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Mycobacterium tuberculosis proteins involved in mycolic acid synthesis and transport localize dynamically to the old growing pole and septum.

Carel C, Nukdee K, Cantaloube S, Bonne M, Diagne CT, Laval F, Daffé M, Zerbib D - PLoS ONE (2014)

Bottom Line: The dynamic localization of FAS-II and of the MA transporter with Wag31, at the old-growing poles and at the septum suggests that the main components of the mycomembrane may potentially be synthesized at these precise foci.Based on the already known polar activities of envelope biosynthesis in mycobacteria, we propose the existence of complex polar machinery devoted to the biogenesis of the entire envelope.As a result, the mycobacterial pole would represent the Achilles' heel of the bacillus at all its growing stages.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France; Université de Toulouse, Université Paul Sabatier, Toulouse, France.

ABSTRACT
Understanding the mechanism that controls space-time coordination of elongation and division of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is critical for fighting the tubercle bacillus. Most of the numerous enzymes involved in the synthesis of Mycolic acid - Arabinogalactan-Peptidoglycan complex (MAPc) in the cell wall are essential in vivo. Using a dynamic approach, we localized Mtb enzymes belonging to the fatty acid synthase-II (FAS-II) complexes and involved in mycolic acid (MA) biosynthesis in a mycobacterial model of Mtb: M. smegmatis. Results also showed that the MA transporter MmpL3 was present in the mycobacterial envelope and was specifically and dynamically accumulated at the poles and septa during bacterial growth. This localization was due to its C-terminal domain. Moreover, the FAS-II enzymes were co-localized at the poles and septum with Wag31, the protein responsible for the polar localization of mycobacterial peptidoglycan biosynthesis. The dynamic localization of FAS-II and of the MA transporter with Wag31, at the old-growing poles and at the septum suggests that the main components of the mycomembrane may potentially be synthesized at these precise foci. This finding highlights a major difference between mycobacteria and other rod-shaped bacteria studied to date. Based on the already known polar activities of envelope biosynthesis in mycobacteria, we propose the existence of complex polar machinery devoted to the biogenesis of the entire envelope. As a result, the mycobacterial pole would represent the Achilles' heel of the bacillus at all its growing stages.

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Polar localization of FAS-II proteins.(A) Enlarged images of wide-field microscopy experiments on GFP-fusion expressing bacteria. Bright field images (BF; leftmost column) together with GFP fluorescence images (GFP, middle column) of Msm expressing GFP fusions with MabA, InhA, KasA or KasB, or GFP alone (Ø) were acquired after 6 hours of induction. The merged images (right columns) allowed visualizing the polar localization of the fusions. Total optical magnification: 630 X. Scale bar: 5 µm. (B) Scatter dot plot representation of polar indexes of Msm strains expressing each GFP fusion after 6 hours of induction. Data were collected from wide-field microscopy images and subsequently expressed and evaluated as described in Materials and Methods. Statistical t-tests were performed to assess the differences between the polar indices of GFP-MabA (black circles) and GFP-InhA (black triangles) and between GFP-KasA (black squares) and GFP-KasB (open diamonds). Each data point corresponds to an individual experiment. Each series of data points for a given fusion represents 500 to1500 bacteria analyzed. The p values of indicated unpaired t-tests are symbolized by asterisks (***, p<0.0001), (*, p = 0.0264). (C) Total fluorescence of Msm cultures expressing GFP fusions. Experimental values are represented as means ± SEM. Fluorescence intensities, expressed in RFU (as defined in the text and in the Materials and Methods) of liquid cultures of Msm expressing either GFP alone (Ø), GFP-MabA (black circles), GFP-KasB (open diamonds), GFP-KasA (black squares) or GFP-InhA (black triangles) were plotted against the length of induction in hours (h). The curve fit was obtained by using a second order polynomial nonlinear regression (quadratic) calculated with GraphPad Prism 5.0 software. (D) Western Blot analysis of GFP-fusions. Total protein content of Msm culture samples expressing either GFP alone or the GFP-fusions with the indicated proteins was analyzed by Western blotting using anti-GFP antibodies. The blots were performed 2 hours (left lanes), 4 hours (middle lanes) or 6 hours (right lanes) after acetamide induction of the same cultures depicted in panel C. The molecular weights of the relevant marker bands are indicated in kDa.
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pone-0097148-g002: Polar localization of FAS-II proteins.(A) Enlarged images of wide-field microscopy experiments on GFP-fusion expressing bacteria. Bright field images (BF; leftmost column) together with GFP fluorescence images (GFP, middle column) of Msm expressing GFP fusions with MabA, InhA, KasA or KasB, or GFP alone (Ø) were acquired after 6 hours of induction. The merged images (right columns) allowed visualizing the polar localization of the fusions. Total optical magnification: 630 X. Scale bar: 5 µm. (B) Scatter dot plot representation of polar indexes of Msm strains expressing each GFP fusion after 6 hours of induction. Data were collected from wide-field microscopy images and subsequently expressed and evaluated as described in Materials and Methods. Statistical t-tests were performed to assess the differences between the polar indices of GFP-MabA (black circles) and GFP-InhA (black triangles) and between GFP-KasA (black squares) and GFP-KasB (open diamonds). Each data point corresponds to an individual experiment. Each series of data points for a given fusion represents 500 to1500 bacteria analyzed. The p values of indicated unpaired t-tests are symbolized by asterisks (***, p<0.0001), (*, p = 0.0264). (C) Total fluorescence of Msm cultures expressing GFP fusions. Experimental values are represented as means ± SEM. Fluorescence intensities, expressed in RFU (as defined in the text and in the Materials and Methods) of liquid cultures of Msm expressing either GFP alone (Ø), GFP-MabA (black circles), GFP-KasB (open diamonds), GFP-KasA (black squares) or GFP-InhA (black triangles) were plotted against the length of induction in hours (h). The curve fit was obtained by using a second order polynomial nonlinear regression (quadratic) calculated with GraphPad Prism 5.0 software. (D) Western Blot analysis of GFP-fusions. Total protein content of Msm culture samples expressing either GFP alone or the GFP-fusions with the indicated proteins was analyzed by Western blotting using anti-GFP antibodies. The blots were performed 2 hours (left lanes), 4 hours (middle lanes) or 6 hours (right lanes) after acetamide induction of the same cultures depicted in panel C. The molecular weights of the relevant marker bands are indicated in kDa.

Mentions: Mtb genes encoding the FAS-II reductases MabA and InhA, and the condensing enzymes KasA and KasB, were merged to the C-terminal end of gfp and expressed under the control of the PamiE inducible promoter in the non-pathogenic bacterium model M. smegmatis mc2155 (Msm). In a first series of experiments, large field fluorescence microscopy images, hereafter called static images, were acquired six hours after induction. GFP alone emitted a bright and diffuse fluorescence throughout the cytoplasm (Figure 2A). Conversely, the reductase fusions yielded intense foci located mainly at one bacterial pole (Figure 2A). In the strains expressing either GFP-KasB or GFP-KasA, polar foci were also detectable although together with a diffuse fluorescence in the cytoplasm (Figure 2A). To quantify foci formation, a polar index (PI) was calculated and defined as the ratio of the number of bacteria containing at least one polar focus reported to the total number of fluorescent bacteria (N). Over one thousand bacteria expressing each fusion were analyzed on several replicates. Two groups were found (Figure 2B). The first group, e.g. MabA (89.47%±1.20, N = 1100) and InhA (63.86%±1.56, N = 2000), had a high PI while the second group, e.g. KasA (13.34%±1.40, N = 900) and KasB (21.02%±2.40, N = 1700), had a lower PI. Due to the inaccuracy of quantifying and comparing static images from different snapshots, the total amount of fluorescence in live bacterial culture was assessed by fluorometry as a function of induction time. This enabled to verify whether PI was correlated or not with fusion expression levels. Eight hours after induction (Figure 2C), the strain containing GFP alone (PI = 0%) emitted the maximum fluorescence (280 RFU). MabA (PI = 89%) and KasB (PI = 21%) yielded intermediate fluorescence levels (45 RFU and 35 RFU, respectively) whereas KasA (PI = 13%) and InhA (PI = 63%) yielded very low levels (below 10 RFU). The absence of correlation between fluorescence levels and PI values (as compared between Figure 2B and Figure 2C) was further confirmed by Western blotting experiments performed on the same cultures (Figure 2D). Western blotting results were perfectly correlated with fluorometry results (see comparison between Figure 2D and Figure 2C) and thus were not correlated with PI values, indicating that foci formation was not due to protein overexpression but rather due to preferential localization of FAS-II at the pole.


Mycobacterium tuberculosis proteins involved in mycolic acid synthesis and transport localize dynamically to the old growing pole and septum.

Carel C, Nukdee K, Cantaloube S, Bonne M, Diagne CT, Laval F, Daffé M, Zerbib D - PLoS ONE (2014)

Polar localization of FAS-II proteins.(A) Enlarged images of wide-field microscopy experiments on GFP-fusion expressing bacteria. Bright field images (BF; leftmost column) together with GFP fluorescence images (GFP, middle column) of Msm expressing GFP fusions with MabA, InhA, KasA or KasB, or GFP alone (Ø) were acquired after 6 hours of induction. The merged images (right columns) allowed visualizing the polar localization of the fusions. Total optical magnification: 630 X. Scale bar: 5 µm. (B) Scatter dot plot representation of polar indexes of Msm strains expressing each GFP fusion after 6 hours of induction. Data were collected from wide-field microscopy images and subsequently expressed and evaluated as described in Materials and Methods. Statistical t-tests were performed to assess the differences between the polar indices of GFP-MabA (black circles) and GFP-InhA (black triangles) and between GFP-KasA (black squares) and GFP-KasB (open diamonds). Each data point corresponds to an individual experiment. Each series of data points for a given fusion represents 500 to1500 bacteria analyzed. The p values of indicated unpaired t-tests are symbolized by asterisks (***, p<0.0001), (*, p = 0.0264). (C) Total fluorescence of Msm cultures expressing GFP fusions. Experimental values are represented as means ± SEM. Fluorescence intensities, expressed in RFU (as defined in the text and in the Materials and Methods) of liquid cultures of Msm expressing either GFP alone (Ø), GFP-MabA (black circles), GFP-KasB (open diamonds), GFP-KasA (black squares) or GFP-InhA (black triangles) were plotted against the length of induction in hours (h). The curve fit was obtained by using a second order polynomial nonlinear regression (quadratic) calculated with GraphPad Prism 5.0 software. (D) Western Blot analysis of GFP-fusions. Total protein content of Msm culture samples expressing either GFP alone or the GFP-fusions with the indicated proteins was analyzed by Western blotting using anti-GFP antibodies. The blots were performed 2 hours (left lanes), 4 hours (middle lanes) or 6 hours (right lanes) after acetamide induction of the same cultures depicted in panel C. The molecular weights of the relevant marker bands are indicated in kDa.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4016276&req=5

pone-0097148-g002: Polar localization of FAS-II proteins.(A) Enlarged images of wide-field microscopy experiments on GFP-fusion expressing bacteria. Bright field images (BF; leftmost column) together with GFP fluorescence images (GFP, middle column) of Msm expressing GFP fusions with MabA, InhA, KasA or KasB, or GFP alone (Ø) were acquired after 6 hours of induction. The merged images (right columns) allowed visualizing the polar localization of the fusions. Total optical magnification: 630 X. Scale bar: 5 µm. (B) Scatter dot plot representation of polar indexes of Msm strains expressing each GFP fusion after 6 hours of induction. Data were collected from wide-field microscopy images and subsequently expressed and evaluated as described in Materials and Methods. Statistical t-tests were performed to assess the differences between the polar indices of GFP-MabA (black circles) and GFP-InhA (black triangles) and between GFP-KasA (black squares) and GFP-KasB (open diamonds). Each data point corresponds to an individual experiment. Each series of data points for a given fusion represents 500 to1500 bacteria analyzed. The p values of indicated unpaired t-tests are symbolized by asterisks (***, p<0.0001), (*, p = 0.0264). (C) Total fluorescence of Msm cultures expressing GFP fusions. Experimental values are represented as means ± SEM. Fluorescence intensities, expressed in RFU (as defined in the text and in the Materials and Methods) of liquid cultures of Msm expressing either GFP alone (Ø), GFP-MabA (black circles), GFP-KasB (open diamonds), GFP-KasA (black squares) or GFP-InhA (black triangles) were plotted against the length of induction in hours (h). The curve fit was obtained by using a second order polynomial nonlinear regression (quadratic) calculated with GraphPad Prism 5.0 software. (D) Western Blot analysis of GFP-fusions. Total protein content of Msm culture samples expressing either GFP alone or the GFP-fusions with the indicated proteins was analyzed by Western blotting using anti-GFP antibodies. The blots were performed 2 hours (left lanes), 4 hours (middle lanes) or 6 hours (right lanes) after acetamide induction of the same cultures depicted in panel C. The molecular weights of the relevant marker bands are indicated in kDa.
Mentions: Mtb genes encoding the FAS-II reductases MabA and InhA, and the condensing enzymes KasA and KasB, were merged to the C-terminal end of gfp and expressed under the control of the PamiE inducible promoter in the non-pathogenic bacterium model M. smegmatis mc2155 (Msm). In a first series of experiments, large field fluorescence microscopy images, hereafter called static images, were acquired six hours after induction. GFP alone emitted a bright and diffuse fluorescence throughout the cytoplasm (Figure 2A). Conversely, the reductase fusions yielded intense foci located mainly at one bacterial pole (Figure 2A). In the strains expressing either GFP-KasB or GFP-KasA, polar foci were also detectable although together with a diffuse fluorescence in the cytoplasm (Figure 2A). To quantify foci formation, a polar index (PI) was calculated and defined as the ratio of the number of bacteria containing at least one polar focus reported to the total number of fluorescent bacteria (N). Over one thousand bacteria expressing each fusion were analyzed on several replicates. Two groups were found (Figure 2B). The first group, e.g. MabA (89.47%±1.20, N = 1100) and InhA (63.86%±1.56, N = 2000), had a high PI while the second group, e.g. KasA (13.34%±1.40, N = 900) and KasB (21.02%±2.40, N = 1700), had a lower PI. Due to the inaccuracy of quantifying and comparing static images from different snapshots, the total amount of fluorescence in live bacterial culture was assessed by fluorometry as a function of induction time. This enabled to verify whether PI was correlated or not with fusion expression levels. Eight hours after induction (Figure 2C), the strain containing GFP alone (PI = 0%) emitted the maximum fluorescence (280 RFU). MabA (PI = 89%) and KasB (PI = 21%) yielded intermediate fluorescence levels (45 RFU and 35 RFU, respectively) whereas KasA (PI = 13%) and InhA (PI = 63%) yielded very low levels (below 10 RFU). The absence of correlation between fluorescence levels and PI values (as compared between Figure 2B and Figure 2C) was further confirmed by Western blotting experiments performed on the same cultures (Figure 2D). Western blotting results were perfectly correlated with fluorometry results (see comparison between Figure 2D and Figure 2C) and thus were not correlated with PI values, indicating that foci formation was not due to protein overexpression but rather due to preferential localization of FAS-II at the pole.

Bottom Line: The dynamic localization of FAS-II and of the MA transporter with Wag31, at the old-growing poles and at the septum suggests that the main components of the mycomembrane may potentially be synthesized at these precise foci.Based on the already known polar activities of envelope biosynthesis in mycobacteria, we propose the existence of complex polar machinery devoted to the biogenesis of the entire envelope.As a result, the mycobacterial pole would represent the Achilles' heel of the bacillus at all its growing stages.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France; Université de Toulouse, Université Paul Sabatier, Toulouse, France.

ABSTRACT
Understanding the mechanism that controls space-time coordination of elongation and division of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is critical for fighting the tubercle bacillus. Most of the numerous enzymes involved in the synthesis of Mycolic acid - Arabinogalactan-Peptidoglycan complex (MAPc) in the cell wall are essential in vivo. Using a dynamic approach, we localized Mtb enzymes belonging to the fatty acid synthase-II (FAS-II) complexes and involved in mycolic acid (MA) biosynthesis in a mycobacterial model of Mtb: M. smegmatis. Results also showed that the MA transporter MmpL3 was present in the mycobacterial envelope and was specifically and dynamically accumulated at the poles and septa during bacterial growth. This localization was due to its C-terminal domain. Moreover, the FAS-II enzymes were co-localized at the poles and septum with Wag31, the protein responsible for the polar localization of mycobacterial peptidoglycan biosynthesis. The dynamic localization of FAS-II and of the MA transporter with Wag31, at the old-growing poles and at the septum suggests that the main components of the mycomembrane may potentially be synthesized at these precise foci. This finding highlights a major difference between mycobacteria and other rod-shaped bacteria studied to date. Based on the already known polar activities of envelope biosynthesis in mycobacteria, we propose the existence of complex polar machinery devoted to the biogenesis of the entire envelope. As a result, the mycobacterial pole would represent the Achilles' heel of the bacillus at all its growing stages.

Show MeSH
Related in: MedlinePlus