Distinct Spatiotemporal Dynamics of Peptidoglycan Synthesis between Mycobacterium smegmatis and Mycobacterium tuberculosis

Abstract

Peptidoglycan (PG), a polymer cross-linked by d-amino acid-containing peptides, is an essential component of the bacterial cell wall. We found that a fluorescent d-alanine analog (FDAA) incorporates chiefly at one of the two poles in Mycobacterium smegmatis but that polar dominance varies as a function of the cell cycle in Mycobacterium tuberculosis: immediately after cytokinesis, FDAAs are incorporated chiefly at one of the two poles, but just before cytokinesis, FDAAs are incorporated comparably at both. These observations suggest that mycobacterial PG-synthesizing enzymes are localized in functional compartments at the poles and septum and that the capacity for PG synthesis matures at the new pole in M. tuberculosis Deeper knowledge of the biology of mycobacterial PG synthesis may help in discovering drugs that disable previously unappreciated steps in the process.IMPORTANCE People are dying all over the world because of the rise of antimicrobial resistance to medicines that could previously treat bacterial infections, including tuberculosis. Here, we used fluorescent d-alanine analogs (FDAAs) that incorporate into peptidoglycan (PG)-the synthesis of which is an attractive drug target-combined with high- and super-resolution microscopy to investigate the spatiotemporal dynamics of PG synthesis in M. smegmatis and M. tuberculosis FDAA incorporation predominates at one of the two poles in M. smegmatis In contrast, while FDAA incorporation into M. tuberculosis is also polar, there are striking variations in polar dominance as a function of the cell cycle. This suggests that enzymes involved in PG synthesis are localized in functional compartments in mycobacteria and that M. tuberculosis possesses a mechanism for maturation of the capacity for PG synthesis at the new pole. This may help in discovering drugs that cripple previously unappreciated steps in the process.

Keywords: cell division; infectious diseases; microbiology; mycobacteria; peptidoglycan; tuberculosis.

FIG 1

Composition of M. tuberculosis peptidoglycan and structures of the fluorescent d-alanine analogs that allow its visualization. (A) Schematic representation of M. tuberculosis peptidoglycan (PG). M. tuberculosis’s PG is composed of strands of a repeating unit of the disaccharide N-acetyl-glucosamine (NAG) and N-acetyl-muramic acid (NAM). The strands are linked by peptide chains composed of l-alanine (l-Ala), d-iso-glutamine (d-iso-Glu, a product of amidation of d-Glu), meso-diaminopimelic acid (meso-DAP), and d-alanine (d-Ala). (B) Chemical structures of the fluorescent d-alanine analogs, HADA and NADA. Ex., excitation; Em., emission.

FIG 2

M. smegmatis elongates chiefly from one of the two poles. (A) Examples of super-resolution photographs of M. smegmatis cells labeled sequentially with HADA for 4 h and then with NADA for 30 min or labeled sequentially with NADA for 4 h and then with HADA for 30 min. Scale bar, 2 μm. Short-pulsed d-alanine analogs incorporated asymmetrically at the two poles. (B) Super-resolution photographs of M. smegmatis labeled sequentially with NADA for 4 h and HADA for 30 min. The middle panel shows the same cells as in the left panel after a 36° rotation around the y axis. Arrowhead, open-ring septum. Inset, enlarged photograph of an open-ring septum. Scale bar, 2 μm. The right panel shows an example of a septum that was labeled with both NADA and HADA. Scale bar, 0.5 μm. (C) Labeling of live or heat-killed M. smegmatis cells with 1 mM HADA and 1 mM HALA. Grey, transmitted-light snapshots. Scale bar, 2 μm. (D) False-colored fluorescence intensities of cells in snapshots presented in panel C. (E) (Top) Fluorescence intensities along the longitudinal axes of 340 cells labeled with 1 mM HADA for 30 min and sorted from the shortest (left) to the longest (right). (Bottom) Absolute lengths of the 340 cells sorted from the shortest (left) to the longest (right). (F) The incorporation index (log₁₀ ii) is the logarithm of the ratio of the fluorescence intensities of the 2 halves of a cell. For short-pulsed cells, the log₁₀ ii documents whether the incorporation of d-alanine analogs at the poles is symmetrical (log₁₀ ii of ~0) or asymmetrical (log₁₀ ii of >0). (G) Log₁₀ ii values of all 340 cells in panel E relative to their lengths. Each dot represents a single cell profiled from 4 independent experiments. The yellow, blue, red, and green shaded areas represent the first, second, third, and fourth size quartiles, respectively. The red dots represent the median values for size and the log₁₀ ii values of all cells in each quartile. The blue line indicates the Spearman correlation. Cells bearing a septum were excluded from the analysis. (H) Cumulative distribution function of the incorporation index. For each quartile color coded in panel G, the curve reports the cumulative frequency cell counts as a function of the log₁₀ ii. This indicates the distribution of the log₁₀ ii values in each quartile and allows comparisons among them. The table includes the following: the Spearman correlation coefficient; its associated P value; the Kruskal-Wallis multiple-comparison test (a significant Kruskal-Wallis test indicates that a minimum of one quartile differs from the others); and the post hoc two-by-two comparison of each quartile with another using a Tukey-Kramer procedure. For the latter statistical test, the null hypothesis is that the data in two quartiles are from the same continuous distribution (value of 0); the alternative hypothesis is that the data in two quartiles are from different continuous distributions (value of 1). The result is 1 if the test rejects the null hypothesis at the 1% significance level and 0 otherwise.

FIG 3

Expression of Wag31-mCherry affects M. smegmatis cells’ elongation. (A) Merodiploid expression of Wag31-mCherry does not alter the growth of M. smegmatis. (B) M. smegmatis Wag31-mCherry cells perfused for 30 min with 1 mM HADA. Scale bar, 2 μm. (C) Super-resolution photographs of M. smegmatis Wag31-mCherry cells labeled sequentially with NADA for 4 h and HADA for 30 min. (Inset) Enlarged photograph, after a 48° rotation around the y axis, of the closed-ring septum boxed in green. Two Wag31-mCherry foci localized to the septum. Scale bar, 2 μm. (D) (Top) Fluorescence intensities along the longitudinal axes of 281 cells, sorted from the shortest (left) to the longest (right). (Bottom) Absolute lengths of the 281 cells, sorted from the shortest (left) to the longest (right). (E) Log₁₀ ii values of all 281 cells in panel D relative to their lengths. The 281 cells profiled are from 3 independent experiments. The yellow, blue, red, and green shaded areas represent the first, second, third, and fourth size quartiles, respectively. The red dots represent the median values for size and the log₁₀ ii values of all cells in each quartile. The blue line indicates the Spearman correlation. Cells bearing a septum were excluded from the analysis. (F) Cumulative distribution functions of the incorporation index. For each quartile color coded in panel E, the curve reports the cumulative frequency cell count as a function of the log₁₀ ii. This indicates the distribution of the log₁₀ ii values in each quartile and allows comparisons among them. Statistical analysis is as described in the legend to Fig. 2H.

FIG 4

Variations in polar dominance of FDAA incorporation as a function of cell cycle in M. tuberculosis. (A) M. tuberculosis growth is not impaired for up to 7 days of perfusion with 1 mM HADA or 1 mM NADA. The final concentration of DMSO in 7H9 medium supplemented with d-alanine analogs was 1%. (B) (Top) M. tuberculosis cells perfused for 4 h with 1 mM HADA. Similar levels of cell-to-cell FDAA incorporation were seen with longer perfusion of FDAAs. (Bottom) Examples of super-resolution photographs of M. tuberculosis cells labeled with 1 mM HADA for 16 h. Scale bar, 2 μm. (C) (Top) Fluorescence intensities along the longitudinal axes of 612 cells labeled with 1 mM HADA for 4 h, sorted from the shortest (left) to the longest (right). (Bottom) Absolute lengths of the 612 cells, sorted from the shortest (left) to the longest (right). (D) Log₁₀ ii values of all 612 cells in panel C relative to their lengths. Each dot represents a single cell profiled from 3 independent experiments. The yellow, blue, red, and green shaded areas represent the first, second, third, and fourth size quartiles, respectively. The red dots represent the median values for size and the log₁₀ ii values for all cells in each quartile. The blue line indicates the Spearman correlation. Cells bearing a septum were excluded from the analysis. (E) Cumulative distribution functions of the incorporation index. For each quartile color coded in panel D, the curve reports the cumulative frequency cell count as a function of the log₁₀ ii. This indicates the distribution of the log₁₀ ii values in each quartile and allows comparisons among them. Statistical analysis is as described in the legend to Fig. 2H. (F) Schematic representation of the modes of elongation of M. smegmatis and M. tuberculosis cells.

FIG 5

Morphotypes of M. smegmatis and M. tuberculosis cells treated with meropenem. (A) High-resolution photographs of M. smegmatis cells untreated or treated with 0.5×, 2×, and 10× MIC of meropenem for 4 h. Scale bar, 2 μm. Arrow, a bacterium that is undergoing branching. (Inset) Two foci, indicative of an open septum. (B) Proportion of untreated or meropenem-treated (0.5 × MIC) M. smegmatis cells that bore a septum. Two independent experiments were quantified. (C) Super-resolution photographs of M. smegmatis cells untreated or treated with 0.5×, 2×, and 10× MIC of meropenem for 4 h. Scale bar, 2 μm. Green arrowhead, open septum; blue arrowhead, closed septum; red arrowhead, clubbed pole. (D) High-resolution photographs of M. tuberculosis cells untreated or treated with 0.5× and 2× MIC of meropenem for 24 h. Scale bar, 2 μm. Blue arrowhead, a shorter cell with one swollen pole; yellow arrowhead, a longer dividing cell with two swollen poles. (E) Super-resolution photographs of M. tuberculosis cells treated with 0.5× MIC of meropenem for 24 h. Scale bar, 2 μm.