A greater understanding of the molecular mechanisms that Borrelia burgdorferi uses to survive during mammalian infection is critical for the development of novel diagnostic and therapeutic tools to improve the clinical management of Lyme disease. By use of an in vivo expression technology (IVET)-based approach to identify B. burgdorferi genes expressed in vivo, we discovered the bb0318 gene, which is thought to encode the ATPase component of a putative riboflavin ABC transport system. Riboflavin is a critical metabolite enabling all organisms to maintain redox homeostasis. B. burgdorferi appears to lack the metabolic capacity for de novo synthesis of riboflavin and so likely relies on scavenging riboflavin from the host environment. In this study, we sought to investigate the role of bb0318 in B. burgdorferi pathogenesis. No in vitro growth defect was observed for the ⌬bb0318 clone. However, the mutant spirochetes displayed reduced levels of survival when exposed to exogenous hydrogen peroxide or murine macrophages. Spirochetes lacking bb0318 were found to have a 100-fold-higher 50% infectious dose than spirochetes containing bb0318. In addition, at a high inoculum dose, bb0318 was found to be important for effective spirochete dissemination to deep tissues for as long as 3 weeks postinoculation and to be critical for B. burgdorferi infection of mouse hearts. Together, these data implicate bb0318 in the oxidative stress response of B. burgdorferi and indicate the contribution of bb0318 to B. burgdorferi mammalian infectivity.

bPertussis toxin (PT) moves from the host cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. The catalytic PTS1 subunit dissociates from the rest of the toxin in the ER and then shifts to a disordered conformation which may trigger its export to the cytosol through the quality control mechanism of ER-associated degradation (ERAD). Functional roles for toxin instability and ERAD in PTS1 translocation have not been established. We addressed these issues with the use of a surface plasmon resonance system to quantify the cytosolic pool of PTS1 from intoxicated cells. Only 3% of surface-associated PTS1 reached the host cytosol after 3 h of toxin exposure. This represented, on average, 38,000 molecules of cytosolic PTS1 per cell. Cells treated with a proteasome inhibitor contained larger quantities of cytosolic PTS1. Stabilization of the dissociated PTS1 subunit with chemical chaperones inhibited toxin export to the cytosol and blocked PT intoxication. ERAD-defective cell lines likewise exhibited reduced quantities of cytosolic PTS1 and PT resistance. These observations identify the unfolding of dissociated PTS1 as a trigger for its ERAD-mediated translocation to the cytosol. The AB toxins are composed of a catalytic A moiety and a cellbinding B moiety. Some AB toxins move by retrograde vesicular transport from the cell surface to the endosomes, from the endosomes to the Golgi apparatus, and from the Golgi apparatus to the endoplasmic reticulum (ER), where A/B subunit dissociation occurs (1). Unfolding of the dissociated A chain places the toxin in a translocation-competent conformation and activates the host mechanism of ER-associated degradation (ERAD) (2). This quality control system exports misfolded proteins from the ER to the cytosol through protein-conducting channels in the ER membrane (3). ERAD substrates are usually degraded by the cytosolic ubiquitinproteasome system, but the A chains of ER-translocating toxins effectively evade this pathway because they lack the lysine residues required for ubiquitin conjugation (4-7).Pertussis toxin (PT) is an AB 5 -type, ER-translocating toxin (8, 9). Its A subunit, PTS1, disrupts host signaling events through the ADP-ribosylation of G␣ proteins. The B subunit is a hetero-oligomer composed of four proteins (PTS2, PTS3, two copies of PTS4, and PTS5) that bind to specific but largely unidentified glycoconjugates on the host cell (10-14). Holotoxin assembly involves noncovalent positioning of PTS1 above and partially within the central pore of the ring-like PTB oligomer (15). As with other ER-translocating toxins, PT transport from the cell surface to the ER appears to be an inefficient process: fluorescence microscopy, immunoelectron microscopy, and subcellular fractionation can detect internalized toxin in the endosomes and Golgi apparatus but not the ER (16-19). The inhibition of PT intoxication with brefeldin A (BfA), a drug that blocks retrograde vesicular transport to the ER, strongly suggests the functional pool of toxin must move from ...

The Lyme disease spirochete Borrelia burgdorferi is dependent on purine salvage from the host environment for survival. The genes bbb22 and bbb23 encode purine permeases that are essential for B. burgdorferi mouse infectivity. We now demonstrate the unique contributions of each of these genes to purine transport and murine infection. The affinities of spirochetes carrying bbb22 alone for hypoxanthine and adenine were similar to those of spirochetes carrying both genes. Spirochetes carrying bbb22 alone were able to achieve wild-type levels of adenine saturation but not hypoxanthine saturation, suggesting that maximal hypoxanthine uptake requires the presence of bbb23. Moreover, the purine transport activity conferred by bbb22 was dependent on an additional distal transcriptional start site located within the bbb23 open reading frame. The initial rates of uptake of hypoxanthine and adenine by spirochetes carrying bbb23 alone were below the level of detection. However, these spirochetes demonstrated a measurable increase in hypoxanthine uptake over a 30-min time course. Our findings indicate that bbb22-dependent adenine transport is essential for B. burgdorferi survival in mice. The bbb23 gene was dispensable for B. burgdorferi mouse infectivity, yet its presence was required along with that of bbb22 for B. burgdorferi to achieve maximal spirochete loads in infected mouse tissues. These data demonstrate that both genes, bbb22 and bbb23, are critical for B. burgdorferi to achieve wild-type infection of mice and that the differences in the capabilities of the two transporters may reflect distinct purine salvage needs that the spirochete encounters throughout its natural infectious cycle. P urine nucleobases are required for the synthesis of DNA and RNA. Consequently, purine biosynthesis and/or transport is a critical process across all kingdoms of life. Moreover, nucleobase transporters represent possible therapeutic targets for cancer and infectious diseases (1).Borrelia burgdorferi, the causative agent of Lyme disease, has a limited genome and lacks the enzymes required for de novo purine biosynthesis (2-6). Therefore, purine salvage from host environments is important for B. burgdorferi survival and pathogenesis (7-9). Recently, our laboratory established that the genes bbb22 and bbb23, located on B. burgdorferi essential circular plasmid 26 (cp26), together encode a purine transport system that is required for hypoxanthine transport and contributes to adenine and guanine transport (7). Spirochetes lacking both bbb22 and bbb23 are noninfectious in mice (7). Conversely, these genes are dispensable for B. burgdorferi growth in nutrient-rich medium in vitro (7).The bbb22 and bbb23 genes encode proteins within cluster COG2252 of the nucleobase cation symporter-2 superfamily (NCS2) (7), which includes permeases found in bacteria, fungi, and plants that are specific for adenine, hypoxanthine, and/or guanine (10-16). The Aspergillus nidulans AzgA transporter, the founding member of this family of transporters, has specif...