Effect of a small molecule Lipid II binder on bacterial cell wall stress

Infection and Drug Resistance Dovepress open access to scientific and medical research S H O RT R E P O RT Infection and Drug Resistance downloaded from https://www.dovepress.com/ by 5.135.254.153 on 13-Jul-2018 For personal use only. Open Access Full Text Article Effect of a small molecule Lipid II binder on bacterial cell wall stress This article was published in the following Dove Press journal: Infection and Drug Resistance 28 February 2017 Number of times this article has been viewed Jakob Malin 1,2 Amol C Shetty 3 Sean C Daugherty 3 Erik PH de Leeuw 1,2 Institute of Human Virology, Department of Biochemistry and Molecular Biology, 3Institute for Genome Sciences, University of Maryland Baltimore School of Medicine, Baltimore, MD, USA 1 2 Introduction Correspondence: Erik PH de Leeuw Institute of Human Virology, University of Maryland Baltimore School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA Tel +1 410 706 3430 Fax +1 410 706 7583 Email Bacteria, and in particular Gram-positive species, surround themselves with cell walls, structures that are essential for survival and growth.1 Interference with the biosynthesis of the cell wall has been a successful antibacterial strategy, and numerous classes of antibiotics have been developed that affect assembly. Penicillin and vancomycin are arguably the two most well-known examples of such therapeutics. Penicillin represents the class of β-lactam agents that interfere with cross-linking of peptide stems of peptidoglycan units outside of the bacterial cell.2 Vancomycin specifically binds to the l-Ala–l-Ala motif of the pentapeptide moiety of Lipid II, an essential precursor of peptidoglycan transport and assembly and acts at the membrane interface. Resistance against agents such as next-generation β-lactams and last-resort antibiotics including vancomycin is evolving at an alarming rate.3 Thus, there is a great need for alternative strategies to combat multidrug-resistant organisms. We recently identified an interaction between a class of antimicrobial peptides in humans, termed defensins, and bacterial Lipid II.4 Based on these initial findings, we further characterized this interaction in molecular detail and identified small molecule Lipid II inhibitors for the first time.5 In this report, we further characterize one small molecule Lipid II inhibitor, termed compound 5107930. Compound 5107930 was examined for its effects on bacterial cell wall stress, binding to Lipid II and antibacterial killing in the context of glycopeptide resistance. 69 submit your manuscript | www.dovepress.com Infection and Drug Resistance 2017:10 69–73 Dovepress © 2017 Malin et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). http://dx.doi.org/10.2147/IDR.S126254 Powered by TCPDF (www.tcpdf.org) Abstract: We have recently identified small molecule compounds that act as binders of Lipid II, an essential precursor of bacterial cell wall biosynthesis. Lipid II comprised a hydrophilic head group that includes a peptidoglycan subunit composed of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) coupled to a short pentapeptide moiety. This headgroup is coupled to a long bactoprenol chain via a pyrophosphate group. Here, we report on the cell wall activity relationship of dimethyl-3-methyl(phenyl)amino-ethenylcyclohexylidene-propenyl3-ethyl-1,3-benzothiazolium iodide (compound 5107930) obtained by functional and genetic analyses. Our results indicate that compounds bind to Lipid II and cause specific upregulation of the vancomycin-resistance associated gene vraX. vraX is implicated in the cell wall stress stimulon that confers glycopeptide resistance. Our small molecule Lipid II inhibitor retained activity against strains of Staphylococcus aureus mutated in genes encoding the cell wall stress stimulon. This suggests the feasibility of developing this new scaffold as a therapeutic agent in view of increasing glycopeptide resistance. Keywords: defensin, Lipid II, antibiotics, bacterial membrane, vancomycin Dovepress Malin et al Infection and Drug Resistance downloaded from https://www.dovepress.com/ by 5.135.254.153 on 13-Jul-2018 For personal use only. Materials and methods Materials Unless stated otherwise, chemicals were obtained from Sigma–Aldrich (St. Louis, MO, USA). Staphylococcus aureus ATCC 29213 was obtained from Microbiologics (St. Cloud, MN, USA). Dimethyl-3-methyl(phenyl)amino-ethenylcyclohexylidene-propenyl-3-ethyl-1,3-benzothiazolium iodide was purchased from Chembridge Ltd. (San Diego, CA, USA). S. aureus strains carrying deletions in the cell wall stress stimulon (CWSS) were generously provided by M. Senn and C. Qublier (Institute for Medicinal Microbiology, University of Zurich) and were described earlier.6 Antibacterial activity assay Determination of the minimal inhibitory concentrations (MICs) by dilution was carried out by broth dilution according to CLSI standards.7 Lipid II purification The sequenced reads were used as input to Bowtie version 0.12.7 to align the reads to the S. aureus 29213 reference genome for each sample.10 The output from Bowtie was obtained as binary alignment map (BAM) format files that consist of information on the alignment of each individual read within the reference genome. In the alignment phase, we allowed two mismatches over the entire read alignment. The BAM alignment files obtained from the Bowtie alignment tool were analyzed to generate the alignment statistics for each sample, namely, the total number of reads, the number of mapped reads, the percent of total mapped reads, and the percentage of reads that mapped to genic and intergenic regions. The coverage analysis aided in determining the success of the sequencing process, the coverage across the reference genome, and the presence of any outliers in the samples. Differential expression analysis Staphylococcus aureus strain 29213 was exposed to compound 5107930 for 30 min at 10 µM or left untreated. Total mRNA was subsequently isolated using the RNEasy mini kit (Qiagen; Germantown, MD, USA) according to manufacturer’s instructions. The alignment BAM files from Bowtie are further utilized to compute gene expression levels and test each gene for differential expression. 11 The reference annotation for S. aureus 29213 in GFF3 format was used. The number of reads that mapped to each gene described in the annotation was c (...truncated)