Thermal Unfolding of the Pertussis Toxin S1 Subunit Facilitates Toxin Translocation to the Cytosol by the Mechanism of Endoplasmic Reticulum-Associated Degradation.

Pertussis 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.

Gene bb0318 Is Critical for the Oxidative Stress Response and Infectivity of Borrelia burgdorferi.

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 Δbb0318 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.

Molecular dissection of a Borrelia burgdorferi in vivo essential purine transport system.

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.