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.