Genetic Manipulation of Borrelia Spp.

Abstract

The spirochetes Borrelia (Borreliella) burgdorferi and Borrelia hermsii, the etiologic agents of Lyme disease and relapsing fever, respectively, cycle in nature between an arthropod vector and a vertebrate host. They have extraordinarily unusual genomes that are highly segmented and predominantly linear. The genetic analyses of Lyme disease spirochetes have become increasingly more sophisticated, while the age of genetic investigation in the relapsing fever spirochetes is just dawning. Molecular tools available for B. burgdorferi and related species range from simple selectable markers and gene reporters to state-of-the-art inducible gene expression systems that function in the animal model and high-throughput mutagenesis methodologies, despite nearly overwhelming experimental obstacles. This armamentarium has empowered borreliologists to build a formidable genetic understanding of the cellular physiology of the spirochete and the molecular pathogenesis of Lyme disease.

Peptide Uptake Is Essential for Borrelia burgdorferi Viability and Involves Structural and Regulatory Complexity of its Oligopeptide Transporter

“…On the other hand, AA uptake by the spirochete has received much less attention and, not surprisingly, is much more poorly understood. Using a state-of-the-art mutagenesis technique ( 43 , 55 , 56 ) to target OppDF, the energy lynchpin of the Opp permease, we definitively demonstrated that peptides are essential for replication of B. burgdorferi in vitro and in vivo and that the Opp system is the principal if not the only means by which the bacterium can acquire them. Indeed, we saw a direct correlation between the concentration of IPTG used for induction of the NBD domain and spirochete replication in vitro .…”

The need to unravel the twisted nature of the Borrelia burgdorferi sensu lato complex across Europe

“…Since then, the development of diverse molecular tools has enabled investigators to probe and uncover some of the underlying pathogenic mechanisms of the spirochete on a molecular genetic level [1]. These include the use of targeted mutagenesis studies for investigating the role of genes in a tick-animal model in a high-throughput manner [49][50][51][52], the use of the cre-lox recombination system for the deletion of a set of genes on a plasmid [53] and the use of reporter genes including cat gene [54], lacZ [55,56], luc [57,58] and GFP [33,59].…”

“…Finally, in the case of gene studies where the role of the gene and the protein it encodes has been deduced by any of the above methods, gene complementation in B. burgdorferi includes the use of shuttle vectors and trans-complementation for the introduction of the gene back into the mutant [64][65][66] to see if it restores the wild-type phenotype. However, genetic manipulation of B. burgdorferi has been greatly hampered by a number of limiting factors, including slow growth, low transformation efficiency, loss of plasmids during laboratory cultivation, the limited number of antibiotic markers for research purposes due to the use of antibiotics such as ampicilin and tertracycline being used to treat Lyme disease patients, and the lack of a minimal defined medium [1,67].…”

“…Targeted mutagenesis and complementation are important methods for studying genes of unknown function among most bacterial pathogens, including the Lyme borreliosis spirochete. Determining specific gene function has been hampered by the intractability of virulent strains of this bacterium to genetic manipulation [1]. To date, almost exclusively, Borrelia burgdorferi sensu stricto (ss) strains have been successfully utilized in genetic studies, since its genome was unveiled in 1997 by Fraser and colleagues [2].…”

Fluorescent Proteins, Promoters, and Selectable Markers for Applications in the Lyme Disease Spirochete Borrelia burgdorferi

“…Technical hurdles have slowed progress in this area. Genetic manipulation of B. burgdorferi is feasible, but the available genetic tools are still limited, and the process remains cumbersome ( 12 , 13 ). Constitutive gene expression is mostly limited to the use of very strong promoters.…”