Progress 03/15/20 to 03/14/25
Outputs
Target Audience:The target audience includes scientists (faculty, postdocs, students) in nutrition and biomedical science, officials form federal agencies, policy makers, dairy industry, journalists, and lay public. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided support for two faculty members, Dr. J. Zempleni (PD) and J. Auchtung (Co-PD) and a postdoctoral associate (Dr. Shu Wang). Dr. Zempleni has acquired new knowledge in studies of microbiology. Dr. Auchtung has acquired new knowledge in milk exosomes nutrition. The postdoctoral associate was new to microbiology and has acquired expertise in this area. How have the results been disseminated to communities of interest?The new knowledge generated in this project has been disseminated to target audiences through publications in peer-reviewed journals, invited seminars, and presentations at local, national, and international conferences (see Products and Other Products). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1. We demonstrated that milk exosomes selected a deletion in the phosphotransferase domain in the ptsG gene ("ptsG _indel"). The ptsG gene facilitates glucose transport in bacteria and is implicated in pathogenicity C. difficile. Exosome-selected C. difficile grew faster and produced less toxin than non-selected controls. Findings were corroborated in ptsG and ptsG_indel overexpression studies. C. difficile in milk exosome-containing media grew 15% to 20% faster than controls cultured in exosome-free media. The production of toxins, tcdA and tcdB, was 60% to 90% lower in the various milk exosome (ME)-containing cultures of C. difficile compared to exosome-free control cultures. We observed that overexpression of ptsG_indel increased growth and reduced toxin production in C. difficile. This observation is consistent with our hypothesis that MEs select for polymorphisms that decrease the pathogenicity of gut pathogens. We discovered that MEs selected a mutation in the DNA binding domain in the catabolite control protein A (ccpA) gene in E. faecalis. The ccpA gene is a transcriptional regulator of carbon catabolite repression and carbon catabolite activation, which ensures optimal energy usage under diverse conditions. Wild-type E. faecalis grew slower with an increased generation time when cultured in ME-free compared to ME-containing media. Conclusion Collectively, we demonstrated that MEs 1) promote the growth of commensal gut pathogens ex vivo and 2) select mutations in genes implicated in carbohydrate metabolism bacterial virulence. Objective 2. Preface The studies depend on feeding ME-defined diets. Briefly, we have developed rodent diets defined by their content of MEs and the RNA content. The diets are based on the AIN-93G formulation. The exosome and RNA-sufficient (ERS) diet contains a nutritionally relevant dose of MEs and RNA cargo (equivalent to 0.5 L milk consumed by an adult), whereas the content of RNA and exosomes is decreased by 8% and up to 99%, respectively, in the exosome and RNA-deficient (ERD) diet. Al other content is identical in ERS and ERD diets. Recap previous reporting periods In Years 1 and 2, we fed C57BL/6 mice ERD and ERS diets for four weeks starting at age 3.5 to 4.5 weeks and challenged the mice with an oral dose of 105 spores from C. difficile. Survival of mice fed the ERD diet decreased by approximately 50% compared to mice fed the ERS diet (P < 0.05). Consistent with this observation, the median clinical sickness scores (B. Fehlhaber et al, Sci. Rep. 2019; 9: 5919; PMC6459866).in mice fed an ERD diet were 3-fold higher than mice fed the ERS (P < 0.01). We assessed bacterial communities in mice fed ERD and ERS diets with C. difficile. challenge) by using 16S rRNA sequencing. We observed significant changes in a small number of taxa between treatment groups on day 3 followingC. difficilechallenge. ERS feeding resulted in an increase ofLachnospiraceae, Bacteroides(5operational taxonomic units, OTUs) andAlistipes(2OTUs), whereas ERD resulted in an increase ofAkkermansia,Escherichia/Shigella,Enterococcaceae, andClostridiumsensustricto. Since Escherichia/ShigellaandEnterococcaceae are associated with increased disease severity, we concluded that the protective effects of ERS diet on the host response might be due to the selection protective taxa and decrease of pathobionts. We observed that myeloperoxidase activity, a marker of neutrophil infiltration was significantly higher in mice fed an ERD diet on day 7 of infection compared to mice fed an ERS diet, pointing to higher levels of inflammation in the absence of milk exosomes. We conducted additional feeding studies and challenged the mice with C.difficile. Seven days post C. difficile, the myeloperoxidase (MPO) activity, which was used to assess the neutrophil/granulocyte infiltration, was higher in cecum and colon from ERD-fed mice than that from ERS-fed mice; IL-17b was also higher in the colon from ERD-fed mice compared to ERS-fed mice. This reporting period (no-cost extension) We investigated the effects of dietary bovine MEs on bacterial translocation and immune responses in a mouse model of Enterococcus faecalis infection. Mice fed a ME-sufficient diet for 4 weeks and treated with cyclophosphamide and a combination of antibiotics (metronidazole, kanamycin and vancomycin). Mice were challenged with 1) wild-type (reference strain), 2) ME-selected, or 3) vehicle (solvent, PBS)-selected E. faecalis strains. Quantitative qPCR analysis using universal 16S rRNA primers revealed no significant differences in bacterial translocation to the spleen among the groups. However, a 2.3-fold reduction in bacterial levels in the liver was observed for the ME-selected E. faecalis strain compared to the wild-type strain (p = 0.0445). Furthermore, we assessed inflammatory responses by quantifying four key markers (TNF-alpha, IL-10, IL-1beta, and MIP-2) associated with E. faecalis infections and mouse immune responses in the cecum. No significant differences in the levels of these markers were detected between treatment groups. These findings suggest that while ME selection may have a minor impact on bacterial localization to the liver, it does not significantly influence systemic bacterial translocation or local inflammatory responses in this model. Conclusion Data are consistent with our proposal's hypotheses that MEs 1) alter bacterial communities in the gut, and 2) enhance the colonization with protective taxa, and 3) have a beneficial effect on hosts challenged with C. difficile, whereas beneficial effects are comparably minor, and limited to reduced bacterial translocation, in E. faecalis. Objective 3. Please see Objective 3. In addition to ERS and ERD diets, we fed mice with AIN-93G. The mice were challenged with C. difficile and pathogenicity was assessed by monitoring the mortality, weight loss over the time, and measuring the C.difficile CFU and toxin activity present in the fecal samples. Results were inconclusive and the feeding study was repeated in Year 3 to increase the sample size. We demonstrated that the minor content of lactose in experimental diets is not a confounder in studies of mice (which are lactose intolerant; see the manuscript by Sukreet et al. in Products). We fed C57/BL6 mice on ERS and ERD diets and challenged them with C. difficile that were selected in the absence or presence of MEs. We observed that when mice fed the ERS diet, mortality was lower in mice challenged with C. difficile grown in the presence of MEs compared to mice C. difficile grown in the absence of MEs (10% versus 22% seven days after the challenge). Weight loss was also lower in mice challenged with C. difficile selected by MEs than in C. difficile not selected by MEs (3.5% versus 11.4% six days after challenge). Conclusion Collectively, our studies to date are consistent with our proposal's hypothesis that a ME-depleted diet (ERD) increases the susceptibility of mice to challenge with C. difficile. The AIN-93G diet did not increase the susceptibility. We speculate that casein in AIN-93G provided contains MEs. Casein is not present in the ERD diet. Beneficial effects are comparably minor, and limited to reduced bacterial translocation, in E. faecalis.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2025
Citation:
Zhou F, Mumtaz PT, Dogan H, Madadjim R, Cui J, Zempleni J. Divergence of gut bacteria through the selection of genomic variants implicated in the metabolism of sugars, amino acids, and purines by small extracellular vesicles in milk. Gut Microbes 17 (1):2449704, 2025
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Setayesh L, Zempleni J. Preserving milk exosome integrity during storage: strategies for minimizing loss. University of Nebraska, Student Research Days, Lincoln, NE, March 27, 2024
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Zempleni J. Milk exosome-driven evolution of antibiotic-resistant gut pathogens. NIFA grantee meeting, Human Health Program (A1343), Purdue University, Lafayette, IN, May 23, 2024
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Zempleni J. Milk exosomes. USDA W-5002 Multistate Group meeting, UC-Davis, CA, May 29, 2024 [talk]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Setayesh L, Zempleni J. Minimizing the loss of extracellular vesicles in human milk. Nutrition 2024 Conference, American Society for Nutrition; Chicago, IL, 6/29 7/2, 2024
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Zaman BW B, Madajim R, Cui J, Zempleni J. Effects of Dairy Consumption on Gene Expression in Gut Bacteria in Adults. Nutrition 2024 Conference, American Society for Nutrition; Chicago, IL, 6/29 7/2, 2024
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Zaman Wahid B, Madadjim R, Cui J, Zempleni J. Effects of dairy consumption on gene expression in gut bacteria in adults. University of Nebraska, University of Nebraska-Lincoln, NPOD 10th Annual Fall Research Symposium, October 8, 2024 [poster]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Mumtaz PT, Zempleni J. Bifidobacterium infantis extracellular vesicles: composition and potential to modulate host biological processes via cross-kingdom communication. University of Nebraska-Lincoln, NPOD 10th Annual Fall Research Symposium, October 8, 2024
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Zempleni J. Milk exosomes in nutrition and drug delivery. Presentation to the Manitoba Interdisciplinary Lactation Centre. February 12, 2025 [invited oral presentation; zoom]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Zaman Wahid B, Madadjim R, Cui J, Zempleni J. Effects of dairy consumption on gene expression in gut bacteria in adults. UNL Research Days, University of Nebraska-Lincoln; Lincoln, NE, March 27, 2024
|
Progress 03/15/23 to 03/14/24
Outputs
Target Audience:The target audience includes scientists (faculty, postdocs, students) in nutrition, biomedical science, policy makers, dairy industry, journalists and lay public. Changes/Problems:The postoc conducting the hands-on work unexpectedly left the lab. WE had to replace her with another postdoc who had to undergo some training. What opportunities for training and professional development has the project provided?The project provides support for two faculty, Drs. J. Zempleni (PD) and J. Auchtung (co-PD), a postdoctoral associate (Dr. S. Wang, replaced with Dr. Qamar Taban in Year 4) and a research technologist. Dr. Zempleni has acquired new knowledge in studies of microbiology, whereas Dr. Auchtung has acquired new knowledge in nutrition research, dairy and milk exosomes. The postdoctoral associate was new to both microbiology and milk exosome research and has acquired expertise in both areas. The technologist was new to milk exosome research and has acquired technical skills in that area. The postdoctoral associate present and Dr. Zempleni findings from this study at scientific conferences though invited seminars and presentations at scientific conferences (see Products). How have the results been disseminated to communities of interest?Results have been disseminated through invited seminars and presentations at scientific conferences, and websites and blogs (see Products and Other Products). What do you plan to do during the next reporting period to accomplish the goals?1. Complete the bioinformatics analysis of RNA-sequencing analysis data to assess ME-dependent gene expression pathways in bacteria. 2. Assess whether a modified stud protocol leads to a stronger phenotype of mutations in E. faecalis than what we have observed in studies completed to date. 3. Manuscript submissions.
Impacts
What was accomplished under these goals?
Objective 1. Recap previous reporting periods Previously, we reported that milk exosomes selected a deletion in the phosphotransferase (PTS_EIIA_1) domain in the ptsG gene ("ptsG _indel"). The ptsG gene facilitates glucose transport in bacteria and is implicated in pathogenicity C. difficile. Exosome-selected C. difficile grew faster and produced less toxin than non-selected controls. C. difficile in milk exosome-containing media grew 15% to 20% faster than controls cultured in exosome-free media. The production of toxins, tcdA and tcdB, was 60% to 90% lower in the various milk exosome (ME)-containing cultures of C. difficile compared to exosome-free control cultures. Toxin production was assessed by using real-time PCR and by using cytotoxic effects in Vero cells (A. Valdiviesco-Garcia et al., 1993; 59: 1981-1983). We have developed strains of C. difficile that overexpress wild-type ptsG or ptsG_indel, and we are working towards developing ptsG knockout C. difficile. Using these strains, we started interrogating effects of ptsG overexpression on growth and toxin production. We observed that overexpression of ptsG_indel increased growth and reduced toxin production in C. difficile. This observation is consistent with our hypothesis that MEs select for polymorphisms that decrease the pathogenicity of gut pathogens. This reporting period In this reporting period we discovered that MEs selected a mutation in the DNA binding domain in the catabolite control protein A (ccpA) gene in E. faecalis. The ccpA gene is a transcriptional regulator of carbon catabolite repression and carbon catabolite activation, which ensures optimal energy usage under diverse conditions. Wild-type E. faecalis grew slower with an increased generation time when cultured in ME-free compared to ME-containing media. When E. faecalis was cultured in ME-defined media prior to transfer and continued culturing in minimal media, the polymorphisms and mutations selected in ME-containing media led to a slower growth in minimal media compared to polymorphisms and mutations selected in ME-free media We observed that E. faecalis that were cultured in ME-supplemented media grew faster and had a shortened generation time compared with E. faecalis that were cultured in ME-free media. We further tested the biofilm production by these E. faecalis after selection. (Biofilm production is associated with virulence and antimicrobial resistance.) We are in the process of completing the bioinformatics analysis of RNA-sequencing analysis data to assess ME-dependent gene expression pathways in bacteria. Interim conclusion Collectively, our studies to date are consistent with our proposal's hypotheses that MEs 1) select polymorphisms and mutations in gut bacteria, and 2) ME-driven selection decreases the virulence of gut pathogens. Objective 2. Preface The studies depend on feeding ME-defined diets. Briefly, we have developed [and published (Sukreet et al, se Products)] rodent diets defined by their content of MEs and the RNA content. The diets a re based on the AIN-93G formulation. The exosome and RNA-sufficient (ERS) diet contains a nutritionally relevant dose of MEs and RNA cargo (equivalent to 0.5 L milk consumed by an adult), whereas the the content of RNA and exosomes is decreased by 8% and up to 99%, respectively, in the exosome and RNA-deficient (ERD) diet. Al other content is identical in ERS and ERD diets. Recap previous reporting periods In Years 1 and 2, we fed C57BL/6 mice ERD and ERS diets for four weeks starting at age 3.5 to 4.5 weeks and challenged the mice with an oral dose of 105 spores from C. difficile. Survival of mice fed the ERD diet decreased by approximately 50% compared to mice fed the ERS diet (P < 0.05). Consistent with this observation, the median clinical sickness scores (B. Fehlhaber et al, Sci. Rep. 2019; 9: 5919; PMC6459866).in mice fed an ERD diet were 3-fold higher than mice fed the ERS (P < 0.01). We assessed bacterial communities in mice fed ERD and ERS diets with C. difficile. challenge) by using 16S rRNA sequencing. We observed significant changes in a small number of taxa between treatment groups on day 3 followingC. difficilechallenge. ERS feeding resulted in an increase ofLachnospiraceae, Bacteroides(5operational taxonomic units, OTUs) andAlistipes(2OTUs), whereas ERD resulted in an increase ofAkkermansia,Escherichia/Shigella,Enterococcaceae, andClostridiumsensustricto. Since Escherichia/ShigellaandEnterococcaceae are associated with increased disease severity, we concluded that the protective effects of ERS diet on the host response might be due to the selection protective taxa and decrease of pathobionts. We observed that myeloperoxidase activity, a marker of neutrophil infiltration was significantly higher in mice fed an ERD diet on day 7 of infection compared to mice fed an ERS diet, pointing to higher levels of inflammation in the absence of milk exosomes. We conducted additional feeding studies and challenged the mice with C.difficile. Seven days post C. difficile, the myeloperoxidase (MPO) activity, which was used to assess the neutrophil/granulocyte infiltration, was higher in cecum and colon from ERD-fed mice than that from ERS-fed mice; IL-17b was also higher in the colon from ERD-fed mice compared to ERS-fed mice. This reporting period In this reporting period, we fed C57BL/6 mice ERD and ERS diets for four weeks starting at age 3.5 to 4.5 weeks and challenged the mice with an oral dose of 1010 CFU from E. faecalis. E. faecalis colonized in the colon and cecum more efficiently in mice fed ERD diets compared to ERS controls. Symptoms of bacteria translocation to blood, spleen, and liver are seldom seen in both groups. Going forward, we will try to collect these measurements. Interim conclusion Collectively, our studies to date are consistent with our proposal's hypotheses that MEs 1) alter bacterial communities in the gut, and 2) enhance the colonization with protective taxa. Objective 3. Recap previous reporting periods Please see Objective 3. In addition to ERS and ERD diets, we fed mice with AIN-93G. The mice were challenged with C. difficile and pathogenicity was assessed by monitoring the mortality, weight loss over the time, and measuring the C. difficile CFU and toxin activity present in the fecal samples. Results were inconclusive and the feeding study was repeated in Year 3 to increase the sample size. We demonstrated that the minor content of lactose in experimental diets is not a confounder in studies of mice (which are lactose intolerant; see the manuscript by Sukreet et al. in Products). This reporting period In Year 3, we fed C57/BL6 mice on ERS and ERD diets and challenged them with C. difficile that were selected in the absence or presence of MEs. We observed that when mice fed the ERS diet, mortality was lower in mice challenged with C. difficile grown in the presence of MEs compared to mice C. difficile grown in the absence of MEs (10% versus 22% seven days after the challenge). Weight loss was also lower in mice challenged with C. difficile selected by MEs than in C. difficile not selected by MEs (3.5% versus 11.4% six days after challenge). Effects of mutations on host health were less severe in E. faecalis than C. difficile. In the upcoming final year of the project, we will repeat studies using a modified exosome feeding protocol in mice. Interim conclusion Collectively, our studies to date are consistent with our proposal's hypothesis that a ME-depleted diet (ERD) increases the susceptibility of mice to challenge with C. difficile. The AIN-93G diet did not increase the susceptibility. We speculate that casein in AIN-93G provided contains MEs. Casein is not present in the ERD diet.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Ngu A. and Zempleni J. Genetically modified bovine milk exosomes (BMEs) evade elimination by murine bone marrow-derived macrophages (BMDMs). University of Nebraska, 14th Annual NPOD Research Retreat, April 25, 2023, Lincoln, NE [poster]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Mumtaz PT, Zempleni J. Bifidobacterium infantis extracellular vesicles: cargo content, internalization by human intestinal Caco-2 cells, and bioavailability and distribution in mice. University of Nebraska, NPOD 14th Annual NPOD Spring Retreat, April 25, 2023, Lincoln, NE [poster]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Mumtaz PT, Zempleni J. Bifidobacterium infantis-derived EVs carry a diverse cargo of compounds and are bioavailable in C57BL/6J mice and internalized by human intestinal Caco-2 cells. University of Nebraska, NPOD 14th Annual Research Retreat April 25, 2023 [poster]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Wang S, Auchtung J, Zempleni J. Milk exosomes select mutations that decrease the toxicity of Clostridioides difficile. University of Nebraska, University of Nebraska, NPOD 14th Annual Research Retreat April 25, 2023 [poster]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Setayesh L, Zempleni J. Impact of storage conditions on the quantity and integrity of exosomes and their cargo in human milk. University of Nebraska, NPOD 14th Annual Research Retreat April 25, 2023 [poster]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Zempleni J. Milk exosomes and their relevance in human nutrition and the delivery of therapeutics. Washinton University School of Medicine, St. Louis, MO, May 11, 2023 [talk]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Zempleni J. Divergence of gut bacteria through the selection of genomic variants by small extracellular vesicles in milk. Annual meeting of the International Society for Extracellular Vesicles, Seattle, WA, May 18, 2023 [poster]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Zempleni J. Milk exosomes in Nutrition and drug delivery. USDA W-4002 Multistate Group meeting, Honolulu, HI, May 25, 2023 [talk]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Zempleni J. Milk exosome-driven evolution of antibiotic-resistant gut pathogens. NIFA1343 grantee virtual meeting, July 5, 2023 [talk]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Zempleni J, Zhou F, Dogan H, Madadjim R, Tajamul Mumtaz P, Cui J. Selection of genomic variants i gut bacteria through by small extracellular vesicles in milk: implications for purine metabolism in the host. International Milk Genomics Consortium 2023 Annual Meeting, Cork, Ireland, September 6-8, 2023 [poster]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Setayesh L, Zempleni J. Preserving Milk Exosome Integrity During Storage: Strategies for Minimizing Loss. University of Nebraska, University of Nebraska, NPOD 9th Annual Fall Research Symposium, September 26, 2023 [poster]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Mumtaz P. T and Zempleni J. Exploring Cross-Kingdom Communication with Microbial Messengers: Bifidobacterium infantis EVs' Diverse Cargo, Bioavailability in Mice, and Interaction with Human Intestinal Cells (Caco 2). University of Nebraska, NPOD 9th Annual Research Symposium, September 26, 2023
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Zempleni J. Milk exosomes and their relevance in human nutrition and the delivery of therapeutics. University of Wisconsin-Madison, October 12, 2023 [invited talk]
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Fratantonio D, Munir J, Shu J, Howard K, Baier SR, Cui J, Zempleni. The RNA cargo in small extracellular vesicles from chicken eggs is bioactive in C57BL/6J mice and human peripheral blood mononuclear cells ex vivo. Front Nutr 10:1162679, 2023
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Khanam, A., Ngu A, Zempleni J. Bioavailability of orally administered small extracellular vesicles from bovine milk in C57BL/6J mice. Int J Pharm 639:122974, 2023
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Sukreet S, Braga CP, Adamec J, Cui J, Zempleni J. The absorption of bovine milk small extracellular vesicles depends on Galectin-3 and galactose ligands in human intestinal cells and C57BL/6J mice. Am J Physiol Cell Physiol 325:C1421-1430, 2023
- Type:
Journal Articles
Status:
Submitted
Year Published:
2023
Citation:
Wu, D, Shu, J, Braga CP, Cui J, Adamec J, Zempleni J. Bovine mRNAs in small extracellular vesicles from cows milk are bioavailable in mice but translation products are not detectable in reticulocyte lysates and human U937 cells
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2023
Citation:
Kuroishi T, Zempleni J. Biotin. In: Modern Nutrition in Health and Disease. Tucker K (editor). Jones & Bartlett Learning, Burlington, MA (in press).
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Munir J, Ngu A, Wang H, Ramirez DMO, Zempleni J. Review: Milk small extracellular vesicles for use in the delivery of therapeutics. Pharm Res 40:909-915, 2023
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Ngu A,* Munir J,* Zempleni J. Milk-borne small extracellular vesicles: kinetics and mechanisms of transport, distribution, and elimination. Extracell Vesicles Circ Nucleic Acids 4:339-346, 2023
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2023
Citation:
Welsh, J, MISEV Consortium [full list of authors on page 42 of the paper]. Minimal information for studies of extracellular vesicles (MISEV2023): from basic to advanced approaches. J Extracell Vesicles
|
Progress 03/15/22 to 03/14/23
Outputs
Target Audience:The target audience includes scientists (faculty, postdocs, students) in nutrition, biomedical science, policy makers, dairy industry, journalists and lay public. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provides support for two faculty, Drs. J. Zempleni (PD) and J. Auchtung (co-PD), a postdoctoral associate (Dr. S. Wang) and a research technologist. Dr. Zempleni has acquired new knowledge in studies of microbiology, whereas Dr. Auchtung has acquired new knowledge in nutrition research, dairy and milk exosomes. The postdoctoral associate was new to both microbiology and milk exosome research and has acquired expertise in both areas. The technologist was new to milk exosome research and has acquired technical skills in that area. The postdoctoral associate present and Dr. Zempleni findings from this study at scientific conferences though invited seminars and presentations at scientific conferences (see Products). How have the results been disseminated to communities of interest?Results have been disseminated through invited seminars and presentations at scientific conferences (see Products). What do you plan to do during the next reporting period to accomplish the goals?1. Challenge mice on ERS, ERD and AIN-93G diets with ptsG knockout C. difficile. 2. Challenge mice on ERS, ERD and AIN-93G diets with selected E. faecalis strains. 3. Complete challenging mice on ERS and ERD diet with E. faecalis. 4. Perform transcriptome analysis on milk exosomes selected and non-selected C. difficile and E. faecalis. 5. Finalize the testing of biofilm production by E. faecalis selected in milk exosome-defined media. 6. Manuscript submissions.
Impacts
What was accomplished under these goals?
Objective 1. Recap previous reporting periods Previously, we reported that milk exosomes selected a deletion in the phosphotransferase (PTS_EIIA_1) domain in the ptsG gene ("ptsG _indel"). The ptsG gene facilitates glucose transport in bacteria and is implicated in pathogenicity C. difficile. Exosome-selected C. difficile grew faster and produced less toxin than non-selected controls. C. difficile in milk exosome-containing media grew 15% to 20% faster than controls cultured in exosome-free media. The production of toxins, tcdA and tcdB, was 60% to 90% lower in the various milk exosome (ME)-containing cultures of C. difficile compared to exosome-free control cultures. Toxin production was assessed by using real-time PCR and by using cytotoxic effects in Vero cells (A. Valdiviesco-Garcia et al., 1993; 59: 1981-1983). We have developed strains of C. difficile that overexpress wild-type ptsG or ptsG_indel, and we are working towards developing ptsG knockout C. difficile. Using these strains, we started interrogating effects of ptsG overexpression on growth and toxin production. We observed that overexpression of ptsG_indel increased growth and reduced toxin production in C. difficile. This observation is consistent with our hypothesis that MEs select for polymorphisms that decrease the pathogenicity of gut pathogens. We have selected polymorphisms in vancomycin-resistant E. faecalis cultures in ME-defined cultures, but the variants were not fully characterized. This reporting period In this reporting period we discovered that MEs selected a mutation in the DNA binding domain in the catabolite control protein A (ccpA) gene in E. faecalis. The ccpA gene is a transcriptional regulator of carbon catabolite repression and carbon catabolite activation, which ensures optimal energy usage under diverse conditions. Wild-type E. faecalis grew slower with an increased generation time when cultured in ME-free compared to ME-containing media. When E. faecalis was cultured in ME-defined media prior to transfer and continued culturing in minimal media, the polymorphisms and mutations selected in ME-containing media led to a slower growth in minimal media compared to polymorphisms and mutations selected in ME-free media We observed that E. faecalis that were cultured in ME-supplemented media grew faster and had a shortened generation time compared with E. faecalis that were cultured in ME-free media. We further tested the biofilm production by these E. faecalis after selection. (Biofilm production is associated with virulence and antimicrobial resistance.) Results were inconclusive regarding the effects of ME selection on biofilm production. We will repeat these studies in Year 4. Interim conclusion Collectively, our studies to date are consistent with our proposal's hypotheses that MEs 1) select polymorphisms and mutations in gut bacteria, and 2) ME-driven selection decreases the virulence of gut pathogens. Objective 2. Preface The studies depend on feeding ME-defined diets. Briefly, we have developed [and published (Sukreet et al, se Products)] rodent diets defined by their content of MEs and the RNA content. The diets a re based on the AIN-93G formulation. The exosome and RNA-sufficient (ERS) diet contains a nutritionally relevant dose of MEs and RNA cargo (equivalent to 0.5 L milk consumed by an adult), whereas the the content of RNA and exosomes is decreased by 8% and up to 99%, respectively, in the exosome and RNA-deficient (ERD) diet. Al other content is identical in ERS and ERD diets. Recap previous reporting periods In Years 1 and 2, we fed C57BL/6 mice ERD and ERS diets for four weeks starting at age 3.5 to 4.5 weeks and challenged the mice with an oral dose of 105 spores from C. difficile. Survival of mice fed the ERD diet decreased by approximately 50% compared to mice fed the ERS diet (P < 0.05). Consistent with this observation, the median clinical sickness scores (B. Fehlhaber et al, Sci. Rep. 2019; 9: 5919; PMC6459866).in mice fed an ERD diet were 3-fold higher than mice fed the ERS (P < 0.01). We assessed bacterial communities in mice fed ERD and ERS diets with C. difficile. challenge) by using 16S rRNA sequencing. We observed significant changes in a small number of taxa between treatment groups on day 3 followingC. difficilechallenge. ERS feeding resulted in an increase ofLachnospiraceae, Bacteroides(5operational taxonomic units, OTUs) andAlistipes(2OTUs), whereas ERD resulted in an increase ofAkkermansia,Escherichia/Shigella,Enterococcaceae, andClostridiumsensustricto. Since Escherichia/ShigellaandEnterococcaceae are associated with increased disease severity, we concluded that the protective effects of ERS diet on the host response might be due to the selection protective taxa and decrease of pathobionts. We observed that myeloperoxidase activity, a marker of neutrophil infiltration was significantly higher in mice fed an ERD diet on day 7 of infection compared to mice fed an ERS diet, pointing to higher levels of inflammation in the absence of milk exosomes. We conducted additional feeding studies and challenged the mice with C.difficile. Seven days post C. difficile, the myeloperoxidase (MPO) activity, which was used to assess the neutrophil/granulocyte infiltration, was higher in cecum and colon from ERD-fed mice than that from ERS-fed mice; IL-17b was also higher in the colon from ERD-fed mice compared to ERS-fed mice. This reporting period In this reporting period, we fed C57BL/6 mice ERD and ERS diets for four weeks starting at age 3.5 to 4.5 weeks and challenged the mice with an oral dose of 1010 CFU from E. faecalis. E. faecalis colonized in the colon and cecum more efficiently in mice fed ERD diets compared to ERS controls. Symptoms of bacteria translocation to blood, spleen, and liver are seldom seen in both groups. Going forward, we will try to collect these measurements. Interim conclusion Collectively, our studies to date are consistent with our proposal's hypotheses that MEs 1) alter bacterial communities in the gut, and 2) enhance the colonization with protective taxa. Objective 3. Recap previous reporting periods Please see Objective 3. In addition to ERS and ERD diets, we fed mice with AIN-93G. The mice were challenged with C. difficile and pathogenicity was assessed by monitoring the mortality, weight loss over the time, and measuring the C.difficile CFU and toxin activity present in the fecal samples. Results were inconclusive and the feeding study was repeated in Year 3 to increase the sample size. We demonstrated that the minor content of lactose in experimental diets is not a confounder in studies of mice (which are lactose intolerant; see the manuscript by Sukreet et al. in Products). This reporting period In Year 3, we fed C57/BL6 mice on ERS and ERD diets and challenged them with C. difficile that were selected in the absence or presence of MEs. We observed that when mice fed the ERS diet, mortality was lower in mice challenged with C. difficile grown in the presence of MEs compared to mice C. difficile grown in the absence of MEs (10% versus 22% seven days after the challenge). Weight loss was also lower in mice challenged with C. difficile selected by MEs than in C. difficile not selected by MEs (3.5% versus 11.4% six days after challenge). Interim conclusion Collectively, our studies to date are consistent with our proposal's hypothesis that a ME-depleted diet (ERD) increases the susceptibility of mice to challenge with C. difficile. The AIN-93G diet did not increase the susceptibility. We speculate that casein in AIN-93G provided contains MEs. Casein is not present in the ERD diet.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Zempleni J. Novel bioactive compounds in milk: exosomes. Harold Hamm Diabetes Center at the University of Oklahoma, Oklahoma City, OK. March 21, 2022 [talk, delivered by Zoom]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Wang S, Auchtung J, Zempleni J. Milk exosomes select mutations that decrease the toxicity of Clostridioides difficile. NPOD Annual Spring Research Retreat, April 19, 2022, Lincoln, NE
- Type:
Journal Articles
Status:
Other
Year Published:
2022
Citation:
Zhou, F, Dogan H, Madadjim R, Mumtaz PT, Cui J, Zempleni J. Divergence of gut bacteria through the selection of genetic variants by small extracellular vesicles in milk (in preparation)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Wang S, Auchtung J, Zempleni J. Milk exosomes select mutations that decrease the toxicity of Clostridioides difficile. Nutrition 2022 Conference (virtual), American Society for Nutrition; 6/14 6/16, 2022 [virtual talk by Zoom]
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Ngu A, Wang S, Wang H, Khanam A, Zempleni J. Milk exosomes in nutrition and drug delivery. Am J Physiol Cell Physiol 322:C865-C874, 2022
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Munir J, Ngu A, Wang H, Ramirez DMO, Zempleni J. Review: Milk Small Extracellular Vesicles for Use in the Delivery of Therapeutics. Pharm Res (in press)
- Type:
Journal Articles
Status:
Other
Year Published:
2022
Citation:
Ngu A,* Munir J,* Zempleni J. Distribution and elimination of milk-borne small extracellular vesicles. (invited review, submitted)
- Type:
Journal Articles
Status:
Other
Year Published:
2022
Citation:
Wang S, Auchtung J, Zempleni J. Milk small extracellular vesicles select genetic variants that decrease the toxicity of Clostridioides difficile (in preparation)
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Sukreet S, Pereira Braga C, An, TT, Adamec J, Cui J, Zempleni J. Ultrasonication of milk decreases the content of exosomes and microRNAs in an exosome-defined rodent diet. J Nutr 152:961-970, 2022
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Progress 03/15/21 to 03/14/22
Outputs
Target Audience:The target audience includes scientists (faculty, postdocs, students) in nutrition, biomedical science, policy makers, dairy industry, journalists and lay public. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provides support for two faculty, Drs. J. Zempleni (PD) and J. Auchtung (co-PD), a postdoctoral associate (Dr. S. Wang) and a research technologist. Dr. Zempleni has acquired new knowledge in studies of microbiology, whereas Dr. Auchtung has acquired new knowledge in nutrition research, dairy and milk exosomes. The postdoctoral associate was new to both microbiology and milk exosome research and has acquired expertise in both areas. The technologist was new to milk exosome research and has acquired technical skills in that area. In future years we intend to have the postdoctoral associate present and Dr. Zempleni findings from this study at scientific conferences, which proved to be a challenge in Year 1 due to the COVID-19 pandemic. Drs. Zempleni and Wang have shared some of the findings though invited seminars and presentations at scientific conferences (see Products). How have the results been disseminated to communities of interest?Results have been disseminated through invited seminars and presentations at scientific conferences (see Products). What do you plan to do during the next reporting period to accomplish the goals?1. Challenge mice on ERS, ERD and AIN-93G diets with C. difficile that overexpress wild-type ptsG or ptsG_indel, and we have developed ptsG knockout C. difficile. 2. Sequence the genomes of E. faecalis selected in milk-exosome-defined media. 3. Perform cultures of gut content and assess milk exosome-dependent changes in the bacterial transcriptome and metabolome. 4.Test effects of defined diet feeding on susceptibility to E. faecalis infection in mice. 5. Repeat the studies in mice fed the AIN-93G diet (see above, Objective 3).
Impacts
What was accomplished under these goals?
Objective 1. Objective 1. Assess the selection of polymorphisms in C. difficile and vancomycin-resistant E. faecalis in milk exosome-defined cultures. This objective tests the hypothesis that the presence of milk exosomes in culture will select for genetic polymorphisms that may enhance or diminish virulence-associated phenotypes that can be measured in vitro compared to bacteria prior to passaging (reference genome). This is our Year 2 report. In Year 1 we reported that milk exosomes selected a deletion in the phosphotransferase (PTS_EIIA_1) domain in the ptsG gene ("ptsG _indel"). The ptsG gene facilitates glucose transport in bacteria and is implicated in pathogenicity C. difficile. Exosome-selected C. difficile grew faster and produced less toxin than non-selected controls (see Objective 3). In this reporting period, we expanded these studies by developing strains of C. difficile that overexpress wild-type ptsG or ptsG_indel, and we have made progress towards developing ptsG knockout C. difficile. Using these strains, we started interrogating effects of ptsG overexpression on growth and toxin production. We observed that overexpression of ptsG_indel increased growth and reduced toxin production in C. difficile. This observation is consistent with our hypothesis that milk exosomes select for polymorphisms that decrease the pathogenicity of gut pathogens. In this reporting period, we also selected polymorphisms of vancomycin-resistant E. faecalis in milk exosome-defined cultures. While we have not yet sequenced the genomes of the isolates, we observed that E. faecalis selected in milk exosome-supplemented media led to increased doubling time in defined medium and biofilm production compared to control E. faecalis strains serially passaged in the absence of exosomes. Objective 2. Objective 2. Assess whether milk exosomes cause changes in the host microbiome that alter the colonization of mice with C. difficile and E. faecalis not selected in exosome cultures. This objective tests the hypothesis that feeding milk exosome and RNA-depleted (ERD) diets and milk exosome and RNA-sufficient (ERS) diets causes changes in microbial communities in the murine gut that alter the susceptibility to challenges with pathogens. In Year 1, we fed C57BL/6 mice milk exosome and RNA-depleted (ERD) diets and milk exosome and RNA-sufficient (ERS) diets for four weeks starting at age 3.5-4.5 weeks and challenged with an oral dose of 105 spores from C. difficile. The survival of mice fed the ERD diet decreased by approximately 50% compared to mice fed the ERS diet (P < 0.05). Consistent with this observation, median clinical sickness scores (B. Fehlhaber et al, Sci. Rep. 2019; 9: 5919; PMC6459866).in mice fed an ERD diet were 3-fold higher than mice fed the ERS (P < 0.01). In this reporting period, we assessed bacterial communities in mice fed ERD and ERS diets, with and without challenge by C. difficile by using 16S rRNA sequencing. We observed significant changes in a small number of taxa between treatment groups on day 3 followingC. difficilechallenge, with ERS diet selecting for increases inLachnospiraceae, Bacteroides(5OTUs) andAlistipes(2OTUs), and ERD diet selecting for increases inAkkermansia,Escherichia/Shigella,Enterococcaceae, andClostridiumsensustricto. Since strains elevated by ERD diet are often associated with increased disease severity (Escherichia/ShigellaandEnterococcaceae), we hypothesize that the primary effects of ERS diet are on the host response to infection as reduced disease severity may allow recovery of protective taxa and limit expansion of pathobionts. We also observed that myeloperoxidase activity, a marker of neutrophil infiltration was significantly higher in mice fed an ERD diet on day 7 of infection compared to mice fed an ERS diet, pointing to higher levels of inflammation in the absence of exosomes. While these results were potentially interesting, we also observed that disease severity differed between mice that were colonized with human microbes and conventional microbes and in mice colonized with human microbes over time, suggesting that interactions between exosomes and specific bacteria present in the microbiome are essential for disease progression. We conducted additional feeding studies and challenged the mice with C.difficile. seven days post C. difficile, the myeloperoxidase (MPO) activity, which was used to assess the neutrophil/granulocyte infiltration, was higher in cecum and colon from ERD-fed mice than that from ERS-fed mice; IL-17b was also higher in the colon from ERD-fed mice compared to ERS-fed mice. In this funding period we also submitted a manuscript detailing the ERD and ERS diets. The manuscript was returned to us for revision (in progress). Objective 3. Objective 3. Assess the pathogenicity of in vitro-selected polymorphisms in mice fed a regular AIN-93G diet. This objective tests the hypothesis that C. difficile and E. faecalis selected in milk exosome-free (E-Minus) cultures cause disease phenotypes that differ in severity compared to phenotypes caused by C. difficile and E. faecalis selected in milk exosome-supplemented (E-Plus) cultures. In Year 1, we selected C. difficile in a variety of milk exosome-defined media and compared growth rate and toxin production to C. difficile cultured in exosome-free media. The growth rate was assessed by measuring the bacteria's doubling time. C. difficile in milk exosome-containing media grew 15% to 20% faster than controls cultured in exosome-free media. However, the production of toxins, tcdA and tcdB, was 60% to 90% lower in the various milk exosome-containing cultures of C. difficile compared to exosome-free control cultures. Toxin production was assessed by using real-time PCR and by using cytotoxic effects in Vero cells (A. Valdiviesco-Garcia et al., 1993; 59: 1981-1983; PMC not available). In this reporting period, we added a dietary treatment group to our mouse feeding studies to include three dietary treatments: mice fed AIN-93G or ERS or ERD diets. Note that the ERD and ERS diets are based on the AIN-93G formulation, except that the ERD diet is depleted o milk exosomes and their RNA cargos. The mice were challenged with C. difficile and pathogenicity was assessed by monitoring the mortality, weight loss over the time, and measuring the C.difficile CFU and toxin activity present in the fecal samples. Results were inconclusive and we will repeat these studies in the next reporting period. That said, studies were successful in demonstrating that the minor content of lactose in experimental diets is not a confounder in studies of mice (which are lactose intolerant; see the manuscript by Sukreet et al. in Products).
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Zempleni J. Biological Activities of Natural Nanoparticles (Exosomes) in Milk. Department of Ophthalmology, Penn State Hershey Medical Center, Hershey, PA. February 17, 2021 [talk, delivered by using Zoom]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Zempleni J. Novel bioactive compounds in milk: exosomes. Department of Animal Science, University of Nebraska-Lincoln, NE. April 6, 2021 [talk, delivered by using Zoom]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Zempleni J. Milk exosome-driven evolution of antibiotic-resistant gut pathogens. NIFA Program Directors Meeting, Kansas City, KS. May 4, 2021 [talk, delivered by using Zoom]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Wang S, Auchtung J, Zempleni J. Milk exosomes select genetic variants that decrease the toxicity of Clostridioides difficile. NPOD 7th Annual Research Symposium, September 13th, 2021, Lincoln, NE [video poster, delivered by Zoom]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Zempleni J, Zhou F, Dogan H, Cui J. Divergence of gut bacteria through the selection of genetic variations by extracellular vesicles in milk. International Society for Extracellular Vesicles. Annual (virtual) conference, 5/18 5/21, 2021 [poster video presentation] J Extracell Vesicles:10 (Suppl. 1):e12083, 2021
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Wang S, Auchtung J, Zempleni J. Milk exosomes protect human microbiota associated-mice against Clostridioides difficile infection. Nutrition 2021 Conference (virtual), American Society for Nutrition; 6/7 6/11/2021
- Type:
Journal Articles
Status:
Accepted
Year Published:
2021
Citation:
Wang H, Wu D, Sukreet S, Delaney A, Belfort MB, Zempleni J. Quantitation of exosomes and their microRNA cargos in frozen human milk. JPGN Reports (in press)
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Ogunnaike M, Wang, H, Zempleni J. Bovine mammary alveolar MAC-T cells afford a tool for studies of bovine milk exosomes in drug delivery. Int J Pharm 610:121263, 2021
- Type:
Journal Articles
Status:
Submitted
Year Published:
2021
Citation:
Zhou, F, Dogan H, Shu J, Fernando SC, Cui J, Zempleni J. Evolution of gut bacteria through the selection of genetic variations by extracellular vesicles in milk (under revision)
- Type:
Journal Articles
Status:
Submitted
Year Published:
2021
Citation:
Sukreet S, Pereira Braga C, An, TT, Adamec J, Cui J, Zempleni J. Modification of the AIN-93G diet by substituting ultrasonicated milk for casein yields a rodent diet deplete of bioavailable milk exosomes and microRNAs (under revision)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Zempleni J, Zhou F, Dogan H, Cui J. Divergence of gut bacteria through the selection of genetic variations by milk exosomes. Keystone Virtual Symposia The Microbiome: From Mother to Child , January 17-21, 2021 [video poster, delivered by using Zoom]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Zempleni J. Invited presentation titled W-4002 progress report Zempleni lab: milk exosomes as part of the remote annual W-4002 Multistate group meeting; Ohio State University, Columbus, OH, January 27th, 2021 [talk]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Zempleni J. Milk exosomes and their microRNA cargos: infants, gut and brain. Department of Nutritional Sciences, University of Michigan, MI. February 10, 2021 [talk, delivered by using Zoom]
|
Progress 03/15/20 to 03/14/21
Outputs
Target Audience:The target audience includes scientists (faculty, postdocs, students) in nutrition, biomedical science, policy makers, dairy industry, journalists and lay public. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provides support for two faculty, Drs. J. Zempleni (PD) and J. Auchtung (co-PD), a postdoctoral associate (Dr. S. Wang) and a research technologist. Dr. Zempleni has acquired new knowledge in studies of microbiology, whereas Dr. Auchtung has acquired new knowledge in nutrition research, dairy and milk exosomes. The postdoctoral associate was new to both microbiology and milk exosome research and has acquired expertise in both areas. The technologist was new to milk exosome research and has acquired technical skills in that area. In future years we intend to have the postdoctoral associate present and Dr. Zempleni findings from this study at scientific conferences, which proved to be a challenge in Year 1 due to the COVID-19 pandemic. Dr. Zempleni has shared some of the findings at invited seminars at Chapman University and the University of Nebraska Medical Center (see Products). How have the results been disseminated to communities of interest?Results have been disseminated through NIFA blogs and Twitter (see Other Products) and invited seminars (see preceding paragraph). What do you plan to do during the next reporting period to accomplish the goals?1. Assess how variations in the ptsG gene alter the virulence of C. difficile. 2. Assess whether milk exosome-evolved C. difficile colonize the murine gut. 3. Assess the uptake of milk exosomes by gut bacteria. 4. Initiate studies in E. faecalis. 5. Perform cultures of gut content and assess milk exosome-dependent changes in the bacterial transcriptome and metabolome.
Impacts
What was accomplished under these goals?
Objective 1.Assess the selection of polymorphisms inC.difficileand vancomycin-resistantE. faecalisin milk exosome-defined cultures. This objective tests the hypothesis that the presence of milk exosomes in culture will select for genetic polymorphisms that may enhance or diminish virulence-associated phenotypes that can be measuredin vitrocompared to bacteria prior to passaging (reference genome). To date we identified a moderate size deletion (~90 bp) in the phosphotransferase (PTS_EIIA_1) domain of the ptsG gene that was consistently present in exosome-evolved strains of Clostridioides difficile that were sequenced (n=6) compared to vehicle-evolved strains (n=3) and parental strain (n=1). We also identified a single base pair variation in the phosphotransferase (PTS_EIIA_1) domain of the ptsG gene in exosome-evolved strains of C. difficile compared to vehicle-evolved strains. The ptsG gene facilitates glucose transport in bacteria. Objective 2.Assess whether milk exosomes cause changes in the host microbiome that alter the colonization of mice withC. difficileandE. faecalisnot selected in exosome cultures. This objective tests the hypothesis that feeding milk exosome and RNA-depleted (ERD) diets and milk exosome and RNA-sufficient (ERS) diets causes changes in microbial communities in the murine gut that alter the susceptibility to challenges with pathogens. We have completed a first study in which we fed C57BL/6 mice milk exosome and RNA-depleted (ERD) diets and milk exosome and RNA-sufficient (ERS) diets for four weeks starting at age 3.5-4.5 weeks and challenged with an oral dose of 105 spores from C. difficile. The survival of mice fed the ERD diet decreased by approximately 50% compared to mice fed the ERS diet (P < 0.05). Consistent with this observation, median clinical sickness scores (B. Fehlhaber et al, Sci. Rep. 2019; 9: 5919; PMC6459866).in mice fed an ERD diet were 3-fold higher than mice fed the ERS (P < 0.01). Objective 3.Assess the pathogenicity ofin vitro-selected polymorphisms in mice fed a regular AIN-93G diet. This objective tests the hypothesis thatC. difficileandE. faecalisselected in milk exosome-free (E-Minus) cultures cause disease phenotypes that differ in severity compared to phenotypes caused byC. difficileandE. faecalisselected in milk exosome-supplemented (E-Plus) cultures. We selected C. difficile in a variety of milk exosome-defined media and compared growth rate and toxin production to C. difficile cultured in exosome-free media. The growth rate was assessed by measuring the bacteria's doubling time. C. difficile in milk exosome-containing media grew 15% to 20% faster than controls cultured in exosome-free media. However, the production of toxins, tcdA and tcdB, was 60% to 90% lower in the various milk exosome-containing cultures of C. difficile compared to exosome-free control cultures. Toxin production was assessed by using real-time PCR and by using cytotoxic effects in Vero cells (A. Valdiviesco-Garcia et al., 1993; 59: 1981-1983; PMC not available).
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Zempleni J. Exosomes and microRNAs in maternal milk are important for growth and gut health during weaning in murine pups. Chapman University, CA. November 11, 2020 [talk, delivered by using Zoom]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Zempleni J. The role of milk exosomes and their RNA cargos in neonatal health. Life Span Diseases Mini Summit in the Child Health Research Institute, University of Nebraska Medical Center, November 13, 2020 [talk, delivered by using Zoom]
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