Bacterial Outer Membrane Vesicles. Mediators of virulence and antibiotic resistance.

Outer membrane vesicles (OMV) have the size of nanoparticles and are released from the outer membrane of most Gram-negative bacteria. The OMV reflect the composition of the bacterial outer membrane, including lipids and virulence factors specific for the pathogen, and are involved in pathogenesis and bacterial survival. Moraxella catarrhalis and Haemophilus influenzae are Gram-negative bacterial species that cause respiratory tract infections such as acute otitis media and exacerbations in chronic obstructive pulmonary disease (COPD). The aim of this thesis was to investigate what role OMV derived from respiratory pathogens such as M. catarrhalis and H. influenzae plays in immune activation and evasion, as well as how the OMV interact with other pathogens of the same colonization niche. In our first study, we determined the protemoic composition of M. catarrhalis OMV, and found them to be composed of mainly outer membrane and periplasmic proteins. We also established that OMV bind to and activate respiratory epithelial cells and cause a pro-inflammatory response both in vitro and in vivo. Considering that more than 97% of all M. catarrhalis strains are positive for β-lactamase, we determined in our second study that OMV from this pathogen contain β-lactamase. We further showed that OMV rescued other amoxicillin susceptible co-pathogens such as non-typeable H. influenzae (NTHi) and Streptococcus pneumoniae from killing. Thereafter, we established in our third study that serum from healthy adults contain anti-Moraxella β-lactamase antibodies. We showed that these antibodies bind to and neutralize β-lactamase in OMV to a certain degree, whereas OMV also act as protective vesicles, shielding the enzyme in the extracellular space. Finally, in the last study we investigated the role of OMV in co-infections with other species dwelling in the respiratory tract. Previous studies have shown treatment failures of group A streptococci (GAS) in pharyngotonsillits even though the GAS are completely susceptible to β-lactams, and that NTHi and M. catarrhalis are often found associated with GAS during treatment failures. We found that OMV from β-lactamase positive NTHi and M. catarrhalis protect GAS from amoxicillin-induced killing, and we suggest OMV secretion to be a mechanism for bacteria in polymicrobial infections to interact and protect each other. In summary, we determined that OMV secreted from Gram-negative bacteria of the upper respiratory tract are mediators of virulence and antibiotic resistance, interacting with host cells as well as other bacterial species found in polymicrobial infections

Galectin-1-Binding Glycoforms of Haptoglobin with Altered Intracellular Trafficking, and Increase in Metastatic Breast Cancer Patients

Sera from 25 metastatic breast cancer patients and 25 healthy controls were subjected to affinity chromatography using immobilized galectin-1. Serum from the healthy subjects contained on average 1.2 mg per ml (range 0.7–2.2) galectin-1 binding glycoproteins, whereas serum from the breast cancer patients contained on average 2.2 mg/ml (range 0.8–3.9), with a higher average for large primary tumours. The major bound glycoproteins were α-2-macroglobulin, IgM and haptoglobin. Both the IgM and haptoglobin concentrations were similar in cancer compared to control sera, but the percentage bound to galectin-1 was lower for IgM and higher for haptoglobin: about 50% (range 20–80) in cancer sera and about 30% (range 25–50) in healthy sera. Galectin-1 binding and non-binding fractions were separated by affinity chromatography from pooled haptoglobin from healthy sera. The N-glycans of each fraction were analyzed by mass spectrometry, and the structural differences and galectin-1 mutants were used to identify possible galectin-1 binding sites. Galectin-1 binding and non-binding fractions were also analyzed regarding their haptoglobin function. Both were similar in forming complex with haemoglobin and mediate its uptake into alternatively activated macrophages. However, after uptake there was a dramatic difference in intracellular targeting, with the galectin-1 non-binding fraction going to a LAMP-2 positive compartment (lysosomes), while the galectin-1 binding fraction went to larger galectin-1 positive granules. In conclusion, galectin-1 detects a new type of functional biomarker for cancer: a specific type of glycoform of haptoglobin, and possibly other serum glycoproteins, with a different function after uptake into tissue cells

Bacterial outer membrane vesicles in disease and preventive medicine.

Gram-negative bacteria have the ability to produce outer membrane-derived vesicles (OMVs) that are released into the extracellular milieu. Even though this intriguing phenomenon is well-known since many years, various aspects of bacterial OMVs are not fully described and are still in the process of being characterized in detail. One major reason for this is that depending on the bacterial species and its respective ecological niche, OMVs exhibit an enormous functional diversity. Research of the past years has clearly shown that OMVs of many pathogenic bacteria contribute to the virulence potential by enriching virulence factors and delivering them over long distances, superseding direct bacterial contact with their host. The subsequent interaction of OMVs with the host can occur at different levels regarding the type of immune response or the target cell type and may lead to different outcomes ranging from non-immunogenic activation or a pro-inflammatory response to cytotoxicity. In contrast to being virulence factors, OMVs are used for vaccination purposes in the combat against bacterial pathogens, and recent research thus is focused on to indirectly aim these versatile bacterial weapons against themselves

Group A streptococci are protected from amoxicillin-mediated killing by vesicles containing β-lactamase derived from Haemophilus influenzae.

Group A streptococci (GAS) cause, among other infections, pharyngotonsillitis in children. The species is frequently localized with the Gram-negative respiratory pathogens non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis, which both produce outer membrane vesicles (OMVs). The aim of this study was to investigate whether OMVs isolated from NTHi contain functional β-lactamase and whether the OMVs hydrolyse amoxicillin and thus protect GAS from killing by the antibiotic

Moraxella catarrhalis Outer Membrane Vesicles Carry β-Lactamase and Promote Survival of Streptococcus pneumoniae and Haemophilus influenzae by Inactivating Amoxicillin▿

Moraxella catarrhalis is a common pathogen found in children with upper respiratory tract infections and in patients with chronic obstructive pulmonary disease during exacerbations. The bacterial species is often isolated together with Streptococcus pneumoniae and Haemophilus influenzae. Outer membrane vesicles (OMVs) are released by M. catarrhalis and contain phospholipids, adhesins, and immunomodulatory compounds such as lipooligosaccharide. We have recently shown that M. catarrhalis OMVs exist in patients upon nasopharyngeal colonization. As virtually all M. catarrhalis isolates are β-lactamase positive, the goal of this study was to investigate whether M. catarrhalis OMVs carry β-lactamase and to analyze if OMV consequently can prevent amoxicillin-induced killing. Recombinant β-lactamase was produced and antibodies were raised in rabbits. Transmission electron microscopy, flow cytometry, and Western blotting verified that OMVs carried β-lactamase. Moreover, enzyme assays revealed that M. catarrhalis OMVs contained active β-lactamase. OMVs (25 μg/ml) incubated with amoxicillin for 1 h completely hydrolyzed amoxicillin at concentrations up to 2.5 μg/ml. In functional experiments, preincubation of amoxicillin (10× MIC) with M. catarrhalis OMVs fully rescued amoxicillin-susceptible M. catarrhalis, S. pneumoniae, and type b or nontypeable H. influenzae from β-lactam-induced killing. Our results suggest that the presence of amoxicillin-resistant M. catarrhalis originating from β-lactamase-containing OMVs may pave the way for respiratory pathogens that by definition are susceptible to β-lactam antibiotics

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

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

Multicomponent Moraxella catarrhalis outer membrane vesicles induce an inflammatory response and are internalized by human epithelial cells.

Moraxella catarrhalis is an emerging human respiratory pathogen in patients with chronic obstructive pulmonary disease (COPD) and in children with acute otitis media. The specific secretion machinery known as outer membrane vesicles (OMVs) is a mechanism by which Gram-negative pathogens interact with host cells during infection. We identified 57 proteins in M. catarrhalis OMVs using a proteomics approach combining two-dimensional SDS-PAGE and MALDI-TOF mass spectrometry analysis. The OMVs contained known surface proteins such as ubiquitous surface proteins (Usp) A1/A2, and Moraxella IgD-binding protein (MID). Most of the proteins are adhesins/virulence factors triggering the immune response, but also aid bacteria to evade the host defence. FITC-stained OMVs bound to lipid raft domains in alveolar epithelial cells and were internalized after interaction with Toll-like receptor 2 (TLR2), suggesting a delivery to the host tissue of a large and complex group of OMV-attributed proteins. Interestingly, OMVs modulated the pro-inflammatory response in epithelial cells, and UspA1-bearing OMVs were found to specifically downregulate the reaction. When mice were exposed to OMVs, a pulmonary inflammation was clearly seen. Our findings indicate that Moraxella OMVs are highly biologically active, transport main bacterial virulence factors and may modulate the epithelial pro-inflammatory response