Construction of Mycoplasma hyopneumoniae P97 Null Mutants

Abstract

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia, a world-wide problem in the pig industry. This disease is characterized by a dry, non-productive cough, labored breathing, and pneumonia. Despite years of research, vaccines are marginally effective, and none fully protect pigs in a production environment. A better understanding of the host-pathogen interactions of the M. hyopneumoniae-pig disease, which are complex and involve both host and pathogen components, is required. Among the surface proteins involved in virulence are members of two gene families called P97 and P102. These proteins are the adhesins directing attachment of the organism to the swine respiratory epithelium. P97 is the major ciliary binding adhesin and has been studied extensively. Monoclonal antibodies that block its binding to swine cilia have contributed extensively to its characterization. In this study we use recombination to construct null mutants of P97 in M. hyopneumoniae and characterize the resulting mutants in terms of loss of protein by immunoblot using monoclonal antibodies, ability to bind purified swine cilia, and adherence to PK15 cells. Various approaches to recombination with this fastidious mycoplasma were tested including intact plasmid DNA, single-stranded DNA, and linear DNA with and without a heterologous RecA protein. Our results indicate that recombination can be used to generate site-specific mutants in M. hyopneumoniae. P97 mutants are deficient in cilia binding and PK15 cell adherence, and lack the characteristic banding pattern seen in immunoblots developed with the anti-P97 monoclonal antibody.

Genome Editing of Veterinary Relevant Mycoplasmas Using a CRISPR-Cas Base Editor System

“…Importing the RecA protein from E. coli improved the obtention of bacterial recombinants in Mycoplasma mycoides subsp. capricolum ( 89 ) and in M. hyorhinis ( 90 ), but not in M. hyopneumoniae ( 91 ). Recently, a RecET-like system from B. subtilis was used for targeted gene inactivation or gene replacement in Mycoplasma pneumoniae ( 25 ) and M. gallisepticum ( 23 ).…”

Mycoplasma hyopneumoniae membrane protein Mhp271 interacts with host UPR protein GRP78 to facilitate infection

“…P97 has received the most attention in the P97 adhesin family due to its special repeat regions 1 and 2 (R1 and R2). It has been demonstrated that the R1 repeats region for P97 with a number varying from 9 to 15 of the R1 repeating units (sequence AAKPV[E]) is essential for its host binding (Clampitt et al, 2021; Hsu & Minion, 1998; Minion et al, 2000). Mhp271, a paralog of P97, is the only other M. hyopneumoniae protein containing R1 and R2 repeats associated with the adhesion process (Deutscher et al, 2010).…”

“…

Porcine enzootic pneumonia (PEP) is caused by Mycoplasma hyopneumoniae (M. hyopneumoniae), which is characterized by dry coughing, severe respiratory distress, and growth reduction (Clampitt et al, 2021;Maes et al, 2018). This infection is highly prevalent, ranging between 38% and 100% in almost all areas of pig production worldwide, and causes significant economic losses (Maes, Sibila, et al, 2021).M.

…”

“…units (sequence AAKPV [E]) is essential for its host binding (Clampitt et al, 2021;Hsu & Minion, 1998;Minion et al, 2000). Mhp271, a paralog of P97, is the only other M. hyopneumoniae protein containing R1 and R2 repeats associated with the adhesion process (Deutscher et al, 2010).…”

Immunogenic characteristics of p113/p126 family in Mycoplasma ovipneumoniae

“…M. ovipneumoniae has the closest genetic relationship withMycoplasma hyopneumoniae (M. hyopneumoniae ) (Clampitt, Madsen, & Minion, 2021). In M. hyopneumoniae , P97 and P102 are not only adhesin molecules that mediate the adhesion, colonization and pathogenesis of host cells, but also the most important immunogen molecules, which can effectively induce the immune response of the body (Raymond et al, 2013; L. K. .…”