In vivo nanoscale analysis of the dynamic synergistic interaction of Bacillus thuringiensis Cry11Aa and Cyt1Aa toxins in Aedes aegypti

Abstract

The insecticidal Cry11Aa and Cyt1Aa proteins are produced by Bacillus thuringiensis as crystal inclusions. They work synergistically inducing high toxicity against mosquito larvae. It was proposed that these crystal inclusions are rapidly solubilized and activated in the gut lumen, followed by pore formation in midgut cells killing the larvae. In addition, Cyt1Aa functions as a Cry11Aa binding receptor, inducing Cry11Aa oligomerization and membrane insertion. Here, we used fluorescent labeled crystals, protoxins or activated toxins for in vivo localization at nano-scale resolution. We show that after larvae were fed solubilized proteins, these proteins were not accumulated inside the gut and larvae were not killed. In contrast, if larvae were fed soluble non-toxic mutant proteins, these proteins were found inside the gut bound to gut-microvilli. Only feeding with crystal inclusions resulted in high larval mortality, suggesting that they have a role for an optimal intoxication process. At the macroscopic level, Cry11Aa completely degraded the gastric caeca structure and, in the presence of Cyt1Aa, this effect was observed at lower toxin-concentrations and at shorter periods. The labeled Cry11Aa crystal protein, after midgut processing, binds to the gastric caeca and posterior midgut regions, and also to anterior and medium regions where it is internalized in ordered "net like" structures, leading finally to cell break down. During synergism both Cry11Aa and Cyt1Aa toxins showed a dynamic layered array at the surface of apical microvilli, where Cry11Aa is localized in the lower layer closer to the cell cytoplasm, and Cyt1Aa is layered over Cry11Aa. This array depends on the pore formation activity of Cry11Aa, since the non-toxic mutant Cry11Aa-E97A, which is unable to oligomerize, inverted this array. Internalization of Cry11Aa was also observed during synergism. These data indicate that the mechanism of action of Cry11Aa is more complex than previously anticipated, and may involve additional steps besides pore-formation activity.

Fig 1. Insecticidal activity of soluble activated toxin, soluble protoxin and crystal inclusions from Cry11Aa and Cyt1Aa, wild type or mutant proteins against 3^rd instar Aedes aegypti larvae after 24 h of exposure.

Each bioassay was repeated three times with five replicates. The LC₅₀ values and 95% confidence intervals (CI) of the samples to larvae were calculated by using Polo Plus Probit and Logit Analysis version 1.0 LeOra software, as follows: Cry11Aa crystals = 329.3 (CI: 203.1–605.6) ng/ml; Cyt1Aa crystals = 679.23 (CI: 554.9–851.1) ng/ml; Cry11Aa- Cyt1Aa 1:1 crystals mixture = 51.53 (CI: 554.9–851) ng/ml; soluble Cry11Aa -Cyt1Aa 1:1 protoxins mixture = 5335 (CI: 4503–6339) ng/ml. LC₅₀ could not be calculated for those samples without larvicidal activity.

Fig 2. Representative images of dissected midgut tissues from Cry11Aa and/or Cyt1Aa-treated Aedes aegypti 4^th instar larvae recorded by clear field microscopy.

Blue arrowheads point to the severely affected gastric caeca region of some midguts. Representative composite images were constructed with Fiji-Image J software. A total of five larvae were dissected for each condition and each assay was performed at least three times.

Fig 3. In vivo localization of labeled Cry11Aa and Cyt1Aa, or mutant Cry11Aa-E97A and Cyt1Aa-V122E proteins, inside the midgut tissue of Aedes aegypti larvae.

Cry11Aa proteins were labeled with Alexa546 showing green fluorescence. Cyt1Aa proteins were labeled with Alexa647 showing red fluorescence. Individuals were fed with of each labeled protein (100 ng/ml or 1000 ng/ml) administrated as soluble activated toxin, soluble protoxin or crystal inclusions for 24 h. A total of five larvae were dissected for each condition and each assay was performed at least three times. Representative images are shown. Midgut tissue was dissected and processed as indicated in Materials and methods. Nucleus was stained with DAPI (showing blue fluorescence) and labeled proteins were observed with confocal laser Olympus FV1000 scanning microscope. Insets show selected images to improve clarity of the images. White arrowhead points to Cry11Aa-E97A activated toxin protein bound to microvilli membrane from medium tissue region. Orange arrowhead points to Cry11Aa protoxin internalized into small vesicles. Yellow arrowheads point to cells of the medium midgut highly brilliant due to Cry11Aa internalization. Red arrowheads point to cells that were already broken down.

Fig 4

In vivo localization of labeled crystal inclusions from Cry11Aa wild type (Panel A) or Cry11AaE97A mutant (Panel B) in the midgut tissue of Aedes aegypti larvae. Individuals were fed (100 ng/ml or 1000 ng/ml) of each labeled crystal inclusions for 3, 6 and 9 h. Cry11Aa proteins were labeled with Alexa546 showing green fluorescence. A total of five larvae were dissected for each condition and each assay was performed at least three times. Representative images are shown. Midgut tissue was dissected and processed as indicated in Materials and methods. Nucleus was stained with DAPI (showing blue fluorescence) and labeled proteins were observed with confocal laser Olympus FV1000 scanning microscope. Clear field microscopy images of caeca region are also shown in panels A and B of this figure. Yellow arrowheads point to highly brilliant midgut cells due to Cry11Aa-Alexa546 internalization. Red arrowheads point to cells that were already broken down and the blue arrow points to the gastric caeca region that was severely affected. Panel C shows the complete midgut tissue and a fragment from the medium midgut region showing the highly brilliant cells and dark cells after feeding with Cry11Aa-Alexa546 crystal inclusions. Yellow arrowheads point to Cry11Aa-brilliant cells. Panel C also shows the intensity of fluorescence measurements of 20 x 20 μm squares selected from the cytoplasm of different cells from all conditions. Data are shown as arbitrary fluorescent units (a.f.u.). Different asterisks indicate statistically significant different data analyzed by using Kruskal-Wallis test (P value < 0.001).

Fig 5. In vivo co-localization of labeled Cry11Aa and Cyt1Aa proteins during their synergism in the midgut tissue of Aedes aegypti larvae fed with different mixtures of the labeled crystal inclusions.

A total of five larvae were dissected for each condition and each assay was performed at least three times. Representative images are shown. Panel A shows mixtures of 1:1 (100 ng/ml) crystal inclusions Cry11Aa-Alexa546 (green fluorescence) with Cyt1Aa-Alexa647 (red fluorescence). Panel B shows Cry11AaE97A-Alexa546 mutant (1000 ng/ml) with Cyt1Aa-Alexa647 (500 ng/ml). Larvae were fed with labeled crystal inclusions proteins for 3 h. Next, larvae were processed, nucleus was stained with DAPI (blue fluorescence) and labeled proteins were observed with confocal laser Olympus FV1000 scanning microscope. Insets show selected images to improve clarity of the images and yellow arrowheads point to highly brilliant midgut cells due to Cry11Aa internalization Panel C shows the intensity of fluorescence measurements of 20 x 20 μm squares selected from the cytoplasm of different cells. Data are shown as arbitrary fluorescent units (a.f.u.). Different asterisks indicate statistically significant different data analyzed by using Kruskal-Wallis test (P value < 0.001).

Fig 6

High resolution SRRF analysis showing the in vivo nanoscale localization of the labeled Cry11Aa-Alexa546 (Panel A), mutant Cry11AaE97A-Alexa546 (Panel B) and Cyt1Aa-Alexa647 (Panel C), in the midgut tissue of Aedes aegypti larvae. Cry11Aa proteins were labeled with Alexa546 showing green fluorescence. Individuals were fed with 1000 ng/ml of each labeled crystal inclusions proteins for 6 h. Next, larvae were processed, nucleus were stained with DAPI (blue fluorescence) and 100 frames of raw spinning disk data were recorded with the confocal Yokogawa spinning disk microscope alternating the laser lines illumination (405, 561 and 640 nm) per-frame basis, and Images were analyzed using NanoJ-core and NanoJ-SRRF plugins of Fiji-Image J software as described in Materials and methods. Panel A, shows one brilliant and one dark cell after Cry11Aa-Alexa546 ingestion. Insets show selected images to improve clarity of the images. Panel D, shows the Ripley’s K-function statistical image analysis of the xy pattern organization observed in selected 10 x 10 μm squares selected from different cells of larvae intoxicated with Cry11Aa-Alexa546. Inset, shows an enlargement of the distal organization. The Cry11Aa-brilliant cells, and Cry11Aa-dark cells were analyzed. Similar analyses were performed for different cells of larvae intoxicated with Cry11AaE97A-Alexa546 (Panel E) or Cyt1Aa-Alexa647 (Panel G). These Ripley’s K-function statistical image analysis compute the average number of particles located within a predefined radius of any typical event, normalized for the event intensity (density). Panel F, shows the Kruskal-Wallis test performed to the distributions with Cry11Aa-Alexa546 or Cry11AaE97A-Alexa546. Different asterisks indicate significant different data (P value = 0.05).

Fig 7. Nanoscopic in vivo organization of Cry11Aa or Cyt1Aa proteins during their synergism in the midgut tissue of Aedes aegypti larvae.

Images were analyzed by high resolution SRRF microscopy. Larvae were fed 3 h with mixtures of crystal inclusions Cry11Aa-Alexa546 with Cyt1Aa-Alexa647 at 100 ng/mL of each protein (Panel A), and mutant Cry11AaE97A-Alexa546 with Cyt1Aa-Alexa647 at 1000 ng/mL + 500 ng/mL, respectively (Panel B). Larvae were processed as explained in Fig 6. Cry11Aa proteins were labeled with Alexa546 showing green fluorescence. Cyt1Aa proteins were labeled with Alexa647, showing red fluorescence. Panel A, shows one complete cell and one cell that is already broken down. Insets show selected images to improve clarity. Red arrowhead points to the specific Cyt1Aa-Alexa647 and Cry11Aa-Alexa546 proteins organization in apical membrane, where Cry11Aa-Alexa546 was located down facing the cell cytoplasm and Cyt1Aa-Alexa647 was found in the upper layer. Yellow arrowhead points to the inverted organization presented by Cry11AaE97A-Alexa546 mutant and Cyt1Aa-Alexa647. Panels C and D, show the quantitative line pattern analysis, where Cyt1Aa protein was arbitrary located at zero value, as described in Materials and methods, from more than 24 rectangles of 2.5 x 5 μm selected from Cry11Aa-Alexa546 with Cyt1Aa-Alexa647 mixture (Panel C), or from Cry11AaE97A-Alexa546 with Cyt1Aa-Alexa647 mixture (Panel D). Panel E, shows the distribution of the maximal distances of Cry11Aa and Cry11AaE97A to Cyt1Aa, which was arbitrarily located at zero value. Panels F and G, show the Ripley’s K-function statistical image analysis of the xy pattern organization observed in 14 selected 10 x 10 μm squares selected from different cells of the larvae intoxicated with the different mixtures of proteins. Panel F, shows the analysis of the fluorescence of Cry11Aa-Alexa546 or mutant Cry11AaE97A-Alexa546 in these mixtures and Panel G, shows the analysis of the fluorescence of Cyt1Aa-Alexa647 in these mixtures. Panels F and G, also show the Kruskal-Wallis test analyses of these distributions, different asterisks indicate significant different data (P value = 0.05), and no asterisks indicate any statistical differences.