Antigenic and Genetic Characterization of Lipoprotein LppQ fromMycoplasma mycoidessubsp.mycoidesSC

Abstract

Lipoprotein LppQ, a predominant 48-kDa antigen, and its corresponding gene, lppQ, were characterized in Mycoplasma mycoides subsp. mycoides SC, the etiological agent of contagious bovine pleuropneumonia. The lppQ gene is specific to M. mycoides subsp. mycoides SC and was found in the type strain and in field strains isolated in Europe, Africa, and Australia, as well as in vaccinal strains. LppQ is encoded as a precursor with a consensus sequence for prokaryotic signal peptidase II and a lipid attachment site. The leader sequence shows significant prominent transmembrane helix structure with a predicted outside-to-inside helix formation capacity. The N-terminal domain of the mature LppQ was shown to be surface exposed. It induced a strong, specific, early, and persistent immune response in naturally and experimentally infected animals. The C-terminal domain of LppQ possesses an integral membrane structure built up of repeated units, rich in hydrophobic and aromatic amino acids, which have a pore formation potential. A recombinant peptide representing the N-terminal domain of LppQ was obtained by site-directed mutagenesis of nine Mycoplasma-specific TGA (Trp) codons into universal TGG (Trp) codons and expression in Escherichia coli hosts. It was used for serodetection of cattle infected with M. mycoides subsp. mycoides SC, in which it was detected postinfection for significantly longer than conventional serological test reactions.Mycoplasma mycoides subsp. mycoides small-colony type (SC) is the etiological agent of contagious bovine pleuropneumonia (CBPP), a highly contagious disease which represents a major threat to raising cattle, particularly in Africa. CBPP is also a problem in other parts of the world, including some European countries, where the disease suddenly reemerged 2 decades ago. CBPP is a disease of major economic concern in the affected countries, not only due to the morbidity and mortality but also due to restrictions on cattle trade imposed by international regulations. Hence, control of the disease is a priority for countries in which it is endemic, in order to eradicate the disease as quickly as possible after outbreaks and to avoid its spreading, as well as for countries which are free of CBPP, in order to keep that status. The main problem in eradication is the frequent occurrence of subacute or asymptomatic infections and the persistence of chronic carriers after the clinical phase. Serological analysis is the most important diagnostic tool for the control of CBPP, but it is significantly hampered by the relatively low sensitivity and specificity of the methods. The complement fixation test (CFT), which is currently the official and most widely used serodiagnostic test, has been shown to be relatively sensitive in the acute phase of the disease, but it levels off rather quickly and is insensitive 3 months after infection (1, 29). In contrast to CFT, immunoglobulin G (IgG) and IgA reactions to many antigens of M. mycoides subsp. mycoides SC are persistent for several months, as shown by immun...

Development of a Novel Cocktail Enzyme-Linked Immunosorbent Assay and a Field-Applicable Lateral-Flow Rapid Test for Diagnosis of Contagious Bovine Pleuropneumonia

“…Eight additional proteins displaying strong serological responses and high disease specificity were also included (15,(18)(19)(20). Among those were lipoprotein Q (LppQ), a highly immunogenic surface protein, previously used for diagnosis of CBPP (15,21). Amino acid sequences for the proteins were retrieved from Mycoplasma mycoides subsp.…”

Antigenic and genetic characterisation of lipoprotein lppC from Mycoplasma mycoides subsp. mycoides SC

“…This could explain why antibodies against the N-terminal domain of LppC cross-react slightly with LppB which is present in the type strain and in African and Australian strains. The C-terminal parts of LppC and LppQ were also similar, consisting of five repeated units, rich in hydrophobic and aromatic amino acids in LppC, or nine of the same repeats in LppQ [2]. In analogy to LppQ, this structure found in LppC gives theoretical evidence for an integral membrane location [23].…”

“…The deduced amino acid sequence of LppC showed similarities with the two other known lipoproteins of M. mycoides subsp. mycoides SC, LppB [29] and LppQ [2]. This could explain why antibodies against the N-terminal domain of LppC cross-react slightly with LppB which is present in the type strain and in African and Australian strains.…”

“…In addition, two other antigenic proteins have been characterised thus far in M. mycoides subsp. mycoides SC: the 72 kDa antigen, also known as LppA [8,16,17] and the 48-kDa protein LppQ [2]. In an attempt to characterise the other major antigenic protein bands, we identified the lppC gene.…”

“…mycoides SC [29]; LppQ, a 48-kDa lipoprotein that appears to be highly specific for the species M. mycoides subsp. mycoides SC [2]; and Vmm, a variable surface protein of 59 amino acids that undergoes reversible phase variation [18].…”