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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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Related in: MedlinePlus

Polar colocalization of FAS-II proteins with Wag31.(A) Enlarged images of wide-field microscopy experiments on GFP-fusion and mCherry-Wag31 fusion-expressing bacteria. GFP fluorescence images (GFP; leftmost column) together with mCherry fluorescence images (Che, middle column) of Msm::mCherry-wag31 expressing GFP fusions with MabA, InhA, KasA, KasB, or GFP alone (Ø) were acquired after 6 hours of induction. The merged images (right columns) allowed visualizing the polar colocalization of the fusions. Magnification and scale bars are identical to those indicated in Figure? 2A. (B) Bar graph representation of GFP polar indices of Msm::mCherry-wag31 strains expressing each GFP fusion after 6 hours of induction. Experimental values are represented as mean ± SD. Data were processes as described in Figure? 2B. Statistical t-tests were performed to determine the differences between the polar indices of GFP-InhA (dark grey bar) and GFP-KasA (light grey bar) and of GFP-KasA and GFP-KasB (white bar). The p values of indicated unpaired t-tests are symbolized by asterisks (*, p = 0.0271 for InhA-KasA), (*, p = 0.0186 for KasA-KasB). (C) Bar graph representation of GFP-mCherry colocalization indices of Msm::mCherry-wag31 strains expressing each GFP fusion after 6 hours of induction. Experimental values are represented as means ± SD. Data were processed as in panel B; no significant differences were found between colocalization indices. (D) Analysis of maximal mCherry-Wag31 (Che-Wag31, left column) and GFP-FAS-II fusion (GFP fusion, right column) fluorescence position within individual Msm::mCherry-wag31 bacteria expressing each GFP fusion after 6 hours of induction. Each type of bacterium was scanned on both channels. Each dot represents the position of maximum fluorescence of the scans expressed in % of the highest values. Each dot was plotted against its position in the bacterium with the length of bacteria reported to 1. The names of the FAS-II proteins fused with GFP are indicated. Ø refers to the Msm::mCherry-wag31 strain containing GFP alone.
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pone-0097148-g004: Polar colocalization of FAS-II proteins with Wag31.(A) Enlarged images of wide-field microscopy experiments on GFP-fusion and mCherry-Wag31 fusion-expressing bacteria. GFP fluorescence images (GFP; leftmost column) together with mCherry fluorescence images (Che, middle column) of Msm::mCherry-wag31 expressing GFP fusions with MabA, InhA, KasA, KasB, or GFP alone (Ø) were acquired after 6 hours of induction. The merged images (right columns) allowed visualizing the polar colocalization of the fusions. Magnification and scale bars are identical to those indicated in Figure? 2A. (B) Bar graph representation of GFP polar indices of Msm::mCherry-wag31 strains expressing each GFP fusion after 6 hours of induction. Experimental values are represented as mean ± SD. Data were processes as described in Figure? 2B. Statistical t-tests were performed to determine the differences between the polar indices of GFP-InhA (dark grey bar) and GFP-KasA (light grey bar) and of GFP-KasA and GFP-KasB (white bar). The p values of indicated unpaired t-tests are symbolized by asterisks (*, p = 0.0271 for InhA-KasA), (*, p = 0.0186 for KasA-KasB). (C) Bar graph representation of GFP-mCherry colocalization indices of Msm::mCherry-wag31 strains expressing each GFP fusion after 6 hours of induction. Experimental values are represented as means ± SD. Data were processed as in panel B; no significant differences were found between colocalization indices. (D) Analysis of maximal mCherry-Wag31 (Che-Wag31, left column) and GFP-FAS-II fusion (GFP fusion, right column) fluorescence position within individual Msm::mCherry-wag31 bacteria expressing each GFP fusion after 6 hours of induction. Each type of bacterium was scanned on both channels. Each dot represents the position of maximum fluorescence of the scans expressed in % of the highest values. Each dot was plotted against its position in the bacterium with the length of bacteria reported to 1. The names of the FAS-II proteins fused with GFP are indicated. Ø refers to the Msm::mCherry-wag31 strain containing GFP alone.

Mentions: Since FAS-II localization appeared to follow the reported localization of the polar protein Wag31 [29], [35], a recognized “old pole marker”, Wag31-FAS-II colocalization was therefore studied. For purposes of clarity, the word colocalization is used in this study to describe the localization of two proteins at the same focus but without implying the existence of any molecular interaction. A strain was constructed bearing a single but ectopic chromosomal copy of a mCherry-Wag31 fusion under the control of the constitutive promoter pHSP60 of the integrative vector pMV361. This type of construct, using the pHSP60 promoter, has been successfully used previously to study Wag31 localization [36]. When this strain was observed in fluorescence microscopy (Figure S3), the mCherry fusion was more visible at one pole as also commonly observed by others [36], [37]. This pole has been shown to represent the old pole (Figure 1) [29]. The fusions were introduced into the mCherry-Wag31 producing strain after which the fluorescence of both GFP and mCherry was analyzed as performed above on static images. As observed in the absence of mCherry-Wag31 (Figure 2A), GFP remained diffuse whereas GFP-MabA and GFP-InhA yielded intense polar foci, mainly at one pole (Figure 4A). GFP-KasA and GFP-KasB still appeared diffuse with a lower extend of polar spotting (Figure 4A). The overall localization pattern of the FAS-II fusions was comparable to that of the previous fusions (Figure 2A). The statistical relevance of the localization (Figure 4B) was analyzed as above on individual bacteria expressing each fusion (N) stemming from several individual slides (n>6). The polar indices of MabA (93.93±0.59 N = 800), KasA (23.87±4.56 N = 500) and KasB (13.49±1.43 N = 500) were not statistically different from the previously observed indices (see comparison between Figure 2B and 4B) whereas the PI of GFP-InhA (35.99±2.14 N = 700) was slightly reduced. While there was no dramatic effect of the presence of the mCherry-Wag31 fusion on the percentage of localization of the FAS-II proteins, there was the presence of colocalization of both Wag31 and FAS-II fusions foci. To quantify this phenomenon on a large number of bacteria a colocalization index was defined as being the percentage of bacteria exhibiting Wag31-GFP colocalization relative to the number of GFP-mCherry-positive bacteria. Colocalization indices (Figure 4C) were very high for all FAS-II proteins: MabA (83.31%±3.27, N = 800), InhA (93.93%±2.60, N = 700), KasA (89.71%±3.37, N = 500) and KasB (89.52%±3.60, N = 500). In order to evaluate the distribution of foci within individual bacteria, a more in-depth analysis was performed on a subset of bacteria by scanning more than 100 organisms of each type. On each scan (Figure 4D), the maximum fluorescence was plotted against the normalized length of the bacteria. A dot represented on a graph can refer to a focus (at the pole) or simply to the maximum of diffuse fluorescence along the cell. Localization of the reductases and colocalization with Wag31 at the old poles were clearly confirmed (Figure 4D). Furthermore, the maximum florescence emitted by KasA and KasB, which displayed the lowest polar indices, were clearly preferentially observed in proximity of the old poles along with Wag31, thus suggesting an actual preference for this localization (Figure 4D). The distribution of the polar foci clouds represented only about 20% of total cell size, i.e. less than 1 µm, probably due to light diffusion. This distribution was similar for FAS-II proteins and for Wag31, already known to be localized close to the polar membranes. In contrast, the maximum accumulation of GFP alone was never found at the old pole suggesting a polar exclusion of GFP alone. These analyses clearly demonstrate that the FAS-II proteins were preferentially located at the old pole with Wag31. Since Wag31 is acknowledged to be an old pole marker, we can conclude that the polar FAS-II proteins were located at the active growing “old pole” of the bacteria.


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 colocalization of FAS-II proteins with Wag31.(A) Enlarged images of wide-field microscopy experiments on GFP-fusion and mCherry-Wag31 fusion-expressing bacteria. GFP fluorescence images (GFP; leftmost column) together with mCherry fluorescence images (Che, middle column) of Msm::mCherry-wag31 expressing GFP fusions with MabA, InhA, KasA, KasB, or GFP alone (Ø) were acquired after 6 hours of induction. The merged images (right columns) allowed visualizing the polar colocalization of the fusions. Magnification and scale bars are identical to those indicated in Figure? 2A. (B) Bar graph representation of GFP polar indices of Msm::mCherry-wag31 strains expressing each GFP fusion after 6 hours of induction. Experimental values are represented as mean ± SD. Data were processes as described in Figure? 2B. Statistical t-tests were performed to determine the differences between the polar indices of GFP-InhA (dark grey bar) and GFP-KasA (light grey bar) and of GFP-KasA and GFP-KasB (white bar). The p values of indicated unpaired t-tests are symbolized by asterisks (*, p = 0.0271 for InhA-KasA), (*, p = 0.0186 for KasA-KasB). (C) Bar graph representation of GFP-mCherry colocalization indices of Msm::mCherry-wag31 strains expressing each GFP fusion after 6 hours of induction. Experimental values are represented as means ± SD. Data were processed as in panel B; no significant differences were found between colocalization indices. (D) Analysis of maximal mCherry-Wag31 (Che-Wag31, left column) and GFP-FAS-II fusion (GFP fusion, right column) fluorescence position within individual Msm::mCherry-wag31 bacteria expressing each GFP fusion after 6 hours of induction. Each type of bacterium was scanned on both channels. Each dot represents the position of maximum fluorescence of the scans expressed in % of the highest values. Each dot was plotted against its position in the bacterium with the length of bacteria reported to 1. The names of the FAS-II proteins fused with GFP are indicated. Ø refers to the Msm::mCherry-wag31 strain containing GFP alone.
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Related In: Results  -  Collection

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

pone-0097148-g004: Polar colocalization of FAS-II proteins with Wag31.(A) Enlarged images of wide-field microscopy experiments on GFP-fusion and mCherry-Wag31 fusion-expressing bacteria. GFP fluorescence images (GFP; leftmost column) together with mCherry fluorescence images (Che, middle column) of Msm::mCherry-wag31 expressing GFP fusions with MabA, InhA, KasA, KasB, or GFP alone (Ø) were acquired after 6 hours of induction. The merged images (right columns) allowed visualizing the polar colocalization of the fusions. Magnification and scale bars are identical to those indicated in Figure? 2A. (B) Bar graph representation of GFP polar indices of Msm::mCherry-wag31 strains expressing each GFP fusion after 6 hours of induction. Experimental values are represented as mean ± SD. Data were processes as described in Figure? 2B. Statistical t-tests were performed to determine the differences between the polar indices of GFP-InhA (dark grey bar) and GFP-KasA (light grey bar) and of GFP-KasA and GFP-KasB (white bar). The p values of indicated unpaired t-tests are symbolized by asterisks (*, p = 0.0271 for InhA-KasA), (*, p = 0.0186 for KasA-KasB). (C) Bar graph representation of GFP-mCherry colocalization indices of Msm::mCherry-wag31 strains expressing each GFP fusion after 6 hours of induction. Experimental values are represented as means ± SD. Data were processed as in panel B; no significant differences were found between colocalization indices. (D) Analysis of maximal mCherry-Wag31 (Che-Wag31, left column) and GFP-FAS-II fusion (GFP fusion, right column) fluorescence position within individual Msm::mCherry-wag31 bacteria expressing each GFP fusion after 6 hours of induction. Each type of bacterium was scanned on both channels. Each dot represents the position of maximum fluorescence of the scans expressed in % of the highest values. Each dot was plotted against its position in the bacterium with the length of bacteria reported to 1. The names of the FAS-II proteins fused with GFP are indicated. Ø refers to the Msm::mCherry-wag31 strain containing GFP alone.
Mentions: Since FAS-II localization appeared to follow the reported localization of the polar protein Wag31 [29], [35], a recognized “old pole marker”, Wag31-FAS-II colocalization was therefore studied. For purposes of clarity, the word colocalization is used in this study to describe the localization of two proteins at the same focus but without implying the existence of any molecular interaction. A strain was constructed bearing a single but ectopic chromosomal copy of a mCherry-Wag31 fusion under the control of the constitutive promoter pHSP60 of the integrative vector pMV361. This type of construct, using the pHSP60 promoter, has been successfully used previously to study Wag31 localization [36]. When this strain was observed in fluorescence microscopy (Figure S3), the mCherry fusion was more visible at one pole as also commonly observed by others [36], [37]. This pole has been shown to represent the old pole (Figure 1) [29]. The fusions were introduced into the mCherry-Wag31 producing strain after which the fluorescence of both GFP and mCherry was analyzed as performed above on static images. As observed in the absence of mCherry-Wag31 (Figure 2A), GFP remained diffuse whereas GFP-MabA and GFP-InhA yielded intense polar foci, mainly at one pole (Figure 4A). GFP-KasA and GFP-KasB still appeared diffuse with a lower extend of polar spotting (Figure 4A). The overall localization pattern of the FAS-II fusions was comparable to that of the previous fusions (Figure 2A). The statistical relevance of the localization (Figure 4B) was analyzed as above on individual bacteria expressing each fusion (N) stemming from several individual slides (n>6). The polar indices of MabA (93.93±0.59 N = 800), KasA (23.87±4.56 N = 500) and KasB (13.49±1.43 N = 500) were not statistically different from the previously observed indices (see comparison between Figure 2B and 4B) whereas the PI of GFP-InhA (35.99±2.14 N = 700) was slightly reduced. While there was no dramatic effect of the presence of the mCherry-Wag31 fusion on the percentage of localization of the FAS-II proteins, there was the presence of colocalization of both Wag31 and FAS-II fusions foci. To quantify this phenomenon on a large number of bacteria a colocalization index was defined as being the percentage of bacteria exhibiting Wag31-GFP colocalization relative to the number of GFP-mCherry-positive bacteria. Colocalization indices (Figure 4C) were very high for all FAS-II proteins: MabA (83.31%±3.27, N = 800), InhA (93.93%±2.60, N = 700), KasA (89.71%±3.37, N = 500) and KasB (89.52%±3.60, N = 500). In order to evaluate the distribution of foci within individual bacteria, a more in-depth analysis was performed on a subset of bacteria by scanning more than 100 organisms of each type. On each scan (Figure 4D), the maximum fluorescence was plotted against the normalized length of the bacteria. A dot represented on a graph can refer to a focus (at the pole) or simply to the maximum of diffuse fluorescence along the cell. Localization of the reductases and colocalization with Wag31 at the old poles were clearly confirmed (Figure 4D). Furthermore, the maximum florescence emitted by KasA and KasB, which displayed the lowest polar indices, were clearly preferentially observed in proximity of the old poles along with Wag31, thus suggesting an actual preference for this localization (Figure 4D). The distribution of the polar foci clouds represented only about 20% of total cell size, i.e. less than 1 µm, probably due to light diffusion. This distribution was similar for FAS-II proteins and for Wag31, already known to be localized close to the polar membranes. In contrast, the maximum accumulation of GFP alone was never found at the old pole suggesting a polar exclusion of GFP alone. These analyses clearly demonstrate that the FAS-II proteins were preferentially located at the old pole with Wag31. Since Wag31 is acknowledged to be an old pole marker, we can conclude that the polar FAS-II proteins were located at the active growing “old pole” of the bacteria.

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