Mycoplasma hyopneumoniae surface-associated proteases cleave bradykinin, substance P, neurokinin A and neuropeptide Y

Veronica Maria Jarocki,Steven Philip Djordjevic,Benjamin Bernard Armando Raymond,Jessica Leigh Tacchi,Matthew Paul Padula

doi:10.1038/s41598-019-51116-w

Veronica Maria Jarocki, Steven Philip Djordjevic + Show 3 more

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https://doi.org/10.1038/s41598-019-51116-w

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Journal: Scientific Reports	Publication Date: Oct 10, 2019
Citations: 10	License type: open-access

Affiliation: University of Technology Sydney

Abstract

Mycoplasma hyopneumoniae is an economically-devastating and geographically-widespread pathogen that colonises ciliated epithelium, and destroys mucociliary function. M. hyopneumoniae devotes ~5% of its reduced genome to encode members of the P97 and P102 adhesin families that are critical for colonising epithelial cilia, but mechanisms to impair mucociliary clearance and manipulate host immune response to induce a chronic infectious state have remained elusive. Here we identified two surface exposed M. hyopneumoniae proteases, a putative Xaa-Pro aminopeptidase (MHJ_0659; PepP) and a putative oligoendopeptidase F (MHJ_0522; PepF), using immunofluorescence microscopy and two orthogonal proteomic methodologies. MHJ_0659 and MHJ_0522 were purified as polyhistidine fusion proteins and shown, using a novel MALDI-TOF MS assay, to degrade four pro-inflammatory peptides that regulate lung homeostasis; bradykinin (BK), substance P (SP), neurokinin A (NKA) and neuropeptide Y (NPY). These findings provide insight into the mechanisms used by M. hyopneumoniae to influence ciliary beat frequency, impair mucociliary clearance, and initiate a chronic infectious disease state in swine, features that are a hallmark of disease caused by this pathogen.

Highlights

Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia, a highly infectious and globally distributed swine respiratory disease
Biochemical characterisation of functionally active rMHJ_0659. rMHJ_0659 was purified from Escherichia coli as a polyhistidine fusion protein and resolved as a single band with a mass of approximately 40 kDa by SDS-PAGE (Fig. 1a). This protein was recovered from the gel, digested with trypsin and confirmed to be MHJ_0659 by LC-MS/MS (46% sequence coverage) (Fig. 1d). rMHJ_0659 was able to remove the N-terminal penultimate proline from BK (Fig. 3), substance P (SP) (Fig. 4) and neuropeptide Y (NPY) (Fig. 5) in a manner that is typical of a putative Xaa-Pro aminopeptidase (PepP) protease
M. hyopneumoniae enters the porcine respiratory tract on mucosal droplets expelled during bouts of coughing from infected animals

Summary

Introduction

Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia, a highly infectious and globally distributed swine respiratory disease. In 1991, M. hyopneumoniae caused an estimated $1 billion economic loss in the USA alone[5] This estimate did not account for the impact incurred by animal waste containing large quantities of multiple antibiotic resistant bacterial populations[6]. The mucociliary escalator is a major innate barrier to all infectious respiratory microorganisms It lines the respiratory tract and is composed of mucus-secreting goblet cells and ciliated epithelium. M. hyopneumoniae avoids mucociliary clearance by disrupting the mucociliary escalator by initiating ciliostasis, loss of cilia function, and epithelial cell death. These sequelae are poorly understood[11]. Host-induced ECM proteolysis represents a mechanism to source nutrients, a process that is vital for genome-reduced organisms, such as M. hyopneumoniae, unable to synthesise amino acids, nucleotides, fatty acids and other macromolecular building blocks[21,22]

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