Screening of Neutrophil Activating Factors from a Metagenome Library of Sponge-Associated Bacteria

marine drugs

Article Screening of Neutrophil Activating Factors from a Metagenome Library of Sponge-Associated Bacteria

Yoshiko Okamura 1,2,* , Hirokazu Takahashi 1,2, Atsuyuki Shiida 2, Yuto Hirata 1, Haruko Takeyama 3 and Katsuhiko Suzuki 4,*

1 Graduate School of Integrated Sciences for Life, Hiroshima University, Higashihiroshima 739-8530, Japan; [email protected] (H.T.); [email protected] (Y.H.) 2 Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashihiroshima 739-8530, Japan; [email protected] 3 Department of Life Science and Medical Bio-Science, Waseda University, Tokyo 162-8480, Japan; [email protected] 4 Faculty of Sport Sciences, Waseda University, Saitama 359-1192, Japan * Correspondence: [email protected] (Y.O.); [email protected] (K.S.); Tel.: +81-4-2947-6898 (K.S.)

Abstract: Marine sponge-associated bacteria are known as bio-active compound produce. We have constructed metagenome libraries of the bacteria and developed a metagenomic screening approach. Activity-based screening successfully identiﬁed novel genes and novel enzymes; however, the efﬁciency was only in 1 out of 104 clones. Therefore, in this study, we thought that bioinformatics could help to reduce screening efforts, and combined activity-based screening with database search. Citation: Okamura, Y.; Takahashi, H.; Neutrophils play an important role for the immune system to recognize excreted bacterial by-products Shiida, A.; Hirata, Y.; Takeyama, H.; as chemotactic factors and are recruited to infection sites to kill pathogens via phagocytosis. These Suzuki, K. Screening of Neutrophil excreted by-products are considered critical triggers that engage the immune system to mount a Activating Factors from a defense against infection, and identifying these factors may guide developments in medicine and Metagenome Library of diagnostics. We focused on genes encoding amino acid ligase and peptide synthetase and selected Sponge-Associated Bacteria. Mar. from an in-house sponge metagenome database. Cell-free culture medium of each was used in a Drugs 2021, 19, 427. https://doi.org/ neutrophil chemiluminescence assay in luminol reaction. The clone showing maximum activity had 10.3390/md19080427 a genomic sequence expected to produce a molecule like a phospho-N-acetylmuramyl pentapeptide

Academic Editors: Ipek Kurtboke, by the metagenome fragment analysis. Leila Tirichine, Wim Vyverman and Detmer Sipkema Keywords: activity-based screening; bioactive compound; metagenome analysis; neutrophil activating peptide Received: 9 June 2021 Accepted: 23 July 2021 Published: 28 July 2021 1. Introduction Publisher’s Note: MDPI stays neutral The immune system plays an important role in the prevention of bacterial infections. with regard to jurisdictional claims in Neutrophils undergo a chemotaxis, which allows them to migrate toward sites of infection published maps and institutional afﬁl- or inﬂammation. Neutrophils recognize chemotactic factors (chemoattractants) via cell sur- iations. face receptors [1]. Detection of chemotactic factors by their receptors triggers the activation of cellular movement and results in quickly congregating at an infection site by attraction by cytokines expressed by activated endothelium, mast cells, and macrophages or by bacterial by-products [2]. In humans, neutrophil chemoattractants belong to four biochemically Copyright: © 2021 by the authors. distinct subfamilies, i.e., chemokine (C-X-C motif) ligand (CXCL) family (CXCL1 to CXCL3 Licensee MDPI, Basel, Switzerland. and CXCL5 to CXCL8 in humans), complement anaphylatoxins (C3a and C5a), chemotactic This article is an open access article lipids (e.g., leukotriene B4 or LTB4), and formyl peptides [3]. A well-known example of a distributed under the terms and formyl peptide is N-formyl-Met-Leu-Pro (fMLP), which was synthesized by mimicking conditions of the Creative Commons bacteria. N-formylmethionine is commonly used for an initial amino acid of bacterial Attribution (CC BY) license (https:// protein; therefore, Schiffman et al. demonstrated N-formylmethioninyl peptides showing creativecommons.org/licenses/by/ chemoattractant activity as for resembling chemotactic factors produced by bacteria [4]. 4.0/).

Mar. Drugs 2021, 19, 427. https://doi.org/10.3390/md19080427 https://www.mdpi.com/journal/marinedrugs Mar. Drugs 2021, 19, 427 2 of 11

In addition to fMLP, there are no other peptide ligands reported for a long time, and the discovery of new ligands is a very important research target in the field of drug discovery. Peptides are generally generated by cleaving proteins or by enzymatic ligation of amino acids. The enzymatic method employs amino acid ligases, which are ATP-dependent enzymes [5]. The enzymatic method is more cost-effective and leads to milder conditions than the solid-phase synthesis method; however, owing to substrate specificity, the variety of peptides is limited by a single kind of amino acid ligase. Therefore, it is necessary to pre- pare many kinds of amino acid ligases for the enzymatic method for industrial production. Amino acid ligases have been discovered mainly from microorganisms. The discovery of new amino acid ligases has been performed by homology search using the sequences of known enzymes or by selecting candidate sequences that have unique ATP-binding domains in the ligase, and finally succeeded in expressing them in host cells such as Escherichia coli for functional analysis [6]. As this approach requires the full-length sequence information of the genes or genomic sequence, it is mainly performed on established strains. However, standard culturing techniques support the establishment of less than 1% of the bacteria found in the environment [7]. More than 99% of the microorganisms in natural environment are reported to be difficult or impossible to culture, so-called ‘visible but non-culturable’(VBNC) bacteria [8]. Therefore, the VBNC bacteria must be ignored and not to be used for a source of novel enzyme screening and a potential source of functional genes. For this reason, metagenome analyses have gained popularity for the screening and identification of genes of interest. It is expected that, with the metagenome library, it would be possible not only to use the genetic information of VBNCs, but also to express them in the host cells such as E. coli and determine the activity to meet the non- elucidated characteristics or undetermined phenotypic properties. In fact, we have reported a novel esterase [9] and a novel gene involved in cadmium accumulation [10] from sponge-associated bacterial metagenome by library screening. We have constructed two types of sponge-associated bacterial metagenome libraries derived from Hyrtios erecta [9] and Stylissa massa [10] because marine sponges are known to be one of the largest producers of sondary metabolites [11] and hold high potential of harboring unique functional genes including those related to heavy metal accumulation [12,13]. On the other hand, we have used 26,496 clones of a plasmid library [9] and 3301 clones of a fosmid library [10] for a functional screening approach, so it is not easy work to cultivate a large number of clones. To solve this problem of inefficiency, we have determined DNA sequences of about 2 kbp at both ends in every single vector from the metagenome library and an established in-house database to combine in silico screening with functional screening. All DNA sequences were analyzed and annotated based on homology and domain, and registered in the in-house database. In this study, at first, we selected 120 candidates from the in-house database for amino acid ligases and peptide synthases using clusters of orthologous groups (COG) analysis or selected by Pfam analysis of the ATPases associated with diverse cellular activities (AAA) domain, an ATP-binding motif that is a conserved domain involved in peptide synthethase, and then the gene cluster activating neutrophils was identified.

2. Results 2.1. Selection of Positive Clones As mentioned above, candidates were selected based on COG and pfam and 120 clones (shown in Supplementary Table S1) were examined for second screening. Neutrophil assay employed boiled culture supernatant to detect chemiluminescent intensity [14]. Commercial chemotactic factor fMLP (Sigma-Aldrich, St. Luis, MO, USA) was used as positive control and supernatant form culture of E. coli harboring empty vector (pHSG398) was used as negative control. As shown in Figure1, there were 18 clones showing greater peak height than 1.3 times the value of the negative control. The positive control (2 ng of fMLP) was 1.3. The assay was repeated ﬁve times or more and 7 out of 120 clones were selected with reproducibility for the next candidates (Figure2). Mar. Drugs 2021, 19, x FOR PEER REVIEW 3 of 11 Mar. Drugs 2021, 19, x FOR PEER REVIEW 3 of 11

vector (pHSG398) was used as negative control. As shown in Figure 1, there were 18 clonesvector showing (pHSG398) greater was peak used heightas negative than 1.3 control. times Asthe shownvalue of in the Figure negative 1, there control. were The 18 Mar. Drugs 2021, 19, 427 3 of 11 pclonesositive showing control (2greater ng of peakfMLP) height was 1.3.than The 1.3 assay times was the repeatedvalue of thefive negative times or control. more and The 7 outpositive of 120 control clones (2 were ng of selected fMLP) withwas 1.3.reproducibility The assay was for repeatedthe next candidatesfive times or (Figure more 2).and 7 out of 120 clones were selected with reproducibility for the next candidates (Figure 2).

. . Figure 1. Neutrophil stimulating activities of selected 120 clones. The activity is shown as relative Figure 1. Neutrophil stimulating activities of selected 120 clones. The activity is shown as relative activity to E. coli harboring empty vector (negative control; NC). N-formyl-Met-Leu-Pro (fMLP) (2 ng) activityFigure 1. to Neutrophil E. coli harboring stimulating empty activities vector (negative of selected control; 120 clones. NC). N The-formyl activity-Met is- Leushown-Pro as (fMLP relative) (2 ng)activitywas was used toused asE. positivecoli as positiveharboring control control empty (PC). (PC). vector Black Black and(negative and red red lines control; lines indicate indicate NC). relative N relative-formyl activities -activitiesMet-Leu of negative- ofPro negative (fMLP control) (2 controlng)and was positive and used positive control,as positive control, respectively. control respectively. (PC). Black and red lines indicate relative activities of negative control and positive control, respectively.

Figure 2. Figure 2. NeutrophilNeutrophil stimulating stimulating activities activities of of further further selected clones at thi thirdrd screening. The activity activityFigureis shown 2. is asNeutrophilshown maximum as maximum stimulating luminol luminol chemiluminescent activities chemiluminescent of further intensity selected intensity (a.u.). clones NC: (a.u.). at HBSS thi rdNC: buffer, screening. HBSS PC: buffer, 2 The ng of PC: fMLP. 2 ngactivityGrey of fMLP. bar: is NCshown Grey (negative asbar: maximum NC control), (negative luminol red control), bar: chemiluminescent PC (positivered bar: PC control). (positive intensity control). (a.u.). NC: HBSS buffer, PC: 2 ng of fMLP. Grey bar: NC (negative control), red bar: PC (positive control). Next,Next, the the culture culture supernatants supernatants of theseof these seven seven clones clones were werepurified purified by HPLC by to HPLC identify to identifythe fractionsNext, the the fractions containing culture containing supernatants neutrophil neutrophil stimulating of these stimulating seven activity. clones However, activity. were However, the purified fractions bythe from HPLCfractions 5 out to fromidentifyof 7 candidates5 out the of fractions 7 candidates did not containing show did not significant neutrophil show significant activities stimulating activities after activity. fractionation after However,fractionation by HPLC. the by fractions HPLC. As the Asfromresults, the 5 out cloneresults, of #987 candidates clone and #115 #98 containeddid and not #115 show significant contained significant activities significant activities compared activities after fractionation with compared the correspondent by with HPLC. the fraction from the negative control (Figure3). The fractions from clone #98 showed less correspondentAs the results, fraction clone #98 from and the #115 negative contained control significant (Figure 3). activities The fractions compared from clonewith #98the activity than the fractions from clone #115. According to Figure3, fraction #5 from clone showedcorrespondent less activit fractiony than from the thefractions negative from control clone (Figure#115. According 3). The fractions to Figure from 3, fraction clone #98 #5 #115 and fraction #6 from clone #98 were chosen and determined time course changes of fromshowed clone less #115 activit andy thanfraction the #6fractions from clone from #98 clone were #115. chosen According and determined to Figure 3, time fraction course #5 luminescence intensity (Figure4). The chemoattractants found in clone #115 and #98 were changesfrom clone of #115luminescence and fraction intensity #6 from (Figure clone 4).#98 The were chemoattractants chosen and determined found in time clone course #115 characterized by an immediate activation of neutrophils followed by a rapid decrease in andchanges #98 wer of luminescencee characterized intensity by an immediate (Figure 4). activation The chemoattractants of neutrophils found followed in cloneby a rapid #115 response. The peak height of fraction #5 in clone #115 was the greatest; therefore, we chose decreaseand #98 wer in e response. characterized The by peak an immediate height of factivationraction #5 of in neutrophils clone #115 followed was the by greatest a rapid; it for further purification. The activity of fraction #6 from clone #98 was unstable, so it was therefore,decrease inwe response. chose it for The further peak purification. height of fraction The activity #5 in cloneof fraction #115 #6was from the clone greatest #98; not applied to further experiment. wtherefore,as unstable we, choseso it was it for not further applied purification. to further experiment. The activity of fraction #6 from clone #98 was unstable, so it was not applied to further experiment.

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Figure 3. Fractionation of culture supernatant including secreted factors from clone #98 and #115 FigureFigure 3. 3. Fractionation Fractionation of of culture culture supernatant supernatant including including s sececretedreted factors factors from from clone clone #98 #98 and and #115 #115 andand neutrophil neutrophil stimulating stimulating activitiesactivities ofof of eacheach each fraction.fraction. fraction. BlueBlue Blue bar:bar: bar: #98,#98, #98, orangeorange orange bar: bar: bar: #115,#115, #115, gray gray gray bar: bar: bar:E. E. E. coli coli(negativecoli (negative (negative control). control). control).

Figure 4. Time course of luminol chemiluminescent intensities in the fraction #5 from clone #115 FigureFigure 4. 4. Time Time course course of of luminol luminol chemiluminescent chemiluminescent intensities intensities in in the the fraction fraction #5 #5 from from clone clone #115 #115 ((AA)) and and fraction fraction #6 #6 from from clone clone #98 #98 #98 ( (B (B).).). Gray Gray Gray color color color showed showed showed the the the activity activity activity of of of same same same fraction fraction fraction from from from E. E.E. coli coli coli (negative(negative control). control). 2.2. Purification of Chemoattractant 2.2.2.2. Pu Purificationrification of of Chemoattractant Chemoattractant Fraction #5 in clone #115 was collected up to 25 mL and lyophilized. The concentrated FractionFraction #5 #5 in in clone clone #115 #115 was was collected collected up up to to 25 25 mL mL and and lyophilized. lyophilized. The The c concentratedoncentrated fraction was prepared by being redissolved in 20 µL of milliQ and applied on a C18 column. fractionfraction was was prepared prepared by by beingbeing redissolvedredissolved in in 20 20 µL µL of of milliQ milliQ and and applied applied on on a a C18 C18 Elution employed a linear gradient of 0–100% acetonitrile (solvent A: 0.l% TFA/water; column.column. Elution Elution employed employed a a linear linear gradient gradient of of 00––100100%% acetonitrileacetonitrile (solvent (solvent A: A: 0.l% 0.l% solvent B: 0.l% TFA/acetonitrile) for 120 min at a flow rate of 0.4 mL/min. Figure5 shows TFATFA/water/water; ; solventsolvent B:B: 0.l%0.l% TFATFA/acetonitrile/acetonitrile)) forfor 120120 minmin atat aa flowflow raterate ofof 0.40.4 mL/min.mL/min. the chromatogram and every peak was collected and assayed. As a result of the assay, Figure 5 shows the chromatogram and every peak was collected and assayed. As a result Figurethe main 5 show bodys the of activity chromatogram was distributed and every into peak three was peaks collected between and 60assayed. and 62 As min. a result Each of the assay, the main body of activity was distributed into three peaks between 60 and 62 ofof the these assay, three the fractions main body was of subjected activity was to Orbi-Trapdistributed MS into analysis; three peaks however, between the 60 fractions and 62 min. Each of these three fractions was subjected to Orbi-Trap MS analysis; however, the minstill. containedEach of these several three substances fractions andwas itsubjected was difficult to Orbi to estimate-Trap MS the analysis de novo; however, sequence the of fractions still contained several substances and it was difficult to estimate the de novo fractionsthe peptide. still Ascontained it was hard several to identify substances the peptideand it was sequence, difficult we to decided estimate to the identify de novo the sequence of the peptide. As it was hard to identify the peptide sequence, we decided to sequenceresponsible of genesthe peptide. of the peptideAs it was synthesis. hard to identify the peptide sequence, we decided to identifyidentify the the responsible responsible genes genes of of the the peptide peptide synthesis. synthesis.

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Figure 5. Further fractionation of culture supernatant from clone #115 and positive fractions (magni- Figure 5. Further fractionation of culture supernatant from clone #115 and positive fractions ﬁed chromatogram) and their activity (inserted bar graph). (magnified chromatogram) and their activity (inserted bar graph). 2.3. Sequence Analysis of Metagenomic Fragment of Clone #115 2.3. Sequence Analysis of Metagenomic Fragment of Clone #115 The nucleotide sequence of the metagenomic fragment of strain #115 originated from HyrtiosThe nucleotide erecta-associated sequence bacteria of the wasmetagenomic determined fragment by primer of strain walking #115 and originated the sequence from of Hyrtios2992 erecta bp was-associated obtained. bacteria The fragment was determined contained by 3 primer ORFs; walking however, and one the promoter sequence region of 2992was bp only was found obtained. before The the fragment last ORF. contained Therefore, 3 itORFs was; considered however, one that promoter the lac promoter region of wasthe only vector found would before be the used last to ORF. express Therefore, former ORFs it was (Supplementary considered that Figure the lac S1A). promoter Moreover, of theunder vectorthe wouldlac promoter, be used to ribosomeexpress former binding ORFs site (Supplement and start codonary Figure derived S1A). from Moreover, the vector underregion the were lac promoter, fused to the ribosome upstream binding of metagenomic site and start fragment codon and derived another from ORF the was vector formed regionby in-frame were fused fusion. to Thus, the upstream four ORFs of inferred metagenomic inside the fragment metagenomic and another fragment, ORF and was ORF1 formedto 3, wereby in assumed-frame fusion. to form Thus, an operon.four ORFs Deduced inferred amino inside acid the sequences metagenomic of each fragment, ORF were andanalyzed ORF1 to by 3, BLASTPwere assumed [15] and to theform homology an operon. is shownDeduced in Tableamino1. acid ORF1 sequence and ORF3s of showed each ORFhighly were homology analyzed withby BLASTP UDP-N -acetylmuramoylalanyl-[15] and the homology iDs -glutamate-2,6-diaminopimelateshown in Table 1. ORF1 and ORF3ligase; showed however, highly ORF1 homology lacked one with out UDP of three-N-acetylmuramoylalanyl domains consisting- ofD-glutamate the homologous-2,6- diaminopimelateprotein. It has beenligase reported; however, that, ORF1 among lacked three one domains, out of three the domains central domain consisting has of an the ATP- homologousbinding function protein. and It has the beenC-terminal reported domain that, hasamong ATP- three and substrate-bindingdomains, the central functions domain [16 ]. hasTherefore, an ATP-binding it was assumedfunction thatand the ORF1 C-terminal might work domain for the has operon. ATP- and substrate-binding functions [16]. Therefore, it was assumed that ORF1 might work for the operon. Table 1. BLASTP sequence similarity search using the NCBI non-redundant protein database. Table 1. BLASTP sequence similarity search using the NCBI non-redundant protein database. Identity ORF Homology Score E-Value Coverage Organisms Accession# Identity(%) ORF Homology Score E-Value Coverage Organisms Accession# UDP-N-acetylmuramoyl-L- (%) ORF1 alanyl-D-glutamate-2,6- 411 Rhodospirillales UDP-N-acetylmuramoyl-L- 91 1 × 10−139 245/473 aa MYE18720.1 ORF1(267aa) diaminopimelate bits(1056)411 Rhodospirillalesbacterium alanyl-D-glutamate-2,6- 91 1 × 10−139 245/473 aa MYE18720.1 (267aa) ligase bits(1056) bacterium ORF2 diaminopimelateNo signiﬁcant similarityligase ------ORF2(151aa) found No significant similarity found ------(151aa) UDP-N-acetylmuramoyl- ORF3 tripeptide-D-alanyl-D- 550 Rhodospirillales UDP-N-acetylmuramoyl- 94 0.0 310/458 aa MXX22569.1 ORF3(311aa) alanine bits(1416)550 Rhodospirillalesbacterium tripeptide-D-alanyl-D-alanine 94 0.0 310/458 aa MXX22569.1 (311aa) ligase bits(1416) bacterium ligasephospho- N- ORF4 448 Rhodospirillales ORF4 phospho-Nacetylmuramoyl--acetylmuramoyl- 448 98 3 × 10−156 231/362Rhodospirillales aa MXX22568.1 (249aa) bits(1153) 98 3 × 10−156 231/362 aa bacterium MXX22568.1 (249aa) pentapeptidepentapeptide-transferase-transferase bits(1153) bacterium

ORF4 is presumed to be a phospho-N-acetylmuramoyl-pentapeptide-transferase, suggesting that it modiﬁes the products produced by ORF1–3. However, ORF4 was truncated so that the conserved domain was not completed (Supplementary Figure S1B).

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Mar. Drugs 2021, 19, 427 ORF4 is presumed to be a phospho-N-acetylmuramoyl-pentapeptide-transferase,6 of 11 suggesting that it modifies the products produced by ORF1–3. However, ORF4 was truncated so that the conserved domain was not completed (Supplementary Figure S1B). Phospho-N-acetylmuramoyl-pentapeptide-transferase has 10 transmembrane regions Phospho-N-acetylmuramoyl-pentapeptide-transferase has 10 transmembrane regions [17], [17], but ORF4 lacked the sequence after the seventh transmembrane region. but ORF4 lacked the sequence after the seventh transmembrane region.

2.4.2.4. Construction Construction of of Recombinant Recombinant Strains Strains Expressing Expressing Combined Combined S Severaleveral ORFs ORFs RecombinantRecombinant strains strains har harboringboring the the expression expression vector vector show shownn in in Supplementa Supplementaryry FigureFigure S S22 were were prepared prepared and and neutrophil neutrophil stimulation stimulation activities activities were were assayed. assayed. The The assay as- resultssay results showed showed that that the the recombinant recombinant retaining retaining all allof ofORF ORF1–41–4 had had the the highest highest values values ((FigureFigure 66).). ORF1–3ORF1–3 retainedretained strainsstrains also also had had relatively relatively high high values, values, but but it wasit was about about half half of ofORF1–4 ORF1–4 retained retained strains. strains. ORF2–4 ORF2–4 retained retained strainsstrains alsoalso showedshowed small activity. This This result result suggestssuggests that that ORF4 was involvedinvolved ininthe the increase increase in in activity activity and and ORF2 ORF2 was was required required for for the thefunction function of ORF4. of ORF4. This This may may be due be todue the to fact the that fact ORF4 that modiﬁesORF4 modifies the products the products of ORF1–3, of ORF1but considering–3, but considering the activity the ofactivity ORF3–4, of ORF3 it is highly–4, it is likely highly that likely ORF4 that modiﬁes ORF4 modifies the products the productsof ORF2–3. of ORF2–3.

Figure 6. Identification of coding genes of neutrophil stimulating activity using the overexpression Figure 6. Identification of coding genes of neutrophil stimulating activity using the overexpression system.system. Protein Protein expression was was induced induced by by lactose. lactose.E. coliE. coliharboring harboring empty empty vector vector (NC) (NC) was alsowas inducedalso inducedby lactose. by Bluelactose. bar: Blue induction, bar: induction, black bar: black non-induction, bar: non-induction, red bar: 5 red ngof bar: fMLP 5 ng for of positivefMLP for control. positive control. 3. Discussion 3. DiscussionAlthough the N-terminal domain of ORF1 and C-terminal domain of ORF4 were deleted, the remaining domains of both ORFs are still functional and the expected pep- Although the N-terminal domain of ORF1 and C-terminal domain of ORF4 were tide was obtained. However, it is not possible to affirm that it is the natural product of deleted, the remaining domains of both ORFs are still functional and the expected peptide the ORF1–4 because of the missing domains. Moreover, ORF1 is required for showing was obtained. However, it is not possible to affirm that it is the natural product of the activity suggesting that both products of ORF1 and ORF3 would be combined to form ORF1–4 because of the missing domains. Moreover, ORF1 is required for showing activity pentapeptide and modified by a phospho-N-acetylmuramoyl-pentapeptide-transferase of suggesting that both products of ORF1 and ORF3 would be combined to form ORF4. ORF2 did not show any significant similarity; however, it would be required for pentapeptide and modified by a phospho-N-acetylmuramoyl-pentapeptide-transferase of ORF4 (Figure6). The genes of ORF1, 3, and 4 showed homologies with the genes involved ORF4. ORF2 did not show any significant similarity; however, it would be required for in peptidoglycan biosynthesis in E. coli cells [18]. Peptidoglycan consists of linear glycan ORF4 (Figure 6). The genes of ORF1, 3, and 4 showed homologies with the genes involved chains interlinked by short peptides. The glycan chains are composed of alternating units inof peptidoglycanN-acetylglucosamine biosynthesis and Nin-acetylmuramic E. coli cells [18]. acid. Peptidoglycan Muramyl consists residues of bear linear short glycan pen- chainstapeptides. interlinked Peptidoglycan by short ispeptides. synthesized The glycan by the keychains enzymes are composed MurA to of MurF. alternating The mur unitsA to ofmur NF-acetylglucosamine genes are all essential and in bacteria. N-acetylmuramic MurE, ATP-dependent acid. Muramyl amino residues acid ligase, bear converts short pentapeptides.UDP-N-acetylmuramyl- PeptidoglycanL-alanine- is synthesizedD-Glutamate by to the UDP- keyN -acetylmuramyl-tripeptide enzymes MurA to MurF. with The murmeso-diaminopimelicA to murF genes are acid. all essential MurF catalyses in bacteria. the MurE, addition ATP of-dependentD-alanyl D -alanineamino acid dipeptide ligase, convertsto UDP- N-acetylmuramyl-tripeptide UDP-N-acetylmuramyl-L-alanine with meso-diaminopimelic-D-Glutamate to UDP acid-N and-acetylmuramyl forms UDP-N-- tripeptideacetylmuramyl-pentapeptide with meso-diaminopimelic [18]. ORF1 acid. would MurF retain catalyses the partthe addition of MurE ofactivity. D-alanyl ORF4 D- alanineshowed dipeptide homology to with UDP MraY,-N-acetylmuramyl which converts-tripeptide UDP-N-acetylmuramyl-pentapeptide with meso-diaminopimelic acidwith andundecaprenyl-phosphate forms UDP-N-acetylmuramyl into undecaprenyl-diphosphate--pentapeptide [18]. ORF1N would-acetylmuramyl-pentapeptide retain the part of MurE activity.and UMP ORF4 [19]. showed homology with MraY, which converts UDP-N-acetylmuramyl- Based on the peptideglycan biosynthetic pathway, the function of the metagenomic fragment was hypothesized (Figure7). As MurE and MurF are homologous to ORF1 and ORF3, respectively, the sequence of the peptide chain might be similar to L-Ala-D-Gln-L- Lys-D-Ala-D-Ala or different. As MraY is homologous to ORF4, it may function to add an Mar. Drugs 2021, 19, x FOR PEER REVIEW 7 of 11

pentapeptide with undecaprenyl-phosphate into undecaprenyl-diphosphate- N- acetylmuramyl-pentapeptide and UMP [19]. Based on the peptideglycan biosynthetic pathway, the function of the metagenomic fragment was hypothesized (Figure 7). As MurE and MurF are homologous to ORF1 and Mar. Drugs 2021 19 , , 427 ORF3, respectively, the sequence of the peptide chain might be similar to L-Ala-D-Gln7 of- 11L- Lys-D-Ala-D-Ala or different. As MraY is homologous to ORF4, it may function to add an undecaprenyl-diphosphate to N-acetylmuramyl-pentapeptide; however, ORF4 is also not undecaprenyl-diphosphatein a complete form, and this to pentapeptideN-acetylmuramyl-pentapeptide; might be secreted from however, the cells ORF4 to the is medium, also not innot a complete bound to form, the celland thiswall. pentapeptide In addition, might as mu berA, secreted murB, frommurC the, and cells m tour theD genes medium, are notnecessary bound to for the peptidoglycan cell wall. In addition, synthesis, as mur theyA, murwereB, mur notC, included and mur D in genes the aremetagenomic necessary forfragment, peptidoglycan and the synthesis, glycol-pentapeptide they were not would included bein in the an metagenomic incomplete fragment,form. It could and the be glycol-pentapeptideconsidered that native would genes be from in an host incomplete E. coli complemented form. It could the be functions. considered A thatresult native from genesde novo from sequence host E. through coli complemented Orbi-trap MS, the however, functions. did A not result correspond from de to novo accurate sequence mass throughof Ala and Orbi-trap Lys, indicating MS, however, that peptide did not sequence correspond would to be accurate different mass from of L Ala-Ala and-D-Gln Lys,-L- indicatingLys-D-Ala- thatD-Ala. peptide According sequence to would Tabata be et different al. [5], amino from L -Ala- acid D ligase-Gln- L is-Lys- knownD-Ala- forD-Ala. low Accordingsubstrate specificity to Tabata et and al. [5 accepts], amino a acid wide ligase variety is known of L-amino for low acids. substrate We consideredspeciﬁcity and the acceptsreason athat wide MS variety fragments of L-amino were acids. shown We to considered be complicated the reason and that did MS not fragments show known were shownaccurate to mass be complicated might be that and several did not sequences show known were contained. accurate mass Based might on the be gene that analysis, several sequencesthe neurophil were contained. activating Based peptide, on the at least,gene analysis, would bethe predicted neurophil activating as the pentapeptide peptide, at least,modified would by beN- predictedacetylmuramic as the acid. pentapeptide modiﬁed by N-acetylmuramic acid.

ORF2 product

ORF1 ORF2 ORF3 ORF4

MurE MurF MraY

L - Ala L - Ala L - Ala L - Ala D - Glu D - Glu D - Glu D - Glu L - Lys L - Lys L - Lys : UDP D - Ala D - Ala

: N-Acetylmuraminic acid DL -- Allaa D - Ala

FigureFigure 7.7. HypothesizedHypothesized productproduct ofof thethe genegene clustercluster encodedencoded inin thethe metagenomemetagenome fragment.fragment.

AlongAlong with with improving improving next next generation generation sequence, sequence, metagenome metagenome determination determination became easierbecame and easier less costly, and less and cost couldly, and be utilized could be for utilized gene screening for gene based screening on the based conserved on the sequenceconserved or sequence homology or search. homology As a result,search. the As identified a result, the genes identified show similar genes characteristics show similar tocharacteristics known genes to and know the chancen genes to and meet the novel chance genes to andmeet enzymes novel genes is less and than enzymes the functional is less screening.than the functional Because screening. of the limitation, Because of we the have limitation, conducted we have activity-based conducted screeningactivity-based for metagenomescreening for analysismetagenome so far. analysis The constructionso far. The construction of a metagenome of a metagenome library and library activity- and basedactivity screening-based screening spent much spent time much and time effort; and however, effort; however to express, to themexpress in thethem host in the cells host of E.cells coli and of E. determine coli and the determine activity can the lead activity to meeting can lead the non-elucidated to meeting the characteristics non-elucidated or undeterminedcharacteristics phenotypicor undetermined properties. phenotypic Moreover, properties. the in-house Moreover, database the in for-house metagenome database librariesfor metagen wasome also libraries established, was thus also we established, were able th tous compare we were the able screening to compare efficiency the betweenscreening general efficiency activity-based between screening general and activity activity-based-based screening screening and combined activity with-based in silico screening, because of the same libraries. As mentioned above, we used 26,496 clones to identify a novel esterase gene [9]. Similarly, a xylanase gene was selected among 40,000 clones from the soil metagenome library [20]. Thus, we estimated the efficiency of activity-based screening was 1 out of 104 clones. In this study, we cultivated only 120 clones by in silico screening and the efforts were reduced by 1/100 and the efficiency increased to 100-fold. In addition to this approach’s merit, we could access the candidates and propagate the recombinants once the libraries were constructed. Mar. Drugs 2021, 19, 427 8 of 11

4. Materials and Methods 4.1. Metagenomic Libraries Metagenomic libraries used in this study have been established in Okamura et al. [9] and Mori et al. [10]. LB medium containing chloramphenicol (Cm; ﬁnal concentration: 20 µg/mL) was used for bacterial culture.

4.2. In Silico Screening For the ﬁrst screening, amino acid ligase or peptide synthase genes were selected from our in-house database. Speciﬁcally, the genes contain an adenylation domain, which is required for activation by ATP to form a peptide bond. COG search and Pfam domain searches were employed to select ligase and AAA domains. The clones that conserved full-length ORFs were selected.

4.3. Sample Preparation for Neutrophil Activity Assay For the second screening, all 120 selected clones were grown in 96-DeepWell plate (maximum volume 2 mL/well) (NuncTM, Nalge Nunc International, New York, NY, USA) with 1 mL of LB (Cm) with continual agitation at 170 rpm in a Bio-Shaker (TITEC, Saitama, Japan) at 37 ◦C for 8 h. After centrifugation at 10,000× g for 5 min, supernatant was boiled at 80 ◦C for 20 min. For the third screening, selected clones were cultured in L-shaped test tube (id: 18 mm, custom-made in Hiroshima University) with 5 mL of LB (Cm) with horizontal shaking at 120 rpm at 37 ◦C for 8 h. After centrifugation and boiling, the same as above, the supernatant was lyophilized, redissolved in milliQ, and concentrated at ﬁvefold.

4.4. Purification The culture supernatant after boiling was lyophilized, redissolved in milliQ, concentrated 5–10 times, and used for HPLC purification. The concentrated supernatant was applied on a C18 column (TSKgel ODS-80Ts, Tosoh, Yamaguchi, Japan) and eluted with a linear gradient of 0–100% acetonitrile (solvent A: 0.l% TFA/water; solvent B: 0.l% TFA/acetonitrile) in 8–40 min for 32 min at a flow rate of 1.0 mL/min. After linear gradient, mobile phases at 100% and 0% acetonitrile were kept for 5 min, respectively. A column oven was used to keep the column at 40 ◦C during the measurement. The eluate was monitored at 230 nm with a MD-2010 multiwavelength photodiode array detector (JASCO Cooperation, Tokyo, Japan). Fractions were obtained at the first 10 min (Fraction #1) and at intervals of 5 min/fraction (Fraction #2–9). For further purification, the same mobile phase was used at a flow rate of 0.4 mL/min in 120 min with linear gradient. The fractions were separated by peak appearance. The obtained fractions were completely dried by decompression drying and re-dissolved in 20 µL of Hanks’ Balanced Salt Solution (HBSS (1×): GIBCO®, Thermo Fisher Scientific, Waltham, MA, USA) for the assay.

4.5. Neutrophil Activating Assay The assay using neutrophils was based on the method of Hasegawa et al. [14]. Whole blood was employed in the assay. The principle is based on the NADPH oxidase activity from neutrophils, which receive the chemoattractants, and the resulting superoxide anion reacts with luminol, thus the activity can be detected as a luminescent intensity. Luminol solution was prepared as follows: 17.72 mg of luminol (SIGMA) was dissolved in 3 mL of 1N NaOH, then 2.5 mL of 1N HCl was added. Then, 30 mL of HBSS (-) (HBSS (1×) without Ca2+ and Mg2+) was added, adjusting pH to pH = 7.4 (37 ◦C) with 0.2 N HCl, and finally mess up to 40 mL with HBSS (1×). For the assay, the luminol solution was diluted five times with HBSS (1×) and used. The blood samples were donated by male and female volunteers in their 20s to 40s. To prevent blood coagulation, 10 µL of 10 U/µL heparin (Wako Pure Chemical Corporation, Tokyo, Japan) dissolved in 50 µL of HBSS (1×) was added to the Falcon in advance. Blood was kept at 37 ◦C as much as possible. A 96-well plate was used for measurement. To all wells, 20 µL of boiling supernatant or purified Mar. Drugs 2021, 19, 427 9 of 11

fraction and 60 µL of reaction mixture of blood and luminol solution in equal volume were added, and the measurement was started immediately. The plate reader was set up so that there was a 10 s shaking step immediately before scanning, and the luminescence in each well was measured for 30 cycles of 3 min each, including a 170 s waiting period after scanning, for an overall measurement time of 90 min. The fluorescence plate reader was ARVO (Perkin Elmer, Waltham, MA, USA) or Fluoroskan Ascent FL (ThermoFisher Scientific, Waltham, MA, USA). Nunclon® 96 well (bottom clear, flat bottom, black, product no. 165305) (ThermoFisher Scientific, Waltham, MA, USA) was used for measurement. The highest value in the assay was used as the peak height for comparison.

4.6. Nucleotide Sequence Analysis of Metagenomic Insertion Fragments The plasmid DNA was extracted from positive clones showing neutrophil activation, and the sequence of the metagenome fragment was sent for sequencing by Takara Bio Inc. (Shiga, Japan). ORFs were predicted by a free software ApE (A plasmid editor, v2.0.61) (https://jorgensen.biology.utah.edu/wayned/ape/, accessed on 10 February 2020). The sequences were analyzed by Basic Local Alignment Search Tool (BLAST) [15]. Promoter regions were predicted using GENETYX Ver.9 (GENETYX CORPORATION, Tokyo, Japan).

4.7. Construction of Expression Vectors and Recombinants In order to determine the responsible gene(s) for neutrophil activation, four ORFs from the metagenome fragment were cloned separately or several clusters into pGEX- 6p-1 expression vector. PCR primers to amplify each ORF or gene cluster were listed in Supplemental Table S2. Restriction enzyme sites within the multi-cloning site were employed, and translation was initiated at the vector start codon. KOD-Plus-Neo (TOY- OBO, Osaka, Japan) was used with 2 µL of the extracted plasmid (10 ng/µL) as template and 1.5 µL of each primer (10 pmol/µL) in 50 µL of total volume. The thermal cycling conditions were as follows: pre-denaturation at 94 ◦C for 2 min, followed by 30 cycles of denaturation at 98 ◦C for 10 s, annealing and extension at 68 ◦C for 30 s for amplicon length <500 bp, 45 s for <800 bp, 60 s for <1 kbp, 75 s for <1.3 kbp, 105 s for <1.7 kbp, or 150 s for 2.3 kbp. The resulting PCR product was ligated into the appropriate sites downstream of the promoter in the pGEX-6p-1 vector and cloned in E. coli DH5a and plated on LB agar medium supplemented with ampicillin at 50 µg/mL. Colony PCR was performed to check for insertion of specific open reading frames (ORFs) in the recombinant clones. KOD Dash (TOYOBO) was used for colony PCR. Then, 0.5 µL of each primer (10 pmol/µL) was used for the reaction, and the total volume was 20 µL. The used primers were pGEX 5’ (5’-GGGCTGGCAAGCCACGTTTGGTG-3’) and pGEX 3’ (5’- CCGGGAGCTGCATGTGTCAGAGG-3’). The thermal cycling conditions were as follows: pre-denaturation at 95 ◦C for 4 min, followed by 35 cycles of denaturation at 98 ◦C for 20 s, annealing at 60 ◦C for 10 s, and extension at 72 ◦C for 2 min, with a final extension at 60 ◦C for 4 min. The PCR products were confirmed by electrophoresis in 1% agarose gel. The resulting transformants were grown in LB medium supplemented with ampicillin (50 µg/mL) and culture supernatant of each transformant was collected to be used for assay.

4.8. Accession Number The sequence of metagenomic fragment was deposited to DDBJ (LC634461).

5. Conclusions Activity-based metagenome library screening combined with in silico screening increased the screening efficiency from 1 out of 100,000 clones to 1 out 120 clones. The positive clone showing neutrophil stimulating activity harbored incomplete peptideglycan biosynthesis genes. The product of the metagenome fragment was expected to be the phospho-N-acetylmuramyl pentapeptide; however, the peptide was not able to be purified and identified. Mar. Drugs 2021, 19, 427 10 of 11

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/md19080427/s1, Figure S1: Arrangement of the open reading frames in the metagenomic fragment of clone #115 (A) and the truncated regions of homologous proteins in ORF1 and ORF4 (B); Figure S2: Expression vector constructs for single ORF expression or co-expression. The inserts were amplified by PCR and cloned into MCS of oGEX-6p-1; Table S1: List of primers designed for gene expression; Table S2: List of selected clones by in silico screening. Author Contributions: Conceptualization, Y.O. and K.S.; formal analysis, Y.O., H.T. (Hirokazu Takahashi), A.S. and Y.H.; investigation, Y.O. and A.S.; writing—original draft preparation, Y.O. and K.S.; writing—review and editing, H.T. (Hirokazu Takahashi) and H.T. (Haruko Takeyama); supervision, Y.O. and H.T. (Haruko Takeyama); project administration, Y.O. and H.T. (Haruko Takeyama); funding acquisition, Y.O. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by JSPS KAKENHI Grant Number 24560964 and Adaptable and Seamless Technology transfer program (A-STEP) of JST [AS232Z02424G]. Data Availability Statement: No new data were created or analyzed in this study. Data sharing is not applicable to this article. Conflicts of Interest: All authors declare no conflict of interest.

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