Susceptibilities of Mycobacterium marinum to Gatifloxacin, Gemifloxacin, Levofloxacin, Linezolid, Moxifloxacin, Telithromycin, and Quinupristin-Dalfopristin (Synercid) Compared to Its Susceptibilities to Reference Macrolides and Quinolones.

The susceptibility pattern of Mycobacterium marinum was determined. Quinupristin-dalfopristin and telithromycin were less active than clarithromycin. Linezolid showed good antimicrobial activity at clinically achievable concentrations. Gatifloxacin, levofloxacin, and moxifloxacin displayed activities similar to those of ciprofloxacin. Gemifloxacin was less active. The Etest method showed variable agreement with the reference method

Human complement control and complement evasion by pathogenic microbes - Tipping the balance

Complement is a central homeotic system of mammals and represents the first defense line of innate immunity. The human complement system is aimed to maintain homeostasis by recognizing and removing damaged or modified self material, as well as infectious foreign microbes. However, pathogenic microbes also control and escape the host complement and immune attack. The increasing resistance of microbial pathogens to either antibiotics or antifungal drugs is a major health problem and is of global interest. Therefore the topic how pathogenic microbes escape human complement and immune control is of high and of central interest. Identifying and defining the action of proteins involved in this intense immune interaction and understanding how these proteins interact is of relevance to design new control strategies. In this review we summarize the complement system of the human host and how this cascade drives effector functions. In addition, we summarize how diverse pathogenic microbes control, modulate and block the complement response of their host. The characterization of pathogen derived virulence factors and complement escape proteins reveals patterns of multiplicity, diversity and redundancy among pathogen encoded proteins. Sequence variability of immune and also complement escape proteins is largely driven by antigenic diversity and adaptive immunity. However common complement escape principles are, emerging in terms of conserved binding repertoire for host regulators and evasion among the large variety of infectious microbes. These conserved and common escape features are relevant and they provide challenging options for new therapeutic approaches. (C) 2013 Elsevier Ltd. All rights reserved

The unique structure of Haemophilus influenzae protein E reveals multiple binding sites for host factors.

Haemophilus influenzae protein E (PE) is a multifunctional adhesin, involved in direct interactions with lung epithelial cells and host proteins, including plasminogen and the extracellular matrix proteins vitronectin and laminin. We recently crystallized PE and successfully collected X-ray diffraction data to 1.8 Å. Here we solved the structure of a recombinant version of PE and analyzed different functional regions. It is a dimer in solution and in the asymmetric unit of the crystals. The dimer has a structure that resembles a flattened β-barrel. It is however not a true β-barrel as there are differences in both the hydrogen bonding pattern and the shape. Each monomer consisted of a 6-stranded antiparallel β-sheet with a rigid α-helix at the C-terminal tethered to the concave side of the sheet by a disulfide bridge. The laminin/plasminogen binding region (residues 41-68) is exposed, while the vitronectin binding region (residues 84-108) is partially accessible in the dimer. The dimerized PE explains the simultaneous interaction with laminin and vitronectin. In addition, we found this unique adhesin being present in many bacterial genera of the family Pasteurallaceae and also orthologues in other unrelated species (Enterobacter cloacae and Listeria monocytogenes). Peptides corresponding to the surface-exposed regions PE24-37, PE74-89, and PE134-156 were immunogenic in the mouse. Importantly, these peptide-based antibodies also recognised PE at the bacterial surface. Taken together, our detailed structure of PE explains how this important virulence factor of H. influenzae simultaneously interacts with host vitronectin, laminin or plasminogen promoting bacterial pathogenesis

Streptococcus pneumoniae endopeptidase O (PepO): a multifunctional plasminogen and fibronectin binding protein, facilitating evasion of innate immunity and invasion of host cells.

Streptococcus pneumoniae infections remain a major cause of morbidity and mortality worldwide. Therefore a detailed understanding and characterization of the mechanism of host cell colonization and dissemination is critical in order to gain control over this versatile pathogen. Here we identified a novel 72 kDa pneumococcal protein endopeptidase O (PepO), as a plasminogen and fibronectin binding protein. Using a collection of clinical isolates, representing different serotypes, we found PepO to be ubiquitously present both at the gene and at the protein level. In addition, PepO protein was secreted in a growth-phase dependent manner to the culture supernatants of the pneumococcal isolates. Recombinant PepO bound human plasminogen and fibronectin in a dose-dependent manner and plasminogen did not compete with fibronectin for binding PepO. PepO bound plasminogen via lysine residues and the interaction was influenced by ionic strength. Moreover, upon activation of PepO bound plasminogen by urokinase-type plasminogen activator, generated plasmin cleaved complement protein C3b thus assisting in complement control. Furthermore, direct binding assays demonstrated the interaction of PepO with epithelial and endothelial cells that in turn blocked pneumococcal adherence. Moreover, a pepO-mutant strain showed impaired adherence to and invasion of host cells compared to their isogenic wild-type strains. Taken together, the results demonstrated that PepO is ubiquitously expressed plasminogen and fibronectin binding protein, which plays role in pneumococcal invasion of host cells and aids in immune evasion

The Moraxella catarrhalis immunoglobulin D-binding protein MID has conserved sequences and is regulated by a mechanism corresponding to phase variation.

The prevalence of the Moraxella catarrhalis immunoglobulin D (IgD)-binding outer membrane protein MID and its gene was determined in 91 clinical isolates and in 7 culture collection strains. Eighty-four percent of the clinical Moraxella strains expressed MID-dependent IgD binding. The mid gene was detected in all strains as revealed by homology of the signal peptide sequence and a conserved area in the 3′ end of the gene. When MID proteins from five different strains were compared, an identity of 65.3 to 85.0% and a similarity of 71.2 to 89.1% were detected. Gene analyses showed several amino acid repeat motifs in the open reading frames, and MID could be called a putative autotransport protein. Interestingly, homopolymeric {polyguanine [poly(G)]} tracts were detected at the 5′ ends within the open reading frames. By flow cytometry, using human IgD and fluorescein isothiocyanate-conjugated anti-IgD polyclonal antibodies, most strains showed two peaks: one high- and one low-intensity peak. All isolates expressing high levels of MID had 1, 2, or 3 triplets of G's in their poly(G) tracts, while strains not expressing MID had 4, 7, 8, or 10 G’s in their poly(G) tracts or point mutations causing a putative preterminated translation. Northern blot analysis revealed that the mid gene was regulated at the transcriptional level. Experiments with nonclumping variants of M. catarrhalis proved that bacteria lost their MID expression by removing a G in their poly(G) tracts. Moraxella strains isolated from the nasopharynx or from blood and sputum specimens expressed MID at approximately the same frequency. In addition, no variation was observed between strains of different geographical origins (Australia, Europe, Japan, or the United States). MID and the mid gene were found solely in M. catarrhalis, whereas related Neisseria and Moraxella species did not express MID. Taken together, MID appears to be a conserved protein that can be found in essentially all M. catarrhalis strains. Furthermore, MID is governed by poly(G) tracts when bacteria undergo phase variation

A fusion protein derived from Moraxella catarrhalis and Neisseria meningitidis aimed for immune modulation of human B cells.

Moraxella IgD-binding protein (MID) is a well characterized trimeric autotransporter that specifically targets the IgD of B cells. We fused the membrane anchor of the meningococcal autotransporter NhhA with the IgD-binding region of MID (aa 962-1200) to create a chimeric protein designated as NID. The aim was to use this specific targeting to provide a better vaccine candidate against meningococci, in particular serogroup B by enhancing the immunogenicity of NhhA. NID was thereafter recombinantly expressed in E. coli. The NID-expressing E. coli bound to peripheral B lymphocytes that resulted in cellular activation. Furthermore, we also successfully expressed NID on outer membrane vesicles, nanoparticles that are commonly used in meningococcal vaccines. This study thus highlights the applicability of the menigococcal-Moraxella fusion protein NID to be used for specific targeting of vaccine components to the IgD B cell receptor

Short Leucine-Rich Proteoglycans Modulate Complement Activity and Increase Killing of the Respiratory Pathogen Moraxella catarrhalis

The respiratory pathogen Moraxella catarrhalis is a human-specific commensal that frequently causes acute otitis media in children and stimulates acute exacerbations in chronic obstructive pulmonary disease patients. The exact molecular mechanisms defining host-pathogen interactions promoting pathogenesis are not clearly understood. Limited knowledge hampers vaccine and immunotherapeutic development required to treat this emerging pathogen. In this study, we reveal in detail a novel antibacterial role displayed by short leucine-rich proteoglycans (SLRPs) in concert with complement. We show that fibromodulin (FMOD), osteoadherin (OSAD), and biglycan (BGN) but not decorin (DCN) enhance serum killing of M. catarrhalis. Our results suggest that M. catarrhalis binding to SLRPs is a conserved feature, as the overwhelming majority of clinical and laboratory strains bound all four SLRPs. Furthermore, we resolve the binding mechanism responsible for this interaction and highlight the role of the ubiquitous surface protein (Usp) A2/A2H in mediating binding to host SLRPs. A conserved immune evasive strategy used by M. catarrhalis and other pathogens is the surface acquisition of host complement inhibitors such as C4b-binding protein (C4BP). We observed that FMOD, OSAD, and BGN competitively inhibit binding of C4BP to the surface of M. catarrhalis, resulting in increased C3b/iC3b deposition, membrane attack complex (MAC) formation, and subsequently decreased bacterial survival. Furthermore, both OSAD and BGN promote enhanced neutrophil killing in vitro, both in a complement-dependent and independent fashion. In summary, our results illustrate that SLRPs, FMOD, OSAD, and BGN portray complement-modulating activity enhancing M. catarrhalis killing, defining a new antibacterial role supplied by SLRPs.</p

Identification of outer membrane Porin D as a vitronectin-binding factor in cystic fibrosis clinical isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a pathogen that frequently colonizes patients with cystic fibrosis (CF) or chronic obstructive pulmonary disease (COPD). Several pathogens are known to bind vitronectin to increase their virulence. Vitronectin has been shown to enhance P. aeruginosa adhesion to host epithelial cells

QseC controls biofilm formation of non-typeable Haemophilus influenzae in addition to an AI-2-dependent mechanism.

Non-typeable Haemophilus influenzae (NTHi) is a common pathogen associated with diseases such as acute otitis media or exacerbations in patients with chronic obstructive pulmonary disease. The biofilm-forming capability substantially contributes to the persistence of NTHi. However, the regulation of biofilm formation is not completely understood. Quorum sensing regulated by autoinducer-2 produced by luxS is until now the only described regulatory mechanism. In this study, we show that the two-component signalling system QseB/C is involved in the biofilm formation of NTHi in vitro. An isogenic NTHi mutant of qseC (Hi3655KR2) showed a significant decrease in biofilm formation under static and semi-static conditions as assessed by crystal violet staining. In addition, under constant flow conditions, Hi3655KR2 formed less biofilm after 48h. The biofilm defects were irrespective of autoinducer-2 levels. Hence, here we suggest for the first time a regulatory circuit in NTHi, which controls biofilm formation by mechanisms other than or in addition to luxS-dependent factors