In vivo gene expression profile of Haemophilus influenzae during human pneumonia.

Haemophilus influenzae is a major cause of community-acquired pneumonia. While studied extensively in various laboratory models, less is known about the cell function while inside the human lung. We present the first analysis of the global gene expression of H. influenzae while the bacteria are in the lung during pneumonia (in vivo conditions) and contrast it with bacterial isolates that have been cultured under standard laboratory conditions (in vitro conditions). Patients with pneumonia were recruited from emergency departments and intensive care units during 2018-2020 (n = 102). Lower respiratory samples were collected for bacterial culture and RNA extraction. Patient samples with H. influenzae (n = 8) and colonies from bacterial cultures (n = 6) underwent RNA sequencing. The reads were then pseudo-aligned to core and pan genomes created from 15 reference strains. While bacteria cultured in vitro clustered tightly by principal component analysis of core genome (n = 1067) gene expression, bacteria in the patient samples had more diverse transcriptomic signatures and did not group with their lab-cultured counterparts. In total, 328 core genes were significantly differentially expressed between in vitro and in vivo conditions. The most highly upregulated genes in vivo included tbpA and fbpA, which are involved in the acquisition of iron from transferrin, and the stress response gene msrAB. The biosynthesis of nucleotides/purines and molybdopterin-scavenging processes were also significantly enriched in vivo. In contrast, major metabolic pathways and iron-sequestering genes were downregulated under this condition. In conclusion, extensive transcriptomic differences were found between bacteria while in the human lung and bacteria that were cultured in vitro. IMPORTANCE The human-specific pathogen Haemophilus influenzae is generally not well suited for studying in animal models, and most laboratory models are unlikely to approximate the diverse environments encountered by bacteria in the human airways accurately. Thus, we have examined the global gene expression of H. influenzae during pneumonia. Extensive differences in the global gene expression profiles were found in H. influenzae while in the human lung compared to bacteria that were grown in the laboratory. In contrast, the gene expression profiles of isolates collected from different patients were found to cluster together when grown under the same laboratory conditions. Interesting observations were made of how H. influenzae acquires and uses iron and molybdate, endures oxidative stress, and regulates central metabolism while in the lung. Our results indicate important processes during infection and can guide future research on genes and pathways that are relevant in the pathogenesis of H. influenzae pneumonia.

TLR4-mediated release of heparin-binding protein in human airways: a co-stimulatory role for IL-26.

Background: Bacterial infection causes accumulation of neutrophils that release antimicrobial proteins including heparin-binding protein (HBP). In human airways, this neutrophil accumulation can be re-capitulated via intrabronchial exposure to lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) agonist, that also causes a local increase in the neutrophil-mobilizing cytokine IL-26. Although LPS is considered a weak stimulus for HBP release ex vivo, its effect on HBP release in human airways in vivo has not been characterized. Methods: We determined whether intrabronchial exposure to LPS causes concomitant release of HBP and IL-26 in human airways, and whether IL-26 can enhance LPS-induced release of HBP in isolated human neutrophils. Results: We found that the concentration of HBP was markedly increased in bronchoalveolar lavage (BAL) fluid 12, 24, and 48 hours after LPS exposure, and that it displayed a strong and positive correlation with that of IL-26. Moreover, the concentration of HBP in conditioned media from isolated neutrophils was enhanced only after co-stimulation with LPS and IL-26. Conclusions: Taken together, our findings indicate that TLR4 stimulation causes concomitant release of HBP and IL-26 in human airways, and that IL-26 may constitute a required co-stimulant for HBP release in neutrophils, thus enabling the concerted action of HBP and IL-26 in local host defense.

A retrospective cohort study of the effect of SARS-CoV-2 point of care rapid RT-PCR at the Emergency Department on targeted admission.

BACKGROUND: To prevent nosocomial transmission of SARS-CoV-2, infection prevention control (IPC) measures are implemented for patients with symptoms compatible with COVID-19 until reliable test results are available. This delays admission to the most appropriate ward based on the medical condition. SARS-CoV-2 rapid antigen detection (RAD) tests and point-of-care (POC) rapid RT-PCR (VitaPCR) were introduced at emergency department (ED) at Skåne University Hospital, Sweden in late 2020, but the consequence on patient flow and targeted admission is unknown. METHODS: Patients presenting at the emergency department of a referral hospital (N = 2940) between 13-Nov-2020 and 12-Jan-2021 were included. The study period was delimited into three periods by the introduction of RAD tests and the VitaPCR. Participant data was collected from hospital records, and outcome variables were Length-of-Stay (LoS), intrahospital transfers and targeted admission to COVID-19 ward. RESULTS: Compared to baseline (RT-PCR only), RAD tests reduced ED Length-of-Stay (LoS) for participants with positive tests. Negative VitaPCR results reduced mean hospital LoS by 1.5 (95% CI 0.3-2.7) days and admissions to COVID-19 wards from 34.5 (95% CI 28.9-40.5) to 14.7 (95% CI 11.1-19.1) per 100 admissions and reduced transfers between hospital wards in the first 5 days from 50.0 (95% CI 45.0-55.0) to 34.0 (95% CI 30.3-37.9) per 100 admissions. CONCLUSION: RAD tests enabled prompt detection of SARS-CoV-2 infection which had pronounced effects on LoS at the ED. Negative VitaPCR enabled cessation of IPC measures and a negative test was associated with increased targeted admissions, reduced intrahospital transfers and shorter LoS at the hospital.

Complex Involvement of Interleukin-26 in Bacterial Lung Infection.

Pneumonia is a global cause of mortality, and this provides a strong incentive to improve the mechanistic understanding of innate immune responses in the lungs. Here, we characterized the involvement of the cytokine interleukin (IL)-26 in bacterial lung infection. We observed markedly increased concentrations of IL-26 in lower airway samples from patients with bacterial pneumonia and these correlated with blood neutrophil concentrations. Moreover, pathogen-associated molecular patterns (PAMPs) from both Gram-negative and -positive bacteria increased extracellular IL-26 concentrations in conditioned media from human models of alveolar epithelial cells, macrophages, and neutrophils in vitro. Stimulation with IL-26 inhibited the inherent release of neutrophil elastase and myeloperoxidase in unexposed neutrophils. This stimulation also inhibited the expression of activity makers in neutrophils exposed to Klebsiella pneumoniae. In addition, priming of human lung tissue ex vivo with exogenous IL-26 potentiated the endotoxin-induced increase in mRNA for other cytokines involved in the innate immune response, including the master Th17-regulator IL-23 and the archetype inhibitory cytokine IL-10. Finally, neutralization of endogenous IL-26 clearly increased the growth of Klebsiella pneumoniae in the macrophage culture. These findings suggest that IL-26 is involved in bacterial lung infection in a complex manner, by modulating critical aspects of innate immune responses locally and systemically in a seemingly purposeful manner and by contributing to the killing of bacteria in a way that resembles an antimicrobial peptide. Thus, IL-26 displays both diagnostic and therapeutic potential in pneumonia and deserves to be further evaluated in these respects.

Chemical and biochemical bleaching of oat hulls: The effect of hydrogen peroxide, laccase, xylanase and sonication on optical properties and chemical composition.

Oat hulls are an excellent dietary fibre source for food supplements due to their rich lignocellulose composition as well as their great abundance as low-value agricultural side stream. For the production of white fibre supplements, a mild, but effective bleaching of the hulls is required. Chemical bleaching with hydrogen peroxide and sodium hydroxide was here found to be a suitable method increasing the CIE L* value (corresponds to a lightness value) above 85. The developed method is mild, retaining the hull's chemical composition. Only a minor decrease in coniferaldehyde structures upon bleaching was detected. Colour and chemical variabilities of oat hulls from different growth seasons did not influence the required bleaching conditions to achieve the desired optical properties. The inclusion of biochemical bleaching steps utilizing the xylanase Pentopan Mono BG, the laccase NS51003 and sonication was industrially not feasible as they could not reduce the required amount of subsequently applied bleaching chemicals significantly.

Heparin-binding protein in lower airway samples as a biomarker for pneumonia.

OBJECTIVES: Ventilator-associated pneumonia (VAP) is difficult to diagnose using clinical criteria and no biomarkers have yet been proved to be sufficiently accurate. The use of the neutrophil-derived Heparin-binding protein (HBP) as a biomarker for pneumonia was investigated in this exploratory case-control study in two intensive care units at a tertiary referral hospital. METHODS: Patients with clinical signs of pneumonia were recruited and bronchoalveolar lavage fluid (BALF) or bronchial wash (BW) samples were collected. Mechanically ventilated and lung healthy subjects were recruited as controls. HBP was measured with enzyme-linked immunosorbent assay. RESULTS: BALF was collected from 14 patients with pneumonia and 14 healthy controls. Median HBP in BALF pneumonia samples was 14,690 ng/ml and controls 16.2 ng/ml (p < 0.0001). BW was collected from 10 pneumonia patients and 10 mechanically ventilated controls. Median HBP in BW pneumonia was 9002 ng/ml and controls 7.6 ng/ml (p < 0.0001). CONCLUSIONS: These data indicate that HBP concentrations is significantly higher in lower airway samples from patients with pneumonia than control subjects and is a potentially useful biomarker for diagnosis of VAP.

Mucin Binding to Moraxella catarrhalis during Airway Inflammation Is Dependent on Sialic Acid.

Chronic obstructive pulmonary disease (COPD) is associated with colonization by bacterial pathogens and repeated airway infections, leading to exacerbations and impaired lung function. The highly glycosylated mucins in the mucus lining the airways are an important part of the host defense against pathogens. However, mucus accumulation can contribute to COPD pathology. Here, we examined whether inflammation is associated with glycosylation changes that affect interactions between airway mucins and pathogens. We isolated mucins from lower airway samples (n = 4-9) from long-term smokers with and without COPD and from never-smokers. The most abundant terminal glycan moiety was N-acetylneuraminic acid (Neu5Ac) among smokers with and without COPD and N-acetyl-hexoseamine among never-smokers. Moraxella catarrhalis bound to MUC5 mucins from smokers with and without COPD. M. catarrhalis binding correlated with inflammatory parameters and Neu5Ac content. M. catarrhalis binding was abolished by enzymatic removal of Neu5Ac. Furthermore, M. catarrhalis bound to α2,6 sialyl-lactose, suggesting that α2,6 sialic acid contributes to M. catarrhalis binding to mucins. Furthermore, we detected more M. catarrhalis binding to mucins from patients with pneumonia than to those from control subjects (n = 8-13), and this binding correlated with C-reactive protein and Neu5Ac levels. These results suggest a key role of inflammation-induced Neu5Ac in the adhesion of M. catarrhalis to airway mucins. The inflammation-induced ability of MUC5 mucins to bind M. catarrhalis is likely a host defense mechanism in the healthy lung, although it cannot be excluded that impaired mucociliary clearance limits the effectiveness of this defense in patients with COPD.

Genomic and Phenotypic Evolution of Achromobacter xylosoxidans during Chronic Airway Infections of Patients with Cystic Fibrosis.

Bacterial pathogens evolve during chronic colonization of the human host by selection for pathoadaptive mutations. One of the emerging and understudied bacterial species causing chronic airway infections in patients with cystic fibrosis (CF) is Achromobacter xylosoxidans. It can establish chronic infections in patients with CF, but the genetic and phenotypic changes associated with adaptation during these infections are not completely understood. In this study, we analyzed the whole-genome sequences of 55 clinical A. xylosoxidans isolates longitudinally collected from the sputum of 6 patients with CF. Four genes encoding regulatory proteins and two intergenic regions showed convergent evolution, likely driven by positive selection for pathoadaptive mutations, across the different clones of A. xylosoxidans. Most of the evolved isolates had lower swimming motility and were resistant to multiple classes of antibiotics, while fewer of the evolved isolates had slower growth or higher biofilm production than the first isolates. Using a genome-wide association study method, we identified several putative genetic determinants of biofilm formation, motility and ß-lactam resistance in this pathogen. With respect to antibiotic resistance, we discovered that a combination of mutations in pathoadaptive genes (phoQ and bigR) and two other genes encoding regulatory proteins (spoT and cpxA) were associated with increased resistance to meropenem and ceftazidime. Altogether, our results suggest that genetic changes within regulatory loci facilitate within-host adaptation of A. xylosoxidans and the emergence of adaptive phenotypes, such as antibiotic resistance or biofilm formation. IMPORTANCE A thorough understanding of bacterial pathogen adaptation is essential for the treatment of chronic bacterial infections. One unique challenge in the analysis and interpretation of genomics data is identifying the functional impact of mutations accumulated in the bacterial genome during colonization in the human host. Here, we investigated the genomic and phenotypic evolution of A. xylosoxidans in chronic airway infections of patients with CF and identified several mutations associated with the phenotypic evolution of this pathogen using genome-wide associations. Identification of phenotypes under positive selection and the associated mutations can enlighten the adaptive processes of this emerging pathogen in human infections and pave the way for novel therapeutic interventions.

Peptidoglycan-Binding Anchor Is a Pseudomonas aeruginosa OmpA Family Lipoprotein With Importance for Outer Membrane Vesicles, Biofilms, and the Periplasmic Shape.

The outer membrane protein A (OmpA) family contains an evolutionary conserved domain that links the outer membrane in Gram-negative bacteria to the semi-rigid peptidoglycan (PG) layer. The clinically significant pathogen Pseudomonas aeruginosa carries several OmpA family proteins (OprF, OprL, PA0833, and PA1048) that share the PG-binding domain. These proteins are important for cell morphology, membrane stability, and biofilm and outer membrane vesicle (OMV) formation. In addition to other OmpAs, in silico analysis revealed that the putative outer membrane protein (OMP) with gene locus PA1041 is a lipoprotein with an OmpA domain and, hence, is a potential virulence factor. This study aimed to evaluate PA1041 as a PG-binding protein and describe its effect on the phenotype. Clinical strains were confirmed to contain the lipoprotein resulting from PA1041 expression with Western blot, and PG binding was verified in enzyme-linked immunosorbent assay (ELISA). By using a Sepharose bead-based ELISA, we found that the lipoprotein binds to meso-diaminopimelic acid (mDAP), an amino acid in the pentapeptide portion of PGs. The reference strain PAO1 and the corresponding transposon mutant PW2884 devoid of the lipoprotein were examined for phenotypic changes. Transmission electron microscopy revealed enlarged periplasm spaces near the cellular poles in the mutant. In addition, we observed an increased release of OMV, which could be confirmed by nanoparticle tracking analysis. Importantly, mutants without the lipoprotein produced a thick, but loose and unorganized, biofilm in flow cells. In conclusion, the lipoprotein from gene locus PA1041 tethers the outer membrane to the PG layer, and mutants are viable, but display severe phenotypic changes including disordered biofilm formation. Based upon the phenotype of the P. aeruginosa PW2884 mutant and the function of the protein, we designate the lipoprotein with locus tag PA1041 as "peptidoglycan-binding anchor" (Pba).

Pseudomonas aeruginosa uses multiple receptors for adherence to laminin during infection of the respiratory tract and skin wounds.

Pseudomonas aeruginosa efficiently adheres to human tissues, including the lungs and skin, causing infections that are difficult to treat. Laminin is a main component of the extracellular matrix, and in this study we defined bacterial laminin receptors on P. aeruginosa. Persistent clinical P. aeruginosa isolates from patients with cystic fibrosis, wounds or catheter-related urinary tract infections bound more laminin compared to blood isolates. Laminin receptors in the outer membrane were revealed by 2D-immunblotting, and the specificities of interactions were confirmed with ELISA and biolayer interferometry. Four new high-affinity laminin receptors were identified in the outer membrane; EstA, OprD, OprG and PA3923. Mutated bacteria devoid of these receptors adhered poorly to immobilized laminin. All bacterial receptors bound to the heparin-binding domains on LG4 and LG5 of the laminin alpha chain as assessed with truncated laminin fragments, transmission electron microscopy and inhibition by heparin. In conclusion, P. aeruginosa binds laminin via multiple surface receptors, and isolates from lungs of cystic fibrosis patients bound significantly more laminin compared to bacteria isolated from the skin and urine. Since laminin is abundant in both the lungs and skin, we suggest that laminin binding is an important mechanism in P. aeruginosa pathogenesis.

Bacterial Outer Membrane Vesicles Induce Vitronectin Release Into the Bronchoalveolar Space Conferring Protection From Complement-Mediated Killing.

Pathogens causing pneumonia utilize the complement regulator vitronectin to evade complement-mediated killing. Although vitronectin is associated with several chronic lung diseases, the role of bronchoalveolar vitronectin in pneumonia has not been studied. This study sought to reveal the involvement of vitronectin in the bronchoalveolar space during pneumonia, to assess the effect of outer membrane vesicles and endotoxin on vitronectin release, and to determine whether bacterial pathogens utilize pulmonary vitronectin for evasion. Vitronectin was analyzed in cell-free bronchoalveolar lavage fluid harvested from patients with pneumonia (n = 8) and from healthy volunteers after subsegmental endotoxin instillation (n = 13). Vitronectin binding by Pseudomonas aeruginosa and Haemophilus influenzae was analyzed, and subsequent complement evasion was assessed by serum challenge. The effects of outer membrane vesicles on vitronectin production in mouse lungs and human type II alveolar epithelial cells (A549) were determined. We detected increased vitronectin concentrations in lavage fluid during pneumonia (p = 0.0063) and after bronchial endotoxin challenge (p = 0.016). The capture of vitronectin by bacteria significantly reduced complement-mediated lysis. Following challenge with vesicles, vitronectin was detected in mouse bronchoalveolar space, and mouse alveolar epithelial cells in vivo as well as A549 cells in vitro contained increased levels of vitronectin. Taken together, outer membrane vesicles and endotoxin from Gram-negative bacteria induce vitronectin, which is released into the bronchoalveolar space, and used for evasion of complement-mediated clearance.

How bacteria hack the matrix and dodge the bullets of immunity.

Haemophilus influenzae, Moraxella catarrhalis and Pseudomonas aeruginosa are common Gram-negative pathogens associated with an array of pulmonary diseases. All three species have multiple adhesins in their outer membrane, i.e. surface structures that confer the ability to bind to surrounding cells, proteins or tissues. This mini-review focuses on proteins with high affinity for the components of the extracellular matrix such as collagen, laminin, fibronectin and vitronectin. Adhesins are not structurally related and may be lipoproteins, transmembrane porins or large protruding trimeric auto-transporters. They enable bacteria to avoid being cleared together with mucus by attaching to patches of exposed extracellular matrix, or indirectly adhering to epithelial cells using matrix proteins as bridging molecules. As more adhesins are being unravelled, it is apparent that bacterial adhesion is a highly conserved mechanism, and that most adhesins target the same regions on the proteins of the extracellular matrix. The surface exposed adhesins are prime targets for new vaccines and the interactions between proteins are often possible to inhibit with interfering molecules, e.g heparin. In conclusion, this highly interesting research field of microbiology has unravelled host-pathogen interactions with high therapeutic potential.

Vibrations of a molecule in an external force field.

The oscillation frequencies of a molecule on a surface are determined by the mass distribution in the molecule and the restoring forces that occur when the molecule bends. The restoring force originates from the atomic-scale interaction within the molecule and with the surface, which plays an essential role in the dynamics and reactivity of the molecule. In 1998, a combination of scanning tunneling microscopy with inelastic tunneling spectroscopy revealed the vibrational frequencies of single molecules adsorbed on a surface. However, the probe tip itself exerts forces on the molecule, changing its oscillation frequencies. Here, we combine atomic force microscopy with inelastic tunneling spectroscopy and measure the influence of the forces exerted by the tip on the lateral vibrational modes of a carbon monoxide molecule on a copper surface. Comparing the experimental data to a mechanical model of the vibrating molecule shows that the bonds within the molecule and with the surface are weakened by the proximity of the tip. This combination of techniques can be applied to analyze complex molecular vibrations and the mechanics of forming and loosening chemical bonds, as well as to study the mechanics of bond breaking in chemical reactions and atomic manipulation.

STM contrast of a CO dimer on a Cu(1 1 1) surface: a wave-function analysis.

We present a method used to intuitively interpret the scanning tunneling microscopy (STM) contrast by investigating individual wave functions originating from the substrate and tip side. We use localized basis orbital density functional theory, and propagate the wave functions into the vacuum region at a real-space grid, including averaging over the lateral reciprocal space. Optimization by means of the method of Lagrange multipliers is implemented to perform a unitary transformation of the wave functions in the middle of the vacuum region. The method enables (i) reduction of the number of contributing tip-substrate wave function combinations used in the corresponding transmission matrix, and (ii) to bundle up wave functions with similar symmetry in the lateral plane, so that (iii) an intuitive understanding of the STM contrast can be achieved. The theory is applied to a CO dimer adsorbed on a Cu(1 1 1) surface scanned by a single-atom Cu tip, whose STM image is discussed in detail by the outlined method.

PRELP Enhances Host Innate Immunity against the Respiratory Tract Pathogen Moraxella catarrhalis.

Respiratory tract infections are one of the leading causes of mortality worldwide urging better understanding of interactions between pathogens causing these infections and the host. Here we report that an extracellular matrix component proline/arginine-rich end leucine-rich repeat protein (PRELP) is a novel antibacterial component of innate immunity. We detected the presence of PRELP in human bronchoalveolar lavage fluid and showed that PRELP can be found in alveolar fluid, resident macrophages/monocytes, myofibroblasts, and the adventitia of blood vessels in lung tissue. PRELP specifically binds respiratory tract pathogens Moraxella catarrhalis, Haemophilus influenzae, and Streptococcus pneumoniae, but not other bacterial pathogens tested. We focused our study on M. catarrhalis and found that PRELP binds the majority of clinical isolates of M. catarrhalis (n = 49) through interaction with the ubiquitous surface protein A2/A2H. M. catarrhalis usually resists complement-mediated serum killing by recruiting to its surface a complement inhibitor C4b-binding protein, which is also a ligand for PRELP. We found that PRELP competitively inhibits binding of C4b-binding protein to bacteria, which enhances membrane attack complex formation on M. catarrhalis and thus leads to increased serum sensitivity. Furthermore, PRELP enhances phagocytic killing of serum-opsonized M. catarrhalis by human neutrophils in vitro. Moreover, PRELP reduces Moraxella adherence to and invasion of human lung epithelial A549 cells. Taken together, PRELP enhances host innate immunity against M. catarrhalis through increasing complement-mediated attack, improving phagocytic killing activity of neutrophils, and preventing bacterial adherence to lung epithelial cells.

Controlled switching of single-molecule junctions by mechanical motion of a phenyl ring.

Mechanical methods for single-molecule control have potential for wide application in nanodevices and machines. Here we demonstrate the operation of a single-molecule switch made functional by the motion of a phenyl ring, analogous to the lever in a conventional toggle switch. The switch can be actuated by dual triggers, either by a voltage pulse or by displacement of the electrode, and electronic manipulation of the ring by chemical substitution enables rational control of the on-state conductance. Owing to its simple mechanics, structural robustness, and chemical accessibility, we propose that phenyl rings are promising components in mechanical molecular devices.

Heparin-Binding Protein Measurement Improves the Prediction of Severe Infection With Organ Dysfunction in the Emergency Department.

OBJECTIVES: Early identification of patients with infection and at risk of developing severe disease with organ dysfunction remains a difficult challenge. We aimed to evaluate and validate the heparin-binding protein, a neutrophil-derived mediator of vascular leakage, as a prognostic biomarker for risk of progression to severe sepsis with circulatory failure in a multicenter setting. DESIGN: A prospective international multicenter cohort study. SETTING: Seven different emergency departments in Sweden, Canada, and the United States. PATIENTS: Adult patients with a suspected infection and at least one of three clinical systemic inflammatory response syndrome criteria (excluding leukocyte count). INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: Plasma levels of heparin-binding protein, procalcitonin, C-reactive protein, lactate, and leukocyte count were determined at admission and 12-24 hours after admission in 759 emergency department patients with suspected infection. Patients were defined depending on the presence of infection and organ dysfunction. Plasma samples from 104 emergency department patients with suspected sepsis collected at an independent center were used to validate the results. Of the 674 patients diagnosed with an infection, 487 did not have organ dysfunction at enrollment. Of these 487 patients, 141 (29%) developed organ dysfunction within the 72-hour study period; 78.0% of the latter patients had an elevated plasma heparin-binding protein level (>30 ng/mL) prior to development of organ dysfunction (median, 10.5 hr). Compared with other biomarkers, heparin-binding protein was the best predictor of progression to organ dysfunction (area under the receiver operating characteristic curve=0.80). The performance of heparin-binding protein was confirmed in the validation cohort. CONCLUSION: In patients presenting at the emergency department, heparin-binding protein is an early indicator of infection-related organ dysfunction and a strong predictor of disease progression to severe sepsis within 72 hours.

Identification of outer membrane Porin D as a vitronectin-binding factor in cystic fibrosis clinical isolates of Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is a pathogen that frequently colonizes patients with cystic fibrosis (CF) or chronic obstructive pulmonary disease (COPD). Several pathogens are known to bind vitronectin to increase their virulence. Vitronectin has been shown to enhance P. aeruginosa adhesion to host epithelial cells. METHODS: We screened clinical isolates from the airways of CF patients and from the bloodstream of patients with bacteremia for binding of vitronectin. Two-dimensional SDS-PAGE and a proteomic approach were used to identify vitronectin-receptors in P. aeruginosa. RESULTS: P. aeruginosa from the airways of CF patients (n=27) bound more vitronectin than bacteremic isolates (n=15, p=0.025). Porin D (OprD) was identified as a vitronectin-binding protein. A P. aeruginosa oprD transposon insertion mutant had a decreased binding to soluble and immobilized vitronectin (p≤0.001). CONCLUSIONS: P. aeruginosa isolates obtained from CF patients significantly bound vitronectin. Porin D was defined as a novel P. aeruginosa vitronectin-receptor, and we postulate that the Porin D-dependent interaction with vitronectin may be important for colonization.

Rotation of a single acetylene molecule on Cu(001) by tunneling electrons in STM.

We study the elementary processes behind one of the pioneering works on scanning tunneling microscope controlled reactions of single molecules [Stipe et al., Phys. Rev. Lett. 81, 1263 (1998)]. Using the Keldysh-Green function approach for the vibrational generation rate in combination with density functional theory calculations to obtain realistic parameters we reproduce the experimental rotation rate of an acetylene molecule on a Cu(100) surface as a function of bias voltage and tunneling current. This combined approach allows us to identify the reaction coordinate mode of the acetylene rotation and its anharmonic coupling with the C-H stretch mode. We show that three different elementary processes, the excitation of C-H stretch, the overtone ladder climbing of the hindered rotational mode, and the combination band excitation together explain the rotation of the acetylene molecule on Cu(100).

Outer membrane vesicles shield Moraxella catarrhalis ß-lactamase from neutralization by serum IgG.

OBJECTIVES: The aim of this study was to detect the presence of IgG against Moraxella catarrhalis ß-lactamase in healthy adults, and to determine whether outer membrane vesicles (OMVs) could protect the enzyme from inhibition by anti-ß-lactamase IgG. METHODS: Transmission electron microscopy was used to detect the presence of ß-lactamase in OMVs. Sera were examined by ELISA for specific IgG directed against recombinant M. catarrhalis ß-lactamase in addition to the outer membrane adhesins MID/Hag, UspA1 and A2. Binding of anti-ß-lactamase IgG from serum to OMVs was analysed by flow cytometry. The chromogenic substrate nitrocefin was used to quantify ß-lactamase enzyme activity. RESULTS: The presence of ß-lactamase was determined in OMVs from a 9-year-old child suffering from M. catarrhalis sinusitis. Furthermore, anti-ß-lactamase IgG was detected in sera obtained from healthy adults. Out of 40 adult blood donors (aged 18-65 years) tested, 6 (15.0%) carried anti-ß-lactamase IgG. No correlation between IgG titres against ß-lactamase and the adhesins was found. Flow cytometry analyses revealed that anti-ß-lactamase IgG from serum bound to ß-lactamase-positive OMVs. By comparing the ß-lactamase activity of intact OMV with OMV that were permeabilized with saponin we found that OMVs shielded active ß-lactamase from the anti-ß-lactamase IgG. CONCLUSIONS: Moraxella catarrhalis ß-lactamase is found in, or associated with, OMVs, providing clinical relevance for the vesicles in the spread of antibiotic resistance. Furthermore, OMVs protect ß-lactamase from specific IgG.