MHJ_0125 is an M42 glutamyl aminopeptidase that moonlights as a multifunctional adhesin on the surface of Mycoplasma hyopneumoniae

Abstract

Bacterial aminopeptidases play important roles in pathogenesis by providing a source of amino acids from exogenous proteins, destroying host immunological effector peptides and executing posttranslational modification of bacterial and host proteins. We show that MHJ_0125 from the swine respiratory pathogen Mycoplasma hyopneumoniae represents a new member of the M42 class of bacterial aminopeptidases. Despite lacking a recognizable signal sequence, MHJ_0125 is detectable on the cell surface by fluorescence microscopy and LC-MS/MS of (i) biotinylated surface proteins captured by avidin chromatography and (ii) peptides released by mild trypsin shaving. Furthermore, surface-associated glutamyl aminopeptidase activity was detected by incubation of live M. hyopneumoniae cells with the diagnostic substrate H-Glu-AMC. MHJ_0125 moonlights as a multifunctional adhesin, binding to both heparin and plasminogen. Native proteomics and comparative modelling studies suggest MHJ_0125 forms a dodecameric, homopolymeric structure and provide insight into the positions of key residues that are predicted to interact with heparin and plasminogen. MHJ_0125 is the first aminopeptidase shown to both bind plasminogen and facilitate its activation by tissue plasminogen activator. Plasmin cleaves host extracellular matrix proteins and activates matrix metalloproteases, generating peptide substrates for MHJ_0125 and a source of amino acids for growth of M. hyopneumoniae. This unique interaction represents a new paradigm in microbial pathogenesis.

Proteolytic processing of the cilium adhesin MHJ_0194 (P123J) inMycoplasma hyopneumoniaegenerates a functionally diverse array of cleavage fragments that bind multiple host molecules

“…None of these cleavage events occur at S/T‐X‐F↓X‐D/E motifs, but cleavage was detected within regions enriched in hydrophobic amino acids consistent with studies describing cleavage events in P159 (Raymond et al ., ) and P216 (Tacchi et al ., ). Furthermore, we have described a surface‐accessible aminopeptidase in M. hyopneumoniae that has a preference for the amino acids that are being targeted in these events (Robinson et al ., ). The N‐terminal peptide derived from P28 J (spot 5) produced the sequence 863 N‐T‐N‐T‐G‐F‐S‐L‐T‐N‐K 873 consistent with the true N‐terminus of P28 J as determined previously by Edman sequencing (Djordjevic et al ., ).…”

P159 from Mycoplasma hyopneumoniae Binds Porcine Cilia and Heparin and Is Cleaved in a Manner Akin to Ectodomain Shedding

“…Several host molecules including fibronectin and plasminogen are known to be sequestered onto the surface of M. hyopneumoniae, and these may provide a scaffold for retaining cleavage fragments. [9][10][11][13][14][15]17,21 Surface-accessible membrane proteins in eukaryotes are known to be released into the extracellular milieu via a process known as ectodomain shedding. 35 Ectodomain shedding is now recognized as a widespread process in eukaryotes, and algorithms have been developed to identify membrane proteins that are targeted by sheddases.…”

“…10,18,20 Surface proteins of M. hyopneumoniae that do not belong to the P97 and P102 paralog families also bind glycosaminoglycans and plasminogen, underscoring the importance of these interactions to the survival of the species. 21 P159 (Mhp494) is not a member of the P97 and P102 paralog families, yet it is known to be a cellular adhesin. 18 The P159 homologue from strain 232 comprises 1410 amino acids and is processed to form N-terminal, central, and C-terminal fragments of 27 (P27), 110 (P110), and 52 kDa (P52) respectively, and these cleavage fragments are presented on the surface of M. hyopneumoniae.…”

Postepizootic Persistence of Asymptomatic Mycoplasma conjunctivae Infection in Iberian Ibex

“…are small bacteria without a cell wall that have a strict parasitic life in association with their hosts, either as commensals or pathogens (1). Mycoplasma has several singular mechanisms for host adaptation and survival (2)(3)(4), which includes one of the highest nucleotide substitution rates among bacteria that provides chances for novel interactions with its hosts (5,6). Mycoplasma infections can therefore involve diverse epidemiological scenarios, resulting either in the development of severe disease or in asymptomatic carriers that may or maybe not further develop clinical symptoms (1,7).…”