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.

Keywords: Mycoplasma hyopneumoniae; P97; adherence assay; adherence protein; immunoblot; null mutant; recombination.

FIGURE 1

Construction of pISM624-626. The regions cloned into pISM624 are shown above the operon along with the base pair locations in the strain 232 chromosome. These fragments were amplified by PCR. The small white box in the C-terminal region of P97 represents the R1 region responsible for cilium binding. Note the HindIII site in the N-terminal P97 fragment used for cloning. The other restriction sites were added to the primers.

FIGURE 2

Immunoblot analysis of M. hyopneumoniae pISM625 transformants. M. hyopneumoniae strain 232 was transformed with pISM625 and pISM626. Transformants were picked, grown in Friis broth and proteins prepared for SDS-PAGE and immunoblot analysis. Transformants from pISM625 are shown above. Each lane represents an individual transformant. 10 μg of protein was loaded per lane. Molecular weight markers are on the right. Conj = conjugate only, no primary antibody; lanes 1−3, dsDNA transformants 232:pISM625.1 Ω1–3(ds); lanes 4−6, ssDNA transformants 232:pISM625.1 Ω1-3(ss); lanes 7−9, linear dsDNA transformants 232:pISM625.1 Ω1–3(lds); C = control wildtype M. hyopneumoniae. Blot was developed with monoclonal F1B6, which binds to the R1 repeat sequence in P97 and blocks cilia binding.

FIGURE 3

Cilia binding assay with M. hyopneumoniae recombinants. The data represent duplicate assays with triplicate wells per assay and are expressed as optical density at 405 nm. Proc GLM ANOVA was performed on the data. Box plots were generated using BoXPlotR (http://shiny.chemgrid.org/boxplotr/). Wt = wildtype control; ds = dsDNA transformant; L = linear dsDNA transformant; and ss = ssDNA transformant. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots n = 6 sample points. Letters indicate values that are statistically different. The three transformant types are significantly different from wildtype (p < 0.0001).

FIGURE 4

PK15 adherence assay with M. hyopneumoniae transformants. The data represent triplicate assays with triplicate wells for each sample and are expressed as log₁₀ percent attached of the starting mycoplasma numbers. Proc GLM ANOVA was performed on the data. Box plots were generated using BoXPlotR (http://shiny.chemgrid.org/boxplotr/). Wt = wildtype control; ds = dsDNA transformant; L = linear dsDNA transformant; and ss = ssDNA transformant. The data is presented as Log₁₀. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots n = 9 sample points. Letters indicate values that are statistically different. The three transformant types are significantly different from wildtype (p < 0.0001).

FIGURE 5

Genetic analysis of M. hyopneumoniae transformants with pISM625. At the top of the figure (A), the P97/P102 operon is shown drawn to scale. The black arrows above the operon represent the regions cloned into pISM625 and pISM626. The large X represents potential cross-over points between the chromosome and plasmid. Single cross-over events can occur either at position X₁ or X₂ resulting in the corresponding structures shown in A1 and A2. (B–D) Agarose gels of PCR reactions with chromosomal DNAs from pISM625 transformants. Templates in all PCRs in all gels except where noted: Lanes 1, 1 kb ladder; 2, M. hyopneumoniae non-transformant wildtype control; 3, linear dsDNA transformant #1; 4, linear dsDNA transformant #2; 5, ssDNA transformant; 6, dsDNA transformant; 7, pISM625 positive control; and 8, water template control. In (C), Lane 8, pMHC9-1 (Maglennon et al., 2013a) positive control; Lane 9, water template control. Primers used were as follows: (B) bla-specific primers (bla_FWD and bla_REV). (C) tetM-specific primers (tetM_FWD and tetM_REV). (D) primers tetM-2X-F and 5-P97-F. (E) primers P102RT-r2 and tetM-2X-F.