Reporter OMVs capture rapid kinetics of vesicle uptake by host cells in real time.

<p>(A) CCF2-AM loaded Hela cells were exposed to OMVs from EHEC carrying ClyA-Bla (red), or vector control (grey) at an MOI of 1000 for 3 hours. Ratio of blue:green fluorescence) over time was plotted as mean ± stdev (n = 3). (B) R_max was determined from data in S2A to visualize speed of uptake and is shown are means ± stdev (n = 3). Significance was determined by analysis of variance, with a Brown Forsythe test to determine equal variance. (**) p≤0.01. (C) Absolute FRET changes after 3 h were determined from data in (A) and plotted as efficiency of OMV uptake. Data shown are means ± stdev (n = 3). Significance was determined by ANOVA, with a Brown Forsythe test to determine equal variance. (**) p≤0.01. (D) CCF2-AM loaded Hela cells were imaged by confocal microscopy and merged blue/green images representative of 15 images (n = 3) are shown. Scale bars, 20 μm. (E) Hela cells incubated with cellmask orange-labelled OMVs (red) for 10 and 60 min and slice views of z-stacks were acquired by confocal microscopy. Scale bars, 10 μm.</p

EHEC OMVs enter host cells more rapidly and efficiently than <i>E</i>. <i>coli</i> K12 OMVs.

<p>(A) CCF2-AM loaded Hela cells were exposed to OMVs from EHEC (red) or <i>E</i>. <i>coli</i> K12 (blue) carrying ClyA-Bla, at an MOI of 1000 for 3 hours. Ratios of blue:green fluorescence over time were plotted as means ± stdev (n = 3). Maximum rates (B) were determined from data in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006760#ppat.1006760.s003" target="_blank">S2 Fig</a> and absolute FRET signal changes after 3 hrs (C) were determined from data in (A) and plotted to visualize overall efficiency of uptake for EHEC (red) and K12 (blue) OMVs. Data shown are means ± stdev (n = 3). Significance was determined by ANOVA, with a Brown Forsythe test to determine equal variance. (***) p≤0.001, (**) p≤0.01.</p

LPS structure affects rate and efficiency of OMV uptake by host cells.

<p>CCF2-AM loaded Hela cells were exposed to ClyA-Bla OMVs isolated from EHEC (serotype O157, A-C), EAEC (serotype O42, B-F) or K12 (serotype O16, G-I) containing O antigen (red), or lacking O antigen (blue), at an MOI of 1000 for 3 hours. Ratios of blue:green fluorescence over time (A, D, G) were plotted as means ± stdev (n = 3). Maximum rates (B, E, H) were extracted from data in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006760#ppat.1006760.s005" target="_blank">S4 Fig</a> and absolute FRET changes after 3 hrs (C, F, I) were determined from data shown in A, D and G. Data shown are means ± stdev (n = 3); Significance was determined using ANOVA, with a Brown Forsythe test to determine equal variance. (***) p≤0.001, (**) p≤0.01, (*) p≤0.05, (ns) not significant.</p

LPS composition determines major route and kinetics of OMV entry into host cells.

<p>Whilst it is well established that pathogenic species utilize OMVs during infection, the specific adaptations which allow OMVs to contribute to pathogenesis require further exploration. This work has developed a new approach to overcome current methodological limitations and provide consistent data for future studies and allow new insights into the interactions of OMVs with host cells during infection. This method has shown the relevance of LPS composition, in particular the presence of O antigen, in determining the entry route and kinetics of OMVs. Further work in this area may reveal targets for inhibition of these processes, and enable attenuation of infections by preventing the OMV-associated delivery of virulence factors.</p

OMVs lacking O antigen are biased towards clathrin-mediated endocytosis, while OMVs with O antigen can efficiently access host cells via lipid rafts.

<p>Hela cells were either left untreated (control, red), or pre-treated with 5 μg/ml papain (lilac), 1 μg/ml chlorpromazine (pink), 5 mM methyl-β-cyclodextrin (light green) or 1 μg/ml filipin (turquoise), and exposed to ClyA-Bla OMVs isolated from EHEC (A), EAEC (B) or K12 (C) with or without O antigen at an MOI of 1000 for 3 hours. Total FRET changes after 3 hrs were determined from data in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006760#ppat.1006760.s007" target="_blank">S6 Fig</a> and data shown are means ± stdev (n = 3). Significance compared to the control group was determined using ANOVA, with a Brown Forsythe test to determine equal variance. (***) indicates p≤0.001, (**) p≤0.01, (*) p≤0.05, (ns) not significant.</p

Genetically encoded Bla probes are enriched in <i>E</i>. <i>coli</i> OMVs and retain their enzymatic activity.

<p>(A) Expression of genetically encoded Bla probes is induced in bacteria and secreted OMVs are isolated for all subsequent experiments. Entry of OMVs containing Bla probes into host cells can be detected using a continuous FRET assay. (B) Whole cell lysate (WCL), supernatant (sup) and outer membrane vesicles (OMV) fractions isolated from EHEC expressing ClyA-Bla, carrying empty vector, or no vector were separated by SDS-PAGE and expression of ClyA-Bla was detected by Western Blotting and probing with α-Bla antibody. (C) Specific enzyme activity in whole cell lysate, supernatant, OMV or solubilized OMV fractions isolated from EHEC expressing ClyA-Bla, Bla-ClyA, or carrying empty vector (data shown are means ± stdev, n = 3).</p

Azetidines Kill Multidrug-Resistant <i>Mycobacterium tuberculosis</i> without Detectable Resistance by Blocking Mycolate Assembly

Tuberculosis (TB) is the leading cause of global morbidity and mortality resulting from infectious disease, with over 10.6 million new cases and 1.4 million deaths in 2021. This global emergency is exacerbated by the emergence of multidrug-resistant MDR-TB and extensively drug-resistant XDR-TB; therefore, new drugs and new drug targets are urgently required. From a whole cell phenotypic screen, a series of azetidines derivatives termed BGAz, which elicit potent bactericidal activity with MIC99 values <10 μM against drug-sensitive Mycobacterium tuberculosis and MDR-TB, were identified. These compounds demonstrate no detectable drug resistance. The mode of action and target deconvolution studies suggest that these compounds inhibit mycobacterial growth by interfering with cell envelope biogenesis, specifically late-stage mycolic acid biosynthesis. Transcriptomic analysis demonstrates that the BGAz compounds tested display a mode of action distinct from the existing mycobacterial cell wall inhibitors. In addition, the compounds tested exhibit toxicological and PK/PD profiles that pave the way for their development as antitubercular chemotherapies

Azetidines Kill Multidrug-Resistant <i>Mycobacterium tuberculosis</i> without Detectable Resistance by Blocking Mycolate Assembly

Tuberculosis (TB) is the leading cause of global morbidity and mortality resulting from infectious disease, with over 10.6 million new cases and 1.4 million deaths in 2021. This global emergency is exacerbated by the emergence of multidrug-resistant MDR-TB and extensively drug-resistant XDR-TB; therefore, new drugs and new drug targets are urgently required. From a whole cell phenotypic screen, a series of azetidines derivatives termed BGAz, which elicit potent bactericidal activity with MIC99 values <10 μM against drug-sensitive Mycobacterium tuberculosis and MDR-TB, were identified. These compounds demonstrate no detectable drug resistance. The mode of action and target deconvolution studies suggest that these compounds inhibit mycobacterial growth by interfering with cell envelope biogenesis, specifically late-stage mycolic acid biosynthesis. Transcriptomic analysis demonstrates that the BGAz compounds tested display a mode of action distinct from the existing mycobacterial cell wall inhibitors. In addition, the compounds tested exhibit toxicological and PK/PD profiles that pave the way for their development as antitubercular chemotherapies