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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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Dynamic localization of MmpL3-N10.(A) Images were extracted from the movie presented as movie S13. The time interval after the starting point of the movie is indicated in hours (h). Images were extracted from three channels (bright field, BF, left column; GFP and mCherry, GFP-Che, middle column; GFP, right column). The dynamics of the localization of the GFP fusions was followed in Msm::mCherry-wag31 bacteria expressing the MmpL3-N10-GFP fusions. The presence of MmpL3-N10 in septa is indicated by white arrows in the right column. The position of mCherry-Wag31 at the poles and septa is clearly visible. Magnification: 630 X. Scale bar: 5 µm. (B) Bar graph representation of the localization of MmpL3 and MmpL3-N10 in Msm strains expressing each GFP fusion after 6 hours of induction. Data are represented as mean ± SEM. Data were collected from wide-field microscopy images (as in Figure? 7 and Figure? 8A) by scanning individual bacteria at the level of the membrane, the cytoplasm, and the poles as described in the Materials and Methods. The y-axis represents the polar accumulation (P) of either the complete (Wt) or the N-terminal portion (N10) of the MmpL3 transporter. A statistical t-test was performed to determine the difference between the polar accumulation of MmpL3-GFP (black bar, P = 5.353±0.3810 N = 192) and the absence of polar accumulation of MmpL3-N10-GFP (white bar, P = 1.160±0.1572 N = 113), with N indicating the number of bacteria analyzed. The unpaired t-test p value is symbolized by asterisks (***, p<0.0001).
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pone-0097148-g008: Dynamic localization of MmpL3-N10.(A) Images were extracted from the movie presented as movie S13. The time interval after the starting point of the movie is indicated in hours (h). Images were extracted from three channels (bright field, BF, left column; GFP and mCherry, GFP-Che, middle column; GFP, right column). The dynamics of the localization of the GFP fusions was followed in Msm::mCherry-wag31 bacteria expressing the MmpL3-N10-GFP fusions. The presence of MmpL3-N10 in septa is indicated by white arrows in the right column. The position of mCherry-Wag31 at the poles and septa is clearly visible. Magnification: 630 X. Scale bar: 5 µm. (B) Bar graph representation of the localization of MmpL3 and MmpL3-N10 in Msm strains expressing each GFP fusion after 6 hours of induction. Data are represented as mean ± SEM. Data were collected from wide-field microscopy images (as in Figure? 7 and Figure? 8A) by scanning individual bacteria at the level of the membrane, the cytoplasm, and the poles as described in the Materials and Methods. The y-axis represents the polar accumulation (P) of either the complete (Wt) or the N-terminal portion (N10) of the MmpL3 transporter. A statistical t-test was performed to determine the difference between the polar accumulation of MmpL3-GFP (black bar, P = 5.353±0.3810 N = 192) and the absence of polar accumulation of MmpL3-N10-GFP (white bar, P = 1.160±0.1572 N = 113), with N indicating the number of bacteria analyzed. The unpaired t-test p value is symbolized by asterisks (***, p<0.0001).

Mentions: Recently, the potential membrane protein MmpL3 has been identified in Mtb as an MA transporter [19], [45]. It is involved in the transport of TMM to the external mycomembrane. Because of the localization of major components of FAS-II observed herein, we hypothesized that MA may be synthesized at these loci to be subsequently transported at the actively growing envelope regions. GFP was therefore merged to the MmpL3 C-terminal given that this domain was predicted to be intra-cytoplasmic (Toppred2, [46], http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html). MmpL3, a RND family protein [47], was predicted to be organized in twelve transmembrane helices. The localization of this fusion was analyzed as above. Upon performing time-lapse experiments with MmpL3-GFP alone (Movie S11), or in the presence of mCherry-Wag31 (Figure 7, extracted from Movie S12), a very clear envelope staining was observed together with preferential labeling of one pole and a very bright and flashing labeling of the septum, likely due to the presence of the double membrane at this position. The first conclusion was that MmpL3 might indeed be in the membrane or at least, in the envelope. Its C-terminal domain, fused with the GFP, could be intra-cytoplasmic because GFP is not fluorescent when it is located in another compartment [48]. As a localization control, a GFP fusion of the first ten transmembrane helices of MmpL3 (MmpL3-N10) was constructed and its localization analyzed in vivo as described above. Upon performing time-lapse experiments with MmpL3-N10-GFP in the presence of mCherry-Wag31 (Figure 8, extracted from Movie S13), the preferential MmpL3 polar localization was not observed. MmpL3-N10, which remained in the membrane, or at least in the envelope, was no more accumulated at the pole. The polar accumulation (P) of MmpL3 was quantified as described in Materials and Methods and compared to that of MmpL3-N10 (Figure 8B). MmpL3-N10 localization was not polar (P = 1.16 ± 0.15 N = 113) whereas MmpL3 was enriched by more than 5 fold at the pole (P = 5.35±0.38 N = 192).


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)

Dynamic localization of MmpL3-N10.(A) Images were extracted from the movie presented as movie S13. The time interval after the starting point of the movie is indicated in hours (h). Images were extracted from three channels (bright field, BF, left column; GFP and mCherry, GFP-Che, middle column; GFP, right column). The dynamics of the localization of the GFP fusions was followed in Msm::mCherry-wag31 bacteria expressing the MmpL3-N10-GFP fusions. The presence of MmpL3-N10 in septa is indicated by white arrows in the right column. The position of mCherry-Wag31 at the poles and septa is clearly visible. Magnification: 630 X. Scale bar: 5 µm. (B) Bar graph representation of the localization of MmpL3 and MmpL3-N10 in Msm strains expressing each GFP fusion after 6 hours of induction. Data are represented as mean ± SEM. Data were collected from wide-field microscopy images (as in Figure? 7 and Figure? 8A) by scanning individual bacteria at the level of the membrane, the cytoplasm, and the poles as described in the Materials and Methods. The y-axis represents the polar accumulation (P) of either the complete (Wt) or the N-terminal portion (N10) of the MmpL3 transporter. A statistical t-test was performed to determine the difference between the polar accumulation of MmpL3-GFP (black bar, P = 5.353±0.3810 N = 192) and the absence of polar accumulation of MmpL3-N10-GFP (white bar, P = 1.160±0.1572 N = 113), with N indicating the number of bacteria analyzed. The unpaired t-test p value is symbolized by asterisks (***, p<0.0001).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0097148-g008: Dynamic localization of MmpL3-N10.(A) Images were extracted from the movie presented as movie S13. The time interval after the starting point of the movie is indicated in hours (h). Images were extracted from three channels (bright field, BF, left column; GFP and mCherry, GFP-Che, middle column; GFP, right column). The dynamics of the localization of the GFP fusions was followed in Msm::mCherry-wag31 bacteria expressing the MmpL3-N10-GFP fusions. The presence of MmpL3-N10 in septa is indicated by white arrows in the right column. The position of mCherry-Wag31 at the poles and septa is clearly visible. Magnification: 630 X. Scale bar: 5 µm. (B) Bar graph representation of the localization of MmpL3 and MmpL3-N10 in Msm strains expressing each GFP fusion after 6 hours of induction. Data are represented as mean ± SEM. Data were collected from wide-field microscopy images (as in Figure? 7 and Figure? 8A) by scanning individual bacteria at the level of the membrane, the cytoplasm, and the poles as described in the Materials and Methods. The y-axis represents the polar accumulation (P) of either the complete (Wt) or the N-terminal portion (N10) of the MmpL3 transporter. A statistical t-test was performed to determine the difference between the polar accumulation of MmpL3-GFP (black bar, P = 5.353±0.3810 N = 192) and the absence of polar accumulation of MmpL3-N10-GFP (white bar, P = 1.160±0.1572 N = 113), with N indicating the number of bacteria analyzed. The unpaired t-test p value is symbolized by asterisks (***, p<0.0001).
Mentions: Recently, the potential membrane protein MmpL3 has been identified in Mtb as an MA transporter [19], [45]. It is involved in the transport of TMM to the external mycomembrane. Because of the localization of major components of FAS-II observed herein, we hypothesized that MA may be synthesized at these loci to be subsequently transported at the actively growing envelope regions. GFP was therefore merged to the MmpL3 C-terminal given that this domain was predicted to be intra-cytoplasmic (Toppred2, [46], http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html). MmpL3, a RND family protein [47], was predicted to be organized in twelve transmembrane helices. The localization of this fusion was analyzed as above. Upon performing time-lapse experiments with MmpL3-GFP alone (Movie S11), or in the presence of mCherry-Wag31 (Figure 7, extracted from Movie S12), a very clear envelope staining was observed together with preferential labeling of one pole and a very bright and flashing labeling of the septum, likely due to the presence of the double membrane at this position. The first conclusion was that MmpL3 might indeed be in the membrane or at least, in the envelope. Its C-terminal domain, fused with the GFP, could be intra-cytoplasmic because GFP is not fluorescent when it is located in another compartment [48]. As a localization control, a GFP fusion of the first ten transmembrane helices of MmpL3 (MmpL3-N10) was constructed and its localization analyzed in vivo as described above. Upon performing time-lapse experiments with MmpL3-N10-GFP in the presence of mCherry-Wag31 (Figure 8, extracted from Movie S13), the preferential MmpL3 polar localization was not observed. MmpL3-N10, which remained in the membrane, or at least in the envelope, was no more accumulated at the pole. The polar accumulation (P) of MmpL3 was quantified as described in Materials and Methods and compared to that of MmpL3-N10 (Figure 8B). MmpL3-N10 localization was not polar (P = 1.16 ± 0.15 N = 113) whereas MmpL3 was enriched by more than 5 fold at the pole (P = 5.35±0.38 N = 192).

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