carried by GroP WTA were found to be required for infection by the ST395-specific bacteriophage Φ187 and to play a crucial role in horizontal gene transfer of S. aureus pathogenicity islands (SaPIs). Notably, ectopic expression of ST395 WTA biosynthesis genes rendered normal S. aureus susceptible to Φ187 and enabled Φ187-mediated SaPI transfer from ST395 to regular S. aureus. We provide evidence that exchange of WTA genes and their combination in variable, mosaic-like gene clusters have shaped the evolution of staphylococci and their capacities to undergo horizontal gene transfer events. IMPORTANCE The structural highly diverse wall teichoic acids (WTA) are cell wall-anchored glycopolymers produced by most Gram-positive bacteria. While most of the dominant Staphylococcus aureus lineages produce poly-ribitol-phosphate WTA, the recently described ST395 lineage produces a distinct poly-glycerol-phosphate WTA type resembling the WTA backbone of coagulase-negative staphylococci (CoNS). Here, we analyzed the ST395 WTA biosynthesis pathway and found new types of WTA biosynthesis genes along with an evolutionary link between ST395 and CoNS, from which the ST395 WTA genes probably originate. The elucidation of ST395 WTA biosynthesis will help to understand how Gram-positive bacteria produce highly variable WTA types and elucidate functional consequences of WTA variation.

Wall teichoic acid structure governs horizontal gene transfer between major bacterial pathogens.

Mobile genetic elements (MGEs) encoding virulence and resistance genes are widespread in bacterial pathogens, but it has remained unclear how they occasionally jump to new host species. Staphylococcus aureus clones exchange MGEs such as S. aureus pathogenicity islands (SaPIs) with high frequency via helper phages. Here we report that the S. aureus ST395 lineage is refractory to horizontal gene transfer (HGT) with typical S. aureus but exchanges SaPIs with other species and genera including Staphylococcus epidermidis and Listeria monocytogenes. ST395 produces an unusual wall teichoic acid (WTA) resembling that of its HGT partner species. Notably, distantly related bacterial species and genera undergo efficient HGT with typical S. aureus upon ectopic expression of S. aureus WTA. Combined with genomic analyses, these results indicate that a 'glycocode' of WTA structures and WTA-binding helper phages permits HGT even across long phylogenetic distances thereby shaping the evolution of Gram-positive pathogens.

Structural characterization of teichoic acids from Lactobacillus brevis.

Teichoic acids are a major constituent of the cell wall of Gram-positive bacteria. Structural characterization of lipoteichoic and teichoic acids isolated from Lactobacillus brevis was undertaken using 1D and 2D NMR experiments as well as chemical methodology. Compositional analysis indicated the presence of high amounts of glycerol, glucose, and alanine. In the case of LTA octadecenoic acid was also detected. The basic LTA/WTA structure was identified as 1,3-poly(glycerol phosphate) nonstoichiometrically substituted at C-2 of the glycerol residues with D-Ala or alpha-D-Glc. In the case of LTA a higher amount of Ala could be detected and partial alanylation at position C-6 of the Glc could also be observed.

Structural characterization of the major glycolipids from Arthrobacter globiformis and Arthrobacter scleromae.

Arthrobacter is a genus of Gram-positive bacteria widely distributed in soil. The ability to catabolize a variety of xenobiotics has shown their potential as a detoxifying agent. Recently, Arthrobacter has been also recognized as an opportunistic pathogen. Glycolipids from A. scleromae, a clinical isolate, and A. globiformis, from soil, were isolated by chloroform-methanol extraction and subsequently purified using column chromatography and high-performance liquid chromatography. Structural studies were carried out utilizing specific chemical degradation, matrix-assisted laser-desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI FT ICR-MS), and 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. The major glycolipids in A. scleromae and A. globiformis were found to be a diglycosylglycerol with the structure alpha-Manp-(1-->3)-alpha-Manp-(1-->3)-Gro (Man A-Man B-Gro; G1), and a monoglycosylglycerol with the structure beta-Galp-(1-->3)-Gro (G2). Glycolipids were acylated at positions 1 of Gro and 6 of Man B in the case of G1 and at positions 1 and 2 of Gro in the case of G2. The distribution of the fatty acids was different in both species. A. scleromae glycolipids contained heptadecanoic acid while in the A. globiformis glycolipids mainly pentadecanoic acid could be detected. The substitution by hexadecanoic acid was proportionally similar in both species. The taxonomical value of major glycolipids from Arthrobacter is also presented.

Monoclonal antibody S60-4-14 reveals diagnostic potential in the identification of Pseudomonas aeruginosa in lung tissues of cystic fibrosis patients.

The lipopolysaccharide (LPS) of Pseudomonas aeruginosa has been identified to contain an inner-core structure expressing a Pseudomonas-specific epitope. This target structure is characterized by a highly phosphorylated and 7-O-carbamoyl-l-glycero-alpha-d-manno-heptopyranose (CmHep) and was found to be present in all human-pathogenic Pseudomonas species of the Palleroni (RNA)-classification I scheme. We raised and selected the monoclonal antibody S60-4-14 (mAb S60-4-14, subtype IgG1) from mice immunized with heat-killed Pseudomonas bacteria. The epitope of this mAb was found to reside in the inner-core structure of P. aeruginosa and, hence, successfully evaluated for the immunohistochemical detection of P. aeruginosa in formalin- or HOPE-fixed (Hepes-glutamic acid buffer-mediated organic solvent protection effect) and paraffin-embedded human lung tissue slices. Lung specimens, mainly from explanted lungs of cystic fibrosis (CF) patients, as well as P. aeruginosa isolates from patients suffering from CF and patients with extrapulmonar Pseudomonas infections were investigated by PCR, immunohistochemistry, and Western blot analysis with mAb S60-4-14. The results revealed an unequivocal coincidence of PCR and immunohistochemistry. Together with the Western blot results mAb S60-4-14 displays a potential diagnostic tool for the specific identification of P. aeruginosa in infected lungs of CF.