Elongation factor Tu is a multifunctional and processed moonlighting protein.

Many bacterial moonlighting proteins were originally described in medically, agriculturally, and commercially important members of the low G + C Firmicutes. We show Elongation factor Tu (Ef-Tu) moonlights on the surface of the human pathogens Staphylococcus aureus (SaEf-Tu) and Mycoplasma pneumoniae (MpnEf-Tu), and the porcine pathogen Mycoplasma hyopneumoniae (MhpEf-Tu). Ef-Tu is also a target of multiple processing events on the cell surface and these were characterised using an N-terminomics pipeline. Recombinant MpnEf-Tu bound strongly to a diverse range of host molecules, and when bound to plasminogen, was able to convert plasminogen to plasmin in the presence of plasminogen activators. Fragments of Ef-Tu retain binding capabilities to host proteins. Bioinformatics and structural modelling studies indicate that the accumulation of positively charged amino acids in short linear motifs (SLiMs), and protein processing promote multifunctional behaviour. Codon bias engendered by an A + T rich genome may influence how positively-charged residues accumulate in SLiMs.

The surface-displayed chaperones GroEL and DnaK of Mycoplasma pneumoniae interact with human plasminogen and components of the extracellular matrix.

Mycoplasma pneumoniae is a common cause of community-acquired infections of the human respiratory tract. The strongly reduced genome of the cell wall-less bacteria results in limited metabolic pathways and a small number of known virulence factors. In addition to the well-characterized adhesion apparatus and the expression of tissue-damaging substances, surface-exposed proteins with a primary function in cytosol-located processes such as glycolysis have been attracting attention in recent years. Due to interactions with host factors, it has been suggested that these bacterial proteins contribute to pathogenesis. Here, we investigated the chaperones GroEL and DnaK of M. pneumoniae as candidates for such moonlighting proteins. After successful expression in Escherichia coli and production of polyclonal antisera, the localization of both chaperones on the surface of bacteria was confirmed. Binding of recombinant GroEL and DnaK to human A549 cells, to plasminogen as well as to vitronectin, fibronectin, fibrinogen, lactoferrin and laminin was demonstrated. In the presence of both recombinant proteins and host activators, plasminogen can be activated to the protease plasmin, which is able to degrade vitronectin and fibrinogen. The results of the study extend the spectrum of surface-exposed proteins in M. pneumoniae and indicate an additional role of both chaperones in infection processes.

Interactions of surface-displayed glycolytic enzymes of Mycoplasma pneumoniae with components of the human extracellular matrix.

Mycoplasma pneumoniae is a major cause of community-acquired respiratory infections worldwide. Due to the strongly reduced genome, the number of virulence factors expressed by this cell wall-less pathogen is limited. To further understand the processes during host colonization, we investigated the interactions of the previously confirmed surface-located glycolytic enzymes of M. pneumoniae (pyruvate dehydrogenase A-C [PdhA-C], glyceraldehyde-3-phosphate dehydrogenase [GapA], lactate dehydrogenase [Ldh], phosphoglycerate mutase [Pgm], pyruvate kinase [Pyk] and transketolase [Tkt]) to the human extracellular matrix (ECM) proteins fibrinogen (Fn), fibronectin (Fc), lactoferrin (Lf), laminin (Ln) and vitronectin (Vc), respectively. Concentration-dependent interactions between Fn and Vc and all eight recombinant proteins derived from glycolytic enzymes, between Ln and PdhB-C, GapA, Ldh, Pgm, Pyk and Tkt, between Lf and PdhA-C, GapA and Pyk, and between Fc and PdhC and GapA were demonstrated. In most cases, these associations are significantly influenced by ionic forces and by polyclonal sera against recombinant proteins. In immunoblotting, the complex of human plasminogen, activator (tissue-type or urokinase plasminogen activator) and glycolytic enzyme was not able to degrade Fc, Lf and Ln, respectively. In contrast, degradation of Vc was confirmed in the presence of all eight enzymes tested. Our data suggest that the multifaceted associations of surface-localized glycolytic enzymes play a potential role in the adhesion and invasion processes during infection of human respiratory mucosa by M. pneumoniae.

Network of Surface-Displayed Glycolytic Enzymes in Mycoplasma pneumoniae and Their Interactions with Human Plasminogen.

In different bacteria, primarily cytosolic and metabolic proteins are characterized as surface localized and interacting with different host factors. These moonlighting proteins include glycolytic enzymes, and it has been hypothesized that they influence the virulence of pathogenic species. The presence of surface-displayed glycolytic enzymes and their interaction with human plasminogen as an important host factor were investigated in the genome-reduced and cell wall-less microorganism Mycoplasma pneumoniae, a common agent of respiratory tract infections of humans. After successful expression of 19 glycolytic enzymes and production of polyclonal antisera, the localization of proteins in the mycoplasma cell was characterized using fractionation of total proteins, colony blot, mild proteolysis and immunofluorescence of M. pneumoniae cells. Eight glycolytic enzymes, pyruvate dehydrogenases A to C (PdhA-C), glyceraldehyde-3-phosphate dehydrogenase (GapA), lactate dehydrogenase (Ldh), phosphoglycerate mutase (Pgm), pyruvate kinase (Pyk), and transketolase (Tkt), were confirmed as surface expressed and all are able to interact with plasminogen. Plasminogen bound to recombinant proteins PdhB, GapA, and Pyk was converted to plasmin in the presence of urokinase plasminogen activator and plasmin-specific substrate d-valyl-leucyl-lysine-p-nitroanilide dihydrochloride. Furthermore, human fibrinogen was degraded by the complex of plasminogen and recombinant protein PdhB or Pgm. In addition, surface-displayed proteins (except PdhC) bind to human lung epithelial cells, and the interaction was reduced significantly by preincubation of cells with antiplasminogen. Our results suggest that plasminogen binding and activation by different surface-localized glycolytic enzymes of M. pneumoniae may play a role in successful and long-term colonization of the human respiratory tract.

Subunits of the Pyruvate Dehydrogenase Cluster of Mycoplasma pneumoniae Are Surface-Displayed Proteins that Bind and Activate Human Plasminogen.

The dual role of glycolytic enzymes in cytosol-located metabolic processes and in cell surface-mediated functions with an influence on virulence is described for various micro-organisms. Cell wall-less bacteria of the class Mollicutes including the common human pathogen Mycoplasma pneumoniae possess a reduced genome limiting the repertoire of virulence factors and metabolic pathways. After the initial contact of bacteria with cells of the respiratory epithelium via a specialized complex of adhesins and release of cell-damaging factors, surface-displayed glycolytic enzymes may facilitate the further interaction between host and microbe. In this study, we described detection of the four subunits of pyruvate dehydrogenase complex (PDHA-D) among the cytosolic and membrane-associated proteins of M. pneumoniae. Subunits of PDH were cloned, expressed and purified to produce specific polyclonal guinea pig antisera. Using colony blotting, fractionation of total proteins and immunofluorescence experiments, the surface localization of PDHA-C was demonstrated. All recombinant PDH subunits are able to bind to HeLa cells and human plasminogen. These interactions can be specifically blocked by the corresponding polyclonal antisera. In addition, an influence of ionic interactions on PDHC-binding to plasminogen as well as of lysine residues on the association of PDHA-D with plasminogen was confirmed. The PDHB subunit was shown to activate plasminogen and the PDHB-plasminogen complex induces degradation of human fibrinogen. Hence, our data indicate that the surface-associated PDH subunits might play a role in the pathogenesis of M. pneumoniae infections by interaction with human plasminogen.

Discovery and genetic characterization of novel caliciviruses in German and Dutch poultry.

Caliciviruses (CV) were identified in the intestinal contents of five chickens and one turkey from various regions in Germany between 2009 and 2011 by degenerate reverse transcription PCR. The full 7,656-nt-long genomic sequence of the turkey CV L11043 was determined. Partial nucleotide sequences were determined for nine chicken strains. Phylogenetic analysis based on partial deduced amino acid sequences of the protease and RNA polymerase and the complete VP1 capsid sequence identified two distinct clusters of avian CVs, the first of which contained chicken CVs that were closely related to strains found in German chickens in Bavaria and that had been proposed to form a novel CV genus (proposed name: Bavovirus). In contrast, the turkey CV strain L11043 and three chicken CV strains formed a genetically distinct second cluster. Distance analysis suggested that the strains of the second cluster may represent members of two distinct genogroups of another novel CV genus (proposed name: Nacovirus). Based on the newly obtained sequence information, two real-time RT-PCR assays were developed and used to identify bavovirus and nacovirus in pooled intestinal contents from 24 chicken farms in Germany and the Netherlands. Of these, 20 (83 %) were positive for bavovirus, 11 (46 %) were positive for nacovirus, and nine (38 %) were positive for both bavovirus and nacovirus. Attempts were made to propagate chicken and turkey CVs from both the bavovirus and nacovirus clusters in primary chicken cecal cells, embryonal liver cells and fibroblast cells, but these attempts were not successful.