Inhibition of Pseudomonas aeruginosa and Mycobacterium tuberculosis disulfide bond forming enzymes.

In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond, including bacterial virulence factors. Thus, proteins involved in disulfide bond formation represent good targets for the development of inhibitors that can act as antibiotics or anti-virulence agents, resulting in the simultaneous inactivation of several types of virulence factors. Here, we present evidence that the disulfide bond forming enzymes, DsbB and VKOR, are required for Pseudomonas aeruginosa pathogenicity and Mycobacterium tuberculosis survival respectively. We also report the results of a HTS of 216,767 compounds tested against P. aeruginosa DsbB1 and M. tuberculosis VKOR using Escherichia coli cells. Since both P. aeruginosa DsbB1 and M. tuberculosis VKOR complement an E. coli dsbB knockout, we screened simultaneously for inhibitors of each complemented E. coli strain expressing a disulfide-bond sensitive ß-galactosidase reported previously. The properties of several inhibitors obtained from these screens suggest they are a starting point for chemical modifications with potential for future antibacterial development.

The presence of tumour-infiltrating neutrophils is an independent adverse prognostic feature in clear cell renal cell carcinoma.

Tumour-promoting inflammation is an emerging hallmark of cancer that is increasingly recognised as a therapeutic target. As a constituent measure of inflammation, tumour-infiltrating neutrophils (TINs) have been associated with inferior prognosis in several cancers. We analysed clinically annotated cohorts of clear cell renal cell carcinoma (ccRCC) to assess the presence of neutrophils within the tumour microenvironment as a function of outcome. We centrally reviewed ccRCC surgical resection and fine-needle aspiration (FNA) specimens, including primary and metastatic sites, from three centres. TINs were scored based on the presence of neutrophils in resection and FNA specimens by two pathologists. TIN count was correlated with tumour characteristics including stage, WHO/ISUP grade, and immunohistochemistry (IHC). In parallel, we performed CIBERSORT analysis of the tumour microenvironment in a cohort of 516 ccRCCs from The Cancer Genome Atlas (TCGA). We included 102 ccRCC cases comprising 65 resection specimens (37 primary and 28 metastatic resection specimens) and 37 FNAs from primary lesions. High TINs were significantly associated with worse overall survival (p = 0.009) independent of tumour grade and stage. In ccRCCs sampled via FNA, all cases with high TINs had distant metastasis, whereas they were seen in only 19% of cases with low TINs (p = 0.0003). IHC analysis showed loss of E-cadherin in viable tumour cells in areas with high TINs, and neutrophil activation was associated with elastase and citrullinated histone H3 expression (cit-H3). In the TCGA cohort, neutrophilic markers were also associated with worse survival (p < 0.0001). TINs are an independent predictor of worse prognosis in ccRCC, which have the potential to be assessed at the time of first biopsy or FNA. Neutrophils act directly on tumour tissue by releasing elastase, a factor that contributes to the breakdown of cell-cell adhesion and to facilitate tumour dissemination.