Identification of a novel zinc metalloprotease through a global analysis of clostridium difficile extracellular proteins

Clostridium difficile is a major cause of infectious diarrhea worldwide. Although the cell surface proteins are recognized to be important in clostridial pathogenesis, biological functions of only a few are known. Also, apart from the toxins, proteins exported by C. difficile into the extracellular milieu have been poorly studied. In order to identify novel extracellular factors of C. difficile, we analyzed bacterial culture supernatants prepared from clinical isolates, 630 and R20291, using liquid chromatography-tandem mass spectrometry. The majority of the proteins identified were non-canonical extracellular proteins. These could be largely classified into proteins associated to the cell wall (including CWPs and extracellular hydrolases), transporters and flagellar proteins. Seven unknown hypothetical proteins were also identified. One of these proteins, CD630_28300, shared sequence similarity with the anthrax lethal factor, a known zinc metallopeptidase. We demonstrated that CD630_28300 (named Zmp1) binds zinc and is able to cleave fibronectin and fibrinogen in vitro in a zinc-dependent manner. Using site-directed mutagenesis, we identified residues important in zinc binding and enzymatic activity. Furthermore, we demonstrated that Zmp1 destabilizes the fibronectin network produced by human fibroblasts. Thus, by analyzing the exoproteome of C. difficile, we identified a novel extracellular metalloprotease that may be important in key steps of clostridial pathogenesis

Abstract P5-11-11: A phase I safety study of topical calcitriol (BPM31543) for the prevention of chemotherapy-induced alopecia (CIA)

Abstract Background: Chemotherapy induced alopecia (CIA) may lead to significant psychosocial and quality of life issues. Currently there are no FDA approved oral or topical agents available to prevent CIA. In murine studies, topical calcitriol reduced CIA, due to arrest of cell cycle in healthy hair follicles, and reduction in the sensitivity of follicular epithelium to chemotherapy. Methods: A prospective dose escalation study is being performed in up to 31 women with breast cancer, gynecologic cancer and sarcomas. Each patient is applying 1mL of BPM31543 to her scalp bid, ≥ 5 days prior to initiation of taxane-based chemotherapy for at least 3 months or until the completion of chemotherapy. The study cohorts are: 5/10/20/40/60/80μg/mL. The first 5 cohorts are completely enrolled and the final cohort is currently being enrolled. Each patient undergoes pk analysis, adverse event (AE) monitoring, patient self-assessment diaries (1-10 scale), and blinded photographic assessments. Efficacy and pK data are still being collected and analyzed for the patients on study, but will be available by December. Results: Twenty-four subjects have been enrolled so far (evaluable at this time, n = 13). Pk data (n = 16; 5-40μg/mL) showed inter-individual variability, but no significant dose-dependent increase in systemic absorption (range, &amp;lt; 20-110 pg/mL). Treatment-related AEs (probably/possibly) were mild/moderate in nature and included scalp pain (n = 1; 5 μg/mL), elevated vitamin D levels in 1 patient (20μg/mL) and passage of renal calculus in another (n = 1; 40μg/mL). All subjects reported changes in overall hair fullness, thickness, and volume of hair during chemotherapy. At the 5/10 μg/mL dose level, ≥ 75% hair loss was reported in 85% of patients. At the ≥ 20 μg/mL dose level, ≥ 75% hair loss was seen only in 43% of patients. Hair loss/ thinning caused all subjects to change their hair style (onset, week 2; peak, weeks 5-6). Conclusions: Data have shown that the twice daily application of BPM31543 in patients receiving taxane-based chemotherapy was safe and well-tolerated. Efficacy data from the preliminary analysis was promising and led to the amendment of the study to evaluate two additional higher dose cohorts: 60 and 80 μg/ml. Citation Format: Goldfarb SB, Konner J, Stevens J, Brouwer S, Narain NR, Ye R, Ravipaty S, Sarangarajan R, Akmaev VR, Jimenez JJ, Belum VR, 'Kitts S, Ciccolini K, Berman B, Lacouture ME. A phase I safety study of topical calcitriol (BPM31543) for the prevention of chemotherapy-induced alopecia (CIA) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-11-11.</jats:p

Secretome Analysis Defines the Major Role of SecDF in <i>Staphylococcus aureus</i> Virulence

<div><p>The Sec pathway plays a prominent role in protein export and membrane insertion, including the secretion of major bacterial virulence determinants. The accessory Sec constituent SecDF has been proposed to contribute to protein export. Deletion of <i>Staphylococcus aureus secDF</i> has previously been shown to reduce resistance, to alter cell separation, and to change the expression of certain virulence factors. To analyse the impact of the <i>secDF</i> deletion in <i>S. aureus</i> on protein secretion, a quantitative secretome analysis was performed. Numerous Sec signal containing proteins involved in virulence were found to be decreased in the supernatant of the <i>secDF</i> mutant. However, two Sec-dependent hydrolases were increased in comparison to the wild type, suggesting additional indirect, regulatory effects to occur upon deletion of <i>secDF</i>. Adhesion, invasion, and cytotoxicity of the <i>secDF</i> mutant were reduced in human umbilical vein endothelial cells. Virulence was significantly reduced using a <i>Galleria mellonella</i> insect model. Altogether, SecDF is a promising therapeutic target for controlling <i>S. aureus</i> infections.</p></div