key: cord-0014182-vzmb9mqn
authors: Lee, Sang-Yeop; Lee, Hayoung; Yun, Sung Ho; Jun, Sangmi; Lee, Yujeong; Kim, Wooyoung; Park, Edmond Changkyun; Baek, Joonyoung; Kwak, Yoonna; Noh, Soojin; Seo, Giwan; Jang, Soojin; Park, Chul Min; Kim, Seung Il
title: Application of multi-omics technology for the elucidation of anti-pneumococcal activity of 3-acyl-2-phenylamino-1,4-dihydroquinolin-4-one (APDQ) derivative against Streptococcus pneumoniae
date: 2020-11-26
journal: Sci Rep
DOI: 10.1038/s41598-020-77694-8
sha: 4900cd62c4cdd13dbca5aa9d4f242375f9d14b2d
doc_id: 14182
cord_uid: vzmb9mqn

Streptococcus pneumoniae is one of Gram-positive pathogen that causes invasive pneumococcal disease. Nowadays, many S. pneumoniae strains are resistant to commonly used antibiotics such as β-lactams and macrolides. 3-Acyl-2-phenylamino-1,4-dihydroquinolin-4-one (APDQ) derivatives are known as novel chemicals having anti-pneumococcal activity against S. pneumoniae. The underlying mechanism of the anti-pneumococcal activity of this inhibitor remains unknown. Therefore, we tried to find the anti-pneumococcal mechanism of APDQ230122, one of the APDQ derivatives active against S. pneumoniae. We performed transcriptomic analysis (RNA-Seq) and proteomic analysis (LC–MS/MS analysis) to get differentially expressed genes (DEG) and differentially expressed proteins (DEP) of S. pneumoniae 521 treated with sub-inhibitory concentrations of APDQ230122 and elucidated the comprehensive expression changes of genes and proteins using multi-omics analysis. As a result, genes or proteins of peptidoglycan biosynthesis and DNA replication were significantly down-regulated. Electron microscopy analysis revealed that the structure of peptidoglycan was damaged by APDQ230122 in a chemical concentration-dependent manner. Therefore, we suggest peptidoglycan biosynthesis is a major target of APDQ230122. Multi-omics analysis can provide us useful information to elucidate anti-pneumococcal activity of APDQ230122.

www.nature.com/scientificreports/ for more than three decades, the currently marketed vaccines do not cover all serotypes, and prevalent serotype replacement and possible species replacement threatens the long-term use of vaccines 4 . Antibiotics can be used to treat pneumococcal infections. However, S. pneumoniae has continuously developed resistance to commonly used antibiotics such as β-lactams and macrolides. The rate of penicillin and macrolide resistance was more than 30% in the United States, Europe and Asian countries 5, 6 . Increased antibiotic resistance is associated with development of multidrug resistance (MDR) in S. pneumoniae. In the United States, 64% of penicillin-nonsusceptible isolates causing invasive pneumococcal diseases were 19A strain, ATCC 700904™, which is highly resistant to antibiotics and exhibits MDR 7 . According to recent data from the United States, 8% of laboratory-confirmed invasive pneumococcal disease (IPD) is serotype 19A. Among children < 5 years of age, the proportion of IPD caused by serotype 19A is 33% 8 . Nasopharyngeal carriage of non-vaccine S. pneumoniae serogroup 15B is increasing, and this serotype may soon become an important cause of pneumococcal disease. Both 15B and 19A were demonstrated to be virulent 9 . Because conjugate vaccines confer imperfect protection against 19F, new antibiotics against S. pneumoniae 19F isolates, which are resistant to multiple β-lactam antimicrobials and erythromycin, are urgently needed 10 .

Using a resazurin-based phenotypic assay for high-throughput screening (HTS) of small molecules based on direct measurement of bacterial growth inhibition, Kim et al. tried to discover novel antibiotics for treating MDR and extensively drug-resistant S. pneumoniae serotypes 15B, 19A, and 19F [11] [12] [13] . Primary screening of 27,000 synthetic small molecules from the Korea Chemical Bank (KCB) revealed that several 3-acyl-2-phenylamino-1,4-dihydroquinolin-4-one (APDQ) derivatives had anti-pneumococcal activities against several S. pneumoniae strains 13 . Through further optimization studies, we found APDQ 230122 which had excellent antipneumococcal activity with MIC 90 values of 0.009, 0.019, and 0.033 μM for ATCC 49619 (19F), ATCC BAA1663 (15B), and ATCC 700904 (19A), respectively. APDQ derivatives have been reported to be potent and selective inhibitors of μ-calpain 14 , suppressors of Myc-dependent proliferation of leukemia cells 15 , and potent inhibitors of MERS-CoV activities 16 . Moreover, APDQ compounds inhibit the phosphoserine phosphatases (PSPs) of Porphyromonas gingivalis (SerB653), which are crucial for host invasion, intracellular persistence, and innate immune suppression 13, 17 .

However, the mechanism underlying the anti-pneumococcal activities of this inhibitor remains unknown. In this study, we used multi-omics technologies (genomic, proteomic, and transcriptomic analysis) to elucidate the anti-pneumococcal mechanism of APDQ230122 in S. pneumoniae. Our results suggest that APDQ230122 inhibits peptidoglycan biosynthesis in this bacterium.

Antibiotic effect of the APDQ derivative APDQ230122 against S. pneumoniae 521. We treated an antibiotic-sensitive strain with APDQ230122 to reveal the mechanism of antibiotic activity. S. pneumoniae 521 (KCTC 43179) is a clinical strain isolated from a Korean hospital; its genome data were deposited on NCBI (accession: CP036529.1). Antibiotic gene screening using ResFinder 3.0 revealed that this strain has no antibiotic resistance genes in its genome. Growth of S. pneumoniae 521 was inhibited by 0.5-8.0 μM APDQ230122 ( Fig. 1a and Supplementary Table S1 ). Cell viability decreased in a dose-dependent manner (Fig. 1b) and the concentrations of MIC 50 and MIC 90 were determined as 0.5 μM and 2.5 μM, respectively (Supplementary Table S2 ). Based on these observations, we concluded that APDQ230122 presents bactericidal activity. Because 1.0 μM APDQ230122 was identified as sub-inhibitory concentration in S. pneumoniae 521, we prepared S. pneumoniae 521 treated with 1.0 μM of APDQ230122 omics analyses.

Transcriptome (mRNA sequencing) and proteome analysis of S. pneumoniae 521. To elucidate the anti-pneumococcal effects of APDQ230122, we performed transcriptomic and proteomic analysis of S. pneumoniae 521. In the transcriptomic analysis, we obtained approximately 25.6 million reads on each sample. Among them, an average of 88.9% of filtered reads mapped uniquely to the S. pneumoniae 521 genome (Supplementary Table S3 ). The mRNA levels of 1882 genes are listed in Supplementary Table S4A . KEGG pathway analysis revealed that glycolysis/gluconeogenesis and terpenoid backbone biosynthesis were the major pathways differentially regulated in APDQ230122-treated S. pneumoniae 521 ( Supplementary Fig. S1 ). We also performed proteomics analysis to identify expressed proteins and determine protein expression levels. LC-MS/MS analysis identified 1152 and 1142 proteins in non-treated and treated samples, respectively (Supplementary Table S4A) .

We combined the proteomic results with the transcriptomic results to search for genes and proteins involved in the antibiotic effects of APDQ230122. For the DEG analysis, 358 genes with p < 0.05 were selected Table S4C ).

from the DEG and DEP analyses, respectively, were subjected to COG analysis (Fig. 2) . In COG analysis using DEG data, we found that many genes involved in energy production and conversion (C), carbohydrate transport and metabolism (G), and replication (L) categories were downregulated in APDQ230122-treated S. pneumoniae 521 (Fig. 2) . COG analysis using DEP data revealed that downregulation of proteins was more comprehensive: among the downregulated groups were amino acid transport and metabolism (E), nucleotide transport and metabolism (F), cell wall/membrane/envelope biogenesis (M), and defense mechanism (V). Because the biological roles of these categories cover basic metabolic activities and proliferation, our results suggested that treatment with APDQ230122 depresses the basic metabolic activities and proliferation of S. pneumoniae 521. www.nature.com/scientificreports/ One exception was that proteins and genes involved in translation (J) were upregulated under APDQ230122 treatment.

Application of DEG and DEP analyses to screening of target candidate genes of APDQ230122. The first step toward elucidating the anti-pneumococcal activities of APDQ230122 was to consider well-known antibiotic mechanisms as possible candidate targets. The following eight classes of antibiotic targets were considered: peptidoglycan biosynthesis, fatty acid biosynthetic process, RNA-elongation, DNAdirected DNA polymerase, DNA topoisomerase, DNA gyrase, SOS response, and tRNA ligase 18 . The results of the DEG and DEP analyses were used as a database for screening of candidate target genes of APDQ230122. We identified DEGs and DEPs belonging to eight classes of antibiotic targets, based on Gene Ontology; these are summarized in Table 1 . We searched for genes that yielded consistent results in DEGs and DEPs, and found six genes that met this criterion: three involved in peptidoglycan biosynthesis, two involved in fatty acid biosynthesis, and one encoding DNA gyrase (Table 1 ). In the fatty acid biosynthesis pathway, all but two genes yielded inconsistent results in the transcriptome and proteome data. However, all three genes involved in peptidoglycan biosynthesis were consistently downregulated at the mRNA and protein levels. Therefore, we considered that these proteins involved in peptidoglycan biosynthesis were directly or indirectly inhibited by APDQ230122, and selected peptidoglycan biosynthesis as a potential candidate APDQ230122 target for further analysis.

Peptidoglycan biosynthesis as a candidate target for APDQ230122 in S. pneumoniae 521. We identified the genes of the peptidoglycan biosynthesis pathway using blastKOALA on the KEGG website (https ://www.kegg.jp/blast koala /). Seventeen genes involved in the peptidoglycan biosynthesis pathway were identified; their proteomic and transcriptomic data are summarized in Fig. 3 and Supplementary Table S4D To investigate whether APDQ230122 interferes with peptidoglycan biosynthesis in S. pneumoniae, we performed TEM to examine the morphology of S. pneumoniae 521 cells before and after treatment with APDQ230122. The thickness of cell wall was definitely reduced at 1 μM and 4 μM (t test p value: 0.008 and 0.006). This results summarized Supplementary Fig. S2 . As shown in Fig. 4a -d, the peptidoglycan of non-treated bacteria had a thickness of 13.5 ± 3.6 nm on average (range 9.5-19.7 nm), indicating that the cell wall morphology was well-maintained. By contrast, in bacteria treated with 1.0 μM APDQ230122 (Fig. 4e-h) , the peptidoglycan was 9.6 ± 2.3 nm thick (range 7.3-13.6 nm), ~ 30% thinner than in non-treated bacteria. This indicates that APDQ230122 weakens the bacterial peptidoglycan. Moreover, the cell wall was partially lysed (red arrows in Fig. 4e -h). Bacteria treated with 4.0 μM APDQ230122 were more seriously damaged ( Fig. 4i-l) ; In most of the bacteria, their cell walls collapsed and the cytoplasm flowed out of the cell (black arrowheads in Fig. 4i ,j,k) 20 . Figure 4l shows that the membranes remained but appeared to be hyperhydrated in the cytoplasm (black arrows) 21, 22 .

APDQ derivatives have shown inhibitory potency on eukaryotic μ-calpain and a few cysteine proteases as well as bacterial phosphoserine phosphatases 14, 17 . Homologues of these enzymes such as peptidase C1 (CGG66885.1) and SerB2 (CJK92665.1) in S. pneumoniae might be inhibited by APDQ230122. However, inhibition of these homologues is unlikely responsible for antipneumococcal activity of APDQ230122 that we observed in this study since the enzymes are either not present in our pneumococcal strain (S. pneumoniae 521) or dispensable for the bacterial survival in in vitro culture condition 13, 23 . Instead, our multi-omics analysis and EM images revealed that APDQ230122 has inhibitory effects on the biosynthesis of peptidoglycan in S. pneumoniae. Until, now, however, we could not clearly pinpoint the molecular target of APDQ230122. The results of this study support several assumptions. First, APDQ230122 may directly inhibit peptidoglycan biosynthesis as β-lactams, β-lactamase inhibitors, glycopeptides, d-cycloserine, etc. do 24 . However, we could not identify a common moiety shared by APDQ230122 and known cell wall biosynthesis inhibitors, suggesting that APDQ230122 may act via different mechanisms, or on different targets, than β-lactam antibiotics. Alternatively, APDQ230122 could induce the downregulation of peptidoglycan biosynthesis at both the translational and transcriptional levels, opening a possibility that transcriptional regulation is the primary target of APDQ230122. Interestingly, APDQ compounds have been reported to inhibit c-Myc transcriptional factor that regulates genes involved in cell-cycle progression and apoptosis in leukemia cells 15 . Proteomic and transcriptomic results revealed that APDQ230122 can also regulate other biological functions, such as replication (Table 1) . Several DNA-directed DNA polymerase subunits and DNA gyrase subunits were downregulated by APDQ230122. These replication-related enzymes seemed to be networked with a response regulator transcriptional factor, COG0745, which was also downregulated by APDQ230122 treatment according to PPI analysis ( Supplementary Fig. S3 ). Elucidating the exact biological function of APDQ230122 will require future study, e.g., a high-throughput target mutagenesis 

To measure the effect of anti-pneumococcal activity against this bacterium, growth kinetics and cell viability were measured as previously described 13 . Briefly, S. pneumoniae 521 (KCTC 43179) was cultivated anaerobically overnight in Tryptic Soy Broth (TSB) at 37.5 °C in an atmosphere containing 5% CO 2 , and then cultured in fresh broth for 7-8 h until mid-log phase (≈OD 600 0.2). The culture media were treated with APDQ230122, a 3-acyl-2-phenylamino-1,4-dihydro quinolin-4-one derivative, at the indicated concentrations (0.5-8.0 μM). After 2 h of treatment, treated and non-treated bacteria were harvested, and RNA and protein extracts were prepared. After 4 h of treatment, cell viability testing was tested using the CellTiter-Blue Cell Viability Assay (Promega, Madison, WI, USA). To monitor growth kinetics, S. pneumoniae 521 was cultured as described above, and the OD 600 was measured at each time point. Sample preparation, in-gel digestion, and proteomic analysis using LC-MS/MS analysis. The harvesting of total protein and preparing for LC-MS/MS were according previously report 29 . Briefly, total extracted protein was fractionated by 12% SDS-PAGE and the fractionated gels were digested by trypsin. The tryptic peptides were enriched and cleaned of chemical contaminants using MGU30-C18 trapping column (LC www.nature.com/scientificreports/ Packings). And peptides were eluted from the column and directed onto a 10 cm × 75 μm ID C18 reverse-phase column (PROXEON, Odense, Denmark) at a flow rate of 300 nl/min. Peptides were eluted with a gradient of 0-65% acetonitrile over 80 min. All MS and MS/MS spectra were acquired on an LTQ-Velos ESI ion trap mass spectrometer (Thermo Scientific, Waltham, MA, USA) in data-dependent mode. Each full MS (m/z range 400-2000) scan was followed by three MS/MS scans of the most abundant precursor ions in the mass spectrum. MASCOT (v.2.4) was used for the protein identification program. To compare the protein abundance in each sample, we calculated differentially expressed proteins (DEPs) using the log ratio of mol% value with a cut-off value of 0.2.

Transmission electron microscopy (TEM). APDQ230122-treated S. pneumoniae 521 were pelleted by centrifugation, resuspended in 2.5% glutaraldehyde solution in phosphate buffer (0.1 M, pH 7.4), and incubated for 2 h at 4 °C. The samples were prepared by a method described previously 30 . The ultrathin plastic sections (80 nm thick) were observed with Zeiss LEO912AB 120 kV TEM (Carl Zeiss) and FEI Tecnai G2 Spirit Twin 120 kV TEM (FEI Company).

Received: 10 July 2020; Accepted: 10 November 2020 

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The authors declare no competing interests.

Supplementary information is available for this paper at https ://doi.org/10.1038/s4159 8-020-77694 -8.Correspondence and requests for materials should be addressed to C.M.P. or S.I.K.Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.