Doctor of Philosophy
School of Biological Sciences
Deutscher, Ania Teresa, Molecular and functional characterisation of p97 and p102 paralogue family members of mycoplasma hyopneumoniae, Doctor of Philosophy thesis, School of Biological Sciences, University of Wollongong, 2012. https://ro.uow.edu.au/theses/3521
Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia, an economically significant and chronic respiratory disease affecting swine. M. hyopneumoniae adheres to ciliated swine respiratory epithelial cells thereby causing ciliostasis and epithelial cell death, making the pigs susceptible to secondary infections. The gene mhp183 encoding the archetype cilium adhesin P97 shares a two-gene operon with mhp182, which encodes P102. Paralogues of P97 and P102 are found within the M. hyopneumoniae genome. P97, P102 and their paralogues are displayed as endoproteolytic cleavage fragments on the cell surface of M. hyopneumoniae, and bind a variety of host molecules such as glycosaminoglycans, plasminogen and fibronectin. These M. hyopneumoniae adhesins are believed to play an important role in colonisation.
M. hyopneumoniae has been detected in organ tissues distal to the respiratory tract. The mechanism of dissemination from the site of colonisation is unknown, though numerous pathogenic bacteria utilise host plasmin, to aid bacterial dissemination. In this study, we show that M. hyopneumoniae binds plasminogen in a dose-dependent and lysine-dependent manner. M. hyopneumoniae surface-bound plasminogen is shown here to be converted to functionally active plasmin by exogenous mammalian activators, such as tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). M. hyopneumoniae coated with plasmin degrades fibrinogen, highlighting the potential for M. hyopneumoniae surface-bound plasmin to promote tissue destruction and facilitate tissue invasion.
The M. hyopneumoniae P97 paralogue Mhp271 contains a C-terminal lysine residue, for which plasminogen typically has a high affinity. P97 contains two repeat regions, R1 and R2, which play a role in the adherence of M. hyopneumoniae to host molecules. Within Mhp271, there are two R1 repeat regions separated by an R2 repeat region; however, the number of tandem R1 repeats is fewer than the eight previously recognised as being required for swine cilial binding. Sequencing of these repeat regions in a set of M. hyopneumoniae strains shows that the number of tandem repeats in the R2 and the second R1 domains is variable. Two recombinant proteins were constructed; one containing the first R1 domain (F1271) and the second containing the C terminal portion of Mhp271, which includes the R2 domain together with the second R1 domain (F2271). In in vitro binding assays, F2271 bound heparin, fibronectin and porcine cilia, while F1271 did not bind these molecules. Heparin reduced the adherence of F2271 to cilia (by ~ 69%), indicating that F2271 binds the heparinase sensitive glycosaminoglycans at the surface of ciliated swine respiratory epithelia. F2271 is an inefficient plasminogen-binder despite containing a C-terminal lysine residue. Cell surface shaving with trypsin combined with two-dimensional liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) revealed that Mhp271 is surfaceexposed; therefore, both the R1 and R2 repeat regions in Mhp271 may contribute to M. hyopneumoniae adherence to host molecules.
Mhp385 is another P97 paralogue possessing a C-terminal lysine residue and R1 repeats. Antisera raised against recombinant protein fragments of Mhp385 reacted with 115 kDa, 88 kDa and 27 kDa proteins in Western blots of whole cell lysates of M. hyopneumoniae cells, which were confirmed by LC-MS/MS as the Mhp385 preprotein P115385 and cleavage fragments P88385 and P27385, respectively. This is the first time a preprotein belonging to the P97 and P102 paralogue families has been identified by mass spectrometry and indicates that Mhp385 is inefficiently cleaved. Cleavage occurs at a novel cleavage site (761L-N-V↓A-V-S766) defined by the semi-tryptic peptide 752IQFELEPISLNV763 denoting the C-terminus of P88385. Trypsin digest studies show that P115385, P88385 and P27385 are surface-exposed. Heparin- and cilium-binding sites were identified within P88385. No primary function was attributed to P27385; however, this molecule contains four tandem R1 repeats with similarity to porcine collagen type VI, which may be an instance of molecular mimicry. The Mhp385 recombinant protein fragments did not bind plasminogen in surface plasmon resonance assays.
The mhp385 gene shares a two-gene operon with mhp384, which encodes the P102 paralogue Mhp384. We show that Mhp384 is efficiently cleaved, at a site that closely conforms to a previously identified cleavage motif of members of the P97 and P102 paralogue families (S/T-X-F↓X-D/E), to create P60384 and P50384. P60384 and P50384 are cell surface-associated and contain heparin- and cilium-binding sites. This study expands the knowledge about the function(s) and endoproteolytic cleavage of members of the P97 and P102 paralogue families, which is integral to understanding how M. hyopneumoniae colonises swine and thereby causes chronic disease.