Our Discoveries

Pioneering Protocols Critical to the Evaluation of Vesicle Production and Function
Characterizing Physiological Benefits of Bacterial Vesicle Production to Bacteria
Vesicle Composition and Engineering
Host Response to and Inflammatory Bacterial Vesicles

Pioneering Protocols Critical to the Evaluation of Vesicle Production and Function

The Scientific Question

We asked the question, “Are these bacterial vesicles really secreted discrete entities, or just products of bacterial lysis?” In 1998, OMVs had been scarcely recognized as secreted entities. As a result, most studies were based on vesicles isolated from simple sedimentation of culture supernatants, which were found to contain bacterial debris, fimbriae, phage, and other non-vesicle material. Although the premise of vesicle-mediated secretion by bacteria remained attractive, using such an impure preparation contaminated the field. 

The Discovery

The Kuehn Lab developed “gold-standard” protocols involving density-gradient purification, biochemical lipid and protein analysis, and cell lysis controls that are critical to the genuine evaluation and characterization of vesicle production and function. As pioneers in this field, we used the model gram-negative organism, E. coli, to identify genes important in regulating and producing vesicles. Through our genetic screening studies, we discovered several envelope features that influence vesicle production (LPS length, Lpp-peptidoglycan crosslinking, NlpI, NlpA, envelope protein overexpression) and revealed that bacterial outer membrane vesicle production is important for bacterial survival under stress.  

Supporting Publications

  1. Chutkan H, MacDonald I, Manning A, Kuehn, M. “Quantitative and qualitative preparations of bacterial outer membrane vescicles.” Bacterial Methods Chapter. 
  2. Vogel R, Coumans FA, Maltesen RG, Böing AN, Bonnington KE, Broekman ML, Broom MF, Buzás EI, Christiansen G, Hajji N, Kristensen SR, Kuehn MJ, Lund SM, Maas SL, Nieuwland R, Osteikoetxea X, Schnoor R, Scicluna BJ, Shambrook M, de Vrij J, Mann SI, Hill AF, Pedersen S. “A standardized method to determine the concentration of extracellular vesicles using tunable resistive pulse sensing.”  J Extracell Vesicles. 2016 5:31242
  3. Schwechheimer C, Kuehn MJ. “Synthetic effect between envelope stress and lack of outer membrane vesicle production in Escherichia coli.” J Bacteriol. 2013.195(18):4161-73.
  4. Schwechheimer C, Kulp A, Kuehn MJ. “Modulation of bacterial outer membrane vesicle production by envelope structure and content.” BMC Microbiol. 2014. 14(1):324.
  5. Schwechheimer C, Rodriguez DL, Kuehn MJ. “NlpI-mediated modulation of outer membrane vesicle production through peptidoglycan dynamics in Escherichia coli.” MicrobiologyOpen. 2015; 4(3):375-89.
  6. Kulp AJ, Sun B, Ai T, Manning AJ, Orench-Rivera N, Schmid AK*, Kuehn MJ* (*equal contribution) “Genome-wide assessment of outer membrane vesicle production in Escherichia coli.” PloS one. 2015; 10(9):e0139200
  7. McMillan HM, Rogers N, Wadle A, Hsu-Kim H, Wiesner MR, Kuehn MJ, Hendren CO. “Microbial vesicle-mediated communication: convergence to understand interactions within and between domains of life. Environ Sci Process Impacts.” Environ Sci Process Impacts. 2021; 23(5):664-677.    

Characterizing Physiological Benefits of Bacterial Vesicle Production to Bacteria

The Scientific Question

We wanted to understand how pathogens utilize vesicles to distribute virulence factors within the host and how they modulate the immune system—with another less obvious question of why vesicles are produced by non-pathogens.

The Discovery

The Kuehn Lab identified that vesicle production is critical to alleviating lethal envelope stress and that it can improve bacterial survival upon exposure to envelope-acting antibiotics and bacteriophage. We found that bacterial vesicles facilitate and maintain OM LPS remodeling that occurs due to environmental shifts. We also discovered that bacterial vesicles can carry cargo that reduce biofilm stability for both the producing strain and for unrelated bystander bacteria.

 

 

Supporting Publications

  1. McBroom AJ, Kuehn MJ. “Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response.” Mol Microbiol. 2007. 63(2):545-58.
  2. Manning AJ, Kuehn MJ. “Contribution of bacterial outer membrane vesicles to innate bacterial defense.” BMC Microbiol. 2011. 11:258.
  3. Macdonald IA, Kuehn MJ. “Stress-induced outer membrane vesicle production by Pseudomonas aeruginosa.”  J Bacteriol. 2013. 195(13):2971-81.
  4. Bonnington, KE, Kuehn MJ. “Outer Membrane Vesicle Production Facilitates LPS Remodeling and Outer Membrane Maintenance in Salmonella during Environmental Transitions.” mBio 2016 7: e01532-16.
  5. Esoda CN, Kuehn MJ. “Pseudomonas aeruginosa Leucine Aminopeptidase Influences Early Biofilm Composition and Structure via Vesicle-Associated Antibiofilm Activity.” mBio. 2019

Vesicle Composition and Engineering

The Scientific Question

We wanted to understand the incorporation of cargo and how host interactions are determined by vesicle cargo. This type of knowledge could benefit the development of vesicles as antigen vectors for vaccines. Vesicles are enriched and depleted in particular populations of both lipid and protein molecules, probably by cargo selection events occurring in the cell envelope. Using molecular hybrids, we gained insight into vesicle cargo selection, and using engineered vesicles that also carry fluorescent cargo, have recorded the trafficking of soluble and membrane-bound vesicle cargo once the vesicles interact with host cells.

The Discovery

The Kuehn Lab has used biochemical methods to investigate the association of heat-labile enterotoxin (LT) with the vesicle and bacterial surface molecule, lipopolysaccharide (LPS). We have also manipulated the composition of vesicles to investigate their function and adaptive immune response to vesicle antigens.

Supporting Publications

  1. Kesty NC, and Kuehn MJ. “Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles.” J Biol Chem 2004. 279:2069-76.
  2. Mudrak B, Rodriguez DL, Kuehn MJ. “Residues of heat-labile enterotoxin involved in bacterial cell surface binding.” J Bacteriol. 2009. 191(9):2917-25.
  3. Muralinath M, Kuehn MJ, Roland KL, Curtiss R 3rd. “Immunization with Salmonella enterica serovar Typhimurium-derived outer membrane vesicles delivering the pneumococcal protein PspA confers protection against challenge with Streptococcus pneumoniae.” Infect Immun. 2011. 79(2):887-94.
  4. McBroom AJ, Kuehn MJ. “Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response.” Mol Microbiol. 2007. 63(2):545-58 
  5. Orench-Rivera N, Kuehn MJ. “Differential Packaging Into Outer Membrane Vesicles Upon Oxidative Stress Reveals a General Mechanism for Cargo Selectivity.” Front Microbiol. 2021 12:561863.

Host Response to Toxic and Inflammatory Bacterial Vesicles

The Question

By definition, outer membrane vesicles are complex mixtures of proteins and lipids. It was anticipated that their ability to stimulate a host response would be more complex, and perhaps synergistic, compared with simply a mixture of soluble, independent components.

The Discovery

The Kuehn lab was the first to formally evaluate this hypothesis, using vesicles isolated and biochemically analyzed from the culture supernatants of a variety of pathogens such as P. aeruginosa, enterotoxigenic E. coli, and H. influenzae. From this, we found that membrane context is critical for the attachment and trafficking of vesicles within the host endocytic pathway, as well as for the consequent toxic and/or innate immune response from the host cell.

For instance, P. aeruginosa vesicles, produced by a strain that was cultured from the lungs of a Cystic Fibrosis patient, adhered better to the lung than to gut epithelial cells. Conversely, a strain isolated from sera showed no such preference for lung cells. It seems that the vesicles stimulate epithelial cells and macrophages, to elicit a cytokine response that is distinct from that of LPS (a major component of the vesicles) alone. We also found that heat-labile enterotoxin (LT)—an important virulence factor of ETEC—is exported from the cells bound to the external surface of vesicles. Presented in this context, the cell is able to mediate the entry of the entire ETEC vesicle into human colorectal tissue culture cells.

Supporting Publications

  1. Bauman SJ, Kuehn M. “Pseudomonas aeruginosa vesicles associate with and are internalized by human lung epithelial cells.” BMC Microbiol. 2009. 9:26.
  2. Ellis TN, Leiman SA, Kuehn MJ. “Naturally produced outer membrane vesicles from Pseudomonas aeruginosa elicit a potent innate immune response via combined sensing of both lipopolysaccharide and protein components.” Infect Immun. 2010. 78(9):3822-31.
  3. Chutkan H, Kuehn MJ. “Context-dependent activation kinetics elicited by soluble versus outer membrane vesicle-associated heat-labile enterotoxin.” Infect Immun. 2011. 79(9):37609.
  4. Finethy R, Luoma S, Orench-Rivera N, Feeley EM, Haldar AK, Yamamoto M, Kanneganti TD, Kuehn MJ, Coers J. “Inflammasome Activation by Bacterial Outer Membrane Vesicles Requires Guanylate Binding Proteins.” mBio. 2017 8: e01188-17.
  5. Rodriguez BV, Kuehn MJ. “Staphylococcus aureus secretes immunomodulatory RNA and DNA via membrane vesicles.” Sci Rep. 2020. 26;10(1):18293
  6. McMillan HM, Zebell SG, Ristaino JB, Dong X, Kuehn MJ. “Protective plant immune responses are elicited by bacterial outer membrane vesicles.” Cell Rep. 2021 Jan 19;34(3):108645.