Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity

Collins, Shannon M.; Nice, Justin B.; Chang, En Hyung; Brown, Angela C.

doi:10.3791/62429

Cited by 12 publications

(12 citation statements)

References 27 publications

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“…The major drawback of the qEV columns, however, was the incomplete separation of a heterogeneous population into distinct subpopulations [35]. In our studies, we packed a longer column with Sephacryl™ S-1000 superfine resin in order to quickly and efficiently purify and separate OMV subpopulations [38]. The results we have reported here demonstrate that separation by SEC was rapid (about 4 hr), resulted in complete separation of OMV subpopulations, and removed free DNA contamination.…”

Section: Discussionmentioning

confidence: 97%

“…More recently, size exclusion chromatography (SEC) has been utilized to remove impurities that pellet with vesicles during ultracentrifugation [35][36][37]. In this study, we used an ultrafine SEC resin to separate OMV subpopulations by size [38] so that we could explore the mechanism driving the selective packaging of LtxA into subpopulations of OMVs. This technique enabled us to efficiently and reproducibly separate the two OMV subpopulations to demonstrate an increased association with LtxA with the larger subpopulation of OMVs.…”

Section: Introductionmentioning

confidence: 99%

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Separation of heterogeneous Aggregatibacter actinomycetemcomitans outer membrane vesicles reveals that toxin sorting is driven by surface-associated DNA

Nice

Collins

Agro

et al. 2022

Preprint

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Aggregatibacter actinomycetemcomitans is a Gram-negative oral bacterium associated with localized aggressive periodontitis as well as some systemic diseases. The strains of A. actinomycetemcomitans most closely associated with periodontal disease tend to produce more of a secreted leukotoxin (LtxA) than isolates from healthy carriers, suggesting a key role for this toxin in disease progression. LtxA is secreted via a type 1 secretion system, from the bacterial cytosol across both the inner and outer membranes in a single step into the supernatant, where it is able to interact with host cells. Upon secretion, some of the toxin associates with the bacterial cell membrane, enabling its release in association with the surface of outer membrane vesicles (OMVs), small, spherical vesicles derived from the outer membrane. We have previously observed that the highly leukotoxic A. actinomycetemcomitans strain JP2 produces two populations of OMVs, a highly abundant population of small (<100 nm in diameter) OMVs, and a less abundant population of large (>300 nm in diameter) OMVs. Here, we have investigated the association of LtxA with the two populations of OMVs varying in size. Our results indicate that surface-associated DNA drives the selective sorting of LtxA to large OMVs.

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Section: Discussionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

Separation of heterogeneous Aggregatibacter actinomycetemcomitans outer membrane vesicles reveals that toxin sorting is driven by surface-associated DNA

Nice

Collins

Agro

et al. 2022

Preprint

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“…We tested both conditions by first pretreating JP2 OMVs collected from the late exponential phase with PMB or DNase I and measuring the resulting changes in the LtxA elution profile by size exclusion chromatography (SEC). We have previously shown that this SEC approach is able to separate large and small A. actinomycetemcomitans OMVs [ 39 , 41 ]; thus, we anticipated that a similar approach could be used to detect variations in the LtxA composition of large and small OMVs after PMB or DNase I treatment.…”

Section: Resultsmentioning

confidence: 99%

Heterogeneity of Size and Toxin Distribution in Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles

Nice,

Collins,

Agro

et al. 2024

Toxins

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Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium associated with localized aggressive periodontitis as well as some systemic diseases. The strains of A. actinomycetemcomitans most closely associated with disease produce more of a secreted leukotoxin (LtxA) than isolates from healthy carriers, suggesting a key role for this toxin in disease progression. LtxA is released into the bacterial cytosol in a free form as well as in association with the surface of outer membrane vesicles (OMVs). We previously observed that the highly leukotoxic A. actinomycetemcomitans strain JP2 produces two populations of OMVs: a highly abundant population of small (<100 nm) OMVs and a less abundant population of large (>300 nm) OMVs. Here, we have developed a protocol to isolate the OMVs produced during each specific phase of growth and used this to demonstrate that small OMVs are produced throughout growth and lack LtxA, while large OMVs are produced only during the exponential phase and are enriched with LtxA. Our results indicate that surface-associated DNA drives the selective sorting of LtxA into large OMVs. This study provides valuable insights into the observed heterogeneity of A. actinomycetemcomitans vesicles and emphasizes the importance of understanding these variations in the context of bacterial pathogenesis.

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“…To investigate size-based heterogeneity, it was crucial to separate the OMVs based on size prior to the Western blot analysis. An ultrafine Sepharose S1000 column has been used previously for size-based OMV separation, which is a simpler alternative to traditional methods like density gradient centrifugation . We aimed to utilize this method to separate A. actinomycetemcomitans OMVs into fractions based on their size.…”

Section: Resultsmentioning

confidence: 99%

“…This lack of research is primarily due to the limited analytical methods currently available for characterizing the heterogeneities within an OMV population. Ensemble assays such as ELISA and Western blots can provide information about the overall composition of OMV populations; however, they are unable to detect individual OMV heterogeneities. ,− In addition, to detect size-based heterogeneities, the OMV populations need to be separated using either the traditional density gradient ultracentrifugation or size exclusion chromatography (SEC) methods which can be time-consuming and can add to the cost of the method. Recently, mass spectrometry has also been employed for OMV proteomics analysis. − However, it is important to note that this technique analyzes OMV ensembles and can be time- and cost-intensive, and it may necessitate specialized instrumentation.…”

Section: Introductionmentioning

confidence: 99%

Multivariate Analysis of Individual Bacterial Outer Membrane Vesicles Using Fluorescence Microscopy

Singh,

Nice,

et al. 2024

Chemical & Biomedical Imaging

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Gram-negative bacteria produce outer membrane vesicles (OMVs) that play a critical role in cell−cell communication and virulence. OMVs have emerged as promising therapeutic agents for various biological applications such as vaccines and targeted drug delivery. However, the full potential of OMVs is currently constrained by inherent heterogeneities, such as size and cargo differences, and traditional ensemble assays are limited in their ability to reveal OMV heterogeneity. To overcome this issue, we devised an innovative approach enabling the identification of various characteristics of individual OMVs. This method, employing fluorescence microscopy, facilitates the detection of variations in size and surface markers. To demonstrate our method, we utilize the oral bacterium Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) which produces OMVs with a bimodal size distribution. As part of its virulence, A. actinomycetemcomitans secretes leukotoxin (LtxA) in two forms: soluble and surface associated with the OMVs. We observed a correlation between the size and toxin presence where larger OMVs were much more likely to possess LtxA compared to the smaller OMVs. In addition, we noted that, among the smallest OMVs (<100 nm diameter), the fractions that are toxin positive range from 0 to 30%, while the largest OMVs (>200 nm diameter) are between 70 and 100% toxin positive.

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Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity

Cited by 12 publications

References 27 publications

Separation of heterogeneous Aggregatibacter actinomycetemcomitans outer membrane vesicles reveals that toxin sorting is driven by surface-associated DNA

Separation of heterogeneous Aggregatibacter actinomycetemcomitans outer membrane vesicles reveals that toxin sorting is driven by surface-associated DNA

Heterogeneity of Size and Toxin Distribution in Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles

Multivariate Analysis of Individual Bacterial Outer Membrane Vesicles Using Fluorescence Microscopy

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