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Keywords:

  • Moraxella catarrhalis;
  • Mannose-binding lectin;
  • Complement activation

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

A hemolytic bystander assay was used to assess the functional serum mannose-binding lectin (MBL) activating capacity of five isolates of Moraxella catarrhalis obtained from children who suffered recurrent acute otitis media episodes. Results showed that this organism is only a poor activator of the lectin pathway of complement activation, with subsequent consequences for the etiology of otitis media by this organism.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Moraxella catarrhalis is a commensal bacterium associated with both the colonization and infection of adults and children. In particular, the bacterium is most often associated with respiratory tract infections, including acute and chronic otitis media and acute exacerbations of chronic obstructive pulmonary disease (COPD) [1]. In terms of the immunological response to M. catarrhalis, research has indicated that colonization/infection leads to the generation of opsonic antibodies against the relevant isolate [2], and that complement resistant isolates (which possess an increased virulence potential) possess the ability to bind vitronectin [3] and/or complement component C4b-binding protein [4]. However, there have as yet been no publications into the possible role of the mannose-binding lectin (MBL) arm of the complement system in the immune response to M. catarrhalis.

Mannose-binding lectin (MBL) is a high-molecular-weight protein, which is present in blood plasma at low concentrations (1.7 μg/ml), and which forms the initial component of the lectin pathway of the complement system. The protein acts by binding to specific sugar residues (i.e., mannose, N-acetylglucosamine, and fucose) present on the surface of microorganisms, leading to the subsequent activation of the (“innate”) MBL arm of the complement cascade system. In man, MBL deficiencies are quite common, with MBL gene polymorphisms leading to differential MBL expression levels, which in turn have been associated with susceptibility to several different types of infection [5]. In this brief report, a functional MBL assay was used to characterize M. catarrhalis as a weak or potent activator of the MBL arm of the complement system.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

2.1Bacterial isolates

Five isolates of M. catarrhalis were tested for MBL activating capacity, all obtained from children who suffered recurrent acute otitis media episodes. Three isolates (isolates 2040-1, 3001-14/20 and 4017-7) had been previously determined to be complement resistant and 2 isolates complement sensitive (isolates 3053-14 and 3007–7) via a simple “spot and drop” test [6,7].

2.2Functional MBL assay

The functional MBL assay used to measure M. catarrhalis-induced MBL activation in this article was based upon that previously published by Kuipers et al. [8], which makes use of microorganism-induced MBL activation in a dilution series of pooled human serum, followed by subsequent C5b6-mediated bystander hemolysis of chicken erythrocytes in the presence of a standardized concentration of MBL-deficient human serum. In this assay, the degree of MBL activation of microorganisms is related to the MBL-activating capacity of a standard isolate of the yeast Saccharomyces cerevisiae (in order to eliminate inter-experimental variation), using pooled human serum containing a known MBL concentration. With regard to M. catarrhalis testing, 12 different dilutions of the five M. catarrhalis isolates to be tested (previously grown on Columbia blood agar and resuspended to an OD660 of 1.0, equivalent to approximately 1.5 × 108 CFU ml−1), or 3 × 105S. cerevisiae cells, were prepared in veronal buffered saline containing Ca2+ and Mg2+ (VSB++). A checkerboard titration was then performed against dilutions of the standard pool of human serum. After incubation, the degree of bystander erythrocyte lysis was translated into the number of active sites per erythrocyte (Z-value) using the equation of Borsos and Rapp [9]. As a control, pre-incubation of the standard human pooled serum with 2.5 mg per well mannose (which binds and blocks MBL binding), followed by repetition of the MBL activation experiments, was performed in order to indicate whether the bystander hemolysis attributable to M. catarrhalis was a consequence of MBL-specific activation. Activation of the alternative pathway of the complement system was excluded by testing in ethylene-glycol bis-amino-tetraacetic acid – veronal buffered saline [10]. The relative MBL activation of M. catarrhalis was read off at a Z value of 0.2 (calculated as: ZM. catarrhalis/ZS. cerevisiae) and M. catarrhalis-mediated MBL activation ranked alongside other pathogenic bacteria using previously published data [8]. S. cerevisiae was used as the standard reference as this was the first reported microorganism to be associated with MBL [11], and the yeast cell wall of this organism is rich in mannan.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

The MBL activating capacity (as a measure of the level of bystander hemolysis of chicken erythrocytes) for the five M. catarrhalis isolates tested is presented in Fig. 1, with no difference being observed in MBL activating capacity between the complement resistant and complement sensitive isolates. A comparison of the M. catarrhalis results with bacterial species previously determined to be weak/potent activators of MBL is shown in Table 1. Experiments using mannose as a competitor for MBL activation showed that pre-incubation with mannose had a negative effect on the ability of M. catarrhalis to activate MBL (Fig. 1), indicating that M. catarrhalis MBL activating activity in the functional assay (though weak) was indeed a specific phenomenon.

image

Figure 1. Composite graph showing average MBL activation by M. catarrhalis (with and without the addition of competing mannose) and the yeast Saccharomyces cerevisiae (used as a standard MBL activating control organism to which organisms are compared in order to eliminate inter-experiment variation). Z value = mean number of active sites per chicken erythrocyte determined by the functional bystander assay. Experiments were performed in triplicate. Note the reduction in MBL activating capacity (Z value) of M. catarrhalis after pre-incubation of standard MBL containing human pooled serum with mannose (indicating the specificity of M. catarrhalis MBL activation). The five M. catarrhalis isolates used were all Dutch isolates, identification numbers 2040-1, 3001-14/20, 4017-7, 3053-14 and 3007–7.

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Table 1.  Comparison of potent and weak MBL activating microbial species (relative to the standard organism S. cerevisiae) as determined using a functional bystander hemolysis assay
 MicrobeZmicrobe (OD660 = 1.0)/ZS.cerevisiae (control)a
  1. aZmicrobe(OD = 1.0)/ZS.cerevisiae (control)= ratio used to rank microorganism MBL activating capacity with respect to the standard reference organism S. cerevisiae, where Z=−ln (1 − fraction erythrocytes lysed) a measure of the mean number of MBL activating sites per chicken erythrocyte [9].

  2. bThe MBL activating capacity of these organisms exceed the maximum limit of this test at an OD660 of 1.0.

PotentNeisseria meningitidis*b
Neisseria gonorrhoeae* 
Salmonella typhimurium* 
Mycobacterium bovis* 
Pseudomonas aeruginosa* 
   
WeakCandida albicans ATCC 140530.80
Yersinia enterocolitica0.77 
Micrococcus lysodeikticus0.62 
Helicobacter pylori0.54 
E. coli ATCC 295220.49 
M. catarrhalis0.21 
Enterococus faecalisNo detectable activity 

These results indicate that M. catarrhalis is only a weak activator of the MBL arm of the complement system. In theory, this lack of MBL activation could be advantageous to the organism in allowing it to remain “hidden” from the MBL arm of the complement system (i.e., protected from lectin pathway-induced complement activation). In this scenario, serum MBL levels would be unlikely to influence M. catarrhalis colonization/infection. However, it should be noted that heavily mannosylated immunoglobulin A (IgA) is also found at tissue surfaces [12], and an interaction between IgA binding and MBL activation in M. catarrhalis mediated infection cannot be discounted at present.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

The authors thank Prof. E.A.M. Sanders, Dr. G. Rijkers (Department of Immunology, UMCU – Wilhelmina Children's Hospital, Utrecht, The Netherlands) and Dr. R. Veenhoven (Department of Pediatrics, Spaaarne Hospital, Haarlem, The Netherlands) for kindly supplying the bacterial isolates used in this study.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References
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