Decision letter: Maturing Mycobacterium smegmatis peptidoglycan requires non-canonical crosslinks to maintain shape (2018)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract In most well-studied rod-shaped bacteria, peptidoglycan is primarily crosslinked by penicillin-binding proteins (PBPs). However, in mycobacteria, crosslinks formed by L,D-transpeptidases (LDTs) are highly abundant. To elucidate the role of these unusual crosslinks, we characterized Mycobacterium smegmatis cells lacking all LDTs. We find that crosslinks generate by LDTs are required for rod shape maintenance specifically at sites of aging cell wall, a byproduct of polar elongation. Asymmetric polar growth leads to a non-uniform distribution of these two types of crosslinks in a single cell. Consequently, in the absence of LDT-mediated crosslinks, PBP-catalyzed crosslinks become more important. Because of this, Mycobacterium tuberculosis (Mtb) is more rapidly killed using a combination of drugs capable of PBP- and LDT- inhibition. Thus, knowledge about the spatial and genetic relationship between drug targets can be exploited to more effectively treat this pathogen. https://doi.org/10.7554/eLife.37516.001 eLife digest Most bacteria have a cell wall that protects them and maintains their shape. Many of these organisms make their cell walls from fibers of proteins and sugars, called peptidoglycan. As bacteria grow, peptidoglycan is constantly broken down and reassembled, and in many species, new units of peptidoglycan are added into the sidewall. However, in a group of bacteria called mycobacteria, which cause tuberculosis and other diseases, the units are added at the tips. The peptidoglycan layer is often a successful target for antibiotic treatments. But, drugs that treat tuberculosis do not attack this layer, partly because we know very little about the cell walls of mycobacteria. Here, Baranowski et al. used genetic manipulation and microscopy to study how mycobacteria build their cell wall. The results showed that these bacteria link peptidoglycan units together in an unusual way. In most bacteria, peptidoglycan units are connected by chemical links known as 4-3 crosslinks. This is initially the same in mycobacteria, but as the cell grows and the cell wall expands, these bonds break and so-called 3-3 crosslinks form. In genetically modified bacteria that could not form these 3-3 bonds, the cell wall became brittle and weak, and the bacteria eventually died. These findings could be important for developing new drugs that treat infections caused by mycobacteria. Baranowski et al. demonstrate that a combination of drugs blocking both 4-3 and 3-3 crosslinks is particularly effective at killing the bacterium that causes tuberculosis. https://doi.org/10.7554/eLife.37516.002 Introduction Peptidoglycan (PG) is an essential component of all bacterial cells (Vollmer et al., 2008a), and the target of many antibiotics. PG consists of linear glycan strands crosslinked by short peptides to form a continuous molecular cage surrounding the plasma membrane. This structure maintains cell shape and protects the plasma membrane from rupture. Our understanding of PG is largely derived from studies on laterally growing model rod-shaped bacteria like Escherichia coli and Bacillus subtilis (Figure 1—figure supplement 1A). In these organisms, new PG is constructed along the lateral side wall by the concerted effort of glycosyltransferases, which connect the glycan of a new PG subunit to the existing mesh, and transpeptidases, which link peptide side chains. An actin-like protein, MreB, positions this multi-protein complex along the short axis of the cell so that glycan strands are inserted circumferentially, creating discontinuous hoops of PG around the cell (Domínguez-Escobar et al., 2011; Garner et al., 2011). This orientation of PG creates a mechanical anisotropy that is responsible for rod shape (Hussain et al., 2018). However, not all rod-shaped bacteria encode MreB. In fact, there are important differences between model bacteria and Actinobacteria like mycobacteria, a genus of rod-shaped bacteria that includes the major human pathogen Mycobacterium tuberculosis (Mtb). In mycobacteria, new PG is inserted at the cell poles (at unequal amounts based on pole age), rather than along the lateral walls (Figure 1A). Additionally, mycobacteria are missing several factors, including MreB, that are important for cell elongation (Kieser and Rubin, 2014). Furthermore, in E. coli and B. subtilis the vast majority (>90%) of the peptide linkages are created by D,D-transpeptidases known as penicillin-binding proteins (PBPs) (Pisabarro et al., 1985). PBPs, the targets of most β-lactams, link the fourth amino acid of one peptide side chain to the third amino acid of another, forming 4–3 crosslinks. Peptidoglycan crosslinks can also be catalyzed by L,D-transpeptidases (LDTs), which link peptide side chains by the third amino acid forming 3–3 linkages (Figure 1—figure supplement 1B). In mycobacteria, these 3–3 crosslinks, are highly abundant, accounting for at least 60% of linkages (Kumar et al., 2012; Lavollay et al., 2008; Wietzerbin et al., 1974). Although there has been extensive characterization of LDTs in vitro (Cordillot et al., 2013; Dubée et al., 2012; Lavollay et al., 2008; Magnet et al., 2007; Mainardi et al., 2005; Mainardi et al., 2007; Triboulet et al., 2013), because PG has been most well studied in bacteria where 3–3 crosslinks are rare, the cellular role of these enzymes and the linkages they create is poorly understood. As is the case with PBPs, there exists many copies of LDTs in the cell - there are five LDTs in Mtb and six in Mycobacterium smegmatis (Msm), a non-pathogenic relative of Mtb (Sanders et al., 2014), making genetic characterization challenging. Also similarly to PBPs, LDT homologues do not appear to functionally overlap completely (Cordillot et al., 2013; Kumar et al., 2017; Schoonmaker et al., 2014). Figure 1 with 4 supplements see all Download asset Open asset FDAAs are incorporated asymmetrically by L,D-transpeptidases. (A) Schematic of mycobacterial asymmetric polar growth. Green, old cell wall; grey, new material; dotted line, septum; large arrows, old pole growth; small arrows, new pole growth. (B) FDAA incorporation in log-phase WT Msm cell after 2 min incubation. Scale bar = 5 µm. Old pole marked with (*). (C) Schematic of Fluorescence Activated Cell Sorting (FACS)-based FDAA transposon library enrichment. An Msm transposon library was stained with FDAAs, the dimmest and brightest cells were sorted, grown, sorted again to enrich for transposon mutants that are unable or enhanced for FDAA incorporation. (D) Results from 1C screen. For each gene, the contribution to low or high staining population was calculated from transposon reads per gene. Plotted is the ratio of the population contribution from the second sort of low FDAA staining (L2) over the second sort of high FDAA staining (H2) cells compared to the Mann-Whitney U p-value. (E) Representative image of FDAA incorporation in log-phase WT, ∆LDT and ∆LDTcomp cells. Scale bar = 5 μm. (F) Profiles of FDAA incorporation in log-phase WT (N = 98), ∆LDT (N = 40), and ∆LDTcomp (N = 77) cells. Thick lines represent mean incorporation profile, thin lines are FDAA incorporation in single cells. https://doi.org/10.7554/eLife.37516.003 Figure 1—source data 1 FDAA FACs screen data used for Figure 1D. https://doi.org/10.7554/eLife.37516.009 Download elife-37516-fig1-data1-v3.xlsx Figure 1—source data 2 FDAA incorporation distribution data used for Figure 1F. https://doi.org/10.7554/eLife.37516.010 Download elife-37516-fig1-data2-v3.xlsx Tuberculosis remains an enormous global health problem, in part, because treating even drug susceptible disease is difficult. The standard regimen includes a cocktail of four drugs given over six months. Treatment of drug-resistant Mtb is substantially longer and includes combinations of up to seven drugs (Global Tuberculosis Report, 2017). While some of the most important anti-mycobacterials target cell wall synthesis, surprisingly, drugs that target PG are not part of the core treatment for either drug-susceptible or drug-resistant disease. However, carbapenems, β-lactam antibiotics that potently inhibit LDTs in vitro (Cordillot et al., 2013; Dubée et al., 2012; Lavollay et al., 2008; Mainardi et al., 2007; Triboulet et al., 2013), are also effective against drug resistant Mtb in vitro and drug-sensitive Mtb in patients (Diacon et al., 2016; Hugonnet et al., 2009). But, why are LDTs important in mycobacteria? To explore this, we constructed a strain of Msm that lacks the ability to form 3–3 crosslinks. We find that 3–3 crosslinks are formed in maturing peptidoglycan and that they are necessary to stabilize the cell wall and prevent lysis. Cells that lose the ability to synthesize 3–3 crosslinks have increased dependence on 4–3 crosslinking. Thus, simultaneous inhibition of both processes results in rapid cell death. Results Fluorescent D-amino acids are incorporated asymmetrically by L,D-transpeptidases PG uniquely contains D-amino acids, which can be conjugated to fluorescent probes (fluorescent D-amino acids, FDAAs) to visualize PG synthesis in live bacterial cells (Kuru et al., 2012). When we incubated Msmwith FDAAs for a short 2 min pulse ( < 2% of Msm’s generation time) we observed incorporation at both poles, the sites of new PG insertion in mycobacteria (Figure 1A,B) (Aldridge et al., 2012). However, we also saw a gradient of fluorescence along the sidewalls, extending from the old pole (the previously established growth pole) that fades to a minimum at roughly mid-cell as it reaches the new pole (the pole formed at the last cell division) (Figure 1B, Figure 1—figure supplement 2). To identify the enzymes responsible for this unexpected pattern of lateral cell wall FDAA incorporation, we performed a fluorescence-activated cell sorting (FACS)-based transposon screen (Figure 1C). Briefly, we stained an Msm transposon library with FDAA and repeatedly sorted the least fluorescent 12.5% of the population by FACS. After each sort we regrew cells, extracted gDNA and used deep sequencing to map the location of the transposons found in the low-staining population. From this screen, we identified three LDTs (ldtA - MSMEG_3528, ldtB - MSMEG_4745, ldtE - MSMEG_0233) (Figure 1D) that appeared primarily responsible for FDAA incorporation. Deleting these three LDTs significantly reduced FDAA incorporation and this defect in incorporation could be partially complemented with constitutive expression of LdtE alone (ldtE-mRFP, Figure 1—figure supplement 3A). To further investigate the physiological role of LDTs, we constructed a strain lacking all 6 LDTs (ΔldtAEBCGF, hereafter ΔLDT). Whole genome sequencing verified all six deletions and did not detect crossover events or chromosomal duplications (see supplemental methods). FDAA incorporation and 3–3 crosslinking are both nearly abolished in ΔLDT cells and can be partially restored by complementation with a single LDT (ldtE-mRFP; ΔLDTcomp) (Figure 1E,F, Figure 1—figure supplements 3B and 4). Thus, as might be the case in Bdellovibrio (Kuru et al., 2017), FDAA incorporation in Msm is primarily LDT-dependent. LDTs have previously been shown to exchange non-canonical D-amino acids onto PG tetrapeptides in Vibrio cholera (Cava et al., 2011). 3–3 crosslinks are required for rod shape maintenance at aging cell wall As deletion of a subset of LDTs in Msm produces morphologic changes (Sanders et al., 2014), we visualized ΔLDT cells by time-lapse microscopy. We observed that a subpopulation of cells loses rod shape progressively over time, resulting in localized spherical blebs (Figure 2A – top row, Figure 2—figure supplement 1A, Figure 2—video 1). Complemented cells are able to maintain rod shape (Figure 2—figure supplement 1B). We reasoned that localized loss of rod shape may occur for two reasons: (1) spatially-specific loss of cell wall integrity and/or (2) cell wall deformation due to uncontrolled, local PG synthesis. If the first hypothesis were true, high osmolarity should protect cells against forming blebs. Indeed, switching cells from iso- to high- osmolarity prevented bleb formation over time (Figure 2A – bottom row, Figure 2—video 2). To test the second hypothesis, we stained ΔLDT or WT cells with an amine-reactive dye, and observed outgrowth of new, unstained material (Figure 2B). Blebs that formed in the ΔLDT cells retained stain, indicating a lack of new cell wall synthesis in the region. WT cells maintained rod shape over time at the stained portion of the bacillus. Collectively, these results indicate that 3–3 crosslinks are required to counteract turgor pressure and maintain rod shape in Msm. This led us to hypothesize that bleb formation is a result of a local defect in cell wall rigidity. Figure 2 with 4 supplements see all Download asset Open asset 3–3 crosslinks are required for rod shape maintenance at aging cell wall. (A) Msm ∆LDT time-lapse microscopy of cells switched from high- to iso- osmolar media (top row, see Figure 2—video 1), or iso- to high osmolar media (bottom row, see Figure 2—video 2). (high = 7H9+150 mM sorbitol; iso = 7H9). t = time in minutes post-osmolarity switch. (B) ∆LDT or WT cells were stained with Alexa 488 NHS-ester (green) to mark existing cell wall, washed, and visualized after outgrowth (unstained material). A, B scale bar = 2 µm. (C) Mean stiffness of WT (N = 73) and ∆LDT (N = 47) Msm cells as measured by atomic force microscopy. Mann-Whitney U p-Value ****<0.0001. (D) Representative profile of cell height and height-normalized stiffness (modulus/height) in a single ∆LDT cell. Pink-shaded portion highlights location of a bleb. (E) Maximum cell width of ∆LDT cell lineages over time. Width of new pole daughters = blue circle; width of old pole daughters = orange circle. Division signs denote a division event. At each division, there are two arrows from the dividing cell leading to the resulting new and old pole daughter cell widths (blue and orange respectively). (F) Model of rod shape loss in old cell wall of ∆LDT cells compared to WT. Green portions of the cell represents old cell wall; grey portion represents new cell wall. The larger arrows indicate more growth from the old pole, while smaller arrows show less relative growth from the new pole. Dotted lines represent septa. op = old pole, np = new pole. https://doi.org/10.7554/eLife.37516.011 Figure 2—source data 1 Modulus (kPa)/Height (nm) for WT and ∆LDT cells used for Figure 2C. https://doi.org/10.7554/eLife.37516.014 Download elife-37516-fig2-data1-v3.xlsx Figure 2—source data 2 Modulus and height for the representative ∆LDT cell corresponding to Figure 2D. https://doi.org/10.7554/eLife.37516.015 Download elife-37516-fig2-data2-v3.xlsx To directly measure cell wall rigidity, we used atomic force microscopy (AFM) on live ΔLDT and WT cells. We measured the rigidity of cells in relation to their height. Generally, WT cells are stiffer than ΔLDT cells (Figure 2C). Blebs in ΔLDT cells can be identified by a sharp increase in height (Figure 2D, pink shaded). Since circumferential stress of the rod measured by AFM is proportional to the radius of the cell, and inversely proportional to the thickness of the cell wall (an immeasurable quantity by AFM), we used cell height, a proxy for radius, to normalize the stiffness measurement. We found that stiffness drops in the area of blebs (Figure 2D, pink shaded). Why does loss of rod shape occur locally and only in a subpopulation of cells? Mycobacterial polar growth and division results in daughter cells with phenotypic differences (Aldridge et al., 2012). For example, the oldest cell wall is specifically inherited by the new pole daughter (Figure 2—figure supplement 2A, Aldridge et al., 2012). We hypothesized that the loss of rod shape might occur in specific progeny generated by cell division. Indeed, the daughter which inherited the new pole from the previous round of division, and the oldest cell wall, consistently lost rod shape over time, while the old pole daughter maintained rod shape (Figure 2E, Figure 2—figure supplement 2B). In addition, blebs localized to the oldest cell wall (Figure 2B), as visualized by pulse-chase labeling of the cell wall. Thus, 3–3 crosslinking is likely occurring in the oldest cell wall, which is non-uniformly distributed along a single cell and in the population via asymmetric polar growth and division. Taken together, these data suggest that LDTs act locally to reinforce aging PG and to maintain rod shape in a subpopulation of Msm cells - specifically, new pole daughters (Figure 2F). Mycobacterium smegmatis is hypersensitive to PBP inactivation in the absence of LDTs Our observations lead to the following model: 4–3 crosslinks made by PBPs are formed at the poles where new PG is inserted and where pentapeptide substrates reside. These newly synthesized 4–3 crosslinks can then be gradually cleaved (by D,D-endopeptidases) as PG ages and moves toward the middle of the cell, leaving tetrapeptide substrates for LDTs to create 3–3 crosslinks. This is consistent with the FDAA incorporation pattern, which reflects the abundance of tetrapeptide substrates available for LDT exchange. Specifically, there are more available tetrapeptides near the poles and fewer near mid-cell, the site of older PG (Figure 1F). In the absence of LDTs to catalyze 3–3 crosslinks, old cell wall loses integrity and turgor pressure causes bleb formation. This model predicts that ΔLDT cells should be even more dependent on 4–3 crosslinking than wild-type cells. To test this hypothesis, we used TnSeq (Long et al., 2015) to identify genes required for growth in cells lacking LDTs (Figure 3A). We found that mutants of two PBPs, pbpA (MSMEG_0031 c) and ponA2 (MSMEG_6201), were recovered at significantly lower frequencies in ΔLDT cells (Figure 3B). Likewise, using allele swapping (Kieser et al., 2015b) (Figure 3C, Figure 3—figure supplement 1), a technique that tests the ability of various alleles to support viability, we found that the transpeptidase (TP) activity of PonA1, which is non-essential in WT cells (Kieser et al., 2015b), becomes essential in ΔLDT cells (Figure 3D). Thus, cells that lack 3–3 crosslinks are more dependent on 4–3 crosslinking enzymes. Figure 3 with 1 supplement see all Download asset Open asset Mycobacterium smegmatis is hypersensitive to PBP inactivation in the absence of LDTs. (A) Fold change in the number of reads for transposon insertion counts in ∆LDT cells compared to WT Msm. p-value is derived from a rank sum test (DeJesus et al., 2015). (B) Transposon insertions per TA dinucleotide in pbpA and ponA2 in WT (grey) and ∆LDT (blue) cells. (C) Schematic of L5 allele swapping experiment. (D) Results of WT or transpeptidase null ponA1 allele swapping experiment in ∆LDT cells. https://doi.org/10.7554/eLife.37516.018 Figure 3—source data 1 ∆LDT Tnseq data used for Figure 3A. Below are the column names with a brief - of gene. - of gene. - of gene. - number of TA sites in gene. Mean - mean insertion over TA sites and for wild-type strain Mean - mean insertion over TA sites and for strain - - sum of insertion counts over TA sites and for wild-type strain - sum of insertion counts over TA sites and for strain - of p-value - of null hypothesis between from p-value - after for Download Figure 3—source data 2 counts per TA site in WT and ∆LDT cells for ponA2 and used in Figure Download Peptidoglycan enzymes to cell wall we hypothesized that enzymes and types of crosslinks should be localized along the of the cell. Specifically, we that 4–3 PBPs at sites of new while 4–3 and 3–3 crosslinking LDTs to sites of older growth PG to the poles, as growth older PG the middle of the cell. To test 4–3 and 3–3 crosslinking enzymes we visualized fluorescent of a PBP and an LDT (Figure both enzymes localized in a gradient pattern along the axis of the cell, not the pattern observed for FDAA incorporation. We found that the distribution pattern of was at the old and new poles, where new PG is inserted (Figure Figure Figure supplement 1A). to the is from the poles, more from the of the in a gradient at the sites of older PG (Figure Figure Figure supplement 1B). Thus, enzymes responsible for 4–3 and 3–3 crosslinks show with to the site of new PG synthesis. This is consistent with the model that these enzymes act on Figure 4 with 6 supplements see all Download asset Open asset Peptidoglycan enzymes to cell wall. (A) Representative fluorescence image of see Figure 1), see Figure and see Figure Scale = 5 µm. (B) (N = (N = or (N = distribution in cells division. (C) Schematic of the in vitro experiment to test and activity of extracted from B. subtilis is first into linear or crosslinked B. subtilis peptidoglycan and then with The are by of the by of with peptidoglycan substrates Figure data 1 Fluorescence used for Figure Download We to a As has been identified in mycobacteria, we used et al., to find to the E. coli an with both and activity et al., we identified a previously shown to have activity in Msm et al., as a to also We and and found that like both and activity on peptidoglycan substrates generated in vitro (Figure Figure supplement We used a for mycobacteria to expression in ΔLDT cells et al., 2017). of the and by led to smaller blebs (Figure supplement Furthermore, localized to from the poles, at sites of older PG (Figure Figure Figure supplement 1C). Taken together, these data are consistent with a model in which blebs are formed in ΔLDT cells due to that bleb formation is not completely by of we that there are in both PBPs and LDTs to Mycobacterium tuberculosis The of 3–3 crosslinks in mycobacteria a While Mtb can be killed by most like which largely target the PBPs, β-lactams, which target both PBPs and LDTs (Kumar et al., 2017; Mainardi et al., 2007; et al., are also effective against Mtb (Diacon et al., 2016; Hugonnet et al., 2009). has been previously and 2013; et al., Kumar et al., 2017; Mainardi et al., that more rapid killing of Mtb could be with drug combinations that target both PBPs and LDTs. Msm Tnseq data that 4–3 transpeptidase activity becomes essential in cells lacking LDTs (Figure the that inhibition of both PBPs and LDTs could mycobacteria very while we could create a strain of Msm lacking all LDTs, previously Mtb Tnseq data that LDTs may be essential in the pathogen (Kieser et al., We Msm and Mtb the from et al., 2012; et al., where can be to growth (Figure supplement 1), to test the combination of and killed Msm or Mtb more rapidly than either drug We found that these drugs together both Msm and Mtb than either alone (Figure Furthermore, this combination in in Mtb not against Msm is as and Materials and Figure Figure supplement 2). This may a in LDT expression or between Msm and Figure 5 with 2 supplements see all Download asset Open asset both PBPs and LDTs mycobacteria more rapidly of Msm (A) and Mtb (B) the from et al., measured via = relative are for drugs were used in combination with 5 Figure data 1 used for Figure Download Discussion The of antibiotics that target like β-lactams, has led to of on this bacterial fluorescent probes have been used to study PG synthesis in live cells of bacterial (Kuru et al., 2012; et al., 2017; et al., 2014). these probes can be incorporated in bacteria and their pattern can mark processes (Kuru et al., 2012). We find that in mycobacteria, FDAA incorporation is primarily LDT-dependent. FDAA incorporation in Msm an unusual gradient pattern et al., 2017), an asymmetric distribution of tetrapeptide for the exchange In to their ability to exchange D-amino acids onto LDTs also catalyze non-canonical 3–3 crosslinks. catalyzed by LDTs are in model rod-shaped bacteria like and B. subtilis are in polar growing bacteria like mycobacteria, and et al., 2012; et al., Kumar et al., 2012; Lavollay et al., 2008; et al., 1985). Here, we find that Msm cells lacking 3–3 crosslinks maintain rod shape at sites of aging cell wall. 4–3 crosslinks made by PBPs appear able to maintain rod shape near the poles, the sites of cell wall (Figure time, as older cell wall moves toward the middle of the cell, it loses and to bleb. The in which rod shape is lost in cells lacking 3–3 crosslinks that cell wall occur to this portion of the with this we find that an that 4–3 crosslinks, the also to sites of old cell wall and of this leads to smaller blebs. Figure 6 Download asset Open asset Model for PG and