Therése Nordström

The emerging pathogen Moraxella catarrhalis interacts with complement inhibitor C4b binding protein through ubiquitous surface proteins A1 and A2

Moraxella catarrhalis ubiquitous surface protein A2 (UspA2) mediates resistance to the bactericidal activity of normal human serum. In this study, an interaction between the complement fluid phase regulator of the classical pathway, C4b binding protein (C4BP), and M. catarrhalis mutants lacking UspA1 and/or UspA2 was analyzed by flow cytometry and a RIA. Two clinical isolates of M. catarrhalis expressed UspA2 at a higher density than UspA1. The UspA1 mutants showed a decreased C4BP binding (37.6% reduction), whereas the UspA2-deficient Moraxella mutants displayed a strongly reduced (94.6%) C4BP binding compared with the wild type. In addition, experiments with recombinantly expressed UspA150–770 and UspA230–539 showed that C4BP (range, 1–1000 nM) bound to the two proteins in a dose-dependent manner. The equilibrium constants (KD) for the UspA150–770 and UspA230–539 interactions with a single subunit of C4BP were 13 μM and 1.1 μM, respectively. The main isoform of C4BP contains seven identical α-chains and one β-chain linked together with disulfide bridges, and the α-chains contain eight complement control protein (CCP) modules. The UspA1 and A2 bound to the α-chain of C4BP, and experiments with C4BP lacking CCP2, CCP5, or CCP7 showed that these three CCPs were important for the Usp binding. Importantly, C4BP bound to the surface of M. catarrhalis retained its cofactor activity as determined by analysis of C4b degradation. Taken together, M. catarrhalis interferes with the classical complement activation pathway by binding C4BP to UspA1 and UspA2.

The Respiratory Pathogen Moraxella catarrhalis Adheres to Epithelial Cells by Interacting with Fibronectin through Ubiquitous Surface Proteins A1 and A2.

Moraxella catarrhalis ubiquitous surface protein (Usp) A1 has been reported to bind fibronectin and is involved in adherence. In this study, using M. catarrhalis mutants derived from clinical isolates, we show that both UspA1 and UspA2 bind fibronectin. Recombinant truncated UspA1/A2 proteins, together with smaller fragments spanning the entire molecule, were tested for binding to fibronectin. Both UspA1 and UspA2 bound fibronectin, and the fibronectin-binding domains were located within UspA1(299-452) and UspA2(165-318). These 2 truncated proteins inhibited binding of M. catarrhalis to Chang conjunctival epithelial cells to an extent similar to that by anti-human fibronectin antibodies. Our observations show that both UspA1 and UspA2 are involved in adherence to epithelial cells via cell-associated fibronectin. The biologically active sites within UspA1(299-452) and UspA2(165-318) have therefore been suggested to be potential candidates to be included in a future vaccine against M. catarrhalis.

Moraxella catarrhalis outer membrane vesicles carry beta-lactamase and promote survival of Streptococcus pneumoniae and Haemophilus influenzae by inactivating amoxicillin.

ABSTRACT 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.

Ionic Binding of C3 to the Human Pathogen Moraxella catarrhalis Is a Unique Mechanism for Combating Innate Immunity

Moraxella catarrhalis ubiquitous surface proteins A1 and A2 (UspA1/A2) interfere with the classical pathway of the complement system by binding C4b-binding protein. In this study we demonstrate that M. catarrhalis UspA1 and A2 noncovalently and in a dose-dependent manner bind both the third component of complement (C3) from EDTA-treated serum and methylamine-treated C3. In contrast, related Moraxella subspecies (n = 13) or other human pathogenic bacteria (n = 13) do not bind C3 or methylamine-treated C3. Experiments with recombinant proteins and M. catarrhalis mutants devoid of UspA1/A2 revealed that UspA1/A2 exert their actions by absorbing and neutralizing C3 from serum and restrain complement activation. UspA2 was responsible for most of the effect, and the Moraxella mutant lacking UspA2 was more sensitive to the lytic effect of human serum compared with the wild type. Interestingly, among the large number of bacteria analyzed, only M. catarrhalis has this unique ability to interfere with the innate immune system of complement by binding C3.

The immunoglobulin D-binding part of the outer membrane protein MID from Moraxella catarrhalis comprises 238 amino acids and a tetrameric structure.

Moraxella catarrhalis IgD-binding protein (MID), a 200-kDa outer membrane protein comprising 2,139 amino acids, has recently been isolated and shown to display a unique and specific affinity for human IgD. To identify the IgD-binding region, MID was digested with proteases. In addition, a series of truncated fragments of MID were manufactured and expressed in Escherichia coli followed by analysis for IgD binding in Western and dot blots. The smallest fragment with essentially preserved IgD binding was comprised of 238 amino acid residues (MID962–1200). Shorter recombinant proteins gradually lost IgD-binding capacity, and the shortest IgD-binding fragment comprising 157 amino acids (MID985–1142) displayed a 1,000-fold reduced IgD binding compared with the full-length molecule. The truncated MID962–1200 was efficiently attracted to a standard IgD serum and to purified myeloma IgD(κ) and IgD(λ) sera but not to IgG, IgM, or IgA myeloma sera. Furthermore, the fragment specifically bound to peripheral blood B lymphocytes, and the binding was inhibited by preincubation with anti-IgD-Fab polyclonal antibodies. Results obtained by introducing five amino acids randomly into MID962–1200 using transposons suggested that α-helix structures were important for IgD binding. Ultracentrifugation experiments and gel electrophoresis revealed that native MID962–1200 was a tetramer. Interestingly, tetrameric MID962–1200 attracted IgD more than 20-fold more efficiently than the monomeric form. Thus, a tetrameric structure of MID962–1200 is crucial for optimal IgD-binding capacity.

The IgD-binding domain of the Moraxella IgD-binding protein MID (MID962-1200) activates human B cells in the presence of T cell cytokines.

Moraxella catarrhalis immunoglobulin D (IgD)‐binding protein (MID) is an outer membrane protein with specific affinity for soluble and cell‐bound human IgD. Here, we demonstrate that mutated M. catarrhalis strains devoid of MID show a 75% decreased activation of human B cells as compared with wild‐type bacteria. In contrast to MID‐expressing Moraxella, the MID‐deficient Moraxella mutants did not bind to human CD19+ IgD+ B cells. The smallest MID fragment with preserved IgD‐binding capacity comprises 238 amino acids (MID962‐1200). To prove the specificity of MID962‐1200 for IgD, a Chinese hamster ovary (CHO) cell line expressing membrane‐anchored human IgD was manufactured. MID962‐1200 bound strongly to the recombinant IgD on CHO cells. Moreover, MID962‐1200 stimulated peripheral blood lymphocyte (PBL) proliferation 5‐ and 15‐fold at 0.1 and 1.0 μg/ml, respectively. This activation could be blocked completely by antibodies directed against the CD40 ligand (CD154). MID962‐1200 also activated purified B cells in the presence of interleukin (IL)‐2 or IL‐4. An increased IL‐6 production was seen after stimulation with MID962‐1200, as revealed by a human cytokine protein array. MID962‐1200 fused to green fluorescent protein (GFP) bound to human B cells and activated PBL to the same degree as MID962‐1200. Taken together, MID is the only IgD‐binding protein in Moraxella. Furthermore, the novel T cell‐independent antigen MID962‐1200 may, together with MID962‐1200–GFP, be considered as promising reagents in the study of IgD‐dependent B cell activation.

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

OBJECTIVES 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. METHODS The antibiotic susceptibility of isolates was determined using the Etest. The resistance genes blaTEM-1 (encoding NTHi β-lactamase), bro-1 (encoding M. catarrhalis β-lactamase) and ftsI (encoding NTHi penicillin-binding protein 3) were searched for by PCR, followed by sequencing. OMVs were isolated by ultracentrifugation and the presence of β-lactamase was detected by western blots including specific rabbit polyclonal antibodies. The chromogenic substrate nitrocefin was used to quantify and compare the β-lactamase enzyme activity in the OMVs. The hydrolysis of amoxicillin by β-lactamase was estimated by an agar diffusion method. RESULTS We showed that OMVs released from β-lactam-resistant M. catarrhalis and NTHi contain functional β-lactamase that hydrolyses amoxicillin and protects GAS from killing by amoxicillin. CONCLUSIONS This is the first report of the presence of β-lactamase in NTHi OMVs. We suggest that OMV-derived β-lactamase from coinfecting pathogens such as NTHi and M. catarrhalis may contribute to the occasional treatment failures seen in GAS tonsillitis.

Immune Evasion of Moraxella catarrhalis Involves Ubiquitous Surface Protein A-Dependent C3d Binding

The complement system plays an important role in eliminating invading pathogens. Activation of complement results in C3b deposition (opsonization), phagocytosis, anaphylatoxin (C3a, C5a) release, and consequently cell lysis. Moraxella catarrhalis is a human respiratory pathogen commonly found in children with otitis media and in adults with chronic obstructive pulmonary disease. The species has evolved multiple complement evasion strategies, which among others involves the ubiquitous surface protein (Usp) family consisting of UspA1, A2, and A2 hybrid. In the present study, we found that the ability of M. catarrhalis to bind C3 correlated with UspA expression and that C3 binding contributed to serum resistance in a large number of clinical isolates. Recombinantly expressed UspA1 and A2 inhibit both the alternative and classical pathways, C3b deposition, and C3a generation when bound to the C3 molecule. We also revealed that the M. catarrhalis UspA-binding domain on C3b was located to C3d and that the major bacterial C3d-binding domains were within UspA1299–452 and UspA2165–318. The interaction with C3 was not species specific since UspA-expressing M. catarrhalis also bound mouse C3 that resulted in inhibition of the alternative pathway of mouse complement. Taken together, the binding of C3 to UspAs is an efficient strategy of Moraxella to block the activation of complement and to inhibit C3a-mediated inflammation.

Outer Membrane Protein OlpA Contributes to Moraxella catarrhalis Serum Resistance via Interaction With Factor H and the Alternative Pathway

Factor H is an important complement regulator of the alternative pathway commonly recruited by pathogens to achieve increased rates of survival in the human host. The respiratory pathogen Moraxella catarrhalis, which resides in the mucosa, is highly resistant to the bactericidal activity of serum and causes otitis media in children and respiratory tract infections in individuals with underlying diseases. In this study, we show that M. catarrhalis binds factor H via the outer membrane protein OlpA. M. catarrhalis serum resistance was dramatically decreased in the absence of either OlpA or factor H, demonstrating that this inhibition of the alternative pathway significantly contributes to the virulence of M. catarrhalis.

Human Siglec-5 Inhibitory Receptor and Immunoglobulin A (IgA) Have Separate Binding Sites in Streptococcal {beta} Protein.

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are receptors believed to be important for regulation of cellular activation and inflammation. Several pathogenic microbes bind specific Siglecs via sialic acid-containing structures at the microbial surface, interactions that may result in modulation of host responses. Recently, it was shown that the group B Streptococcus (GBS) binds to human Siglec-5 (hSiglec-5), an inhibitory receptor expressed on macrophages and neutrophils, via the IgA-binding surface β protein, providing the first example of a protein/protein interaction between a pathogenic microbe and a Siglec. Here we show that the hSiglec-5-binding part of β resides in the N-terminal half of the protein, which also harbors the previously determined IgA-binding region. We constructed bacterial mutants expressing variants of the β protein with non-overlapping deletions in the N-terminal half of the protein. Using these mutants and recombinant β fragments, we showed that the hSiglec-5-binding site is located in the most N-terminal part of β (B6N region; amino acids 1–152) and that the hSiglec-5- and IgA-binding domains in β are completely separate. We showed with BIAcoreTM analysis that tandem variants of the hSiglec-5- and IgA-binding domains bind to their respective ligands with high affinity. Finally, we showed that the B6N region, but not the IgA-binding region of β, triggers recruitment of the tyrosine phosphatase SHP-2 to hSiglec-5 in U937 monocytes. Taken together, we have identified and isolated the first microbial non-sialic acid Siglec-binding region that can be used as a tool in studies of the β/hSiglec-5 interaction.