Unmarked insertional mutagenesis in the bovine pathogen Mycoplasma mycoides subsp. mycoides SC: characterization of a lppQ mutant

Abstract

Mycoplasma mycoides subspecies mycoides small colony (SC) is the aetiologic agent of contagious bovine pleuropneumonia (CBPP), a respiratory disease causing important losses in cattle production. The publication of the genome sequence of M. mycoides subsp. mycoides SC should facilitate the identification of putative virulence factors. However, real progress in the study of molecular mechanisms of pathogenicity also requires efficient molecular tools for gene inactivation. In the present study, we have developed a transposon-based approach for the random mutagenesis of M. mycoides subsp. mycoides SC. A PCR-based screening assay enabled the characterization of several mutants with knockouts of genes potentially involved in pathogenicity. The initial transposon was further improved by combining it with the transposon cd TnpR/res recombination system to allow the production of unmarked mutations. Using this approach, we isolated a mutant free of antibiotic-resistance genes, in which the gene encoding the main lipoprotein LppQ was disrupted. The mutant was found to express only residual amounts of the truncated N-terminal end of LppQ. This approach opens the way to study virulence factors and pathogen-host interactions of M. mycoides subsp. mycoides SC and to develop new, genetically defined vaccine strains.

Changes in pathogenicity and immunogenicity of Mycoplasma mycoides subsp. mycoides strains revealed by comparative genomics analysis

“…2 ). Lipoproteins of Mmm have previously been characterized as strong major antigens, e.g., LppB ( Ben1_0562 ) 14 , LppQ ( Ben1_1077 ) 15 16 , and variable surface protein Vmm ( Ben1_0422 ) 17 . In addition to these, we identified LppA ( Ben1_0014 ), LppC ( Ben1_1059 ), LppD ( Ben1_0685 ), and seven variable surface protein (VSP) genes in the genome ( Supplementary Table 3 ).…”

Baseline analysis of Mycoplasma mycoides subsp. mycoides antigens as targets for a DIVA assay for use with a subunit vaccine for contagious bovine pleuropneumonia

“…In a study to establish a DIVA diagnostic test to be used in combination with the CBPP live-attenuated vaccine, a very immunogenic antigen, LppQ was characterized and an LppQ-knock-out derivative of the T1/44 live attenuated vaccine strain, the T1LppQ-MT1 was created [ 18 – 20 ]. It was suggested that LppQ could be used in a DIVA assay for discriminating animals vaccinated with the depleted T1LppQ-MT1 strain from those infected with the strains from the field [ 21 , 22 ].…”

Protein-Specific Analysis of Humoral Immune Responses in a Clinical Trial for Vaccines against Contagious Bovine Pleuropneumonia

“…These could be identified by using larger groups of cattle or by measuring other immune response components such as IgM, IgA, or cellular responses. LppQ is known to be a marker of CBPP (1), but its usefulness as a vaccine is questionable based on recent studies (19,27). The results of this study indicate that MSC_1046 (LppQ) is the most significant component of the T1/44-induced response ( Fig.…”