Borrelia (B.) burgdorferi sensu lato, including the tick-transmitted agents of human Lyme borreliosis, have particularly complex genomes, consisting of a linear main chromosome and numerous linear and circular plasmids. The number and structure of plasmids is variable even in strains within a single genospecies. Genes on these plasmids are known to play essential roles in virulence and pathogenicity as well as host and vector associations. For this reason, it is essential to explore methods for rapid and reliable characterisation of molecular level changes on plasmids. In this study we used three strains: a low passage isolate of B. burgdorferi sensu stricto strain B31(−NRZ) and two closely related strains (PAli and PAbe) that were isolated from human patients. Sequences of these strains were compared to the previously sequenced reference strain B31 (available in GenBank) to obtain proof-of-principle information on the suitability of next generation sequencing (NGS) library construction and sequencing methods on the assembly of bacterial plasmids. We tested the effectiveness of different short read assemblers on Illumina sequences, and of long read generation methods on sequence data from Pacific Bioscience single-molecule real-time (SMRT) and nanopore (Oxford Nanopore Technologies) sequencing technology. Inclusion of mate pair library reads improved the assembly in some plasmids as did prior enrichment of plasmids. While cp32 plasmids remained refractory to assembly using only short reads they were effectively assembled by long read sequencing methods. The long read SMRT and nanopore sequences came, however, at the cost of indels (insertions or deletions) appearing in an unpredictable manner. Using long and short read technologies together allowed us to show that the three B. burgdorferi s.s. strains investigated here, whilst having similar plasmid structures to each other (apart from fusion of cp32 plasmids), differed significantly from the reference strain B31-GB, especially in the case of cp32 plasmids. Short read methods are sufficient to assemble the main chromosome and many of the plasmids in B. burgdorferi. However, a combination of short and long read sequencing methods is essential for proper assembly of all plasmids including cp32 and thus, for gaining an understanding of host- or vector adaptations. An important conclusion from our work is that the evolution of Borrelia plasmids appears to be dynamic. This has important implications for the development of useful research strategies to monitor the risk of Lyme disease occurrence and how to medically manage it.

The plasminogen system is essential for dissolution of fibrin clots, and in addition, it is involved in a wide variety of other physiological processes, including proteolytic activation of growth factors, cell migration, and removal of protein aggregates. On the other hand, uncontrolled plasminogen activation contributes to many pathological processes, e.g. tumor cells’ invasion in cancer progression. Moreover, some virulent bacterial species, e.g. Streptococci or Borrelia, bind human plasminogen and hijack the host’s plasminogen system to penetrate tissue barriers. Thus, the conversion of plasminogen to the active serine protease plasmin must be tightly regulated. Here, we show that human lactoferrin, an iron-binding milk glycoprotein, blocks plasminogen activation on the cell surface by direct binding to human plasminogen. We mapped the mutual binding sites to the N-terminal region of lactoferrin, encompassed also in the bioactive peptide lactoferricin, and kringle 5 of plasminogen. Finally, lactoferrin blocked tumor cell invasion in vitro and also plasminogen activation driven by Borrelia. Our results explain many diverse biological properties of lactoferrin, and also suggest that lactoferrin may be useful as a potential tool for therapeutic interventions to prevent both invasive malignant cells and virulent bacteria from penetrating host tissues.

The reactivity of human IgM with the outer surface protein C (OspC) of Borrelia burgdorferi sensu lato is frequently used to detect Borrelia specific IgM in commercial immunoassays, and such antibodies usually occur in the early phase of the infection. We identified a group of individuals with persistent Borrelia IgM without symptoms of Lyme borreliosis.