Domain analysis of lipoprotein LppQ in Mycoplasma mycoides subsp. mycoides SC

Abstract

The lipoprotein LppQ is the most prominent antigen of Mycoplasma mycoides subsp. mycoides small colony type (SC) during infection of cattle. This pathogen causes contagious bovine pleuropneumonia (CBPP), a devastating disease of considerable socio-economic importance in many countries worldwide. The dominant antigenicity and high specificity for M. mycoides subsp. mycoides SC of lipoprotein LppQ have been exploited for serological diagnosis and for epidemiological investigations of CBPP. Scanning electron microscopy and immunogold labelling were used to provide ultrastructural evidence that LppQ is located to the cell membrane at the outer surface of M. mycoides subsp. mycoides SC. The selectivity and specificity of this method were demonstrated through discriminating localization of extracellular (i.e., in the zone of contact with host cells) vs. integral membrane domains of LppQ. Thus, our findings support the suggestion that the accessible N-terminal domain of LppQ is surface exposed and such surface localization may be implicated in the pathogenesis of CBPP.

Evidence for the Cytoplasmic Localization of the L-α-Glycerophosphate Oxidase in Members of the “Mycoplasma mycoides Cluster”

“…The separated proteins were either directly subjected to Coomassie brilliant blue staining or transferred to nitrocellulose membranes using the Trans-Blot SD semi-dry transfer system (Bio-rad) at 18 V in Towbin transfer buffer (25 mM Tris, 192 mM Glycine and 20% (v/v) methanol). After membrane blocking using 5% milk powder or 2% bovine serum albumin (BSA), the membranes were probed with either rabbit anti-GlpO serum (Pilo et al, 2005) diluted at 1:5,000, rabbit anti-LppQ primary antibody (Bonvin-Klotz et al, 2008) diluted at 1:2,000 or rabbit anti-beta Galactosidase polyclonal antibody diluted at 1:5,000 followed by incubation with a horseradish peroxidase (HRP) labeled donkey anti-rabbit IgG (H+L) (Thermo Fisher Scientific) diluted at 1:10,000. Probed protein bands were detected using chemiluminescence (Thermo Fisher Scientific) and were documented using a Vilber-FusionFX-Machine (Vilber Lourmat SAS, France).…”

Identification of genes coding for B cell antigens of Mycoplasma mycoides subsp. mycoides Small Colony (Mmm SC) by using phage display

“…GapN, in contrast, may play a role in transcription [ 30 ] and apoptosis [ 31 ]. Membrane lipoproteins that interact with host cells can stimulate the release of pro-inflammatory cytokines [ 32 ] and are major antigens [ 23 , 33 , 34 ]. The lipoprotein (LppB) identified by the phage display is found in African and Australian strains of Mmm SC, but not in the less virulent European strains [ 22 ].…”

Detection of Mycoplasma mycoides subsp. mycoides SC in bronchoalveolar lavage fluids of cows based on a TaqMan real-time PCR discriminating wild type strains from an lppQ− mutant vaccine strain used for DIVA-strategies

“…mycoides SC as it is serologically specific to this organism. It has a particularly strong antigenic N-terminal part that is located on the outer surface of the membrane ( Bonvin-Klotz et al, 2008 ) and that elicits a specific and early immune response in naturally and experimentally infected animals ( Bruderer et al, 2002 ). The C-terminal part of LppQ possesses an integral membrane structure built up of repeated units that are suspected to be involved in membrane anchoring ( Abdo et al, 2000; Bonvin-Klotz et al, 2008 ).…”