The Direct Anti-Virulence but Not Bactericidal Activity of Human Neutrophil Elastase against Moraxella catarrhalis.

Neutrophil elastase (NE) contributes to innate antibacterial defense at both the intracellular (phagocytosis) and extracellular (degranulation, NETosis) levels. Moraxella catarrhalis, a human respiratory pathogen, can exist in an inflammatory milieu which contains NE. No data are available on the action of NE against M. catarrhalis or on the counteraction of NE-dependent host defenses by this pathogen. Using time-kill assays we found that bacteria are able to survive and replicate in the presence of NE. Transmission electron microscopy and flow cytometry studies with NE-treated bacteria revealed that while NE admittedly destabilizes the outer membrane leaflet, it does not cause cytoplasmic membrane rupture, suggesting that the enzyme does not target components that are essential for cell integrity. Using LC-MS/MS spectroscopy we determined that NE cleaved at least three virulent surface proteins in outer membrane vesicles (OMVs) of M. catarrhalis, including OMP CD, McaP, and TbpA. The cleavage of OMP CD contributes to the significant decrease in resistance to serum complement in the complement-resistant strain Mc6. The cleavage of McaP did not cause any sensitization to erythromycin nor did NE disturb its drug action. Identifying NE as a novel but subtle anti-virulence agent together with its extracellularly not-efficient bactericidal activity against M. catarrhalis may facilitate the pathogen's existence in the airways under inflammation.

Interspecies Outer Membrane Vesicles (OMVs) Modulate the Sensitivity of Pathogenic Bacteria and Pathogenic Yeasts to Cationic Peptides and Serum Complement.

The virulence of bacterial outer membrane vesicles (OMVs) contributes to innate microbial defense. Limited data report their role in interspecies reactions. There are no data about the relevance of OMVs in bacterial-yeast communication. We hypothesized that model Moraxella catarrhalis OMVs may orchestrate the susceptibility of pathogenic bacteria and yeasts to cationic peptides (polymyxin B) and serum complement. Using growth kinetic curve and time-kill assay we found that OMVs protect Candida albicans against polymyxin B-dependent fungicidal action in combination with fluconazole. We showed that OMVs preserve the virulent filamentous phenotype of yeasts in the presence of both antifungal drugs. We demonstrated that bacteria including Haemophilus influenza, Acinetobacter baumannii, and Pseudomonas aeruginosa coincubated with OMVs are protected against membrane targeting agents. The high susceptibility of OMV-associated bacteria to polymyxin B excluded the direct way of protection, suggesting rather the fusion mechanisms. High-performance liquid chromatography-ultraviolet spectroscopy (HPLC-UV) and zeta-potential measurement revealed a high sequestration capacity (up to 95%) of OMVs against model cationic peptide accompanied by an increase in surface electrical charge. We presented the first experimental evidence that bacterial OMVs by sequestering of cationic peptides may protect pathogenic yeast against combined action of antifungal drugs. Our findings identify OMVs as important inter-kingdom players.

Neuropeptides SP and CGRP Diminish the Moraxella catarrhalis Outer Membrane Vesicle- (OMV-) Triggered Inflammatory Response of Human A549 Epithelial Cells and Neutrophils.

Neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP) play both pro- and anti-inflammatory activities and are produced during infection and inflammation. Moraxella catarrhalis is one of the leading infectious agents responsible for inflammatory exacerbation in chronic obstructive pulmonary disease (COPD). Since the airway inflammation in COPD is connected with activation of both epithelial cells and accumulated neutrophils, in this study we determined the in vitro effects of neuropeptides on the inflammatory potential of these cells in response to M. catarrhalis outer membrane vesicle (OMV) stimulant. The various OMV-mediated proinflammatory effects were demonstrated. Next, using hBD-2-pGL4[luc2] plasmid with luciferase reporter gene, SP and CGRP were shown to inhibit the IL-1ß-dependent expression of potent neutrophil chemoattractant, hBD-2 defensin, in transfected A549 epithelial cells (type II alveolar cells) upon OMV stimulation. Both neuropeptides exerted antiapoptotic activity through rescuing a significant fraction of A549 cells from OMV-induced cell death and apoptosis. Finally, CGRP caused an impairment of specific but not azurophilic granule exocytosis from neutrophils as shown by evaluation of gelatinase-associated lipocalin (NGAL) or CD66b expression and elastase release, respectively. Concluding, these findings suggest that SP and CGRP mediate the dampening of proinflammatory action triggered by M. catarrhalis OMVs towards cells engaged in lung inflammation in vitro.

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

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.

Defensive and Offensive Cross-Reactive Antibodies Elicited by Pathogens: The Good, the Bad and the Ugly.

Understanding how immunity to pathogens develops is crucial for progress in the quest for effective vaccines. Intraspecies and interspecies cross-reacting antibodies are produced in high frequency against immune-relevant and shared microbial epitopes. It has been confirmed that cross-reactive antigens may have a crucial role in natural epidemiology to a particular infection and that cross-protection may influence the outcome of natural infections. On the other hand, the action of cross-reactive antibodies may be very harmful for the host. In this review we discuss both the defensive and offensive capabilities of cross-reactive antibodies. The defensive properties are discussed with regard to the beneficial cross-protective interaction of these antibodies against various microorganisms including viruses, bacteria, fungi and protozoan parasites. We summarize the current knowledge of numerous effector functions of these antibodies such as agglutination, neutralization of infectivity, complement activation, phagocytosis enhancement, and antibody-dependent cellular cytotoxicity. We also discuss the offensive action of cross-reactive antibodies including their detrimental effects in exacerbation of the infective diseases, as well as autoimmune diseases and allergy as a result of inappriopriate or deleterious inflammatory response associated with host tissue destruction. The factors influencing cross-protective capacity of antibodies are also presented.