Processive dynamics of the usher assembly platform during uropathogenic Escherichia coli P pilus biogenesis.

Uropathogenic Escherichia coli assemble surface structures termed pili or fimbriae to initiate infection of the urinary tract. P pili facilitate bacterial colonization of the kidney and pyelonephritis. P pili are assembled through the conserved chaperone-usher pathway. Much of the structural and functional understanding of the chaperone-usher pathway has been gained through investigations of type 1 pili, which promote binding to the bladder and cystitis. In contrast, the structural basis for P pilus biogenesis at the usher has remained elusive. This is in part due to the flexible and variable-length P pilus tip fiber, creating structural heterogeneity, and difficulties isolating stable P pilus assembly intermediates. Here, we circumvent these hindrances and determine cryo-electron microscopy structures of the activated PapC usher in the process of secreting two- and three-subunit P pilus assembly intermediates, revealing processive steps in P pilus biogenesis and capturing new conformational dynamics of the usher assembly machine.

Persistently elevated abnormal B-cell subpopulations and anti-core antibodies in patients co-infected with HIV/HCV who relapse.

Hepatitis C (HCV) treatment for patients coinfected with human immunodeficiency virus (HIV) and HCV is associated with modest rates of sustained virologic response (SVR) and an increased rate of relapse when compared to HCV monoinfected patients. As patients who attain SVR and patients who relapse are clinically indistinguishable during treatment, where both groups have fully suppressed HCV viral load, it has not been possible to identify in advance those who will relapse. Biomarkers that may distinguish patients with differential treatment response may be clinically useful and provide insight into mechanisms of relapse. In this retrospective study, serum and PBMCs were obtained from 41 HIV/HCV co-infected patients and 17 healthy volunteers. Changes in antibody titers to various regions of the HCV proteome during treatment for HCV were determined using a novel luciferase immunoprecipitation assay. Changes in B-cell subtypes in patients with differential treatment response as well as healthy volunteers were compared. This study demonstrates that elevated anti-HCV core antibody titers persisted during HCV treatment in patients who relapsed when compared to those who attained SVR. Furthermore, characterization of B cells in patients who relapsed demonstrated an abnormal B-cell phenotype distribution characterized by elevated frequencies of exhausted B cells among relapsers at baseline, which persisted despite suppression of HCV viremia at 24 weeks, along with increased frequencies of plasmablasts. These data suggest that anti-HCV specific B cells may be responding to ongoing subclinical HCV replication in patients who will relapse.

The pmr gene, encoding a Ca2+-ATPase, is required for calcium and manganese homeostasis and normal development of hyphae and conidia in Neurospora crassa.

The pmr gene is predicted to encode a Ca(2+)-ATPase in the secretory pathway. We examined two strains of Neurospora crassa that lacked PMR: the Δpmr strain, in which pmr was completely deleted, and pmr(RIP), in which the gene was extensively mutated. Both strains had identical, complex phenotypes. Compared to the wild type, these strains required high concentrations of calcium or manganese for optimal growth and had highly branched, slow-growing hyphae. They conidiated poorly, and the shape and size of the conidia were abnormal. Calcium accumulated in the Δpmr strains to only 20% of the wild-type level. High concentrations of MnCl(2) (1 to 5 mM) in growth medium partially suppressed the morphological defects but did not alter the defect in calcium accumulation. The Δpmr Δnca-2 double mutant (nca-2 encodes a Ca(2+)-ATPase in the plasma membrane) accumulated 8-fold more calcium than the wild type, and the morphology of the hyphae was more similar to that of wild-type hyphae. Previous experiments failed to show a function for nca-1, which encodes a SERCA-type Ca(2+)-ATPase in the endoplasmic reticulum (B. J. Bowman, S. Abreu, E. Margolles-Clark, M. Draskovic, and E. J. Bowman, Eukaryot. Cell 10:654-661, 2011). The pmr(RIP) Δnca-1 double mutant accumulated small amounts of calcium, like the Δpmr strain, but exhibited even more extreme morphological defects. Thus, PMR can apparently replace NCA-1 in the endoplasmic reticulum, but NCA-1 cannot replace PMR. The morphological defects in the Δpmr strain are likely caused, in part, by insufficient concentrations of calcium and manganese in the Golgi compartment; however, PMR is also needed to accumulate normal levels of calcium in the whole cell.