Enhanced expression of codon optimized Mycobacterium avium subsp. paratuberculosis antigens in Lactobacillus salivarius

Abstract

It is well documented that open reading frames containing high GC content show poor expression in A+T rich hosts. Specifically, G+C-rich codon usage is a limiting factor in heterologous expression of Mycobacterium avium subsp. paratuberculosis (MAP) proteins using Lactobacillus salivarius. However, re-engineering opening reading frames through synonymous substitutions can offset codon bias and greatly enhance MAP protein production in this host. In this report, we demonstrate that codon-usage manipulation of MAP2121c can enhance the heterologous expression of the major membrane protein (MMP), analogous to the form in which it is produced natively by MAP bacilli. When heterologously over-expressed, antigenic determinants were preserved in synthetic MMP proteins as shown by monoclonal antibody mediated ELISA. Moreover, MMP is a membrane protein in MAP, which is also targeted to the cellular surface of recombinant L. salivarius at levels comparable to MAP. Additionally, we previously engineered MAP3733c (encoding MptD) and show herein that MptD displays the tendency to associate with the cytoplasmic membrane boundary under confocal microscopy and the intracellularly accumulated protein selectively adheres to the MptD-specific bacteriophage fMptD. This work demonstrates there is potential for L. salivarius as a viable antigen delivery vehicle for MAP, which may provide an effective mucosal vaccine against Johne's disease.

Under the microscope: From pathogens to probiotics and back

“…62 These physiologically enhanced probiotics function as ideal drug and vaccine delivery platforms; 63 targeting difficult to treat pathogens such as Clostridium difficile, 64 Acinetobacter baumannii 65 and Mycobacteria. [66][67][68] In addition to the obvious clinical applications, described above, genetic manipulation of probiotics has provided fundamental insights into their natural life cycle within the host. In Cronin et al, 69 for example, we describe the development of a non-invasive luciferase-based reporter system (pLuxMC1) for real-time tracking of Bifidobacterium species in vivo.…”

Knowledge gaps that hamper prevention and control ofMycobacterium aviumsubspeciesparatuberculosisinfection

“…Subunit vaccines have been reported to provide incomplete protection in murine models (Stabel, Barnhill, Bannantine, Chang, & Osman, 2012) or ruminant models (calves and goats) of infection (Kathaperumal et al, 2008(Kathaperumal et al, , 2009Koets et al, 2006). In addition, other subunit vaccines (Facciuolo & Mutharia, 2014;Gurung, Begg, Purdie, & Whittington, 2013;Johnston et al, 2014) are being proposed as having potential to prevent infection. Most vaccines have been tested in a preventive pre-exposure setting.…”

Pathogenesis, Molecular Genetics, and Genomics of Mycobacterium avium subsp. paratuberculosis, the Etiologic Agent of Johne’s Disease

“…In the context of vectored subunit vaccines, success has been achieved in the expression of MAP antigens in Lactobacillus salivarius using a codon optimization strategy ( 151 , 152 ). Multi-antigen virally vectored vaccines were also engineered and tested in mice and cattle.…”