Escherichia coli induced secretion of proinflammatory factors by uninfected Mφ. Inflammatory responses induced by exosomes derived from Mφ infected with heat-inactivated E. coli or lipopolysaccharide were significantly weaker than those elicited by outer membrane vesicles (OMVs) released from live E. coli. Proteome analysis of exosomes from Mφ infected with live or heat-inactivated E. coli revealed that E. coli proteins OmpA, GroL1, DegP, CirA, and FepA are candidate triggers of exosome-mediated inflammatory responses. OMVs from a cirA-deleted strain suppressed exosome-mediated inflammatory responses by uninfected Mφ. The C terminus of the CirA protein (residues 158 to 633), which was relayed from E. coli-derived OMV to Mφ-derived exosomes, promoted exosome-mediated inflammatory responses by uninfected Mφ. These results suggest an alternative mechanism by which extracellular vesicles from E. coli OMV-elicited Mφ transmit proinflammatory responses to uninfected Mφ.Extracellular vesicles are considered to be an inflammatory factor in several acute and chronic inflammatory diseases. The present study shows that exosomes from macrophages (Mφ) infected with live  Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus are the most common causative microorganisms of community-acquired cases of adult sepsis  at a multiplicity of infection (MOI) of 5. Both live and heat-inactivated E. coli led to a strong increase in expression of inducible NO synthase (iNOS) and COX-2 proteins; however, expression of these proteins did not increase in noninfection  for 18 h was 2.26-fold or 2.40-fold higher than that in exosomes from uninfected cells (none-exo)  and Cox-2  significantly compared with exosomes from untreated Mφ  . Expressone-exo) . In addione-exo) . When RAnd COX-2 . In addieated Mφ . In contE. coli rather than the bacteria themselves; this is because we did not prepare OMVs from heat-inactivated E. coli. To examine whether OMVs from live E. coli trigger inflammatory responses by recipient Mφ via donor Mφ-derived exosomes, we prepared OMV pellets from E. coli culture media by ultracentrifugation at 100,000 × g (E. coli cultured for 3 to 24 h peaked at 12 h (E. coli cultured for 12 h (E. coli cultured for 12 h. After incubation of donor Mφ with E. coli culture medium (med) for 9 h, the supernatant (sup), or the pellet (OMVs) , the Mφ medium was exchanged with fresh medium. Exosomes were then collected again from Mφ culture medium after 9 h. We found that expression of iNOS and COX-2 proteins by donor Mφ treated with med, sup, or the OMV pellet was higher than that by untreated Mφ  treated with a low concentration of OMVs (0.5 μg/7 mL) were the same as in Mφ (8 × 106 cells) treated with a large concentration of OMVs (25 μg/7 mL) , which deleted the two secondary acyl chains of the normally hexa-acylated LPS, in recipient cells. ΔLPS-OMVs increased the expression of iNOS and COX-2 proteins by donor Mφ  see . These rt of LPS  and b. Lt of LPS . In conte medium . We examdonor Mφ . Exosome10.1128/mbio.03051-22.1FIG S1E. coli K-12 culture medium. RAW264.7 cells were incubated with OMVs at the indicated concentration for 9 h. Culture media were replaced with media containing antibiotics, and Mφ were cultured for 1 h. After we changed the new media again, Mφ were cultured for 9 h. Exosomes isolated from culture medium of donor Mφ. RAW 264.7 cells were incubated with exosomes (0.7 μg/mL) for 24 h. The expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in cell lysates of donor Mφ (a) and recipient Mφ (b) was measured by Western blotting. β-Actin was used as loading control. CS Analyzer 3.0 was used as image analysis software. Data are the mean ± SD (n = 3). ANOVA and Mann-Whitney U test were used for statistical analysis. *, P < 0.05; ***, P < 0.005; ****, P < 0.001. Download FIG S1, PDF file, 2.4 MB.Exosomes caused inflammatory responses in OMVs dose dependently. Mouse RAW264.7 macrophages (Mφ) were incubated with OMVs (0 to 25 μg/7 mL) derived from Copyright © 2023 Imamiya et al.2023Imamiya et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03051-22.2FIG S2E. coli K-12 strain (MOI of 5) for 9 h. Culture medium was replaced with medium containing antibiotics, and Mφ were cultured for 1 h. After we changed the new medium again, Mφ were cultured for 9 h. Exosomes were isolated from the culture medium of donor Mφ. (b) RAW 264.7 cells were incubated with exosomes (0.7 μg/mL) for 24 h. (c) RAW264.7 cells were incubated with 0 to 10 ng/mL LPS or live-exo (0.7 μg/mL). The protein expression of iNOS and COX-2 in cell lysates of Mφ was measured by Western blotting. β-Actin was used as loading control. (d) LPS concentration in culture medium of recipient Mφ, which were treated with 0.7 μg/mL live-exo, were analyzed by using Limulus amebocyte lysate (LAL). (e) ΔLPS-OMVs from 0.5 mL of culture medium of ClearColi were prepared by ultracentrifugation. The protein expression of OmpA and CirA was detected by Western blotting. (f) RAW264.7 cells were incubated with or without ΔLPS OMVs (10 μg/7 mL) for 9 h. After the culture medium was replaced with fresh medium containing antibiotics, cells were incubated for 1 h. After we changed the fresh medium again, cells were incubated for 9 h. The protein expression of iNOS and COX-2 in whole-cell lysates was measured by Western blotting. β-Actin was used as loading control. (g) RAW264.7 cells were incubated with exosomes (0.7 μg/mL) for 12 h. The mRNA expression of iNos, Cox-2, tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), and IL-6 was analyzed by quantitative real-time PCR and normalized to 18S rRNA. Data are the mean ± SD (n = 3). A t test was used for statistical analysis. *, P < 0.05; **, P < 0.01. Download FIG S2, PDF file, 2.5 MB.LPS was not associated with inflammatory responses by macrophage-derived exosomes. (a) Mouse RAW264.7 macrophages (Mφ) were incubated with 100 ng/mL lipopolysaccharide (LPS) or a heat-inactivated (HI) Copyright © 2023 Imamiya et al.2023Imamiya et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli-derived OMVs enhanced inflammatory responses. E. coli-derived OMVs increased the expression of iNOS and COX-2 proteins by donor Mφ of BMDM  treated with  of BMDM . Mouse B of BMDM .10.1128/mbio.03051-22.3FIG S3iNos, Cox-2, tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), and IL-6 were analyzed by quantitative real-time PCR and normalized to 18S rRNA. Data are the mean ± SD (n = 3). A t test was used for statistical analysis. *, P < 0.05. Download FIG S3, PDF file, 1.9 MB.Exosomes from mouse BMDMs treated by OMVs upregulated inflammatory mediator. (a) DMDMs were incubated with or without OMVs (10 μg/7 mL) for 9 h. After the culture medium was replaced with fresh medium containing antibiotics, cells were incubated for 1 h. After we changed fresh medium again, cells were incubated for 9 h. The protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in whole-cell lysates was measured by Western blotting. β-Actin was used as loading control. (b) Exosomes isolated from culture medium of donor Mφ. The expression of exosome markers inducing heat shock protein 90 (HSP90) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in exosomes were detected by Western blotting. (c) Mouse BMDMs were incubated with exosomes (0.7 μg/mL) for 12 h. The mRNA expression of Copyright © 2023 Imamiya et al.2023Imamiya et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli were treated with proteinase K, DNase, or RNase in the presence or absence of Triton X-100. Live-exo treated with proteinase K in the presence or absence of Triton X-100 failed to increase expression of iNOS protein and mRNA significantly  obtained from the Keio library triggered exosome-mediated inflammatory responses. Since groE1 is a lethal gene, we did not examine it in this study. Moreover, strain ΔompC was used as a negative control because OmpC is an abundant protein in OMVs. Density gradient ultracentrifugation was used to confirm expression of the OmpA, FepA, CirA, DegP, and OmpC proteins in OMVs. All proteins were most abundant in fraction 9  (ΔcirA strain (ΔcirA-exo) decreased 0.51-fold in comparison with treatment by OMVs from the WT strain (WT-exo). Expression of iNOS by ΔcirA-exo decreased 0.52-fold compared with that by OMVs from the WT strain, albeit not significantly. Similar to protein levels, the increase in expression of iNos and Cox-2 mRNA decreased 0.56-fold and 0.32-fold, respectively, in recipient Mφ exposed to ΔcirA-exo compared to WT-exo . In addition, expression of TNF-α and IL-1β was decreased by ΔcirA-exo to a greater extent than by WT-exo  ). In cono WT-exo . The incy WT-exo . These r10.1128/mbio.03051-22.4FIG S4E. coli K-12 strain by density gradient ultracentrifugation. Gradient fractions were collected and analyzed by Western blotting with anti-OmpA, anti-FepA, anti-CirA, anti-DegP, and anti-OmpC antibodies. (b) After OMVs were separated on a 5 to 40% iodixanol gradient, the densities of 12 fractions were calculated by measuring the absorbance at a wavelength of 244 nm. (c) OMVs from a single gene-deleted strain  were analyzed by Western blotting. (d) The number of OMVs is shown. Data are the mean ± SD. Download FIG S4, TIF file, 0.7 MB.Density of each fraction after centrifugation of density gradient. (a) OMVs were isolated from the culture medium of Copyright © 2023 Imamiya et al.2023Imamiya et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03051-22.5FIG S5cirA-deleted E. coli suppressed the mRNA levels of proinflammatory factors by macrophage-derived exosomes. Mouse RAW264.7 cells  were incubated with exosomes from donor cells (5 × 105 cells) for 24 h. The mRNA expression levels of tumor necrosis factor alpha (TNF-α) (a), interleukin 1 beta (IL-1β) (b), and IL-6 (c) were analyzed by quantitative real-time PCR and normalized to 18S rRNA. Values are expressed as the mean ± SD (n = 4) of at least three independent biological replicates. One-way ANOVA and Tukey’s test were used for statistical analysis; *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001. Download FIG S5, PDF file, 0.2 MB.Copyright © 2023 Imamiya et al.2023Imamiya et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coliΔcirA strain. In bacterial cells and OMVs, the protein levels of CirA in the cirA gene-complementary strain (ΔcirA/cirA) were the same as those in the WT strain  The number of OMVs is shown. (b) RAW264.7 cells  were incubated with exosomes from donor cells (5 × 105 cells) for 24 h. The mRNA expression levels of tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), and IL-6 were analyzed by quantitative real-time PCR and normalized to 18S rRNA. Values are expressed as the mean ± SD (n = 4) of at least three independent biological replicates. One-way ANOVA and Tukey’s test were used for statistical analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001. Download FIG S6, PDF file, 0.1 MB.Complementation of the 5Copyright © 2023 Imamiya et al.2023Imamiya et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the ΔcirA strains (ΔcirA/N-cirA or ΔcirA/C-cirA) transformed with the pTV-N-cirA or pTV-C-cirA strains was observed in bacterial cells and OMVs , ΔcirA/N-cirA , and ΔcirA/C-cirA  strains  was as same as that by WT-exo, while that by ΔcirA-exo  and ΔcirA/N-cirA-exo  was lower  and a C-terminal fragment comprising amino acids 158 to 633 (C-CirA) . Expressand OMVs . The inc strains . In recias lower . These rE. coli-derived OMVs, and exosomes derived from infected Mφ, rely on inflammatory responses to naive Mφ. Expression of iNOS and COX-2 by naive Mφ increased after exposure to exosomes from live E. coli-infected Mφ, but not Mφ infected by dead (heat-inactivated) E. coli , which allows E. coli to breach the blood-brain barrier  and found that OMVs from the ΔompC strain strongly increased expression of iNOS and COX-2. We considered that high concentrations of E. coli-secreted molecules, including LPS, were contained within OMVs from the ΔompC strain. TonB-dependent outer membrane transporters include FecA, the ferric citrate transporter; BtuB, the vitamin B12 receptor; FhuA, the ferrochrome receptor; and CirA-FepA, the ferric-enterochelin receptor (E. coli-derived OMVs. CirA and FepA share the highest sequence similarity (30.8%) within the TonB-dependent group. Moreover, the association between CirA and FepA could be extended to encompass a close evolutionary relationship (3+) complexed with siderophores. CirA was identified as a receptor of microcin that is of a lower molecular weight than colicins  to prepare OMVs. Iron depletion increased expression of CirA in E. coli, resulting in the taking up of CirA into OMVs. E. coli K-12 strain-derived OMVs, which were analyzed by Reimer et al., did not contain the TonB-dependent transporters, including CirA  were incubated with exosomes from donor cells (5 × 105 cells) for 24 h. The mRNA expression of tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), and IL-6 was analyzed by quantitative real-time PCR and normalized to 18S rRNA. Values are expressed as the mean ± SD (n = 4) of at least three independent biological replicates. One-way ANOVA and Tukey’s test were used for statistical analysis; *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001. Download FIG S7, PDF file, 0.1 MB.Complementation of N- and C-terminal Copyright © 2023 Imamiya et al.2023Imamiya et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03051-22.8FIG S8E. coli K-12 strain and Salmonella enterica serovar Enteritidis IID604 strain were cultured with DMEM containing 0 to 100 μM FeCl3 at 37°C for 12 h. Bacteria lysates were analyzed by Western blotting. Download FIG S8, PDF file, 0.3 MB.Iron concentration and CirA expression. The Copyright © 2023 Imamiya et al.2023Imamiya et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli-derived OMVs induced NLRP3 activation, resulting in activation of caspase-1 and pyroptosis via processing gasdermin D. Caspase-1 was activated in mouse BMDM after stimulation by E. coli-derived OMVs -2,5-diphenyl-2H-tetrazolium bromide (MTT) assay resulted in an increase by E. coli-derived OMVs (E. coli-derived OMVs (data not shown). These data suggest that the dose of E. coli-derived OMVs used in this study caused little pyroptosis. In fact, more than 50 μg of OMVs was reported to cause pyroptosis  for 9 h. After culture medium was replaced with fresh medium containing antibiotics, cells were incubated for 1 h. After replacement with fresh media containing antibiotics again, cells were incubated for 9 h.  The protein expressions of caspase-1 and cleaved caspase-1 in whole-cell lysates were measured by Western blotting.  The protein expression of caspase-11 and cleaved caspase-11 in whole-cell lysates was measured by Western blotting.  The protein expression of gasdermin D and cleaved gasdermin D in whole-cell lysates was measured by Western blotting. β-Actin was used as loading control.  Luciferase activity was measured as the amount of ATP by using FilterMax F5 multimode microplate reader.  Cell viability was analyzed by WST assay. Absorbance (Abs) was measured at 450 nm as the number of viable cells by using FilterMax F5 multimode microplate reader. A t test was used for statistical analysis. *, P < 0.05. Download FIG S9, TIF file, 0.6 MB.Activation of inflammasome and cell death in OMV-treated macrophages. Mouse RAW264.7 macrophages (Mφ) (a to e) and mouse bone marrow-derived Mφ (f to j) were incubated with each Copyright © 2023 Imamiya et al.2023Imamiya et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli is incorporated into Mφ-derived exosomes and increases protein expression of inflammatory mediators. However, proteins other than CirA may play roles in exosome-mediated inflammatory responses. In particular, increased expression of iNOS was partially attenuated by the ΔcirAE. coli strain. Other factors will be clarified in a future study.We demonstrate that the CirA protein in the OMVs from live E. coli K-12 BW25113 (wild type [WT]) and single gene-deleted mutant strains  were provided by National BioResource Project . The competent cell BL21(DE3) strain and DH5α strain were purchased from TaKaRa . LB broth was purchased from Sigma/Merck . The plasmid pET22b(+) was purchased from Novagen/Merck . pTV118N vector was gifted by Ishijima . Ex Taq, Mighty TA-cloning kit containing pMD T vector, DNA ligation kit Mighty Mix, in-fusion HD cloning kit, isopropyl β-d-1-thiogalactopyranoside (IPTG), and TB Green premix Ex Taq II were purchased from TaKaRa . Ni-nitrilotriacetic acid (NTA) agarose was purchased from Qiagen . cOmplete Mini protease inhibitor was purchased from Roche/Nippon Gene . Anti-OmpC antibody was purchased from MyBioSource . Isogen II was purchased from Nippon Gene . Can Get Signal and ReverTra Ace quantitative PCR (qPCR) reverse transcription (RT) kit were purchased from Toyobo .RAW264.2 cells were purchased from ATCC. All plasmids and bacterial strains used in this study are shown in E. colicirA gene (GenPept accession no. NP_416660) was amplified using Ex Taq with the primers pMD-cirANdeI-s and pMDcirANotI-a and E. coli (WT) genome DNA as the template. The pMD-cirA plasmid was ligated with the amplified fragments and pMD T vector by using Ligation Mighty Mix (TaKaRa). The pET-cirA plasmid was ligated with the fragments cirANdeI-NotI of pMD-cirA digested by NdeI and NotI restriction enzymes and the NdeI-NotI-digested pET-22b(+) plasmid by using ligation mix (TaKaRa). The cirA-6×His fragments were amplified with the primers pTV-cirANcoI-s and pTV-cirANcoI-a and pET-cirA as the template. The pTV-cirA plasmid was ligated with the amplified fragment of cirA-6×His and NcoI-digested pTV plasmid by the In-Fusion HD. The fragment of N/C-cirA was amplified with plasmids pET-NcirANcoI-s and pET-NcirANotI-a, or pET-CcirANcoI-s and pET-CcirANotI-a, and pET-cirA plasmid as the template. The pET-N/CcirA plasmids were ligated with amplified fragments N/CcirA, and NcoI-digested pET plasmid by In-Fusion HD. The N/CcirA-6×His fragments were amplified with the primers pTV-N/CcirANcoI-s and pTV-N/CcirANcoI-a and pET-N/CcirA as the template. The pTV-N/CcirA plasmids were ligated with the amplified fragment of N/CcirA-6×His and NcoI-digested pTV plasmid by In-Fusion HD. Cloning of the E. colidegP gene (GenPept accession no. NP_414703) was amplified with the primers pMD-degPNdeI-s and pMD-degPHindIII-a and E. coli (WT) genome DNA as the template. The pMD-degP plasmid was ligated with the amplified fragments and pMD T vector by using Ligation Mighty Mix. The pET-degP plasmid was ligated with the fragments degPNdeI-HindIII of pMD-degP digested by NdeI and HindIII restriction enzymes and the NdeI-HindIII-digested pET-22b(+) plasmid by using ligation mix. Cloning of the E. coliompA gene (GenPept accession no. NP_415477) and fepA gene (GenPept accession no. NP_415116) were amplified with the primers pMD-ompANdeI-s and pMD-ompAXhoI-a, or pMD-fepANdeI-s and pMD-fepAXhoI-a, and E. coli (WT) genome DNA as the template. The plasmid of pET-ompA or pET-fepA was ligated with the amplified fragment of ompA or fepA and NcoI-digested pET plasmid by In-Fusion HD. Each gene-containing coding region was designed to allow the expression of a C-terminal, 6× His-tagged variant of recombinant proteins following ligation into the pET-22b(+) plasmid. After construction, the integrity of some expression vectors was confirmed by DNA sequencing. The gene-complementary strains, ΔcirA/cirA, ΔcirA/NcirA, and ΔcirA/CcirA, were obtained through the transformation of pTV-cirA, pTV-NcirA, and pTV-CcirA into ΔcirA strain using electroporation . These strains were cultured in LB broth with 25 μg/mL for kanamycin and 100 μg/mL for ampicillin (Wako).Cloning of the ompA, pET-fepA, pET-cirA, and pET-degP) were transformed into the competent E. coli strain BL21(DE3) strain. Bacteria were incubated in LB broth with 100 μg/mL ampicillin at 37°C with shaking until the optical density at 600 nm (OD600) reached about 0.6. After incubation of bacteria at 22°C overnight with 0.1 mM IPTG, the bacterial cells were pelleted at 2,000 × g at 4°C for 30 min, and the supernatant was removed. The cells were lysed by lysis buffer  and were completely disrupted by sonication. After the centrifugation at 2,000 × g at 4°C for 30 min, the supernatant was filtrated through a 0.45-μm filter . The filtrate was loaded onto an Ni-NTA agarose column. The column was washed with binding buffer  and wash buffer . The recombinant protein was eluted with elution buffer .The expression vectors  were subcutaneously injected with 10 μg of each recombinant protein  mixed with Freund’s incomplete adjuvant  twice at 2-week intervals. Mouse serum 21 days after the second immunization was used to analyze each recombinant protein or E. coli DH5α strain, E. coli K-12 BW25113 strain (WT), and E. coli deletion mutants were cultured at 37°C in LB broth. Bacteria were prepared at an OD600 of 0.0008 by Dulbecco's modified Eagle medium (DMEM) containing 4.5 g/L glucose and 0.2 μM Fe3+  and then were cultured at 37°C for 12 h under 5% CO2 conditions. ClearColi (E. coli BL21(DE3) mutant lacking the oligosaccharide chain of LPS; Lucigen, Buenos Aires, Argentina) was gifted from Sohkichi Matsumoto . Similarly, ClearColi bacteria were cultured at 37°C for 27 h under 5% CO2 conditions. After centrifugation at 2,000 × g for 30 min, the bacterial culture medium was collected as supernatant. After we centrifuged the bacterial culture medium at 10,000 × g at 4°C for 30 min, the supernatant was filtrated by a 0.2-μm filter (Kurabo). After we ultracentrifuged the filtrate (Ec-med) using a 50.2 Ti rotor (Beckman) at 100,000 × g at 4°C for 3 h, the supernatant (Ec-sup) was removed, and phosphate-buffered saline (PBS) was added in pellets. After we ultracentrifuged the filtrate at 100,000 × g at 4°C for 2 h, pellets (OMVs) were suspended by PBS.The g at 4°C for 3 h, pellets suspended by PBS were used as OMVs. Moreover, the OMVs were purified using an OptiPrep  density gradient. A discontinuous iodixanol gradient was prepared by diluting a stock solution of OptiPrep  with 10 mM Tris, pH 7.5, containing 0.25 M sucrose to generate 40%, 20%, 10%, and 5% (wt/vol) iodixanol solutions. The discontinuous iodixanol gradient was generated by sequentially layering 3.0 mL of 40% (wt/vol) iodixanol solutions, 3.0 mL of 20% (wt/vol) iodixanol solutions, 3.0 mL of 10% (wt/vol) iodixanol solutions, and 2.5 mL of 5% (wt/vol) iodixanol solutions in a centrifuge 344060 tube (Beckman). A 0.3-mL volume of OMVs in PBS was overlaid on discontinuous iodixanol gradient and ultracentrifuged using an SW40 Ti rotor (Beckman) at 160,000 × g at 4°C for 16 h. Twelve fractions of 1 mL each were collected from the top of the iodixanol solution and used for analysis such as Western blotting.After we ultracentrifuged the filtrate at 100,000 × OMV samples were applied to the carbon film TEM grids, which were created by carbon evaporation using VE-2020 . After staining with 1% uranyl acetate, samples were observed in a Talos F200C G2 (Thermo).6 cells) were cultured in 7 mL of DMEM containing 10% fetal bovine serum  and 100 units/mL penicillin and 100 μg/mL streptomycin . After preincubation for 24 h, the medium was changed to fresh DMEM without antibiotics. Bone marrow was flushed from the femur and tibia of C57BL/6 mice , and cells were plated in dishes with DMEM containing 10% fetal bovine serum (FBS), 4.5 g/L glucose, and 20% conditioned medium from the supernatants of L929 (LC14) fibroblasts secreting macrophage colony-stimulating factor. Bone marrow cells were differentiated into macrophages in 7 to 10 days and were then used in experiments  were spread in a 96-well plate and cultured for 24 h at 37°C. After E. coli-derived OMVs (0.143 μg/0.1 mL/well) were added to cells for 9 h, culture media were replaced with fresh DMEM medium containing antibiotics, and the cells were incubated for 1 h. After the medium was replaced again with fresh DMEM medium containing antibiotics, cells were incubated for 9 h. After the addition of CellTiterGlo  into each well, luciferase activity was measured as the amount of ATP by using a FilterMax F5 multimode microplate reader .RAW264.7 cells and mouse bone marrow-derived macrophages (BMDMs) (3 × 103 cells/well) were spread in a 96-well plate and cultured for 24 h at 37°C. After E. coli-derived OMVs (0.143 μg/0.1 mL/well) were added to cells for 9 h, culture media were replaced with fresh DMEM medium containing antibiotics, and the cells were incubated for 1 h. After replacement again with fresh DMEM medium containing antibiotics, cells were incubated for 9 h. After addition of cell counting kit-8 solution  into each well, absorbance (Abs) was measured for 4 h at 450 nm as the number of viable cells by using a FilterMax F5 multimode microplate reader . The ratio was calculated as [Abs(2h) − Abs(1h) of OMVs-treated cells]/[Abs(2h) − Abs(1h) of untreated cells] from absorbance of change for 1 h.RAW264.7 cells and mouse BMDMs  from Mφ, which were infected with live E. coli for 18 h, were incubated with or without 0.01% Triton X-100 at room temperature for 15 min. After incubating the samples with 150 μg/mL proteinase K (Thermo), 0.3 U/mL DNase, or 100 μg/mL RNase at 37°C for 30 min, these were added to the cells (1 × 105 cells/0.3 mL).The culture medium from donor cells was centrifuged at 2,000 × The exosomes prepared by ultracentrifugation were analyzed by using a NanoSight  to establish the concentration, size, and intensity. PBS used for dilutions was also measured by using NanoSight to further ensure that there was no contamination. Once the desired concentration of exosomes was reached, the sample was injected into the sample chamber of the NanoSight, and the particle size distribution was obtained through nanoparticle tracking analysis.Limulus amebocyte lysate (LAL) assay .The concentration of LPS in exosomes prepared by ultracentrifugation was analyzed by RAW264.7 cells were cultured by DMEM containing 10% FBS and antibiotics. After preincubation for 24 h, the medium was changed to fresh maintenance medium, and exosomes were added. For each experiment, the concentrations of exosomes in recipient cells are shown in figure legends. After incubation for 12 h, the cells were analyzed for the expression of mRNA by real-time PCR. After incubation for 24 h, the cells were analyzed for the expression of proteins by immunoblotting.l-phenylalanine chloromethyl ketone, 100 nM tosyl-l-lysyl-chloromethane hydrochloride, 150 mM NaCl, 0.05 M NaF, 25 mM β-glycerophosphate pentahydrate, 1 mM Na3VO4, and 1 tablet/10 mL of cOmplete Mini). The cell lysate was centrifuged at 5,000 × g for 5 min at 4°C, and the supernatants were used to analyze the expression of proteins by Western blotting. The bacteria were lysed by lysis buffer  and were completely disrupted by sonication. After centrifugation at 2,000 × g at 4°C for 30 min, the supernatants were used to analyze the expression of proteins by Western blotting. The samples were suspended in SDS buffer  and heated at 95°C for 5 min. Each sample was applied on 7.5 to 15% polyacrylamide gels and immunoblotted against iNOS , COX-2 , β-actin , integrin β1 , HSP90 , CD63 , GAPDH , CD9 , caspase-1 , cleaved caspase-1 , caspase-11 , gasdermin D , 6×His , OmpA , CirA , DegP , FepA , and OmpC  diluted in Can Get Signal or 0.5% skim milk. Blotted proteins were visualized with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or goat anti-mouse IgG antibody and Immobilon Western chemiluminescent HRP substrate. The amount of protein detected by some antibodies was measured using a computed image analysis system . ImageJ and CS Analyzer 3.0 (Atto) were used for image analysis.The cells were lysed by BugBuster  or radioimmunoprecipitation assay (RIPA) buffer  analysis. Real-time PCR was performed at 95°C for 5 min, followed by 45 cycles at 95°C for 10 s, 60°C for 10 s, and 72°C for 10 s. Primers are shown in Total RNA (0.5 μg), extracted from cells by using Isogen II according to the manufacturer’s instructions, was transcribed into cDNA with the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover in a total volume of 10 μL according to the manufacturer’s instructions. cDNAs were then used as the templates for PCR amplification using TB Green premix Ex Taq II with LightCycler 96 . Interpolated values for each sample were divided by the corresponding values for 18S rRNA as the housekeeping gene, and the results obtained were expressed as fold changes for a specific gene/18S rRNA using a threshold cycle  of at least three independent biological replicates. Comparisons among groups were performed using a one-way analysis of variance (ANOVA) followed by Tukey’s test or a two-way ANOVA followed by the Bonferroni test. Statistical analyses were performed with GraphPad Prism (Version 9.0). Differences were significant at a


Malania oleifera Chum is a unique renewable plant resource in China, its fruit oil has a high content of 15-tetracosenic acid, and it is a good raw material for the synthesis of cyclopentadecanone and cyclopentadecanolide. A Novel synthesis method of cyclopentadecanone and cyclopentadecanolide from vegetable oil was designed, and the synthesis yields of c cyclopentadecanone and cyclopentadecanolide were 38.5% and 63.0%, respectively. The effect of different ester groups on cyclization of α,ω-difatty acid alkyl ester in cyclopentadecanone synthesis, and effect of catalysts on cyclization ofω-hydroxycarboxylic acid triglyceride in cyclopentadecanolide synthesis were investigated. The designed synthesis strategy has the characteristics of less synthesis steps and high utilization efficiency of 15-tetracosenic acid, which has simple and economic.The online version contains supplementary material available at 10.1186/s13065-022-00840-y. Macrocyclic musk is an expensive flavor, it widely used in perfumes, cosmetics, food, and medicine. Cyclopentadecanolide of macrocyclic lactones and cyclopentadecanone of macrocyclic ketoneare two important macrocyclic musk , they coMalania oleifera Chun and S. K. Lee  is a wild woody plant, mainly distributed in Guangxi and Yunnan Province of China [Malania oleifera Chum. However, it is difficult to separate and purify 15-tetracosenoic acid from the fatty acids mixture [of China . Its fruof China   has been collected by the herbarium of Guangxi Institute of Botany, China, No.: IBK00373464. Malania oleifera Chum oil was extracted from fruit of Malania oleifera Chun harvested in Guangxi province of China. Plant species identified by Professor Lai Jiaye, botany expert . The consists of oils and fats 53.2%(w/w), in which the fats mainly contain 15-tetracosenic acid 46.7%, 9-octadecenoic acid 27.9%, and erucic acid 12.5%, other fatty acids 12.9%.Standard cyclopentadecanone was purchased from Aldrich Co. (USA), purity: 98.0%. Standard cyclopentadecanolide was purchased from Askrich Chemical Company Inc. (Japan), purity: 98%. In order to In order to further verify the correctness of the synthetic product, the spectra  of the synthetic product and the standard were compared. Hexane, Shanghai Adamas-β Co. (China), purity: 97.0%. Acetic acid, Shanghai Adamas-β Co. (China), purity: 99.5%.IR spectra were recorded by a SHIMAZHU FT-IR8400S spectrometer (Japan). Mass spectra were determined by SHIMAZHU GC-MS/QP5050A (Japan). NMR spectra were recorded by AVANCE III HD 600 NMR .Malania oleifera Chum. It included the crushed Malania oleifera Chum and 8 h Soxhlet reflux in petroleum ether of boiling range 60–80 ℃. Preparation of fatty acid methyl esters used a slightly modified method based on Simoneau and Vicario et al. [The extraction followed Soxhlet method using 50 g sample of o et al. , 32.− 1. Initial oven temperature was set at 150 ℃, held for 1 min, ramped at 4 ℃ min− 1 to 270 ℃ and held for further 5 min with heated capillary transfer line maintained at 270 ℃. Splitless injection was carried out at 270 ℃ and 0.2 µL of sample was injected. In the GC/MS full scan mode, m/z 40 to m/z 450 was recorded. Chromatographic peaks were identified with NIST mass spectral data library and the retention times were compared with standard compounds listed in NIST 2008 Mass Spectral Libraries V2.2  equipped with USA J&W Co. DB-1 column (30.0 m × 0.25 mm × 0.25 m) was used for compound identification. Helium was employed as carrier gas at a constant flow rate of 1.5 mL minMalania olceifera Chum oil (40.0 g) in hexane (300 mL) and acetic acid (90 mL) was ozonized at 0 ℃ for 4 h, and then H2O2  was added dropwisely for another 3 h period at room temperature. The reaction mixture was added to ice water, filtered and washed with water to obtain solid. The solid was dried to give 30 g mixed products P1.A solution of P1 30 g, Methanol (270 g), and sulfuric acid (6 g) was mixed and refluxed for 4 h. The mixture was cooled, extracted with diethyl ether(100 mL 2 times), distilled to provide 30 g mixture of products P2.P2 30 g in xylene (40 mL) was added to pulverized sodium (10 g) in refluxing xylene (500 mL) under nitrogen during 1 h. The mixture was refluxed for 1 h. Then, 150 mL ethanol was added slowly to the reactor at 80 ℃, after cooling to room temperature. Acetic acid (100 mL) was added to reactor, followed by 150 mL water. The xylene solution was separated from reaction mixture, and then treated with water. Xylene was evaporated under reduced pressure. The residue was distilled to give 16.0 g acyloin.Hydrochloric acid (16 mL) was added to the mixture of crude acyloin (16.0 g) and Zn powder (4 g) during 1 h at 110 ℃. After the reaction maintained for another 30 min, the mixture was cooled to room temperature, extracted with benzene (200 mL), and washed with water.The mixture was distilled, separated by vacuum distillation (or vacuum distillation of glycerin as entrainer) and recrystalled with ethanol to obtain cyclopentadecanone 16.0 g with purity of 97.4%.A solution of Malania olceifera Chum oil (200.0 g) in hexane (200 mL) and ethanol (200 mL) was ozonized at 0 ℃ for 4 h, and then reactant was added dropwisely 50 g potassium borohydride was dissolved in 500 mL aqueous solution at 10 ℃ for 3 h. Then neutralize to neutrality with hydrochloric acid for 2 h, filtration, washing with water for 3 times, and drying to obtain ω- hydroxycarboxylic acid triglyceride 135 g.Put ω- hydroxycarboxylic acid triglyceride (5) (100 g) and catalyst into 1000 mL three bottles, then slowly add glycerol and heating up to make glycerol distillate at vacuum degree is < 755 mmHg, and mixture products 75 g was obtained when reaction is 40 h. The mixture was fractionating by distillation and recrystallized by ethanol, and cyclopentadecanolide 47 g were obtained.1H NMR  and 13C NMR , and compared with their standards.The structures of cyclopentadecanone and cyclopentadecanolide were determined by IR (KBr), GC-MS, Although there are many preparation methods for cyclopentadecanone , 19, the2)13COOMe or HOOC(CH2)13COOH. Cyclopentadecanolide and cyclopentadecanone have similar carbon structure. Its commercial synthesis method is based on 15-hydroxypentadecarboxylic acid [Malania oleifera Chum oil (1), and synthesized cyclopentadecanolide. The synthetic route is shown in Fig. Therefore, the most important difficulty in the synthesis of cyclopentanone is how to obtain raw material MeOOC is only about 10% from Malania oleifera Chum oil.Although on chemical synthesis, the preparation of mixed fats of is simple from Malania oleifera Chum oil (1) resources and shorten the synthesis route of cyclopentadecanone (4) and cyclopentadecanolide (7), we proposed a synthesis strategy, as shown in Fig. In order to make efficient use of Firstly, we conducted a typical synthesis method experiment, and the synthesis route is shown in Fig. Malania oleifera Chum oil is low, the yield of cyclopentadecanone is only 12.8%.Since the yield of 15-tetracosenic acid with a purity of more than 90% obtained from Then, we carried out the synthesis experiment according to the synthesis strategy Fig. a. It is Malania oleifera Chum oil is high.Table 1H NMR  and 13C NMR  are identical.Comparing the prepared cyclopentadecanone with the standard cyclopentadecanone, IR (KBr), GC-MS, − 1. 1H NMR  δ 2.43 , 1.65 , 1.43–1.32 , 1.31 . 13 C NMR  δ 212.81, 212.81, 42.10, 27.58, 26.78, 26.73, 26.66, 26.44, 26.30, 23.46. GC-MS (EI): m/z = 224.4 (M+).Cyclopentadecanone is white solid, m.p. 60–61 ℃. IR (KBr): 3411, 2928, 2855, 1710, 1459, 1446, 1408, 1367, 1286, 1260, 1215, 1210, 1152, 1126, 1078, 729, 720, 570 cmMalania oleifera Chum is a renewable plant resource, and using its oil to synthesize cyclopentadecanolide has good application value.When cyclopentadecanolide was synthesized according to the preparation method of Fig. In order to make efficient use of resources, we designed a synthesis strategy, in the synthesis strategy Fig.b, alkali3ONa/ NaOH, it is high compared with catalyst single NaOH or CH3ONa. Compared with the synthesis route in Fig. Table 1H NMR  and 13C NMR  are identical.Comparing the prepared cyclopentadecanolide with the standard cyclopentadecanolide, IR (KBr), GC-MS, − 1. 1H NMR  δ 4.18–4.11 , 2.38–2.30 , 1.65 , 1.49–1.39 , 1.35 . 13C NMR  δ 174.05, 63.98, 34.42, 28.38, 27.76, 27.13, 27.10, 26.90, 26.66, 26.34, 26.03, 25.92, 25.84, 25.11, 24.93. GC-MS (EI): m/z = 240.2 (M+).Cyclopentadecanolide is white solid, m.p. 36–37 ℃. IR (KBr): 3468, 2925, 2856, 2685, 1738, 1462, 1377, 1350, 1285, 1248, 1234, 1166, 1109, 1071, 1061, 1054, 1014, 963, 879, 723 cmMalania oleifera Chum oil of a renewable plant resource. A novel synthesis method of cyclopentadecanone and cyclopentadecanolide were developed. Preparation method of cyclopentadecanone is three steps process which consists of ozonization, oxidation and esterification, and total yield is 38.5% when in terms of 15-tetracosenic acid. The effects of ester groups on cyclopentadecanone were explored. Preparation method of cyclopentadecanolide is three steps process which consists of ozonization and reduction reaction, cyclization, separation and 63% yield of cyclopentadecanolide was obtained. The effect of catalysts on cyclization of ω-hydroxycarboxylic acid triglyceride was investigated. The synthesis strategy is a very short technological route, and it is easy to industrialize.Musk compounds cyclopentadecanone and cyclopentadecanolide were synthesized from Additional file 1: Appendix. Ms of cyclopentadecanolide and cyclopentadecanone.


Rhodococcus pyridinivorans BSRT1-1, from the soil in Thailand. However, this strain’s PHA synthase (phaCRp) gene has not yet been characterized. Thus, this study aims to synthesize PHA using a newly engineered bacterial strain, Cupriavidus necator PHB−4/pBBR_CnPro-RpphaC, which harbors the phaCRp from strain BSRT1-1, and characterize the properties of PHA for skin tissue engineering application. To the best of our knowledge, this is the first study on the characterization of the PhaC from R. pyridinivorans species. The results demonstrated that the expression of the phaCRp in C. necator PHB−4 had developed in PHA production up to 3.1 ± 0.3 g/L when using 10 g/L of crude palm kernel oil (CPKO) as a sole carbon source. Interestingly, the engineered strain produced a 3-hydroxybutyrate (3HB) with 2 mol% of 3-hydroxyhexanoate (3HHx) monomer without adding precursor substrates. In addition, the 70 L stirrer bioreactor improved P(3HB-co-2 mol% 3HHx) yield 1.4-fold over the flask scale without altering monomer composition. Furthermore, the characterization of copolymer properties showed that this copolymer is promising for skin tissue engineering applications.Polyhydroxyalkanoates (PHAs) are biodegradable polymers synthesized by certain bacteria and archaea with functions comparable to conventional plastics. Previously, our research group reported a newly PHA-producing bacterial strain,  Skin tissue engineering is a promising alternative to replacing conventional dressings and autologous skin transplants for wound-healing purposes . It aimsco-3-hydroxyhexanoate) [P(3HB-co-3HHx)] copolymer is one of the most attractive copolymers applied in biomedical applications, especially bone and skin tissue engineering applications , 0.062 g/L of CaCl2, 0.39 g/L of MgSO4, and 1 mL/L of trace element (TE) solution [4·7H2O, 2.4 g/L; FeSO4·7H2O, 15 g/L; MnSO4·H2O, 2.4 g/L; and CuSO4·5H2O, 0.48 g/L in 0.1 M HCl. The pH of MM was adjusted to 6.8 before sterilization. The carbon sources, precursors, urea, CaCl2, MgSO4, and TE solution were sterilized separately and added to the MM before bacterial culture inoculation.The mineral medium (MM) for PHA biosynthesis was comprised of 4.0 g/L of NaHsolution . The TE C. necator PHB−4/pBBR_CnPro-RpphaC was prepared by transferring three full loops of a single colony in 50 mL of NR medium containing kanamycin (50 µg/mL) and incubated at 30 °C with shaking at 200 rpm for 8 h. After that, the 3% v/v of prepared inoculum was transferred into the PHA production medium (MM) and incubated at 30 °C with 200 rpm for 48 h. The bacterial cells were collected by transferring the bacterial culture to a 50 mL centrifuge tube and centrifuged at 8000 rpm, 4 °C for 10 min. After that, the supernatant was discarded, and the cell pellets were washed with distilled water. Next, the cell pellets were transferred to a pre-weighed bijoux bottle, then frozen at −20 °C for overnight, and then lyophilized using a freeze dryer. Lastly, the lyophilized cells were weighed to calculate the DCW (g/L). The PHA content and monomer composition of PHA were analyzed by gas chromatography (GC) analysis [The inoculum of analysis .co-3HHx) yield by C. necator PHB−4/pBBR_CnPro-RpphaC. The seed culture was prepared in NR media supplemented with kanamycin (50 µg/mL). The bioreactor containing MM media was sterilized at 121 °C for 30 min, cooled, and then inoculated with 3% (v/v) of seed culture. Batch cultivation was performed at 30 °C with a controlled pH of 6.8. A pH controller maintained the culture media by adding 3M of NaOH or H3PO4. The aeration rate and agitation speed were fixed at 0.25 vvm and 200 rpm, respectively. The PHA content and cell biomass were analyzed at 12 h time intervals for 72 h of fermentation. The fermentation experiments were performed in triplicates, and average values were calculated.The fermentations were carried out in a 70 L stirred-tank bioreactor  with a working volume of 50 L to improve biomass and P at 30 °C. About 3 mg of the purified PHA was dissolved in 1 mL of deuterated chloroform (CDCl3) at a concentration of 25 mg/mL. Tetramethysilane was used as an internal chemical shift reference and NMR analysis was proceeded at 500 MHz with 64 scans. The spectra were calculated for each monomer by comparing with a previous publication.The Tg) and melting temperature (Tm) were observed from the DSC thermogram.The thermal characteristic of the produced polymer was determined using a DSC-60  instrument. The flow rate of nitrogen was fixed at 30 mL/min. Approximately 5 mg of purified PHA was filled into an aluminum pan and heated from 25 to 200 °C with a 15 °C/min heating rate. The heated samples were then maintained at 200 °C for 2 min and subjected to rapid quenching to −40 °C. They were then heated again from −40 to 200 °C at a 15 °C/min heating rate. The glass transition temperature  . About 1 mg purified polymer was dissolved in 1 mL chloroform and analyzed at a column temperature of 40 °C. Polystyrene standards were used to generate a calibration curve. Molecular weight was reported as polystyrene equivalents.The dry cell weight (DCW) of the bacterial cell was determined following the method described by Trakunjae et al. (2021) . The anav/v sulfuric acid and 85% v/v methanol). The tubes were heated at 100 °C for 140 min and then cooled at room temperature. After that, 1 mL of distilled water was added, then vortexed for 1 min to achieve the layers of phase separation. The lower layer, containing hydroxyacyl methyl esters, was transferred into the tube containing sodium sulfate anhydrous to eliminate residue water. Then, the 500 mL of hydroxyacyl methyl esters solution and 500 mL of 0.2% v/v of caprylic acid methyl ester (CME) solution were mixed in a GC vial. In this case, the CME solution was used as an internal standard. The sample was analyzed by GC2014  equipped with an AOC-20i auto-injector , Restek RTX-1 capillary column, and a flame ionization detector (FID). The injector temperature was set at 270 °C, and the temperature of column was set at 70 °C and slowly increased at a rate of 10 °C/min to 280 °C, while the detector temperature was established at 280 °C. Nitrogen was used as a carrier gas.The PHA content and monomers composition of the harvested cell were analyzed by gas chromatography (GC) according to the method described by Braunegg et al. (1978) . The 15–All experiments were performed in triplicates. The obtained experimental data are indicated as the mean value of the triplicate with standard error.RpphaC, a newly isolated PHA-producing strain, was obtained and used for the initial BLAST search. The nucleotide and amino acid length of RpphaC were 1755 bp and 584 amino acids, respectively. According to the BLASTn analysis, the RpphaC revealed significant similarity with the phaC of R. rodochrous ATCC BAA870 (CP032675.1) (97.60%), R. rodochrous EP4 (CP032221.1) (97.60%), R. pyridinivorans DNHP-S2 (CP088975.1) (95.20%), R. pyridinivorans YC-MTN (CP095851.1) (95.20%), and R. pyridinivorans SB3094 (CP006996.1) (94.85%), respectively. The determination of the taxonomic position of RpphaC was carried out by the construction of phylogenetic analysis to compare its amino acid sequence with that of other bacterial species. The RpphaC formed a coherent clade with strain R. pyridinivorans KG-16 (CAA47035.1) in the NJ phylogenetic tree (RpphaC formed a cluster with the phaC of R. pyridinivorans SB3094 (AHD20144.1), R. aetherivorans I24 (phaC1) (AED02507), and R. ruber (CAA47035.1), respectively.PhaC is the crucial enzyme for the synthesis of PHA, where the monomers are polymerized of PHA into PHA polymers. The substrate specificity of the PhaC determines the types of monomers incorporated into the PHA polymer ,33. In ttic tree . In addiMW) between 61 and 68 kDa [RpphaC is a consistent clade with the PhaC of those bacteria in class I PHA synthases , and palm oil, CPKO was found to be the best carbon source for cell growth and PHA biosynthesis by C. necator PHB−4/pBBR_CnPro-RpphaC  directly from palm oil has been reported by Budde et al. (2011) using recombinant Ralstonia eutropha strains expressing heterologous PhaCs from the bacterium R. aetherivorans I24 [R. aetherivorans I24 was extensively studied for PHA production among the Rhodococcus species. Budde et al. [co-3HHx) by R. eutropha (C. necator) Re2000 and Re2001, which were engineered bacterial strains containing RaphaC1 and RaphaC2, respectively. The results revealed that strains Re2000 and Re2001 produced 5.98 and 1.09 g/L of P(3HB-co-3HHx) incorporated with 1.1 ± 0.3 and 1.5 ± 0.2 mol% when using palm oil as a sole carbon source, respectively. The P(3HB-co-3HHx) production from strain Re2000 was slightly higher than in this study, in which C. necator PHB-4/pBBR_CnPro-RpphaC obtained 3.1 g/L of P(3HB-co-3HHx). However, this strain produced a molar fraction of 2 mol% 3HHX incorporated in P(3HB-co-3HHx), which was higher than those produced by strains Re2000 and Re2001.Plant oils have been reported as an excellent carbon source for PHA production by many bacterial strains. For example, the biosynthesis of P copolymer including high 3HHx monomer fraction by C. necator strain Re2160/pCB113 was studied using CPKO as a carbon substrate [co-3HHx) using the recombinant C. necator strain Re2058/pCB113 were reported by Muragan et al. (2016) [Furthermore, the production of P(3HB-ubstrate . In addi. (2016) . Therefoco-3HHx) production and improve the ability of C. necator PHB−4/pBBR_CnPro-RpphaC for synthesizing different types of PHA monomers. The 2 g/L of five different precursors, i.e., sodium 4-hydroxybutyrate, Ɣ–Butyrolactone, sodium valerate, 1,4-Butanediol, sodium heptanoate, and sodium hexanoate, were supplemented in the MM. The fermentations were carried out in a 250 mL flask at 30 °C for 48 h, using 10 g/L of CPKO as the primary carbon substrate. The results revealed that 3HHx monomers were successfully incorporated by C. necator PHB−4/pBBR_CnPro-RpphaC in all experimental runs  production by C. necator PHB−4/pBBR_CnPro-RpphaC was carried out with a batch cultivation in a 70 L stirred-tank bioreactor containing 50 L of MM. A total of 10 g/L of CPKO was applied as a sole carbon source. The temperature, pH, aeration rate, and agitation speed were fixed at 30 °C, 6.8 and 0.25 vvm, and 200 rpm, respectively. During the first 48 h of fermentation, the growth of C. necator PHB−4/pBBR_CnPro-RpphaC gradually increased, then remained constant until 60 h; after that, it slightly decreased at 72 h (The enhancement of P(3HB- at 72 h . Similar at 72 h .co-3HHx) was at 48 h when the biomass, PHA content, and 3HHx mol% were 7.7 ± 0.6 g/L, 56 ± 5 wt%DCW, and 2 mol%, respectively  from CO2 by engineered C. necator MF01∆B1/pBBP-ccrMeJ4a-emd. These strains can synthesize P(3HB-co-3HHx) containing 47.7 ± 6.2 mol% of 3HHx from fructose using a complete mineral medium and a substrate gas mixture (H2/O2/CO2). However, the composition of 3HHx in P(3HB-co-3HHx) was controlled to 11 mol%, which is more suitable for practical applications. Furthermore, Thinagaran and Sudesh [co-21 mol% 3HHx) using emulsified sludge palm oil by an engineered C. necator Re2058/pCB113. The 9.7 g/L of dried cells mass of C. necator Re2058/pCB113 with 74 wt% of PHA content was obtained [co-3HHx), which incorporated 16–27 mol% of 3HHx composition using different waste animal fats by C. necator Re2058/pCB113 [Previously, Tanaka et al.  reportedd Sudesh  have stuobtained . Further8/pCB113 .1H NMR was performed to confirm 3HB and 3HHx units in the polymer fraction of PHA copolymer synthesized by the strain C. necator PHB−4/pBBR_CnPro-RpphaC. 1H NMR spectrum of H4 and H6, which represent the C4 methylene groups and C6 methyl-group in the 3HHx unit, respectively, implying the actual formation of the P(3HB-co-3HHx) copolymer [The opolymer ,43. Howeco-2 mol% 3HHx) copolymers produced by C. necator PHB−4/pBBR_CnPro-RpphaC was analyzed by gel permeation chromatography (GPC). The weight-average molecular weight (Mw) of 6.27 × 105 Da was obtained with the polydispersity (Mw/Mn) of 1.70. Typically, the molecular weight of the PHA varies for several reasons, for instance, the availability of precursors for PHA synthesis, the type of PhaC, the expression level of PhaCs synthases, and the availability of enzymes that hydrolyze PHA [In this study, the molecular weight of the P(3HB-lyze PHA .co-3HHx), which has a high 3HHx monomer percentage, is typically significantly lower than that of P(3HB) homopolymer synthesized by wild-type C. necator H16. [The molecular weight of P(3HB-tor H16. co-3HHx) copolymer synthesized by C. necator PHB−4/pBBR_CnPro-RpphaC were analyzed using DSC. Defining the polymer thermal stability is essential for understanding the chemical recycling of polymer materials [Tm) and glass transition temperature (Tg) of P(3HB-co-3HHx) were 160.18 and 6.04 °C, respectively (co-3HHx) copolymers are commonly lesser than those of P(3HB). However, there was no significant correlation between these parameters and 3HHx molar fractions [Thermal properties of P are promising biomaterials for the application in tissue engineering because they are biodegradable, biocompatible, and low cytotoxic.PHAs have been reported for application for wound repair and skin tissue engineering applications ,49,50,51co-2 mol% 3HHx) copolymer was produced by a newly engineered strain of C. necator PHB−4/pBBR_CnPro-RpphaC, which possesses physical and chemical properties to be applied for wound repair or skin tissue engineering application.In this study, P(3HB-co-3HHx) and P(3HB)/P(3HB-co-4HB) copolymers in several ratios for fabricating the nanofiber 3D scaffolds and matrices cast solution for testing with human epidermal cells (keratinocytes) [co-5 mol% 3HHx), poly(3HB-co-7 mol% 4HB), and poly(3HB-co-97 mol% 4HB) copolymers [Previously, Li et al. (2008) studied the blending of P(3HB)/P(3HB-nocytes) . Investinocytes) . Furtherpolymers . They fopolymers .R. pyridinivorans species. The strain C. necator PHB−4/pBBR_CnPro-RpphaC, a newly engineered strain of C. necator PHB−4 with RpphaC. CPKO and sodium hexanoate were found to be the best carbon source and precursor for P(3HB-co-3HHx) copolymer production by this strain, respectively. Under fermentation conditions, this strain can accumulate 56 ± 5 wt%DCW of PHA containing 2 mol% of 3HHx and produce 7.7 ± 0.6 g/L of biomass using a 70 L stirred-tank bioreactor. Furthermore, the properties of the P(3HB-co-3HHx) copolymer have demonstrated that this copolymer is promising for skin tissue engineering applications.This is the first study on the characterization of PhaC of


C. felis, FeLV, FHV-1 and FCV were 103, 104, 103, 103, 103, 104, 104, 105 and 105 copies/µL, respectively. Moreover, the specificity of the two mPCRs was high. In summary, the two newly mPCR methods provide available, specific, sensitive and inexpensive tools for detecting pathogens.Coinfection with multiple pathogens is common among feline respiratory tract and intestinal diseases. In clinical cases, cats may exhibit similar symptoms between respiratory and intestinal disease, making it difficult to accurately identify and distinguish pathogens. This study aimed to establish two novel multiplex polymerase chain reactions (mPCRs) for simultaneous detection of pathogens associated with the feline respiratory and intestinal tracts. Regrading sensitivity, the detection limits for FeKoV, FPV, FeAstV, FCoV, IAV, Chlamydia felis (C. felis) and influenza A virus (IAV)). The results of sensitivity analysis revealed that the detection limits for FeKoV, FPV, FeAstV, FCoV, IAV, C. felis, FeLV, FHV-1 and FCV were 103, 104, 103, 103, 103, 104, 104, 105 and 105 copies/µL, respectively. Moreover, the specificity of the two mPCRs was high. When the two mPCRs were applied to clinical samples, the assay worked well. In conclusion, we established two mPCR methods that provide an excellent tool for the diagnosis and monitoring of pathogens associated with the feline respiratory and intestinal tracts.Respiratory tract and intestinal diseases are common threats to feline health. Coinfection with multiple pathogens is not rare among clinical infectious cases. Rapid diagnosis of these coinfections is of great significance for timely and effective clinical treatment. In this study, two novel multiplex polymerase chain reactions (mPCRs) were established for simultaneous detection of four pathogens associated with the feline intestinal tract (feline coronavirus (FCoV), feline astrovirus (FeAstV), feline panleukopenia virus (FPV) and feline kobuvirus (FeKoV)) and five pathogens associated with the respiratory tract , feline herpesvirus 1 (FHV-1), feline leukemia virus (FeLV),  The health problems associated with respiratory and intestinal tract infections are a great concern in cats. In clinical cases, cats are often infected by multiple pathogens and exhibit similar respiratory or intestinal symptoms, making it difficult to accurately identify and distinguish pathogens.Chlamydia felis (C. felis) [C. felis mainly causes pneumonia and conjunctivitis [It is a widely held view that feline upper respiratory tract disease (URTD) is an important cause of morbidity and mortality in kittens . URTD is. felis) . FCV oft. felis) . FHV-1 cctivitis ,5. In adctivitis ,9,10,11.ctivitis ,13. Diffctivitis . TherefoFeline panleukopenia virus (FPV) is a crucial pathogen that causes feline diarrhea . In addiC. felis and IAV) and four types of pathogens associated with intestinal tracts .Due to the complexity of pathogens associated with feline respiratory and intestinal tracts, rapid diagnosis and detection of these pathogens are important for disease prevention, epidemiological investigation and clinical treatment. Although there are many multiplex polymerase chain reaction (mPCR) methods for feline intestinal and respiratory pathogens, most of these methods can detect only between approximately two and four pathogens, and emerging pathogens are not covered ,24,25,26The genome of rabies virus was extracted from the Nobivac Rabies vaccine. Escherichia coli (ATCC-29522) and Salmonella enterica (ATCC-14028) are kept in our laboratory.C. felis, IAV, FCoV, FeAstV, FPV and FeKoV. Primers were designed based on conserved sequences using Primer 5.0 software . All specific primer pairs for mPCRs satisfied the following requirements: similar annealing temperatures; lack of primer dimer formation, hairpin loop formation or self-complementarity; and minimized degenerate bases. The primers  was used to compare conserved sequences of FCV, FHV-1, FeLV,  primers  were synThe RaPure viral RNA/DNA Kit  was used to extract nucleic acids from clinical samples. The extracted total nucleic acids were used for RNA reverse transcription (RT) using HiScriptⅡ1st Strand cDNA Synthesis Kit  and amplification. The reaction system volume of RT was 20 µL, consisting of 10 µL of 2 × RT Mix, 1 µL of random hexamers, 7 µL of total RNA and 2 µL of HiScript Ⅱ Enzyme Mix. Finally, the cDNA was stored at −20 °C.2O. The single PCR program was as follows: 98 °C for 3 min; 36 cycles of 98 °C for 20 s, 57 °C for 1 min, and 72 °C for 1 min 30 s; and 72 °C for 5 min. The products of PCR were detected with 2% agarose gel electrophoresis. The PCR amplification fragments of FCV (1940 bp), FHV-1 (1014 bp), FeLV (608 bp), C. felis (500 bp), IAV (155 bp), FCoV (726 bp), FeAstV (418 bp), FPV (337 bp) and FeKoV (198 bp) were ligated into the pMD 18-T vector . The ligation products were transformed and cultured, and then the positive plasmids were extracted. Additionally, the positive test plasmids were sent to the Beijing Genomics Institute  for sequencing.The reaction system volume of mPCR for PAIT and PAFR was 20 µL, including 10 µL of 2 × Taq Master Mix , 1 µL of template, 1 µL of the forward primer (10 mM), 1 µL of reverse primer (10 mM) and 7 µL of ddH10 copies/µL of the mixed template . The reaction system volume for PAFR was 30 µL, consisting of 15 µL of Taq PCR StarMix with Loading Dye , 1 µL of mixed forward primers (FCV-F (0.13 µL), FHV-1-F (0.10 µL), FeLV-F (0.24 µL), C.felis-F (0.19 µL) and IAV-F (0.34 µL)), 1 µL of mixed reverse primers (FCV-R (0.13 µL), FHV-1-R (0.10 µL), FeLV-R (0.24 µL), C.felis-R (0.19 µL) and IAV-R (0.34 µL)), 1 µL of 1010 copies of the mixed template  and 12 µL ddH2O. Both mPCR procedures were as follows: 98 °C for 3 min; 36 cycles of 98 °C for 20 s, gradient annealing temperature of 50~60 °C for 1 min and 72 °C for 1 min; and 72 °C for 5 min.To establish the mPCR methods, two mixtures of plasmid standards were used to verify the optimum annealing temperature and the ratio of each primer. The reaction system volume for PAIT was 20 µL, including 10 µL of 2× Taq Master Mix , 1 µL of mixed forward primers (FCoV-F(0.26 µL), FeAstV-F(0.16 µL), FPV-F(0.16 µL) and FeKoV-F(0.42 µL)), 1µL of mixed reverse primers (FCoV-R(0.26 µL), FeAstV-R (0.16 µL), FPV-R (0.16 µL) and FeKoV-R (0.42 µL)) and 1 µL of 10The products of mPCR (15 μL) were detected with 2% agarose gel electrophoresis.We used a feline kidney cell line  and other pathogens  as templates to verify the specificity of the mPCR methods. Single plasmids and mixed plasmids were also detected. Furthermore, the negative control for mPCR was empty pMD 18-T vector.10 copies/μL to 1 × 101 copies/μL. The diluted plasmids were subsequently used to verify the sensitivity of the mPCR methods.The plasmid mixtures and individual plasmids were diluted in a gradient from 1 × 10Twenty-three anal swabs and twenty nasal swabs from cats with respiratory or intestinal disease were collected with the consent of the cat owners. Clinical samples were kept at −80 °C prior to nucleic acids extraction. The template was 1 μL of cDNA obtained by RT. Eventually, each cDNA was used for clinical testing via the mPCR methods.C. felis and IAV  as templates to verify the specificity of the mPCR. No amplicons were observed in the lanes for the negative controls, Salmonella enterica, rabies virus, and Escherichia coli, while the lanes for FCoV, FeAstV, FPV and FeKoV A and FCV and IAV B showed 3  of 1326 feline clinical samples tested positive for at least one virus. The positivity rates for FeLV, FPV, FHV-1, FCV and FIPV were 59.6%, 19.2%, 16.3%, 14.2% and 0.5%, respectively . TherefoIn this study, we included FeLV and IAV in mPCR detection. FeLV is prevalent in cats worldwide. FeLV can be transmitted through oropharyngeal secretions . TherefoC. felis, IAV, FCoV, FeAstV, FPV and FeKoV). In this study, we firstly included FeLV and IAV in the mPCR method to provide detection tools for assessing their prevalence and coinfection with other pathogens. This study provides an efficient tool for clinical diagnosis and laboratory epidemiological monitoring.In summary, the two newly established mPCR methods provide available, specific, sensitive and inexpensive tools for detecting pathogens (FCV, FHV-1, FeLV,


Streptococcus pneumoniae, the rise of antimicrobial resistance and pneumococcal disease caused by strains not covered by the vaccine creates a need for developing novel antimicrobial strategies. N,N-dimethyldithiocarbamate (DMDC) was found to be a potent copper-dependent antimicrobial against several pathogens, including S. pneumoniae. Here, DMDCs efficacy against Streptococcal pathogens Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus anginosus was tested using bactericidal and inductively coupled plasma - optical emission spectrometry. After confirming DMDC as broad-spectrum streptococcal antimicrobial, DMDC was derivatized into five compounds. The derivatives’ effectiveness as copper chelators using DsRed2 and as copper-dependent antimicrobials against S. pneumoniae TIGR4 and tested in bactericidal and animal models. Two compounds, sodium N-benzyl-N-methyldithiocarbamate and sodium N-allyl-N-methyldithiocarbamate (herein “Compound 3” and “Compound 4”), were effective against TIGR4 and further, D39 and ATCC® 6303™ _(a type 3 capsular strain). Both Compound 3 and 4 increased the pneumococcal internal concentrations of copper to the same previously reported levels as with DMDC and copper treatment. However, in an in vivo murine pneumonia model, Compound 3, but not Compound 4, was effective in significantly decreasing the bacterial burden in the blood and lungs of S. pneumoniae-infected mice. These derivatives also had detrimental effects on the other streptococcal species. Collectively, derivatizing DMDC holds promise as potent bactericidal antibiotics against relevant streptococcal pathogens.Despite the availability of several vaccines against multiple disease-causing strains of  Streptococcus pneumoniae  is a Gram-positive bacterium that is a leading cause of pneumonia, otitis media, meningitis, and sepsis. As such, the pneumococcus causes a significant disease burden in the pediatric and elderly populations despite effective vaccines against the most common strains  approval for other indications  were investigated, with DMDC being identified as a potent copper-dependent antimicrobial against S. pneumoniae, Coccidioides immitis, and Schistosoma mansoni  has been used to treat alcohol dependence for decades, despite a significant decrease in clinical use over time. In the body, disulfiram is rapidly converted to its metabolite diethyldithiocarbamate (DETDC), which is complexed with divalent metal zinc and copper ions . Recentlarasites . In 2021arasites . In that mansoni . Since t mansoni .Mechanistically, DMDC significantly increases the intrabacterial concentration of copper, putting stress on the bacteria to export copper before mismetallation inactivates or reduces the efficiency of key bacterial enzymes, eventually leading to bacterial death . FurtherStreptococcus genus has many significant pathogens. Most notably are Streptococcus pyogenes , Streptococcus agalactiae  and Streptococcus anginosus   . While dnginosus , howeveralactiae .in vitro copper-dependent antimicrobial efficacy of these compounds (named “Compound 1” through “Compound 5”) against S. pneumoniae TIGR4 (a virulent strain), D39 (a virulent derivative of the Avery strain), and Type 3 (a hyper-encapsulated strain). The zinc-dependent toxicity of these compounds was also tested against TIGR4 and found to be bacteriostatic. Next, the in vivo efficacy of these compounds was tested in a murine pneumonia model demonstrating that they reduced the bacterial burden in the lungs. Finally, the ability of these compounds to enhance in vitro macrophage-mediated killing was tested, finding the combination of Compound 3 and copper to be cleared more efficiently. Overall, this study identifies novel compounds in the dithiocarbamate class that are broadly anti-streptococcal in vitro and effective in vivo.In this study, DMDC was successfully able to kill different Streptococcal species and because of this, dithiocarbamate compounds were then derived from the starting compound, DMDC. Derivatives tested the 2  were added to the solution successively. The resulting mixture was stirred at room temperature for 2 h, and then the solvent was reduced under vacuum. The first compound, Compound 1 , was synthesized. 1H NMR  δ 4.34–4.22 , 3.51–3.39 . 13C NMR  δ 215.02, 66.58, 50.01.The general synthetic procedure used was with a solution of KOH  in EtOH  was cooled to 0°C, then morpholine  and CSp-tolyl)piperazine-1-carbodithioate, LRS01-084) 1H NMR  δ 4.20 . 13C NMR  δ 214.14, 49.51.Following the general procedure, the title compound was synthesized in 57% as a white solid to Compound 2  1H NMR  δ 6.98 , 6.80 , 4.41 , 2.96 , 2.16 . 13C NMR  δ 214.73, 149.41, 129.78, 127.97, 116.15, 49.25, 49.09, 20.49.Following the general procedure using NaOH  as a base, the title compound was synthesized in 83% as a light yellow solid to synthesize Compound 3 . 1H NMR  δ 7.28–7.19 , 7.19–7.12 , 5.43 , 3.24 . 13C NMR  δ 215.70, 139.58, 128.42, 127.66, 126.75, 57.85, 40.99.Following the general procedure using NaOH as a base, the title compound was synthesized in 73% as a white solid for Compound 4 -1-ethoxy-3-methyl-1-oxopentan-2-yl) carbamodithioate, LRS01-072). 1H NMR  δ 5.83–5.70 , 5.08–5.02 , 5.02–4.97 , 4.73 , 3.25 . 13C NMR  δ 214.76, 135.06, 116.16, 57.57, 40.88.Following the general procedure using NaOH as a base, the title compound was synthesized in 56% as a white solid to Compound 5  Smith and Sherman emend. Whiley and Beighton 33,397™. For S. pneumoniae TIGR4, D39, and Type 3, S. agalactiae ATCC 13813, and S. anginosus M17 media  was prepared according to the manufacturer’s instructions. Briefly, 37.25 g of powder was suspended in 950 ml of Milli-Q grade water (≥18.0 MΩ cm−1) and autoclaved at 121°C for 15 min before cooling to 50°C and adding 50 ml of a sterile 10% lactose  solution. For S. pyogenes, the culture medium was grown in Difco Todd Hewitt Broth (ThyB). For plating, Tryptic Soy Agar  was dissolved in Milli-Q water and autoclaved. After cooling autoclaved TSA, 5% defibrillated sheep’s blood (HemoStat Laboratories) of final volume and 20 μg/ml neomycin  was added to the solution (for S. pneumoniae TIGR4 only). These plates (blood agar plates—BAP) were used for routine culture on solid media and “killing curve” serial dilution CFU counting. Bacteria from freshly streaked plates were placed into M17 and grown at 37°C in 5% CO2, to an optical density (OD or OD600) of 0.125 for growth curve assays and to an OD of ~0.300 for killing curve assays. To prepare working stocks, growing cultures are resuspended in fresh media +20% v/v glycerol and stored at –80°C. Aliquot viability and CFU counts were determined before use in experiments. Glycerol stock aliquots were diluted 1:5 into M17 with indicated copper and compound concentrations for assays.Strains used here are 4 pentahydrate  in Milli-Q water. Stock solutions of 100 mM Zn2+ were prepared from ZnSO4 heptahydrate . Stock solutions of 1 mM Compound compounds were prepared in Milli-Q water from solute derived and purified by the University of Arizona Center for Drug Discovery (ACDD). Sterile, individually wrapped clear 96 well polystyrene plates  were arranged to test a range of concentrations combinations of Cu2+, Zn2+, and compounds. Frozen aliquots of bacteria were thawed and diluted five-fold into fresh M17 before adding 20 μl per well into a total well volume of 200 μl . Assay plates were loaded into a Biotek Cytation5  pre-equilibrated to 37°C and 4% CO2. Gas control settings were modified for an elevation of 720 m according to the manufacturer’s directions. The protocol-maintained temperature and CO2, while measuring OD absorbance at 600 nm every 30 min for 16–20 h.Growth curve assay as described in 4, Compound compounds, Zn2+, and Mn2+ . After exposure to the indicated conditions, bacteria were incubated at 37°C in 5% CO2 for the stated time. Samples were serially diluted, plated on BAP, incubated overnight at 37°C in 5% CO2, and counted to determine viable CFU. Colonies on each plate were counted and multiplied by the appropriate dilution factor based on which dilution it was to determine CFU/ml. For plates in which no colonies were visualized, this was deemed to be below the limit of detection (LoD) and is noted with a data point below the LoD line.Killing curve assay as described in via an affinity nickel column and buffer exchanged via a desalting column. The fractions were concentrated using a 10,000 mw CO (Millipore). The protein was left to mature overnight at room temperature. Concentration was calculated by sequenced and absorbance at 280 nm. Purity was assessed by 12% SDS PAGE. The protein was stored at 4°C and used within 2 weeks.As performed previously  BL21 cel4). In a Biotek Cytation5, the absorbance at 585 nm was monitored for 3 h to establish chelation of copper by the compounds and restore DsRed2 fluorescence.A relative affinity test was performed using the reversible fluorescence quenching of DsRed2 by copper . The fol2 until an OD of ~0.400 was reached. The culture was split into the indicated treatments and control. Incubation with treatments was performed at 37°C and 5% CO2 for 30 min. Samples were quenched in a −3°C water bath to slow metabolism, followed by two washes of cold buffer , and centrifugation 3,500 ×g for 10 min at 4°C. Cold decanted samples were resuspended in 70% HNO3, followed by overnight incubation at 65°C. After incubation, the samples were diluted to 2.5% HNO3 using Milli-Q water at 18.1 MΩ. Bacterial plate counts were performed in TSA + 5% Sheep’s Blood through serial dilutions, as described above. Samples were analyzed for metal content using an iCAP PRO XDUO ICP-OES with a wavelength 324.8 nm copper. Standards were made using the iCap Series Multi-element test solution ICAP 6000 series Validator from Thermo scientific, and metal content of the washed samples was calculated using the Qtegra software. Experiments were performed in triplicate.To measure the internal metal concentration of the bacteria, strains were initially cultured on M17 + 5 mM lactose and frozen at −80°C in 20% glycerol. These glycerol stocks were used as the seed stock to inoculate 150 ml of M17 + 5 mM lactose. The bacterial culture was incubated at 37°C under 5% CO7 CFU viable S. pneumoniae in 25 μl of Tris-buffered saline . Cohort controls were given 25 μl of TBS. At 4-, 8-, or 24-h post-infection, mice were treated with doses of intranasal Compound 3 or Compound 4 (approximately 1.6 mg/kg) in 25 μl of TBS. Mice were sacrificed by CO2 asphyxiation and immediately dissected for lung and blood collection 48 h post-infection. Lung tissue was collected into 1.5 ml tubes containing 500 ml of phosphate-buffered saline  after a brief initial wash in 500 μl of PBS to remove any excess blood during dissection. The tissue was then homogenized and centrifuged for 30 s at 400 ×g. Blood samples (5 μl volume) were placed in a 45-μl volume PBS solution with heparin (10 UI/ml). Both lung and blood samples were then serially diluted at 1: 10 and plated on blood agar plates and incubated overnight at 37°C and 5% CO2 for growth. The resulting bacterial colonies were counted for quantification and comparison.All mouse studies were conducted with prior approval and under the guidelines of the IACUC at the University of Arizona  as previously performed , 2022. A2 incubator with Dulbecco’s Modified Eagle’s Medium  containing fetal bovine serum , glutamine , penicillin , streptomycin , and NaHCO3 (0.015%). Cells were grown to 90% confluence in 12-well tissue culture plates  in 1 ml/well of growth medium. On the morning of the experiment, macrophages were washed twice with 1 ml PBS, and resuspended in 1 ml of “Serum-Free DMEM” growth medium without antibiotics, glutamine, NaHCO3 or FBS but supplemented with 5 ng/ml IFN-γ , 400 ng/ml LPS  for “priming of macrophages” experiment. For “post hoc killing efficiency” experiment, macrophages were resuspended in 1 ml of Serum-Free DMEM containing 32 μM Compound 3, 5 ng/ml IFN-γ, and 400 ng/ml LPS.J774A.1 macrophages  were maintained in a 37°C, 5% CO2 for 12 h. Glycerol stocks of TIGR4 S. pneumoniae kept at OD 0.3 are removed from-80°C storage, diluted into four 15 ml conical tubes of 5 ml total M17 + Lactose containing no additives (“Untreated”), 32 μM Compound 3, 250 μM CuSO4, and 250 μM CuSO4 + 32 μM Compound 3, respectively, for pre-treatment of bacteria experiment. Prior to incubation of bacteria in 37°C, 5% CO2, an inoculum plate is made by serial diluting 100 μl from the no additives conical. After 15 min of incubation, bacteria are centrifuged at 4500 ×g for 10 min and resuspended in DMEM without antibiotics, glutamine, NaHCO3 or FBS. Macrophages are removed from incubation, media is removed, washed with 1 ml PBS twice and then infected with 100 μl of S. pneumoniae solutions for both experiment types, corresponding to a multiplicity of infection (MOI) of 10 bacteria per macrophage. The 12-well tissue culture plates were centrifuged at 200 ×g for 2 min to facilitate co-culturing.Macrophages were incubated at 37°C, 5% CO2 incubator in DMEM containing gentamicin (50 μg/ml). Macrophages were lysed in 0.02% SDS by pipetting up and down in ddH2O and serially diluted to determine the counts of viable intracellular bacteria. Data were normalized to the level of killing observed for the untreated TIGR4 bacteria for each assay.Wells were then washed twice with PBS at the given time-points ; each wash was followed by a 5-min incubation in a 37°C, 5% COt-test , two-way ANOVA, or one-way ANOVA and Dunnett’s multiple comparisons test . The p-values were as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.Statistical significance was analyzed using Student S. pneumoniae TIGR4, D39 and Type 3  after 4 h with S. pyogenes . Compound 3 and Compound 4 showed statistically significant growth defects for the combination treatments in comparison to an untreated control in TIGR4 S. pneumoniae  of S. pneumoniae TIGR4, then at 8 h later were given Compound 3 or 4. Then, the bacterial burden was measured after 48 h. A significant decrease in bacterial titers in the blood and lungs 48 h post-infection in 8-week-old BALB/c mice given 25 μl of 10 mM Compound 3 was observed  and a serotype type 3 strain that is highly encapsulated . These strains offer a small by representative sample of the more than 100 serotypes that exist for the pneumococcus. Compound 3 and Compound 4 were bactericidal in combination with copper against the D39 and the Type 3 strain, and more so  than the effects of DMDC versus these strains as seen previously . DMDC was previously identified as a potent copper-dependent antibiotic against S. pneumoniae, S. aureus, C. immitis, and S. mansoni, and here, was effective against other pathogenic streptococcal species   . Compounnginosus , 8. It win vivo administration of dithiocarbamates such as Compound 3 and DMDC may be able to mimic the in vitro copper-dependent antimicrobial effect. Further investigation into the dithiocarbamates mechanism (s) of action are areas of active investigation.It has been shown in previous studies utilizing murine models of infection that the concentration of copper increases within the blood and lungs up to four-fold . Contrib2 group characteristic of dithiocarbamates facilitates complex formation with transition metal cations and enzyme inhibitory activity  make additional derivatives, (2) use these derivatives against other pathogenic bacteria, and (3) determine the rules that govern the efficacy of a given ionophore. Point 3 can likely be accomplished by understanding the metal flux of copper into different organisms as a function of the ionophores’ capacity to chelate copper and the homology of metal importers that could be functioning to take in the complex.S. pneumoniae and pathogenic Streptococcal species. In addition, it was shown that both of those compounds exhibit zinc-dependent toxicity, and one of the compounds, Compound 3, exhibits potent in vivo efficacy as an antibiotic for aiding murine S. pneumoniae clearance. Further investigation into dithiocarbamate derivatives warrants further study for developing novel antimicrobials in the fight against antimicrobial-resistant pathogens.Overall, data here supports that from a small-molecule screen of compounds derived from DMDC, that Compound 3 and Compound 4 are potent copper-dependent antibiotics against several strains of The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by IACUC at the University of Arizona.MJ, SM, and YS-R contributed to the conception and design of the study. SM, YS-R, CP, HO’B, KO, MD, NH, and RH provided experimental design. RL, FG, and WW provided compound design and quality control. MJ and SM wrote the manuscript. All authors contributed to the article and approved the submitted version.http://n2t.net/addgene:54608; RRID:Addgene_54608).This study was supported by NIH grant 1R35128653. Additionally, SM’s training was supported by an F30 Ruth L. Kirschstein Individual Predoctoral NRSA Fellowship from NIGMS (5F30GM139246-02). DsRed2-pBAD was a gift to MJ (Addgene plasmid # 54608; The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.