NlpI-mediated modulation of outer membrane vesicle production through peptidoglycan dynamics in Escherichia coli.

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Outer membrane vesicles (OMVs) are ubiquitously secreted from the outer membrane (OM) of Gram-negative bacteria. These heterogeneous structures are composed of OM filled with periplasmic content from the site of budding. By analyzing mutants that have vesicle production phenotypes, we can gain insight into the mechanism of OMV budding in wild-type cells, which has thus far remained elusive. In this study, we present data demonstrating that the hypervesiculation phenotype of the nlpI deletion mutant of Escherichia coli correlates with changes in peptidoglycan (PG) dynamics. Our data indicate that in stationary phase cultures the nlpI mutant exhibits increased PG synthesis that is dependent on spr, consistent with a model in which NlpI controls the activity of the PG endopeptidase Spr. In log phase, the nlpI mutation was suppressed by a dacB mutation, suggesting that NlpI regulates penicillin-binding protein 4 (PBP4) during exponential growth. The data support a model in which NlpI negatively regulates PBP4 activity during log phase, and Spr activity during stationary phase, and that in the absence of NlpI, the cell survives by increasing PG synthesis. Further, the nlpI mutant exhibited a significant decrease in covalent outer membrane (OM-PG) envelope stabilizing cross-links, consistent with its high level of OMV production. Based on these results, we propose that one mechanism wild-type Gram-negative bacteria can use to modulate vesiculation is by altering PG-OM cross-linking via localized modulation of PG degradation and synthesis.





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Schwechheimer, Carmen, Daniel L Rodriguez and Meta J Kuehn (2015). NlpI-mediated modulation of outer membrane vesicle production through peptidoglycan dynamics in Escherichia coli. Microbiologyopen, 4(3). pp. 375–389. 10.1002/mbo3.244 Retrieved from

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Margarethe Joanna Kuehn

Associate Professor of Biochemistry

Enterotoxigenic E. coli (ETEC) causes traveler's diarrhea and infant mortality in underdeveloped countries, and Pseudomonas aeruginosa is an opportunistic pathogen for immunocompromised patients. Like all gram negative bacteria studied to date, ETEC and P. aeruginosa produce small outer membrane vesicles that can serve as delivery "bombs" to host tissues. Vesicles contain a subset of outer membrane and soluble periplasmic proteins and lipids. In tissues and sera of infected hosts, vesicles have been observed to bud from the pathogen and come in close contact with epithelial cells. Despite their association with disease, the ability of pathogenic bacteria to distribute an arsenal of virulence factors to the host cells via vesicles remains relatively unexplored.

In our lab, we focus on the genetic, biochemical and functional features of bacterial vesicle production. Using a genetic screen, we have identified genes essential in the vesiculation process, we have identified specific proteins that are enriched in vesicles, and we have identified critical molecules that govern the internalization of vesicles into host cells. Using biochemical analysis of purified vesicles from cell-free culture supernatants, we have found that heat-labile enterotoxin, an important virulence factor of ETEC, is exported from the cells bound to the external surface of vesicles. Presented in this context, it is able to mediate the entry of the entire ETEC vesicle into human colorectal tissue culture cells. We have also discovered that the ability of vesicles to bind to specific cell types depends on their strain of origin: for example, P. aeruginosa vesicles produced by a strain that was cultured from the lungs of a patient with Cystic Fibrosis adhered better to lung than to gut epithelial cells, whereas a strain that was isolated from sera showed no such preference for lung cells. 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.

These studies will provide new insights into the membrane dynamics of gram-negative bacteria and consequently aid in the identification of new therapeutic targets for important human pathogens.

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