Binding capacity of mannose-binding lectin (MBL) is associated with the severity of chronic Chagas cardiomyopathy

Chagas disease (CD) is a global problem. Currently, it affects approximately 15 million individuals in Latin America. It is well know that the human immune response is related to different clinical manifestations. Mannose binding lectin (MBL) plays an important role in innate immunity, and it mediates the phagocytosis and complement-mediated destruction of pathogens. The binding capacity is enhanced by the oligomerization of MBL. In this study, we evaluated the serum concentration and the binding capacity of MBL in patients with chronic chagasic cardiomyopathy. A total of 77 patients with chronic CD were included with indeterminate (n?=?19), mild cardiac (n?=?29) and severe cardiac (n?=?29) forms. The serum concentration and the binding capacity were measured using enzyme-linked immunosorbent assays (ELISA). There was no significant difference in the serum MBL levels between the groups of patients. However, we found a relationship between the binding capacity and the groups studied. Our results suggest that binding capacity of MBL could be an indicator of clinical manifestation in Chronic Chagas cardiomyopathy. Furthermore, combined with the Mannose Binding Index results in a useful clinical tool for management of Chronic Chagas Patients.     

Complement resistance is a virulence factor of Branhamella (Moraxella) catarrhalis

Abstract The purpose of this study was to investigate complement resistance in Branhamella (Moraxella) catarrhalis isolated from healthy schoolchildren or sputum-producing adult patients. Two techniques were used: a serum bactericidal assay as the gold standard and an easier ‘culture and spot’ test. Children (age 4–13; n = 303) and patients ( n = 1047) showed high colonization/infection rates with B. catarrhalis (31% and 19%, respectively). Complement resistance or intermediate sensitivity occurred frequently in patient isolates (62% and 27%, respectively) and less often in children (33% and 8.5%, respectively; P ⪡ 0.0001). In young children (age 4–5 years), the proportion of complement-resistant strains was around 50%. Complement resistance in B. catarrhalis is associated with illness and may hence be considered a virulence factor.     

Attachment of Moraxella catarrhalis to pharyngeal epithelial cells is mediated by a glycosphingolipid receptor

Abstract Moraxella catarrhalis is one of the major pathogens of respiratory infections and has the ability to attach to the pharyngeal cells via fimbriae. We characterized the epithelial cell receptor to which fimbriate M. catarrhalis binds. Neuraminidase pretreatment of pharyngeal epithelial cells resulted in a significant decrease of M. catarrhalis attachment, suggesting interaction with the sialic acid component. The attachment was not decreased in M. catarrhalis pretreated with 2 and 1 mg/ml of fucose, N -acetyl-neuraminic acid, N -acetyl-glucosamine, N -acetyl-galactosamine, acetyl-salicylic acid and colominic acid. However, M. catarrhalis treated with gangliosides M1, M2, D1a, D1b and T1a at a concentration of 2.5 μg/ml had significantly decreased the attachment compared to the control. In contrast treatment with gangliosides M3 and asialoganglioside M1 did not decrease the attachment of M. catarrhalis and thereby provided evidence for specificity of the inhibition. Concentration dependent effects of ganglioside M2 on the attachment were also observed. Other fimbriate isolates of M. catarrhalis showed decrease in attachment after treatment with ganglioside M2. However there was no effect on attachment when a nonfimbriate isolate was treated with ganglioside M2. This study indicates that the receptor of fimbriate M. catarrhalis on pharyngeal epithelial cells resides in the sequences of ganglioside M2.     

Unveiling electrotransformation of Moraxella catarrhalis as a process of natural transformation

The human respiratory tract pathogen Moraxella catarrhalis is a naturally competent microorganism. However, electrotransformation has long been used to introduce foreign DNA into this organism. This study demonstrated that electrotransformants obtained with linear or circular nonreplicating plasmid DNA originated exclusively from natural transformation processes taking place during the recovery phase after the application of current. Only replicating plasmid DNA could be introduced into M. catarrhalis by electrotransformation, in a type IV pilus-independent manner. Electrotransformation with homologous genomic DNA indicated that restriction of double-stranded DNA was independent of type III restriction-methylation systems. Nontransformability of M. catarrhalis by electrotransformation was observed using double- as well as single-stranded DNA. In addition, the study showed that natural competence is a very constant feature of M. catarrhalis.     

Moraxella (Branhamella) catarrhalis: Restriction enzyme analysis typing with HinfI, HaeIII and PstI

Abstract Restriction enzyme analysis typing with Hin fI, Hae III and Pst I was performed on Moraxella (Branhamella) catarrhalis strains consecutively collected from children suspected of respiratory tract infection and the type strain. Use of Hin fI gave the most distinct patterns. Great polymorphism was seen among strains.     

Identification of a Moraxella catarrhalis gene that confers adherence to various human epithelial cell lines in vitro

Moraxella catarrhalis is a pathogen of the human airways. We found that expression of the M. catarrhalis gene mcmA by Escherichia coli increases adherence to epithelial cells 100-fold. Furthermore, we discovered that disrupting mcmA decreases M. catarrhalis adherence to laryngeal and lung cells, which are relevant to pathogenesis by the bacterium.     

Transmission electron microscopy studies of Moraxella (Branhamella) catarrhalis

A trypsin-sensitive 200-kDa protein has been reported to be exclusively associated with haemagglutinating isolates of Moraxella (Branhamella) catarrhalis. Transmission electron microscopy studies revealed that haemagglutination by M. catarrhalis to both human and rabbit erythrocytes was mediated by a trypsin-sensitive outer fibrillar coat. This fibrillar layer was absent on non-haemagglutinating isolates examined. Immuno-electron microscopy, using a polyclonal antiserum containing antibodies to the 200-kDa protein as a probe, showed that the 200-kDa protein is present on the outer fibrillar layer of the bacterium. These findings suggest that the haemagglutinin of M. catarrhalis is a 200-kDa protein present on the outer fibrillar coat.     

Near-planar solution structures of mannose-binding lectin oligomers provide insight on activation of lectin pathway of complement

The complement system is a fundamental component of innate immunity that orchestrates complex immunological and inflammatory processes. Complement comprises over 30 proteins that eliminate invading microorganisms while maintaining host cell integrity. Protein-carbohydrate interactions play critical roles in both the activation and regulation of complement. Mannose-binding lectin (MBL) activates the lectin pathway of complement via the recognition of sugar arrays on pathogenic surfaces. To determine the solution structure of MBL, synchrotron x-ray scattering and analytical ultracentrifugation experiments showed that the carbohydrate-recognition domains in the MBL dimer, trimer, and tetramer are positioned close to each other in near-planar fan-like structures. These data were subjected to constrained modeling fits. A bent structure for the MBL monomer was identified starting from two crystal structures for its carbohydrate-recognition domain and its triple helical region. The MBL monomer structure was used to identify 10-12 near-planar solution structures for each of the MBL dimers, trimers, and tetramers starting from 900 to 6,859 randomized structures for each. These near-planar fan-like solution structures joined at an N-terminal hub clarified how the carbohydrate-recognition domain of MBL binds to pathogenic surfaces. They also provided insight on how MBL presents a structural template for the binding and auto-activation of the MBL-associated serine proteases to initiate the lectin pathway of complement activation.     

Activation of the alternative complement pathway by mannose-binding lectin via a C2-bypass pathway

MBL is a serum lectin that activates the lectin pathway of the complement system. MBL forms complexes with three types of MASPs. Upon binding to Salmonella serogroup C-specific oligosaccharide, MBL activates the alternative pathway via a C2-bypass pathway without involving MASP-2, C2 or C4. We demonstrate that mannan-bound MBL activates the alternative pathway via a C2-bypass pathway that requires MASP-2 and C4. Thus, depending on the ligands to which MBL binds, there may be two distinct MBL-mediated C2-bypass pathways.     

Polymorphism of the major surface epitope of the CopB outer membrane protein of Moraxella catarrhalis

The CopB outer membrane protein has been considered a vaccine candidate for the prevention of infections due to Moraxella catarrhalis. Monoclonal antibody 10F3 recognizes whole cells of about 70% of clinical isolates, suggesting that this epitope is reasonably conserved. To determine whether CopB has other surface epitopes, we analyzed M. catarrhalis isolates using polyclonal sera against recombinant CopB proteins from a 10F3 positive isolate and a 10F3 negative isolate, and polyclonal sera against synthetic peptides that contained the sequence corresponding to the 10F3 epitope region of three different isolates. Extensive cross-reactivity was observed with the anti-CopB sera towards purified recombinant CopB proteins in Western blot and antigen ELISA, implying that antigenic regions common to both proteins were present. However, anti-CopB sera resembled anti-CopB peptide sera in exhibiting similar binding specificity to whole cells, segregating M. catarrhalis isolates into four CopB groups. We subsequently cloned and sequenced the copB genes from representative isolates. The deduced CopB amino acid sequences and the degree of sequence identity also demonstrated the existence of the same four CopB groups. Each of the four groups had a unique sequence in the 10F3 epitope region and a fifth group had the epitope deleted. The polymorphism of the major surface epitope prompts further consideration regarding the utility of CopB as a vaccine component as well as the design of an efficacious CopB-based vaccine to achieve broad protection against Moraxella infection.     

cDNA cloning and characterization of a mannose-binding lectin fromZingiber officinaleRoscoe (ginger) rhizomes

Using RNA extracted fromZingiber officinale rhizomes and primers designed according to the conservative regions of monocot mannose-binding lectins, the full-length cDNA ofZ. officinale agglutinin (ZOA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA ofzoa was 746 bp and contained a 510 bp open reading frame (ORF) encoding a lectin precursor of 169 amino acids with a signal peptide. ZOA was a mannose-binding lectin with three typical mannose-binding sites (QDNY). Semi-quantitative RT-PCR analysis revealed thatzoa expressed in all the tested tissues ofZ. officinale including leaf, root and rhizome, suggesting it to be a constitutively expressing form. ZOA protein was successfully expressed inEscherichia coli with the molecular weight expected. To our knowledge, this is the first mannose-binding lectin cDNA cloned from the family Zingiberaceae. Our results demonstrate that monocot mannose-binding lectins also occur within the family Zingiberaceae     

Moraxella catarrhalis induces CEACAM3‐Syk‐CARD9‐dependent activation of human granulocytes

The human restricted pathogen Moraxella catarrhalis is an important causal agent for exacerbations in chronic obstructive lung disease in adults. In such patients, increased numbers of granulocytes are present in the airways, which correlate with bacteria‐induced exacerbations and severity of the disease. Our study investigated whether the interaction of M. catarrhalis with the human granulocyte‐specific carcinoembryonic antigen‐related cell adhesion molecule (CEACAM)‐3 is linked to NF‐κB activation, resulting in chemokine production. Granulocytes from healthy donors and NB4 cells were infected with M. catarrhalis in the presence of different inhibitors, blocking antibodies and siRNA. The supernatants were analysed by enzyme‐linked immunosorbent assay for chemokines. NF‐κB activation was determined using a luciferase reporter gene assay and chromatin‐immunoprecipitation. We found evidence that the specific engagement of CEACAM3 by M. catarrhalis ubiquitous surface protein A1 (UspA1) results in the activation of pro‐inflammatory events, such as degranulation of neutrophils, ROS production and chemokine secretion. The interaction of UspA1 with CEACAM3 induced the activation of the NF‐κB pathway via Syk and the CARD9 pathway and was dependent on the phosphorylation of the CEACAM3 ITAM‐like motif. These findings suggest that the CEACAM3 signalling in neutrophils is able to specifically modulate airway inflammation caused by infection with M. catarrhalis.     

Tissue culture adherence and haemagglutination characteristics of Moraxella (Branhamella) catarrhalis

The haemagglutination and tissue culture adherence properties of 20 isolates of Moraxella catarrhalis obtained from the sputum of elderly patients with lower respiratory tract infections were compared with those of 20 isolates of M. catarrhalis obtained from the nasopharynx of elderly persons colonised by the organism. Eighty percent of isolates from the infected group as opposed to 5% of isolates from the colonised group haemagglutinated human erythrocytes (P < 0.001), indicating that the haemagglutinin might be a marker of pathogenicity for M. catarrhalis. There was a significant difference in the adherence to HEp-2 cells of isolates from the infected group in comparison to isolates from the colonised group (P = 0.03). Haemagglutination and tissue culture adherence properties were unrelated, indicating that separate adhesin systems are involved. The adherence of M. catarrhalis to HEp-2 cells was unaffected following pronase and trypsin treatment, however, sodium periodate pre-treatment of the bacteria significantly reduced the tissue culture adherence index, indicating that the adhesin by which the bacteria bind to HEp-2 cells may have a carbohydrate moiety. Transmission electron microscopy studies revealed that adherence of M. catarrhalis to HEp-2 cells was mediated by trypsin-resistant 'tack-/spicule-like' structures protruding from the surface of the bacteria.     

Mannose-binding lectin (MBL)-associated serine protease (MASP)-1 contributes to activation of the lectin complement pathway

The complement system plays an important role in innate immunity. In the lectin complement pathway, mannose-binding lectin (MBL) and ficolins act as recognition molecules, and MBL-associated serine protease (MASP) is a key enzyme. It has been suggested that MASP-2 is responsible for the activation of C4. Other serine proteases (MASP-1 and MASP-3) are also associated with MBL or ficolins; however, their functions are still controversial. In this study, a MASP-1- and MASP-3-deficient mouse model (MASP1/3(-/-)) was generated by a gene targeting strategy to investigate the roles of MASP-1 and MASP-3 in the lectin pathway. Serum derived from MASP1/3(-/-) mice showed significantly lower activity of both C4 and C3 deposition on mannan-agarose, and this low activity was restored by the addition of recombinant MASP-1. MASP-1/3-deficient serum showed a significant delay for activation of MASP-2 compared with normal serum. Reconstitution of recombinant MASP-1 in MASP-1/3-deficient serum was able to promote the activation of MASP-2. From these results, we propose that MASP-1 contributes to the activation of the lectin pathway, probably through the activation of MASP-2.     

Comparative study of mannose-binding C-type lectin isolated from channel catfish (Ictalurus punctatus) and blue catfish (Ictalurus furcatus)

Mannose-binding C-type lectin (MBL) was isolated from channel catfish (Ictalurus punctatus) NWAC 102 and 103 strains, blue catfish (Ictalurus furcatus) D+B and Rio Grande strains, hybrid catfish (channel catfish female NWAC 103 x blue catfish male D+B) sera, and purified by affinity chromatography from channel catfish Norris strain serum. Reduction of purified channel catfish MBL with 2-ME yielded a single band of 62 kDa by SDS-PAGE and Western blot analysis using guinea pig anti-MBL IgG as primary antibody. Channel catfish NWAC 102 strain, channel catfish NWAC 103 strain and hybrid catfish sera had molecular masses of 63 kDa for MBL. Blue catfish (D+B strain) serum MBL had a molecular mass of 66 kDa. Rio Grande blue catfish serum MBL had a molecular mass of 65 kDa. Amino acid composition analysis (mol%) of the affinity-purified channel catfish MBL found a high content of serine present. Functional binding studies of channel catfish and blue catfish MBLs binding to Edwardsiella ictaluri were done using a dot-immunoblot ELISA method. A dot-immunoblot ELISA binding assay was done to compare nine different strains and species of channel catfish and blue catfish for their levels of serum MBL. Blue catfish had higher levels of MBL than did the various strains of channel catfish tested. MBL could be used as a genetic marker for selection of disease resistance in the different strains of catfish used in aquaculture. This study describes the presence of serum MBL in catfish and evidence for a C-type lectin complement pathway of innate immunity.     

Mannan-binding lectin (MBL) in women with tumours of the reproductive system

Mannan-binding lectin (MBL) is an important factor of innate immunity contributing to the clearance of microorganisms. Recently, an antitumourigenic role of MBL has been suggested. We investigated mbl2 genotypes, MBL concentrations, and MBL-MASP-2 complex activity in patients with ovarian cancer. The expression of both mbl2 and masp-2 genes were investigated in ovarian tissue sections. Additionally, samples from patients with other malignant and benign tumours of the reproductive tract were tested. A significantly higher incidence of MBL deficiency/insufficiency-associated genotypes was found among patients with malignant disease compared to age-matched controls. Unexpectedly, no differences in median MBL level or MBL-MASP-2 complex activity were found between the groups. This was partly a reflection of higher MBL concentrations and MBL-MASP-2 activity in cancer patients compared with healthy women carrying corresponding genotypes. MBL-specific mRNA expression was detected in several normal and malignant ovarian tissues, as well as in ovarian epithelial cell lines. Intracellular staining with MBL-specific antibodies demonstrated the presence of MBL in ovarian cell lines, and in normal as well as malignant ovarian tissue sections. In contrast, MASP-2-specific mRNA expression was detected only in the ovary tissues of patients with malignant disease. No significant changes in MBL concentration during 3 months of chemotherapy were noticed. MBL was detected in ascites and in the fluid of benign ovarian cysts. Our findings may reflect anti-tumourigenic activity of MBL protein which might suggest potential therapeutic application. However, it cannot be excluded that mbl-2 mutant alleles may be in linkage disequilibrium with an unidentified tumour susceptibility gene(s).     

Moraxella (Branhamella) catarrhalis Adherence to Human Bronchial and Oropharyngeal Cells: The Role of Adherence in Lower Respiratory Tract Infections

To study the role of Moraxella (subgenus Branhamella) catarrhalis (B. catarrhalis) adherence to airway cells in lower respiratory tract infections, the in vitro attachments of B. catarrhalis to upper airway (oropharyngeal) and lower airway (bronchial) epithelial cells were compared. The adherence of 4 strains (1 nonfimbriated and 3 fimbriated) of B. catarrhalis to respiratory tract epithelial cells collected from 11 patients with chronic pulmonary disease (CPD) and 11 healthy individuals was evaluated. Both the fimbriated and nonfimbriated strains showed increased attachment to oropharyngeal cells in the CPD patients (mean ± SEM; 25.0 ± 3.2/cell; P < 0.01) when compared to the control subjects (12.1 ± 1.1/cell). On the average, the attachment to bronchial cells was 6.1 to 13.6 times greater per surface area (bacteria/μ²) than the attachment to oropharyngeal cells. The fimbriated strains tended to adhere in higher numbers to bronchial cells (19.0 ± 1.8/cell) than the nonfimbriated strain (8.7 ± 1.2/cell), although there was no difference between the CPD and control groups. In conclusion, the attachment of B. catarrhalis to oropharyngeal cells may be an enhancing factor for colonization in the upper respiratory tract in patients with CPD, and elevated adherence of the bacteria to bronchial cells may suggest pathogenic importance when mucociliary function is impaired.     

Heterocomplexes of mannose-binding lectin and the pentraxins PTX3 or serum amyloid P component trigger cross-activation of the complement system

The long pentraxin 3 (PTX3), serum amyloid P component (SAP), and C-reactive protein belong to the pentraxin family of pattern recognition molecules involved in tissue homeostasis and innate immunity. They interact with C1q from the classical complement pathway. Whether this also occurs via the analogous mannose-binding lectin (MBL) from the lectin complement pathway is unknown. Thus, we investigated the possible interaction between MBL and the pentraxins. We report that MBL bound PTX3 and SAP partly via its collagen-like domain but not C-reactive protein. MBL-PTX3 complex formation resulted in recruitment of C1q, but this was not seen for the MBL-SAP complex. However, both MBL-PTX3 and MBL-SAP complexes enhanced C4 and C3 deposition and opsonophagocytosis of Candida albicans by polymorphonuclear leukocytes. Interaction between MBL and PTX3 led to communication between the lectin and classical complement pathways via recruitment of C1q, whereas SAP-enhanced complement activation occurs via a hitherto unknown mechanism. Taken together, MBL-pentraxin heterocomplexes trigger cross-activation of the complement system.