Virulence factors of Moraxella catarrhalis outer membrane vesicles are major targets for cross-reactive antibodies and have adapted during evolution

Abstract

Moraxella catarrhalis is a common human respiratory tract pathogen. Its virulence factors associated with whole bacteria or outer membrane vesicles (OMVs) aid infection, colonization and may induce specific antibodies. To investigate pathogen-host interactions, we applied integrated bioinformatic and immunoproteomic (2D-electrophoresis, immunoblotting, LC-MS/MS) approaches. We showed that OMV proteins engaged exclusively in complement evasion and colonization strategies, but not those involved in iron transport and metabolism, are major targets for cross-reacting antibodies produced against phylogenetically divergent M. catarrhalis strains. The analysis of 31 complete genomes of M. catarrhalis and other Moraxella revealed that OMV protein-coding genes belong to 64 orthologous groups, five of which are restricted to M. catarrhalis. This species showed a two-fold increase in the number of OMV protein-coding genes relative to its ancestors and animal-pathogenic Moraxella. The appearance of specific OMV factors and the increase in OMV-associated virulence proteins during M. catarrhalis evolution is an interesting example of pathogen adaptation to optimize colonization. This precisely targeted cross-reactive immunity against M. catarrhalis may be an important strategy of host defences to counteract this phenomenon. We demonstrate that cross-reactivity is closely associated with the anti-virulent antibody repertoire which we have linked with adaptation of this pathogen to the host.

Figure 1

Phylogenetic relationships between Moraxella and Acinetobacter genomes. MrBayes tree based on the concatenated alignments of sequences from 739 orthologous groups represented 31 Moraxella genomes and three members of Acinetobacter genus. The part of tree marked by the grey rectangle is shown on the left in the larger scale. Numbers at nodes, in the order shown, correspond to: posterior probabilities estimated in MrBayes (MB), support values obtained by the approximate likelihood ratio test based on a Shimodaira-Hasegawa-like procedure (Anisimova and Gascuel O. 2006) calculated in morePhyML (mPh) and bootstrap values obtained in PhyML (Phb). Values of the posterior probabilities, SH-like branch supports and bootstrap percentages lower than 0.50 and 50%, respectively, were omitted or indicated by a dash “−”.

Figure 2

Immunoproteomic analyses of M. catarrhalis OMV preparations. After 2D-gel electrophoresis OMV proteins were visualized by Coomassie staining (A,E). 2D immunoblots probed with homologous antisera (B,F), stronger cross-reactive antisera (C,G) and weaker cross-reactive antisera (D,H). Molecular size markers are indicated on the left. The exposure times for chemiluminescent substrate were exactly the same for each presented blot. Gel and blot images are cropped and brightness/contrast-adjusted for better readability. Original images are shown in Supplementary Fig. S2.

Figure 3

Heatmap of immunoreactive spot intensities. Reactivity of murine post-immunization sera with OMVs proteins was estimated semi-quantitatively on a quartile scale. Only proteins that reacted with at least one antisera are shown.

Figure 4

Distribution of orthologous group for OMV proteins (A) and total proteins (B) found with OrthoMCL across three sets of genomes: Acinetobacter, Moraxella catarrhalis and other Moraxella species. The group was included if it was represented by at least one genome in the set. The unique proteins not classified into groups were also included.

Figure 5

Gains and losses of genes for OMV proteins (A) and all annotated protein sequences (B) during evolution of Moraxella and Acinetobacter genomes. The number of gene gains and losses per phylogenetic lineage were presented by bars. Ancestral gene contents in genomes were shown by numbers at tree branches and the current gene contents were presented by numbers in parenthesis at individual genomes.

Figure 6

Changes in gene content during evolution of Moraxella and Acinetobacter genomes. Number of genes for total proteins and OMV proteins estimated for ancestral genomes (A) in subsequent internal nodes from 1 to 8 in the phylogenetic tree presented in (C). Percentage of OMV proteins coded in the ancestral genomes from subsequent internal nodes (B).