Identification and analysis of small molecule inhibitors of FosB from Staphylococcus aureus.

Antimicrobial resistance (AMR) poses a significant threat to human health around the world. Though bacterial pathogens can develop resistance through a variety of mechanisms, one of the most prevalent is the production of antibiotic-modifying enzymes like FosB, a Mn2+-dependent l-cysteine or bacillithiol (BSH) transferase that inactivates the antibiotic fosfomycin. FosB enzymes are found in pathogens such as Staphylococcus aureus, one of the leading pathogens in deaths associated with AMR. fosB gene knockout experiments establish FosB as an attractive drug target, showing that the minimum inhibitory concentration (MIC) of fosfomycin is greatly reduced upon removal of the enzyme. Herein, we have identified eight potential inhibitors of the FosB enzyme from S. aureus by applying high-throughput in silico screening of the ZINC15 database with structural similarity to phosphonoformate, a known FosB inhibitor. In addition, we have obtained crystal structures of FosB complexes to each compound. Furthermore, we have kinetically characterized the compounds with respect to inhibition of FosB. Finally, we have performed synergy assays to determine if any of the new compounds lower the MIC of fosfomycin in S. aureus. Our results will inform future studies on inhibitor design for the FosB enzymes.

Inhibition of Pseudomonas aeruginosa Alginate Synthesis by Ebselen Oxide and Its Analogues.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is frequently found in the airways of cystic fibrosis (CF) patients due to the dehydrated mucus that collapses the underlying cilia and prevents mucociliary clearance. During this life-long chronic infection, P. aeruginosa cell accumulates mutations that lead to inactivation of the mucA gene that results in the constitutive expression of algD-algA operon and the production of alginate exopolysaccharide. The viscous alginate polysaccharide further occludes the airways of CF patients and serves as a protective matrix to shield P. aeruginosa from host immune cells and antibiotic therapy. Development of inhibitors of alginate production by P. aeruginosa would reduce the negative impact from this viscous polysaccharide. In addition to transcriptional regulation, alginate biosynthesis requires allosteric activation by bis (3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding to an Alg44 protein. Previously, we found that ebselen (Eb) and ebselen oxide (EbO) inhibited diguanylate cyclase from synthesizing c-di-GMP. In this study, we show that EbO, Eb, ebsulfur (EbS), and their analogues inhibit alginate production. Eb and EbS can covalently modify the cysteine 98 (C98) residue of Alg44 and prevent its ability to bind c-di-GMP. However, P. aeruginosa with Alg44 C98 substituted with alanine or serine was still inhibited for alginate production by Eb and EbS. Our results indicate that EbO, Eb, and EbS are lead compounds for reducing alginate production by P. aeruginosa. Future development of these inhibitors could provide a potential treatment for CF patients infected with mucoid P. aeruginosa.

Myeloid arginase-1 controls excessive inflammation and modulates T cell responses in Pseudomonas aeruginosa pneumonia.

Regulatory properties of macrophages associated with alternative activation serve to limit the exaggerated inflammatory response during pneumonia caused by Pseudomonas aeruginosa infection. Arginase-1 is an important effector of these macrophages believed to play an essential role in decreasing injury and promoting repair. We investigated the role of arginase-1 in the control of inflammatory immune responses to P. aeruginosa pneumonia in mice that exhibit different immunologic phenotypes. C57BL/6 mice with conditional knockout of the arginase-1 (Arg1) gene from myeloid cells (Arg1ΔM) or BALB/c mice treated with small molecule inhibitors of arginase were infected intratracheally with P. aeruginosa. Weight loss, mortality, bacterial clearance, and lung injury were assessed and compared, as were the characterization of immune cell populations over time post-infection. Myeloid arginase-1 deletion resulted in greater morbidity along with more severe inflammatory responses compared to littermate control mice. Arg1ΔM mice had greater numbers of neutrophils, macrophages, and lymphocytes in their airways and lymph nodes compared to littermate controls. Additionally, Arg1ΔM mice recovered from inflammatory lung injury at a significantly slower rate. Conversely, treatment of BALB/c mice with the arginase inhibitor S-(2-boronoethyl)-l-cysteine hydrochloride (BEC) did not change morbidity as defined by weight loss, but mice at day 10 post-infection treated with BEC had gained significantly more weight back than controls. Neutrophil and macrophage infiltration were similar between groups in the lung parenchyma, and neutrophil migration into the airways was reduced by BEC treatment. Differences seem to lie in the impact on T cell subset disposition. Arg1ΔM mice had increased total CD4+ T cell expansion in the lymph nodes, and increased T cell activation, IFNγ production, and IL-17 production in the lymph nodes, lung interstitium, and airways, while treatment with BEC had no impact on T cell activation or IL-17 production, but reduced the number of T cells producing IFNγ in the lungs. Lung injury scores were increased in the Arg1ΔM mice, but no differences were observed in the mice treated with pharmacologic arginase inhibitors. Overall, myeloid arginase production was demonstrated to be essential for control of damaging inflammatory responses associated with P. aeruginosa pneumonia in C57BL/6 mice, in contrast to a protective effect in the Th2-dominant BALB/c mice when arginase activity is globally inhibited.

Characterization of a Unique Interrupted Adenylation Domain That Can Catalyze Three Reactions.

Interrupted adenylation (A) domains contain auxiliary domains within their structure and are a subject of growing interest in the field of nonribosomal peptide biosynthesis. They have been shown to possess intriguing functions and structure as well as promising engineering potential. Here, we present the characterization of an unprecedented type of interrupted A domain from the columbamides biosynthetic pathway, ColG(AMsMbA). This interrupted A domain contains two back-to-back methylation (M) domains within the same interruption site in the A domain, whereas previously, naturally occurring reported and characterized interrupted A domains harbored only one M domain. By a series of radiometric and mass spectrometry assays, we show that the first and second M domains site specifically methylate the side-chain oxygen and backbone nitrogen of l-Ser after the substrate is transferred onto a carrier thiolation domain, ColG(T). This is the first reported characterization of a dimethylating back-to-back interrupted A domain. The insights gained by this work lay the foundation for future combinatorial biosynthesis of site specifically methylated nonribosomal peptides.