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 UspA1(299-452) and UspA2(165-318). 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.

Purification of alpha1-antichymotrypsin from human plasma with recombinant M. catarrhalis ubiquitous surface protein A1.

Alpha 1-antichymotrypsin (ACT) inhibits chymotrypsin-like enzymes, particularly neutrophil cathepsin G. Moreover, ACT in its native form suppresses chemotaxis of neutrophils and decreases neutrophil production of superoxide radicals. We recently showed that Moraxella catarrhalis ubiquitous surface protein (Usp) A1 is able to specifically bind ACT in the context of a novel virulence mechanism. In this study, we report that recombinant UspA1(557-704) coupled to CNBr-Sepharose can be used in a simple one-step purification of ACT from human plasma. UspA1(557-704)-purified ACT remains intact and active as shown by binding to M. catarrhalis and a chymotrypsin inhibition assay. The novel method for ACT isolation from plasma has important advantages in simplicity and time as compared to conventional methods.

Moraxella-dependent alpha 1-antichymotrypsin neutralization: a unique virulence mechanism.

The acute phase reactant and protease inhibitor alpha(1)-antichymotrypsin is considered to play a protective role in the airways, but whether it interacts with respiratory bacteria is not known. We analyzed whether the common respiratory pathogens Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and other bacterial species interact with antichymotrypsin. M. catarrhalis was the only species that bound antichymotrypsin among 25 bacterial species tested by flow cytometry and direct binding assay. We compared a series of clinical isolates in addition to wild-type and ubiquitous surface protein-deficient Moraxella to study the nature of antichymotrypsin binding by the bacteria. Experiments with Moraxella mutants revealed that ubiquitous surface proteins A1 and A2 were responsible for the interaction, and using recombinant fragments, a consensus sequence within ubiquitous surface proteins A1 and A2 was defined. Binding of iodine-labeled antichymotrypsin was dose dependent and strong (dissociation constant [K(d)] 24.9-44.8 nM). Moreover, a chymotrypsin activity assay showed that antichymotrypsin, when bound to the bacterial surface, was neutralized. Moraxella antichymotrypsin neutralization is a novel microbial virulence mechanism that may induce excessive inflammation resulting in more exposed extracellular matrix that is beneficial for bacterial colonization.

Continuous acetonitrile degradation in a packed-bed bioreactor.

A 20-l packed-bed reactor filled with foamed glass beads was tested for the treatment of acetonitrile HPLC wastes. Aeration was provided by recirculating a portion of the reactor liquid phase through an aeration tank, where the dissolved oxygen concentration was kept at 6 mg/l. At a feeding rate of 0.77 g acetonitrile l(-1) reactor day(-1), 99% of the acetonitrile was removed; and 86% of the nitrogen present in acetonitrile was released as NH3, confirming that acetonitrile volatilization was not significant. Increasing the acetonitrile loading resulted in lower removal efficiencies, but a maximum removal capacity of 1.0 g acetonitrile l(-1) reactor day(-1) was achieved at a feeding rate of 1.6 g acetonitrile l(-1) reactor day(-1). The removal capacity of the system was well correlated with the oxygenation capacity, showing that acetonitrile removal was likely to be limited by oxygen supply. Microbial characterization of the biofilm resulted in the isolation of a Comamonas sp. able to mineralize acetonitrile as sole carbon, nitrogen and energy source. This organism was closely related to C. testosteroni (91.2%) and might represent a new species in the Comamonas genus. This study confirms the potential of packed-bed reactors for the treatment of a concentrated mixture of volatile pollutants.