1. Microbiology and Infectious Disease
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Maturing Mycobacterium smegmatis peptidoglycan requires non-canonical crosslinks to maintain shape

  1. Catherine Baranowski
  2. Michael A Welsh
  3. Lok-To Sham
  4. Haig A Eskandarian
  5. Hoong Chuin Lim
  6. Karen J Kieser
  7. Jeffrey C Wagner
  8. John D McKinney
  9. Georg E Fantner
  10. Thomas R Ioerger
  11. Suzanne Walker
  12. Thomas G Bernhardt
  13. Eric J Rubin  Is a corresponding author
  14. E Hesper Rego  Is a corresponding author
  1. Harvard TH Chan School of Public Health, United States
  2. Harvard Medical School, United States
  3. National University of Singapore, Singapore
  4. Swiss Federal Institute of Technology in Lausanne, Switzerland
  5. Texas A&M University, United States
  6. Yale University School of Medicine, United States
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Cite this article as: eLife 2018;7:e37516 doi: 10.7554/eLife.37516

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
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

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
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

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
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

Peptidoglycan synthesizing enzymes localize to differentially aged cell wall

Given our model, we hypothesized that enzymes catalyzing and processing different types of crosslinks should be differentially localized along the length of the cell. Specifically, we postulated that 4–3 generating PBPs would localize at sites of new PG, while 4–3 cleaving D,D-endopeptidases and 3–3 crosslinking LDTs would localize to sites of older PG. Polar growth segregates newer PG to the poles, and, as growth occurs, older PG migrates towards the middle of the cell. To test whether 4–3 and 3–3 crosslinking enzymes localize differently, we visualized fluorescent fusions of a PBP (PonA1), and an LDT (LdtE), (Figure 4A). Intriguingly, both enzymes localized in a gradient pattern along the long axis of the cell, not unlike the pattern observed for FDAA incorporation. We found that the distribution pattern of PonA1-RFP was highest at the old and new poles, where new PG is inserted (Figure 4A,B, Figure 4—video 1, Figure 4—figure supplement 1A). Compared to PonA1-RFP, the LdtE-mRFP localization is highest farther from the poles, more inward from the ends of the bacillus (albeit in a similar gradient pattern), at the sites of older PG (Figure 4A,B, Figure 4—video 2, Figure 4—figure supplement 1B). Thus, enzymes responsible for 4–3 and 3–3 crosslinks show distinctive subcellular localizations with respect to the site of new PG synthesis. This is consistent with the model that these enzymes act on differentially aged PG.

Figure 4 with 6 supplements see all
Peptidoglycan synthesizing enzymes localize to differentially aged cell wall.

(A) Representative fluorescence image of PonA1-RFP (magenta, see Figure 4—video 1), LdtE-mRFP (cyan, see Figure 4—video 2), and DacB2-mRFP (green, see Figure 4—video 3). Scale bars = 5 µm. (B) Average PonA1-RFP (N = 24), LdtE-mRFP (N = 23) or DacB2-mRFP (N = 23) distribution in cells before division. (C) Schematic of the in vitro experiment to test D,D-carboxy- and D,D-endopeptidase activity of DacB2 (top). Lipid II extracted from B. subtilis is first polymerized into linear (using SgtB) or crosslinked (using B. subtilis PBP1) peptidoglycan and then reacted with DacB2. The reaction products are analyzed by LC-MS. Extracted ion chromatograms of the reaction products produced by incubation of DacB2 with peptidoglycan substrates (bottom).

https://doi.org/10.7554/eLife.37516.022

We next sought to localize a D,D-endopeptidase. As no D,D-endopeptidase has been clearly identified in mycobacteria, we used HHPRED (Zimmermann et al., 2018) to find candidates. By homology to the E. coli protein AmpH, an enzyme with both D,D- carboxy- and endopeptidase activity (González-Leiza et al., 2011), we identified DacB2 (MSMEG_2433), a protein previously shown to have D,D-carboxypeptidase activity in Msm (Bansal et al., 2015), as a candidate to also harbor D,D-endopeptidase capability. We expressed and purified DacB2 and found that it, like AmpH, had both D,D-carboxypeptidase and D,D-endopeptidase activity on peptidoglycan substrates generated in vitro (Figure 4C, Figure 4—figure supplement 1A–C). We used a recently developed CRISPRi system for mycobacteria to knockdown dacB2 expression in ΔLDT cells (Rock et al., 2017). Induction of the sgRNA and dCas9 by anhydro-tetracycline (aTc) led to smaller blebs (Figure 4—figure supplement 3). Furthermore, DacB2-mRFP localized closer to LDT-mRFP, farther from the poles, at sites of older PG (Figure 4A,B, Figure 4—video 3, Figure 4—figure supplement 1C). Taken together, these data are consistent with a model in which blebs are formed in ΔLDT cells due to unchecked D,D-endopeptidase activity. Given that bleb formation is not completely rescued by knockdown of dacB2, we speculate that there are additional D,D-endopeptidases in M. smegmatis.

Drugs targeting both PBPs and LDTs synergize to kill Mycobacterium tuberculosis

The importance of 3–3 crosslinks in mycobacteria suggests a unique vulnerability. While Mtb can be killed by most non-carbapenem (N-C) β-lactams like amoxicillin, which largely target the PBPs, carbapenem β-lactams, which target both PBPs and LDTs (Kumar et al., 2017; Mainardi et al., 2007; Papp-Wallace et al., 2011) are also effective against Mtb (Diacon et al., 2016; Hugonnet et al., 2009). It has been previously proposed (Gonzalo and Drobniewski, 2013; Gupta et al., 2010; Kumar et al., 2017; Mainardi et al., 2007) that more rapid killing of Mtb could be achieved with drug combinations that target both PBPs and LDTs. Msm Tnseq data suggests that typically dispensable 4–3 transpeptidase activity becomes essential in cells lacking LDTs (Figure 3), supporting the notion that inhibition of both PBPs and LDTs could kill mycobacteria very successfully. Interestingly, while we could create a strain of Msm lacking all LDTs, previously published Mtb Tnseq data suggests that LDTs may be essential in the pathogen (Kieser et al., 2015a).

We utilized Msm and Mtb strains expressing the luxABCDE operon from Photorhabdus luminescens (Andreu et al., 2012; Andreu et al., 2010), where light production can be correlated to growth (Figure 5—figure supplement 1), to test if the combination of amoxicillin (a penam) and meropenem (a carbapenem) killed Msm or Mtb more rapidly than either drug alone. We found that these drugs together kill both Msm and Mtb faster than either alone (Figure 5A,B). Furthermore, this combination exhibits synergism in minimal inhibitory concentration in Mtb but, not against Msm (where synergism is defined as Σ Fractional Inhibitory Concentration <0.5 (‘Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Materials and methods,’ 2016), Figure 5B, Figure 5—figure supplement 2). This may reflect a difference in LDT expression or essentiality between Msm and Mtb.

Figure 5 with 2 supplements see all
Drugs targeting both PBPs and LDTs kill mycobacteria more rapidly when combined (A, B).

Killing dynamics of Msm (A) and Mtb (B) (expressing the luxABCDE operon from Photorhabdus luminescens [Andreu et al., 2010]) measured via luciferase production (RLU = relative light units). Amoxicillin (AM) (Msm-1.25; Mtb-3.125 µg/mL); Meropenem (MR) (Msm-10; Mtb-6.25 µg/mL); Amoxicillin + Meropenem: Msm-1.25 µg/mL AM +10 µg/mL MR; Mtb-3.125 µg/mL AM +6.25 µg/mL MR). Biological triplicate are plotted for Mtb. All drugs were used in combination with 5 µg/mL clavulanate.

https://doi.org/10.7554/eLife.37516.032

Discussion

The success of antibiotics that target PG, like β-lactams, has led to decades of research on this critical bacterial polymer. Recently developed fluorescent probes (FDAAs) have been used extensively to study PG synthesis in live cells of numerous bacterial species (Kuru et al., 2012; Kuru et al., 2017; Liechti et al., 2014). Intriguingly, these probes can be incorporated through diverse pathways in different bacteria and thus, their pattern can mark distinct processes (Kuru et al., 2012). We find that in mycobacteria, FDAA incorporation is primarily LDT-dependent. FDAA incorporation in Msm shows an unusual gradient pattern (Botella et al., 2017), suggesting an asymmetric distribution of tetrapeptide substrate for the LDT-dependent exchange reaction. In addition to their ability to exchange D-amino acids onto tetrapeptides, LDTs also catalyze non-canonical 3–3 crosslinks.

Crosslinks catalyzed by LDTs are rare in model rod-shaped bacteria like E.coli and B. subtilis but, are abundant in polar growing bacteria like mycobacteria, Agrobacterium tumefaciens and Sinorhizobium meliloti (Brown et al., 2012; Cameron et al., 2015; Kumar et al., 2012; Lavollay et al., 2008; Pisabarro et al., 1985). Here, we find that Msm cells lacking 3–3 crosslinks cannot 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 newer cell wall (Figure 6A). Over time, as older cell wall moves toward the middle of the cell, it loses structural stability, and begins to bleb. The gradual manner in which rod shape is lost in cells lacking 3–3 crosslinks suggests that cell wall processing must occur to de-stabilize this portion of the rod. Consistent with this idea, we find that an enzyme that cleaves 4–3 crosslinks, the D,D-endopeptidase/D,D-carboxypeptidase DacB2, also localizes to sites of old cell wall and knockdown of this enzyme leads to smaller blebs.

Model for PG enzyme and substrate distribution as governed by polar growth and PG segregation by age.

(A) A model for PG age, PG enzyme and crosslink segregation via polar growth in mycobacteria. First, 4–3 crosslinks are made by PBPs at site of new growth, where the pentapeptide substrate resides. Then, these 4–3 crosslinks can be cleaved by D,D-endopeptidases (END). This action would leave a free tetrapeptide. Lastly, LDTs can utilize this tetrapeptide to generate 3–3 crosslinks. As this is occurring over time and during polar growth, the aging cell wall moves toward mid-cell (new growth at the poles moves away from the existing cell wall). (B) Schematic of PG segregation by age (top). 2 min FDAA pulse (cyan), 45 min outgrowth, followed by 2 min FDAA chase (magenta) in WT Msm cells (bottom). Newest cell wall (magenta), older cell wall (cyan). Scale bar = 5 μm.

https://doi.org/10.7554/eLife.37516.037

Why would Msm cells create 4–3 crosslinks to eventually cleave them? There are many possibilities. For example, perhaps in the absence of lateral cell wall synthesis, the creation of substrate for LDTs through the destruction of 4–3 crosslinks allows the cell to engage the PG along the lateral cell body. This could be important for altering the thickness of the PG layer or anchoring it to the membrane at sites of otherwise ‘inert’ cell wall. Additionally, it may be that as PG ages, it is being manicured or marked for septal synthesis. Supporting this idea, we find that the gradient localization patterns of fluorescently tagged PonA1, LdtE and DacB2, (as well as FDAAs) all have local minima at mid-cell closer to the new pole- a location that correlates with the asymmetric site of division in mycobacteria (Aldridge et al., 2012; Santi et al., 2013; Eskandarian et al., 2017). The lack of localization of PG synthesis enzymes and FDAAs suggests a lack of penta- and tetra- peptide substrates. This implies that this region of the cell may be more abundantly crosslinked, as crosslinking utilizes these peptide species. Could 3–3 crosslinking be a signal for septal placement? Mycobacteria are missing known molecular septal placement mechanisms like the Noc and the Min system (Hett and Rubin, 2008). The major septal PG hydrolase is RipA, a D,L-endopeptidase which cleaves the bond between the second and third amino acid of PG side chains, a substrate available on 3–3 crosslinked material (Böth et al., 2011; Vollmer et al., 2008b). While LdtE-mRFP does not itself strongly localize to the site of division, the crosslinks it synthesizes could migrate toward the mid-cell through polar elongation. Transmitting information from the tip to mid-cell through polar growth was recently described in mycobacteria: atomic force microscopy revealed cell-envelope deformations formed at the pole of Msm travel to mid-cell through polar growth, marking the future site of division (Eskandarian et al., 2017). Thus, it is intriguing to speculate that 3–3 crosslinks found at aging cell wall could be important for localizing cell division machinery.

In well-studied rod-shaped bacteria like E.coli and B. subtilis, shape is maintained by MreB-directed PG synthesis along the lateral cell body (Garner et al., 2011; Hussain et al., 2018; Ursell et al., 2014). On the other hand, mycobacteria maintain shape in the absence of an obvious MreB homolog, and in the absence of lateral cell wall elongation. Furthermore, in contrast to lateral-elongating bacteria, in which new and old cell wall are constantly intermingled during growth, polar growth segregates new and old cell wall (Figure 6B). We find that mycobacteria appear to utilize 3–3 crosslinks at asymmetrically distributed aging cell wall to provide stability along the lateral body, something that may not be required in the presence of MreB-directed PG synthesis.

New drug combinations for TB are desperately needed. There has been a renewed interest in repurposing FDA-approved drugs for TB treatment (Diacon et al., 2016). Some of that interest has focused on β-lactams, the oldest class of antibiotics which are the therapeutic bedrock for most other infections. We find that the protein targets of two different classes of β-lactams – enzymes which do very similar chemistry – PG crosslinking – are distributed differentially in a single cell and across the population. In the absence of 3–3 crosslinks, 4–3 crosslinks become more important for cell viability. These data predict that a drug combination which inhibits both PBPs and LDTs will work synergistically to more quickly kill Mtb, a prediction we verified in vitro. Interestingly, meropenem combined with amoxicillin/clavulanate resulted in early clearance of Mtb from patient sputum (Diacon et al., 2016). In fact, the combination might be key to accelerated killing of Mtb (Gonzalo and Drobniewski, 2013).

Materials and methods

Key resources table
Reagent type
(species)
or resource
DesignationSource or
reference
IdentifiersAdditional information
Strain (Mycobacterium smegmatis)KB85; (WT Msm)this workMycobacterium smegmatis mc2155Wildtype M. smegmatis
Strain (M. smegmatis)KB134this workmc2155∆ldtA::loxP
Strain (M. smegmatis)KB156this workmc2155∆ldtA::loxP + ∆ldtE:: zeoR
Strain (M. smegmatis)KB200 (∆ldtAEB)this workmc2155∆ldtA::loxP ∆ldtE:: zeoR + ∆ldtB:: hygR
Strain (M. smegmatis)KB209this workmc2155∆ldtA::loxP ∆ldtE::loxP ∆ldtB::loxP + ∆ldtC:: hygR
Strain (M. smegmatis)KB222this workmc2155∆ldtA::loxP ∆ldtE::loxP ∆ldtB::loxP ∆ldtC:: hygR ∆ldtG:: zeoR
Strain (M. smegmatis)KB303 (∆LDT)this workmc2155∆ldtA::loxP ∆ldtE::loxP ∆ldtB::loxP ∆ldtC:: loxP ∆ldtG:: loxP ∆ldtF:: hygR
Strain (Escherichia coli XL1-Blue)KB302this workpTetO-ldtE(MSMEG_0233)-Gly-Gly-Ser linker-mRFP
Strain (M. smegmatis)KB316 (∆LDTcomp)this work[mc2155∆ldtA::loxP ∆ldtE::loxP ∆ldtB::loxP ∆ldtC:: loxP ∆ldtG:: loxP ∆ldtF:: hygR]+KB302
Strain (M. smegmatis)KK311this work; plasmid from Kieser et al. (2015a)mc2155 + TetO-ponA1-RFP (Kieser et al., 2015b)
Strain (Escherichia coli Top10)KB380this workpTetO-dacB2 (MSMEG_2433)-glycine-glycine-serine linker-mRFP
Strain (M. smegmatis)KB414this workmc2155 + KB380
Strain (M. smegmatis)HR583this workKB303 (∆LDT)+CRISPRi vector (Rock et al., 2017) with dacB2 targeting sgRNAPlasmid from Dr. Sarah Fortune (Harvard School of Public Health) and Dr. Jeremy Rock (Rockefeller University)
Strain (E. coli BL21)KB428this workE.coli BL21 + pET28 b (dacB2)Plasmid pET28b from Dr. Suzanne Walker

Bacterial strains and culture conditions

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Unless otherwise stated, M. smegmatis (mc2155) was grown shaking at 37°C in liquid 7H9 media consisting of Middlebrook 7H9 salts with 0.2% glycerol, 0.85 g/L NaCl, ADC (5 g/L albumin, 2 g/L dextrose, 0.003 g/L catalase), and 0.05% Tween 80 and plated on LB agar. M. tuberculosis (H37Rv) was grown in liquid 7H9 with OADC (oleic acid, albumin, dextrose, catalase) with 0.2% glycerol and 0.05% Tween 80. Antibiotic selection for M. smegmatis and M. tuberculosis were done at the following concentrations in broth and on agar: 25 μg/mL kanamycin, 50 μg/mL hygromycin, 20 μg/mL zeocin and 20 μg/mL nourseothricin and, twice those concentrations for cloning in E.coli (TOP10, XL1-Blue and DH5α).

Strain construction

ΔLDT

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M. smegmatis mc2155 mutants lacking ldtABECFG (ΔLDT) was constructed using recombineering to replace endogenous copies with zeocin or hygromycin resistance cassettes flanked by lox sites as previously described (Boutte et al., 2016). Briefly, about 500 base pairs of upstream and downstream sequence surrounding the gene of interest were amplified via PCR (KOD XtremeTM Hot Start DNA polymerase (EMD Millipore, Billerica, MA)). These flanking regions were amplified with overlaps to either a zeocin or hygromycin resistance cassette flanked by loxP sites and these pieces were assembled into a deletion construct via isothermal assembly (Gibson et al., 2009). Each deletion cassette was transformed into Msm expressing inducible copies of RecET for recombination (Murphy et al., 2015). Once deletions were verified by PCR and sequencing, the antibiotic resistance cassettes were removed by the expression of Cre recombinase. The order of deletion construction in the ΔLDT strain was as follows (where arrows represent transformation of a Cre-recombinase plasmid, followed by curing of the Cre-recombinase plasmid as it contains the sacB gene for sucrose counter selection on LB supplemented with 10% sucrose, and strain names are listed in parenthesis). This resulted in the removal of antibiotic cassettes flanked by loxP sites:

1) mc2155ΔldtA:: zeoR (KB103)→ mc2155ΔldtA::loxP (KB134)

Sequence flanking ldtA upstream was amplified with KB208/209; downstream flanking sequence was amplified with KB210/211

2) mc2155ΔldtA::loxP +ΔldtE:: zeoR (KB156)

Sequence flanking ldtE upstream was amplified with KB220/221; downstream flanking sequence was amplified with KB222/223

3) mc2155ΔldtA::loxP ΔldtE:: zeoR + ΔldtB:: hygR (KB200) → mc2155ΔldtA::loxP ΔldtE::loxP ΔldtB::loxP (KB207)

Sequence flanking ldtB upstream was amplified with KB444/445; downstream flanking sequence was amplified with KB446/447

4) mc2155ΔldtA::loxPΔldtE::loxP ΔldtB::loxP + ΔldtC:: hygR (KB209)

Sequence flanking ldtC upstream was amplified with KB216/448; downstream flanking sequence was amplified with KB449/219

5) mc2155ΔldtA::loxP ΔldtE::loxP ΔldtB::loxP ΔldtC:: hygR ΔldtG:: zeoR (KB222)→ mc2155ΔldtA::loxP ΔldtE::loxP ΔldtB::loxP ΔldtC:: loxP ΔldtG:: loxP (KB241)

Sequence flanking ldtG upstream was amplified with KB228/454; downstream flanking sequence was amplified with KB455/231

6) mc2155ΔldtA::loxP ΔldtE::loxP ΔldtB::loxP ΔldtC:: loxP ΔldtG:: loxP ΔldtF:: hygR

Sequence flanking ldtF upstream was amplified with KB224/452; downstream flanking sequence was amplified with KB453/227

(KB303 referred to as ΔLDT).

Mtb and Msm Lux

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M. tuberculosis H37Rv was transformed with a vector expressing the codon optimized Photorhabdus luminescens luxABCDE operon (pMV306hsp + LuxG13 –Addgene #26161; RRID:SCR_005907) (Andreu et al., 2010). This strain is referred to as Mtb Lux. The same plasmid was transformed into Msm and this strain is referred to as Msm Lux.

ΔLDTcomp

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To complement ΔLDT (KB303) we placed a copy of ldtE (MSMEG_0233) under the constitutive TetO promoter (a UV15 derivative within a pMC1s plasmid that is inducible with anhydro-tetracycline in the presence of a tet-repressor TetR, which the ΔLDTcomp strain lacks [Kieser et al., 2015b]) on vector that integrates at the L5 phage integration site of the chromosome of the ΔLDT strain (the vector is marked with kanamycin resistance). A glycine, glycine, serine linker was cloned between ldtE and mRFP in this complementation construct. LdtE lacking a stop codon, with a glycine-glycine-serine linker was amplified with primers 323A/351. The fluorescent protein mRFP was amplified with primers KB352/353 with overlaps to the linker and the vector backbone.

DacB2 expression strain

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A truncated MSMEG_2433 was cloned into pET28b for isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible expression in E. coli BL21 (DE3). MSMEG_2433(29-296) amplified using the primers KB662/663 with overlaps to NdeI digested pET28b. This PCR product was assembled with the digested vector using isothermal assembly. The resulting vector was transformed into E. coli BL21.

PonA1-RFP

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PonA1-RFP was amplified from Kieser et al. (2015a) using primers KK1/KK2 and KK3/KK4, digested with NdeI and cloned into the same vector as ldtE-mRFP (see above).

DacB2-mRFP

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Using the same cloning strategy as for LdtE-mRFP, dacB2 (MSMEG_2433) was amplified lacking a stop codon, with a gly-gly-ser linker using primers KB626/627. The fluorescent protein mRFP was amplified with primers KB628/353 containing overlaps to dacB2, the linker and the vector backbone. The resulting vector was transformed into WT Msm.

PonA1 transpeptidase essentiality L5 allele swapping

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To test essentiality of transpeptidation by PonA1 in the ΔLDT cells, L5 allele swapping as described in Kieser et al., 2015b was performed. The plasmids used in this experiment were previously published in Kieser et al., 2015b. Briefly, a wild-type copy of PonA1 (TetO driven expression, L5 integrating and kanamycin marked) was transformed into ΔLDT. Then, the endogenous copy of ponA1 was replaced with zeocin using the above mentioned recombineering technique (amplifying the construct from a previously published deletion mutation of ponA1 [Kieser et al., 2015b]). Swapping efficiency of either wildtype or transpeptidase mutant PonA1 marked with nourseothricin was tested with a transformation into ΔLDT//L5-TetO-ponA1 (WT)-kanamycin.

Whole genome sequencing

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Whole genome sequencing was performed on wild-type mc2155 as well as the ΔLDT mutant. Sequencing was done on an Illumina HiSeq 4000 (RRID:SCR_016386) with 150 bp paired-end reads. There was a mean depth of coverage of 148x. All 6 LDT genes were verified as deleted. Furthermore, there was no evidence of any duplications or cross-over events based on a coverage plot.

The sequencing has been uploaded to NCBI’s SRA (details for sample identifiers are provided below).

STUDY: PRJNA451029 (SRP141343)

ΔLDT SAMPLE: deltaLdtAEBCGF (SRS3442031)

ΔLDT EXPERIMENT: deltaLdtAEBCGF (SRX4275943)

ΔLDT RUN: deltaLdtAEBCGF_R2.fastq (SRR7403831)

WT SAMPLE: Msmeg-KB (SRS3442032)

WT EXPERIMENT: Msmeg-KB (SRX4275944)

WT RUN: Msmeg-KB_R2.fastq (SRR7403830)

M. tuberculosis and M. smegmatis minimum inhibitory concentration (MIC) determination

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Mtb or Msm Lux was grown to log phase and diluted to an OD600 = 0.006 in each well of non-treated 96-well plates (Genesee Scientific) containing 100 μL of meropenem (Sigma Aldrich) and/or amoxicillin (Sigma Aldrich) diluted in 7H9 + OADC + 5 μg/mL clavulanate (Sigma Aldrich). Msm media contained ADC rather than OADC. Cells were incubated in drug at 37°C shaking for 7 days (Mtb) or 1 day (Msm), 0.002% resazurin (Sigma Aldrich) was added to each well, and the plates were incubated for 24 hr before MICs were determined. Pink wells signify metabolic activity and blue signify no metabolic activity. (Kieser et al., 2015a) Checkerboard MIC plates and fractional inhibitory concentrations were calculated as described in (Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods, 2016).

M. tuberculosis and M. smegmatis drug killing assays

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Mtb Lux was grown to log phase (kanamycin 25 μg/mL) and diluted in 30 mL inkwells (Corning Lifesciences) to an OD600 = 0.05 in 7H9 + OADC + 5 μg/mL clavulanate with varying concentrations of amoxicillin, meropenem, or both. 100 μL of these cultures were pipetted in duplicate into a white 96-well polystyrene plate (Greiner Bio-One) and luminescence was measured in a Synergy H1 microplate reader from BioTek Instruments, Inc. using the Gen5 Software (2.02.11 Installation version). The correlation between relative light units (RLU) and CFU is shown in Msm in Figure 5—figure supplement 1.

Msm Lux was grown to log phase and diluted into white 96-well polystyrene plates to an OD600 = 0.05. Plates were sealed with 4titude Moisture Barrier Seals and shaken continuously at 37°C. Luminescence measurements (RLU) were taken at 15-min intervals integrated over 1000 ms in a TECAN Spark 10M plate reader for 18 hr.

Fluorescent D-amino acid labeling

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NADA (3-[7-nitrobenzofurazan]-carboxamide-D-Alanine), HADA (3-[7-hydroxycoumarin]-carboxamide-D-Alanine) and TADA (3-[5-carboxytetramethylrhodamine]-carboxamide-D-Alanine) were synthesized by Tocris following the published protocol (Kuru et al., 2015). To 1 mL of exponentially growing cells 0.1 mM of FDAA final was added and incubated for 2 min before washing in 7H9 twice. For still imaging, after the second wash, cells were fixed in 7H9 + 1% paraformaldehyde before imaging. For pulse chase experiments, cells were stained, washed with 7H9 and allowed to grow out for 40 min before being stained with a second dye and imaged.

Flow cytometry

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An M. smegmatis transposon library was grown to mid-log phase, and stained with 2 µg/mL NADA for 2 min. Cells were centrifuged and half of the supernatant was discarded. The pellet was resuspended in the remaining supernatant, passed through a 10 µm filter and taken to be sorted (FACSAria; Excitation: 488 nm; Emission filter: 530/30; RRID:SCR_009839). Two bins were drawn at the lowest and highest staining end of the population, representing 12.5% of the population. 600,000 cells from these bins were sorted into 7H9 medium. Half of this was directly plated onto LB agar supplemented with kanamycin to select for cells harboring the transposon. The remaining 300,000 cells were grown out in 7H9 to log phase, stained with FDAA and sorted again to enrich the populations.

Transposon sequencing, mapping and FDAA flow cytometry enrichment analysis

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Genomic DNA (gDNA) was harvested from the sorted transposon library colonies and transposon-gDNA junction libraries were constructed and sequenced using the Illumina Hi-Seq platform (Long et al., 2015). Reads were mapped on the M. smegmatis genome, tallied and reads at each TA site for the bins (low/high incorporating sort 1 and 2) were imported into MATLAB and processed by a custom scripts as described in Rego et al. (2017). Source code for this analysis can be found on GitHub at: https://github.com/hesperrego/baranowski_2018 (copy archived at https://github.com/elifesciences-publications/baranowski_2018).

Sequencing data are available in NCBI’s SRA with accession number SRP141343.

Microscopy

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Both still imaging and time-lapse microscopy were performed on an inverted Nikon TI-E microscope at 60x magnification. Time-lapse was done using a CellASIC ONIX2 Microfluidic System (Millipore Sigma, B04A plate) with constant liquid 7H9 flow in a 37°C chamber. For turgor experiment (Figure 2A), cells were grown in either 7H9 or 7H9 500 mM sorbitol overnight, and then switched to either 7H9 with 150 mM sorbitol (high osmolar) or to 7H9 alone (iso-osmolar).

Atomic force microscopy

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AFM experimentation was conducted as previously(Eskandarian et al., 2017). In short, polydimethylsiloxane (PDMS) – coated coverslips were prepared by spin-coating a mixture of PDMS at a ratio of 15:1 (elastomer:curing agent) with hexane (Sigma 296090) at a ratio of 1:10 (PDMS:hexane) (Koschwanez et al., 2009; Thangawng et al., 2007). A 50 µl filtered (0.5 µm pore size PVDF filter – Millipore) aliquot of bacteria grown to mid-exponential phase and concentrated from 2 to 5 ml of culture was deposited onto the hydrophobic surface of a PDMS-coated coverslip and incubated for ~20 min to increase surface interactions between bacteria and the coverslip. 7H9 medium (~3 ml) was supplied to the sample so as to immerse the bacterial sample and the AFM cantilever in fluid. The AFM imaging mode, Peak Force QNM, was used to image bacteria with a Nanoscope five controller (Veeco Metrology) at a scan rate of 0.5 Hz and a maximum Z-range of 12 µm. A ScanAsyst fluid cantilever (Bruker) was used. Height, peak force error, DMT modulus, and log DMT modulus were recorded for all scanned images in the trace and retrace directions. Images were processed using Gwyddion (Department of Nanometrology, Czech Metrology Institute). ImageJ was used for extracting bacterial cell profiles in a tabular form.

Correlated optical fluorescence and AFM

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Correlated optical fluorescence and AFM images were acquired as described (Eskandarian et al., 2017). Briefly, optical fluorescence images were acquired with an electron-multiplying charge-coupled device (EMCCD) iXon Ultra 897 camera (Andor) mounted on an IX81 inverted optical microscope (Olympus) equipped with an UPLFLN100XO2PH x100 oil immersion objective (Olympus). Transmitted light illumination was provided by a 12V/100W AHS-LAMP halogen lamp. An U-MGFPHQ fluorescence filter cube for GFP with HQ-Ion-coated filters was used to detect GFP fluorescence. The AFM was mounted on top of the inverted microscope, and images were acquired with a Dimension Icon scan head (Bruker) using ScanAsyst fluid cantilevers (Bruker) with a nominal spring constant of 0.7 N m−1 in Peak Force QNM mode at a force setpoint ~1 nN and typical scan rates of 0.5 Hz. Indentation on the cell surface was estimated to be ~10 nm in the Z-axis. Optical fluorescence microscopy was used to identify Wag31-GFP puncta expressed in a wild-type background (Santi et al., 2013) in order to distinguish them from cells of the ∆LDT mutant strains.

Calculating cell surface rigidity

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A cell profile was extracted from AFM Height and DMT Modulus image channels as sequentially connected linear segments following the midline of an individual cell. A background correction was conducted to by dividing the DMT modulus values of the cell surface by the mean value of the PDMS surface and rescaled to compare the cell surface rigidity between individual cells from different experiments. The DMT modulus reflects the elastic modulus (stress-strain relationship) for each cross-sectional increment along the cell length.

Analysis of fluorescent protein distribution

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Using a segmented line, profiles of cells from new to old pole were created at the frame ‘pre-division’ based on physical cell separation of the phase image. A custom FIJI (Schindelin et al., 2012) script was run to extract fluorescence line profiles of each cell and save them as. csv files. These. csv files were imported to Matlab where a custom script was applied to normalize the fluorescence line profile to fractional cell length and to interpolate the fluorescence values to allow for averaging. Source code for this analysis can be found on GitHub at:https://github.com/hesperrego/baranowski_2018

Analysis of cell wall distribution

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Cells were stained with AlexaFluor 488 NHS ester (ThermoFisher Scientific) as described previously (Aldridge et al., 2012) and followed via time-lapse microscopy in the CellASIC device. Briefly, 1 mL of log phase cells was pelleted at 8000 rpm for 1 min and washed with 1 mL PBST. The pellet was resuspended in 100 uL of PBST and 10 uL Alexa Fluor 488 carboxylic acid succinimidyl ester was added for a final concentration of 0.05 mg/mL. This was incubated for 3 min at room temperature. Stained cells were pelleted for 1 min at 13,000 rpm and washed with 500 μL PBST. They were spun again and resuspended in 7H9 for outgrowth observation over time in the CellASIC device.

Analysis of FDAAs

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Images were analyzed using a combination of Oufti (Paintdakhi et al., 2016) (RRID:SCR_016244) for cell selection followed by custom coded Matlab scripts to plot FDAA fluorescence over normalized cell length, calculate cell length and bin cells by existence of an FDAA labeled septum. This code and a manual for its use has been included as a source code file with this manuscript (Source Code-Instructions and code for FDAA image analysis in Figure 1).

Generation of transposon libraries

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M. smegmatis cells were transduced at (OD6001.1–1.7) with φMycoMarT7 phage (temperature sensitive) that has a Kanamycin marked Mariner transposon as previously described (Long et al., 2015). Briefly, mutagenized cells were plated at 37°C on LB plates supplemented with Kanamycin to select for phage transduced cells. Roughly 100,000 colonies per library were scraped, and genomic DNA was extracted. Sequencing libraries were generated specifically containing transposon disrupted DNA. Libraries were sequenced on the Illumina platform. Data were analyzed using the TRANSIT pipeline (DeJesus et al., 2015) (RRID:SCR_016492).

Sequencing data are available in NCBI’s SRA with accession number SRP141343.

Peptidoglycan isolation and analysis

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600 mL of wild-type and ΔLDT cells were grown to log phase and collected via centrifugation at 5000 x g for 10 min at 4°C. The resulting pellet was resuspended in PBS and cells were lysed using a cell disruptor at 35,000 psi twice. Lysed cells were boiled in 10% SDS (sodium dodecyl sulfate) for 30 min and peptidoglycan was collected via centrifugation at 17,000 x g. Pellets were washed with 0.01% DDM(n-Dodecyl β-D-maltoside) to remove SDS and resuspended in 1XPBS + 0.01% DDM. PG was digested with alpha amylase (Sigma A-6380) and alpha chymotrypsin (Amersco 0164) overnight. The samples were again boiled in 10% SDS and washed in 0.01% DDM. The resulting pellet was resuspended in 400 μL 25 mM sodium phosphate pH6, 0.5 mM MgCl2, 0.01% DDM. 20 μL of lysozyme (10 mg/mL) and 20 μL 5 U/μL mutanolysin (Sigma M9901) were added and incubated overnight at 37°C. Samples were heated at 100°C and centrifuged at 100,000 x g. 128 μL of ammonium hydroxide was added and incubated for 5 hr at 37°C. This reaction was neutralized with 122 μL of glacial acetic acid. Samples were lyophilized, resuspended in 300 μL 0.1% formic acid and subjected to analysis by LC-MS/MS. Peptide fragments were separated with an Agilent Technologies 1200 series HPLC on a Nucleosil C18 column (5 μm 100A 4.6 × 250 mm) at 0.5 mL/min flow rate with the following method: Buffer A = 0.1% Formic Acid; Buffer B = 0.1% Formic Acid in acetonitrile; 0% B from 0 to 10 min, 0–20% B from 10 to 100 min, 20% B from 100 to 120 min, 20–80% B from 120 to 130 min, 80% B from 130 to 140 min, 80–0% B from 140 to 150 min, 0% B from 150 to 170 min. MS/MS was conducted in positive ion mode using electrospray ionization on an Agilent Q-TOF (6520).

Expression and purification of MSMEG_2433 (DacB2)

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MSMEG_2433 was expressed and purified using a modified method for purification of low-molecular-weight PBPs that was previously published (Qiao et al., 2014). An N-terminally truncated MSMEG_2433(29-296) was cloned into the pET28b vector for isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible expression in E. coli BL21 (DE3) (see strain construction notes above). 10mLs of overnight culture grown in LB with Kanamycin (50 μg/mL) were diluted 1:100 into 1 L of LB with Kanamycin (50 μg/mL) and grown at 37°C until an OD600 of 0.5. The culture was cooled to room temperature, induced with 0.5 mM IPTG, and shaken at 16°C overnight. Cells were pelleted via centrifugation at 4000 rpm for 20 min at 4°C. The pellet was suspended in 20 mL binding buffer (20 mM Tris pH 8, 10 mM MgCl2, 160 mM NaCl, 20 mM imidazole) with 1 mM phenylmethylsulfonylfluoride (PMSF) and 500 μg/mL DNase. Cells were lysed via three passage through a cell disrupter at ≥10,000 psi. Lysate was pelleted by ultracentrifugation (90,000 × g, 30 min, 4°C). To the supernatant, 1.0 mL washed Ni-NTA resin (Qiagen) was added and the mixture rocked at 4°C for 40 min. After loading onto a gravity column, the resin was washed twice with 10 mL wash buffer (20 mM Tris pH 8, 500 mM NaCl, 20 mM imidazole, 0.1% Triton X-100). The protein was eluted in 10 mL of elution buffer (20 mM Tris pH8, 150 mM NaCl, 300 mM imidazole, 0.1% reduced Triton X-100) and was concentrated to 1 mL with a 10kD MWCO Amicon Ultra Centrifuge Filter. The final protein concentration was measured by reading absorbance at 280 nm and using the estimated extinction coefficient (29459 M−1cm−1) calculate concentration. The protein was diluted to 200 μM in elution buffer with 10% glycerol, aliquoted, and stored at −80°C.

Proper folding of purified MSMEG_2433(29-296) was tested via Bocillin-FL binding. Briefly, 20 μM of purified protein was added to penicillin G (100, 1000 U/mL in 20 mM K2HPO4, 140 mM NaCl, pH7.5) in a 9 μL reaction. The reaction was incubated at 37°C for 1 hr. 10 μM Bocillin-FL was added and incubated at 37°C for 30 min. SDS loading dye was added the quench the reaction and samples were loaded onto a 4–20% gel. MSMEG_2433(29-296) bound by Bocillin-FL was imaged using a Typhoon 9400 Variable Mode Imager (GE Healthcare) (Alexa Excitation-488nm Emission-526nm).

Lipid II extraction

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B. subtilis Lipid II was extracted as previously published (Qiao et al., 2017).

SgtB purification

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S. aureus SgtB was purified as previously published (Rebets et al., 2014).

Purification of B. subtilis PBP1

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Purification of B. subtilis PBP1 was carried out as previously described (Lebar et al., 2014).

In vitro Lipid II polymerization and crosslinking

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20 μM purified BS Lipid II was incubated in reaction buffer (50 mM HEPES pH 7.5, 10 mM CaCl2) with either 5 μM PBP1 or 0.33 μM SgtB for 1 hr at room temperature. The enzymes were heat denatured at 95°C for 5 min. Purified MSMEG_2433(29-296) was added (20 uM, final) and the reaction was incubated at room temperature for 1 hr. Mutanolysin (1 μL of a 4000 U/mL stock) was added and incubated for 1.5 hr at 37°C (twice). The resulting muropeptides were reduced with 30 μL of NaBH4 (10 mg/mL) for 20 min at room temperature with tube flicking every 5 min to mix. The pH was adjusted to ~4 using with 20% H3PO4 and the resulting product was lyophilized to dryness. The residue was resuspended in 18 μL of water and analyzed via LC-MS as previously reported (Welsh et al., 2017).

Experimental replicates

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Biological replicates – independent cultures; Technical replicates – the same culture in replicate.

Microscopy for Figure 1E,F and Figure 1—figure supplement 2 was done once and analyzed. The data shown in Figure 1—figure supplement 3 was done once in technical triplicate. The graph shows one replicate.

Time-lapse experiment in Figure 2A was done twice (biological duplicate on separate days). Included for this figure are videos of full fields of view of the time-lapse experiments (Figure 2—video 1- full field; Figure 2—video 2- full field). Microscopy for Figure 2B was done in biological triplicate on three separate days. The time-lapse phenotype highlighted Figure 2—figure supplement 1 was observed in biological triplicate on 3 independent days. AFM data in Figure 2D and E was derived from two independent experiments done on separate days.

Allele swapping experiment in Figure 3C was done once.

Time-lapse microscopy for Figure 4A was performed in biological duplicate. The graph in Figure 4B represents data from one experiment. Figure 4C is representative data from two technical replicates (the same protein and substrate preparations were used). Microscopy and quantification of bleb size in dacB2 CRISPRi knock-down (Figure 4—figure supplement 3) was done twice (biological duplicate on separate days).

Luciferase Msm data in Figure 5A was performed once. Luciferase Mtb survival data in Figure 5B was done in biological triplicate and technical triplicate. Biological triplicates are plotted. Minimum inhibitory concentrations (MIC) were determined in biological duplicate (two separate cultures on two separate days) and technical duplicate for Figure 5B. Combination MIC for Figure 5—figure supplement 2 was determined once for Mtb and twice for Msm strains.

Fluorescent D-amino acid pulse-chase for Figure 6B was done on two independent days (biological duplicate).

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Decision letter

  1. Wendy S Garrett
    Senior Editor; Harvard TH Chan School of Public Health, United States
  2. Bavesh D Kana
    Reviewing Editor; University of the Witwatersrand, South Africa
  3. Michel Arthur
    Reviewer; INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, France

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Maturing Mycobacterial Peptidoglycan Requires Non-canonical Crosslinks to Maintain Shape" for consideration by eLife. Your article has been reviewed by four peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Wendy Garrett as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Michel Arthur (Reviewer #2); Clif Barry (Reviewer #3); Gyanu Lamichhane (Reviewer #4).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

The manuscript by Dr. Rego and colleagues explores peptidoglycan (PG) metabolism in mycobacteria. The biosynthetic pathway for the PG polymer in these organisms represents several departures from well-known paradigms, most notably; mycobacteria extend their cell surfaces by incorporating new material at the cell tips. Also, the composition of cross-linked mycobacterial PG comprises a large proportion of 3-3 crosslinks, which are relatively rare in model organisms wherein PG has been intensely studied. This form of cross linking is mediated by the activity of L,D-transpeptidases (LDTs) and the authors focus their efforts on describing the function of these enzymes in Mycobacterium smegmatis. Using fluorescent D-amino acids (FDAAs) to visualise PG synthesis, they report strong staining of an FDAA at the cell poles, with a gradient of staining that extends to mid-cell, from the old pole. To determine which enzymes drive this pattern of staining the authors carry out saturating transposon mutagenesis, together with fluorescence associated cell sorting, to identify cells where staining was reduced. This analysis identified three LDTs (LDTA, B, and E) and combinatorial deletion of these resulted in reduced FDAA uptake. The authors proceed to delete all six LDTs (ΔLDT) and note similar effects, indicating that FDAA incorporation in mycobacteria is LDT-dependent. Next, the authors characterise the morphological defects that occur upon combinatorial deletion of all LDTs and report that a proportion of cells lose rod shape, which could be rescued by high osmolarity, suggesting that loss of cell wall rigidity mediated shape defects, a notion supported by measurement of cell rigidity using atomic force microscopy. Time-lapse microscopy revealed that the daughter cell that lost rod shape inherited the new pole and oldest cell wall, with spherical blebs localized in the cell retaining the old cell wall. This suggested that LDTs stabilize old cell wall material. After this, the authors demonstrate that in the absence of LDTs, mycobacteria depend on PbpA and PonA2-mediated 4-3 crosslink formation to maintain cell wall integrity. The authors then demonstrate that PG synthesizing and remodelling enzymes locate to distinct sites of the cell. Combinatorial inhibition of both 4-3 and 3-3 crosslink formation by antibiotics illustrated the potential tractability of targeting PG for tuberculosis drug treatment.

Central conclusions:

1) LDT-mediated 3-3 crosslinks are required for maintenance of rod shape in M. smegmatis.

2) The different types of cross-links, 4-3 versus 3-3, are most likely spatially distributed to distinct locations in the mycobacterial cell, with mature PG locating to the sidewall in a 3-3 crosslinked conformation.

3) In the absence of the ability to generate 3-3 crosslinks, mycobacteria become critically reliant of 4-3 crosslinks generated by HMW PBPs.

The study is carefully conducted and opens new avenues to decipher the mechanism underlying septum localization in mycobacteria and to identify strategies for synergistic killing of mycobacteria by drug combinations.

Essential revisions:

1) In Figure 1B, how do the authors determine the new from the old pole? This most likely related to birth size and intensity of staining at the cell pole, but some illustration of this would be useful in the figure and is required to understand the rest of the work.

2) Data demonstrating how the genotype of the Msm strains lacking all 6 (and 3) LDT-encoding genes was verified is necessary. It could be Southern blotting or sequencing. Sequencing is mentioned in the Materials and methods section, but it is not clear what was done to ensure that there were no unintended cross-over events elsewhere in the chromosome as well.

3) Why just complement with LDTE? Were the other three LDTs enriched/identified in the screen? Was the sextuple mutant necessary to completely eliminate FDAA staining? In this context, for rigour, genetic complementation of the LDT triple mutant is necessary, this seems relatively easy. The same construct used to complement the ΔLDT mutant can be used in this case.

4) The study unambiguously shows that DacB2 has endopeptidase activity (cleavage of 4-3 crosslinks) and that DacB2 co-localizes with LDTE. The authors conclude that the endopeptidase activity of DacB2 is responsible for the formation of blebs in the absence of LDTs. Direct evidence of the proposed model should be obtained by constructing a dacB2 ΔLDT mutant. As dacB2 was deemed to be nonessential in genome wide inactivation studies in M. tuberculosis and direct gene deletion (Microb Pathog. 2012 52:109-16), this experiment should be feasible and is required to support the conclusions of the study. Genetic complementation of the resulting combinatorial mutant (dacB2 ΔLDT mutant) is also required.

5) The MS analysis of peptidoglycan structure appearing in "Figure 1—figure supplement 3B" raises numerous issues including the absence of any definition of the molecular ions that were detected and the inversion of the donor and acceptor stems between the sets of 4-3 and 3-3 crosslinked dimers. There are inconsistencies between the observed mass and the proposed structures, e.g. peaks 7 and 14 display the same mass but contain 2 and 4 NH2, respectively. Please, be consistent and list the number of NH2 rather than a mixed designation with OH for certain peaks. More importantly, peak 10 disappeared upon deletion of the LDT genes although it is reported to contain a 4-3 crosslink (formed by PPBs). These shortfalls must be addressed.

6) The experiments showing the cellular localization of the various proteins are not really as clear. Figure 4, subsection “Peptidoglycan synthesizing enzymes localize to differentially aged cell wall”, first paragraph: While it is clear that LDTE-mRFP localized farther from the poles, it appears PonA1-RFP localizes throughout the cell wall as the staining is clearly visible throughout the cell.

Subsection “Peptidoglycan synthesizing enzymes localize to differentially aged cell wall”, last paragraph: Figure 4A, bottom panel, shows prominent staining in the poles as well. Therefore, the conclusion that 'DacB2-mRFP localized closer to LDT-mRFP' is not entirely supported by the data.

Similarly, from the Discussion (end of third paragraph) to conclude that 3-3 cross-linkages are responsible for localizing the biosynthetic machinery seems inadequate from the location of only one of these proteins. These shortfalls can be addressed by attempting localization of another LDT homologue and providing clearer pictures of how PonA1-RFP localizes. Please ensure that any quantification of localization intensity is done for a sufficient number of cells to ensure robustness.

7) Figure 6A, subsection “Mycobacteria are hypersensitive to PBP inactivation in the absence of LDTs”, first paragraph: It is proposed that D,D-endopeptidase generates tetrapeptide substrates. Data/citation to support this claim is missing. The figure shows tetrapeptide as a substrate. Please address this.

8) Subsection “Drugs targeting both PBPs and LDTs synergize to kill Mtb”: Clavulanic acid at 5 μg/mL was used for this assay. Therefore, it is important to include this detail here. It is not possible to conclude that amoxicillin and meropenem exhibit synergism as the experiment included another agent (clavulanic acid). Or, the assay needs to be repeated in the absence of clavulanic acid to draw this conclusion. Also, previous work (Kumar P et al., 2017) describes the relationship between various B-lactams and transpeptidases of Mtb. The concluding sentence of this paper is that combinations of two different b-lactams would be most optimal to treat TB. This paper is directly relevant to parts of this manuscript and should be considered.

9) The synergy experiments in M. tuberculosis are a (wholly unnecessary) distraction from the importance of the study. It would be far more useful to see the synergy (and or lack of) with the various M. smegmatis mutants. None of the other studies were done in Mtb and there is not even an attempt to discuss the relevant enzymes or orthologs in Mtb. Please address this.

10) Currently, the figures are highly complex and the manuscript would benefit from an improved lead in their content. Figure 6 is limited to substrate and enzyme localization. The relation of the central part to panels A and B is unclear. Considering this, Figure 6 is not at the high quality level of the rest of the manuscript. Address this. A related minor point is that Figure 6 is introduced before Figure 3 to 5. As eLife caters to a broad readership base, improved flow of your text, clear figures demonstrating key conclusions and a carefully synthesized graphic of the central conclusions would substantively enhance the value of your submission.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Maturing Mycobacterium smegmatis Peptidoglycan Requires Non-canonical Crosslinks to Maintain Shape" for further consideration at eLife. Your revised article has been favorably evaluated by Wendy Garrett (Senior Editor), a Reviewing Editor, and one reviewer.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

1) With regards to the previous requests to correct the mass spectroscopy data, the ion designation is still incorrect [M+H] 1+ and [M+2H] 2+. Should the m/z ratio in panel C not be 1012.469?

2) The structure of the ions that correspond to the m/z 903.4 and 904.4 (and 974.5 and 975.5) should be provided.

3). Neutral mass = calculated monoisotopic mass. Observed mass [M+H] should be replaced by the observed monoisotopic mass (or observed m/z [M+H] 1+ if preferred). m/z should be italicized. A brief explanation of the techniqueshould be introduced in the legend.

https://doi.org/10.7554/eLife.37516.041

Author response

Essential revisions:

1) In Figure 1B, how do the authors determine the new from the old pole? This most likely related to birth size and intensity of staining at the cell pole, but some illustration of this would be useful in the figure and is required to understand the rest of the work.

We thank the reviewers for this suggestion. We performed time-lapse microscopy with both FDAA and Alexa488 NHS ester to determine which pole stains brighter with FDAA and from which pole the gradient originates. These data can now be found in Figure 1—figure supplement 2 and are mentioned in the first paragraph of the subsection “Fluorescent D-amino acids are incorporated asymmetrically by L,D99 transpeptidases”.

2) Data demonstrating how the genotype of the Msm strains lacking all 6 (and 3) LDT-encoding genes was verified is necessary. It could be Southern blotting or sequencing. Sequencing is mentioned in the Materials and methods section, but it is not clear what was done to ensure that there were no unintended cross-over events elsewhere in the chromosome as well.

We performed whole genome sequencing on wild-type mc2155 as well as the ΔLDT mutant. Sequencing was done on an Illumina HiSeq 4000 with 150 bp paired-end reads. There was a mean depth of coverage of 148x.

All 6 LDT genes were verified as deleted. Furthermore, there was no evidence of any duplications or cross-over events based on a coverage plot.

As often occurs with strains that have been manipulated extensively, there were unrelated SNPs between the strains (listed below).

–MSMEG_0210:G328V (lprO)

–MSMEG_0946:T35T

–MSMEG_3529:S204S

–MSMEG_3627:M76I (ureA)

–MSMEG_4303:V283D (methyltransferase)

–MSMEG_5447:+c in aa 28/516 (dolichyl-phosphate-mannosyltransferase)

–MSMEG_6454:D190A

The sequencing has been uploaded to NCBI’s SRA (details for sample identifiers are provided below).

STUDY: PRJNA451029 (SRP141343)

ΔLDT SAMPLE: deltaLDTAEBCGF (SRS3442031)

ΔLDT EXPERIMENT: deltaLDTAEBCGF (SRX4275943)

ΔLDT RUN: deltaLDTAEBCGF_R2.fastq (SRR7403831)

WT SAMPLE: Msmeg-KB (SRS3442032)

WT EXPERIMENT: Msmeg-KB (SRX4275944)

WT RUN: Msmeg-KB_R2.fastq (SRR7403830)

These data are mentioned in the subsection “3-3 crosslinks are required for rod shape maintenance at aging cell wall” and methods were added in the subsection “Expression and purification of MSMEG_2433 (DacB2)”).

3) Why just complement with LDTE? Were the other three LDTs enriched/identified in the screen? Was the sextuple mutant necessary to completely eliminate FDAA staining? In this context, for rigour, genetic complementation of the LDT triple mutant is necessary, this seems relatively easy. The same construct used to complement the ΔLDT mutant can be used in this case.

Our screen predicted that the loss of LDTE would result in the most substantial loss of FDAA incorporation. For this reason, we chose to complement with LDTE alone. As the reviewer suggested, we expressed the same construct we have used to complement the phenotypes of the sextuple ΔLDT mutant, in the triple ΔLDTAEB mutant, and found that it partially restores the loss of FDAA incorporation in that genetic background as well. These data are shown in Figure 1—figure supplement 3A and mentioned in the last paragraph of the subsection “Fluorescent D-amino acids are incorporated asymmetrically by L,D-transpeptidases”). These data suggest that additional expression of either LDTA or LDTB may be required for full complementation, something we have not tested.

Additionally, FDAA staining in the sextuple mutant is roughly three fold lower than in the ΔLDTAEB mutant, showing that deletion of additional LDT genes leads to additional reduction in FDAA staining.

4) The study unambiguously shows that DacB2 has endopeptidase activity (cleavage of 4-3 crosslinks) and that DacB2 co-localizes with LDTE. The authors conclude that the endopeptidase activity of DacB2 is responsible for the formation of blebs in the absence of LDTs. Direct evidence of the proposed model should be obtained by constructing a dacB2 ΔLDT mutant. As dacB2 was deemed to be nonessential in genome wide inactivation studies in M. tuberculosis and direct gene deletion (Microb Pathog. 2012 52:109-16), this experiment should be feasible and is required to support the conclusions of the study. Genetic complementation of the resulting combinatorial mutant (dacB2 ΔLDT mutant) is also required.

We thank the reviewers for suggesting this experiment. We have attempted to knock-out dacB2 in the ΔLDT strain, but were unsuccessful. The biological meaning of this is unclear, as the ΔLDT strain already grows quite slowly, and even more so when expressing the RecET recombination machinery. To address this same point, we used a recently-developed mycobacterial CRISPRi system. In the ΔLDT background, we constructed a strain that expresses both a dacB2-specific guide RNA and dCas9 upon addition of anhydro-tetracycline (aTc). Addition of aTc led to smaller blebs compared to the blebs formed by the same strain not induced by aTc (Figure 4—figure supplement 3). These data are consistent with our model, in which bleb formation in ΔLDT is due to unchecked endopeptidase activity. However, as knockdown of dacB2 did not completely eliminate bleb formation in ΔLDT, we hypothesize that there are additional M. smegmatis D,D-endopeptidases. The text for this experiment can be found in the second paragraph of the subsection “Peptidoglycan synthesizing enzymes localize to differentially aged cell wall”.

5) The MS analysis of peptidoglycan structure appearing in "Figure 1——figure supplement 3B" raises numerous issues including the absence of any definition of the molecular ions that were detected and the inversion of the donor and acceptor stems between the sets of 4-3 and 3-3 crosslinked dimers. There are inconsistencies between the observed mass and the proposed structures, e.g. peaks 7 and 14 display the same mass but contain 2 and 4 NH2, respectively. Please, be consistent and list the number of NH2 rather than a mixed designation with OH for certain peaks. More importantly, peak 10 disappeared upon deletion of the LDT genes although it is reported to contain a 4-3 crosslink (formed by PPBs). These shortfalls must be addressed.

We have re-analyzed these data and created a new, more informative and more clearly labeled figure (Figure 1—figure supplement 4). We have provided EICs (extracted ion chromatograms) for 3-3 crosslinked species that are readily detectable. We do not detect any significant 3-3 crosslinks in the ΔLDT strain. The disappearance of peak 10 is curious. However, since we have deleted 6 cell wall enzymes in this strain, we expected there to be other changes in peptidoglycan besides disappearance of 3-3 crosslinks. For instance, the LDTs may play important roles as partners in complexes responsible for creating specific 4-3 crosslinks.

6) The experiments showing the cellular localization of the various proteins are not really as clear. Figure 4, subsection “Peptidoglycan synthesizing enzymes localize to differentially aged cell wall”, first paragraph: While it is clear that LDTE-mRFP localized farther from the poles, it appears PonA1-RFP localizes throughout the cell wall as the staining is clearly visible throughout the cell.

Subsection “Peptidoglycan synthesizing enzymes localize to differentially aged cell wall”, last paragraph: Figure 4A, bottom panel, shows prominent staining in the poles as well. Therefore, the conclusion that 'DacB2-mRFP localized closer to LDT-mRFP' is not entirely supported by the data.

Similarly, from the Discussion (end of third paragraph) to conclude that 3-3 cross-linkages are responsible for localizing the biosynthetic machinery seems inadequate from the location of only one of these proteins. These shortfalls can be addressed by attempting localization of another LDT homologue and providing clearer pictures of how PonA1-RFP localizes. Please ensure that any quantification of localization intensity is done for a sufficient number of cells to ensure robustness.

Upon rereading this section, we agree with the reviewers that it is not written clearly and we have carefully rewritten this section (subsection “Peptidoglycan synthesizing enzymes localize to differentially aged cell wall”). There is a substantial amount of heterogeneity in the localization of all three enzymes. We agree that, for certain cells, PonA1 is localized more uniformly than for other cells. To account for this heterogeneity, we have quantified and averaged the distributions for approximately 25 cells for each strain. This quantification (Figure 4B) clearly shows that PonA1 is most abundant near the poles. While we have not rigorously explored the underlying source of the heterogeneity in the localization of these enzymes, it appears that it could be dynamic in nature. For this reason, we have provided videos of the time-lapse microscopy for these localization studies as well as a frame-by-frame distribution of PonA1-RFP, LDTE-mRFP and DacB2-mRFP in the figure supplement (Figure 4—figure supplement 1). Lastly, we have attempted to localize LDTA-mRFP and LDTB-mRFP using the same cloning scheme as LDTE-mRFP without success.

7) Figure 6A, subsection “Mycobacteria are hypersensitive to PBP inactivation in the absence of LDTs”, first paragraph: It is proposed that D,D-endopeptidase generates tetrapeptide substrates. Data/citation to support this claim is missing. The figure shows tetrapeptide as a substrate. Please address this.

The figure has been modified to clearly show that the 4-3 crosslink generated by the PBP is the substrate for the D,D-endopeptidase reaction. Our in vitro DacB2 experiment (Figure 4C, Figure 4—figure supplement 2) shows that 4-3 crosslinks are cleaved and result in tetrapeptides. As one peptide stem participating in a 4-3 crosslink is a tetrapeptide, when the crosslink is cleaved there must remain a tetrapeptide.

8) Subsection “Drugs targeting both PBPs and LDTs synergize to kill Mtb”: Clavulanic acid at 5 μg/mL was used for this assay. Therefore, it is important to include this detail here. It is not possible to conclude that amoxicillin and meropenem exhibit synergism as the experiment included another agent (clavulanic acid). Or, the assay needs to be repeated in the absence of clavulanic acid to draw this conclusion. Also, previous work (Kumar P et al., 2017) describes the relationship between various B-lactams and transpeptidases of Mtb. The concluding sentence of this paper is that combinations of two different b-lactams would be most optimal to treat TB. This paper is directly relevant to parts of this manuscript and should be considered.

We apologize for missing this critical literature citation and thank the reviewers for this information. We have cited this important work in the Introduction, and in the subsection “Drugs targeting both PBPs and LDTs synergize to kill Mtb Mycobacterium tuberculosis”.

Because clavulanic acid is maintained identically in all conditions, it is controlled for in this experiment and therefore is unlikely to be a confounder for these results.

9) The synergy experiments in M. tuberculosis are a (wholly unnecessary) distraction from the importance of the study. It would be far more useful to see the synergy (and or lack of) with the various M. smegmatis mutants. None of the other studies were done in Mtb and there is not even an attempt to discuss the relevant enzymes or orthologs in Mtb. Please address this.

We thank the reviewers for this important suggestion and agree that, as the manuscript is currently organized, there is a leap to Mtb experiments. We have addressed this by including experiments in Msm (Figure 5A, Figure 5—figure supplement 2) and also by including relevant information regarding LDTs in Mtb (subsection “Drugs targeting both PBPs and LDTs synergize to kill Mycobacterium tuberculosis”).

We do feel, however, that direct testing in Mtb is worthwhile as this has consequences for drug therapy. We already know that there are differences in essentiality of LDTs in Msm and Mtb (Kieser et al.,2015); thus, the lack of synergy by MIC in Msm does not necessarily reflect the potential of this treatment for tuberculosis. The kinetic data shown in Figure 5 (and Figure 5—figure supplement 2) illustrates that the combination of these drugs kills Msm more rapidly than either drug alone. Given this, we reasoned that it was necessary to attempt these drug synergy experiments in the pathogen itself.

10) Currently, the figures are highly complex and the manuscript would benefit from an improved lead in their content. Figure 6 is limited to substrate and enzyme localization. The relation of the central part to panels A and B is unclear. Considering this, Figure 6 is not at the high quality level of the rest of the manuscript. Address this. A related minor point is that Figure 6 is introduced before Figure 3 to 5. As eLife caters to a broad readership base, improved flow of your text, clear figures demonstrating key conclusions and a carefully synthesized graphic of the central conclusions would substantively enhance the value of your submission.

The manuscript has been heavily edited to better assist the reader with the figures. We agree that the central panel of Figure 6 (a summary of the left panel) is not necessary, and actually is quite confusing. We have re-made Figure 6 to address these points.

https://doi.org/10.7554/eLife.37516.042

Article and author information

Author details

  1. Catherine Baranowski

    Department of Immunology and Infectious Disease, Harvard TH Chan School of Public Health, Boston, United States
    Contribution
    Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0407-8609
  2. Michael A Welsh

    Department of Microbiology and Immunobiology, Harvard Medical School, Boston, United States
    Contribution
    Investigation, Visualization, Methodology, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8268-6285
  3. Lok-To Sham

    1. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, United States
    2. Department of Microbiology and Immunology, National University of Singapore, Singapore, Singapore
    Contribution
    Formal analysis, Investigation, Methodology
    Competing interests
    No competing interests declared
  4. Haig A Eskandarian

    1. School of Life Sciences, Swiss Federal Institute of Technology in Lausanne, Lausanne, Switzerland
    2. School of Engineering, Swiss Federal Institute of Technology in Lausanne, Lausanne, Switzerland
    Contribution
    Formal analysis, Investigation, Methodology, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0610-0550
  5. Hoong Chuin Lim

    Department of Microbiology and Immunobiology, Harvard Medical School, Boston, United States
    Contribution
    Software, Formal analysis, Visualization, Methodology
    Competing interests
    No competing interests declared
  6. Karen J Kieser

    Department of Immunology and Infectious Disease, Harvard TH Chan School of Public Health, Boston, United States
    Contribution
    Resources, Writing—review and editing
    Competing interests
    No competing interests declared
  7. Jeffrey C Wagner

    Department of Immunology and Infectious Disease, Harvard TH Chan School of Public Health, Boston, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  8. John D McKinney

    School of Life Sciences, Swiss Federal Institute of Technology in Lausanne, Lausanne, Switzerland
    Contribution
    Resources, Supervision, Funding acquisition
    Competing interests
    No competing interests declared
  9. Georg E Fantner

    School of Engineering, Swiss Federal Institute of Technology in Lausanne, Lausanne, Switzerland
    Contribution
    Resources, Supervision, Funding acquisition
    Competing interests
    No competing interests declared
  10. Thomas R Ioerger

    Department of Computer Science and Engineering, Texas A&M University, Texas, United States
    Contribution
    Data curation, Formal analysis, Writing—review and editing
    Competing interests
    No competing interests declared
  11. Suzanne Walker

    Department of Microbiology and Immunobiology, Harvard Medical School, Boston, United States
    Contribution
    Resources, Supervision, Funding acquisition
    Competing interests
    No competing interests declared
  12. Thomas G Bernhardt

    Department of Microbiology and Immunobiology, Harvard Medical School, Boston, United States
    Contribution
    Resources, Supervision, Funding acquisition, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3566-7756
  13. Eric J Rubin

    1. Department of Immunology and Infectious Disease, Harvard TH Chan School of Public Health, Boston, United States
    2. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, United States
    Contribution
    Conceptualization, Resources, Supervision, Funding acquisition, Writing—review and editing
    For correspondence
    erubin@hsph.harvard.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5120-962X
  14. E Hesper Rego

    Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, United States
    Contribution
    Conceptualization, Resources, Software, Formal analysis, Supervision, Investigation, Writing—review and editing
    For correspondence
    hesper.rego@yale.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2973-8354

Funding

National Institutes of Health (U19 AI107774)

  • Thomas R Ioerger
  • Eric J Rubin

Burroughs Wellcome Fund (Career Award at the Scientific Interface)

  • E Hesper Rego

American Heart Association (14POST18480014)

  • Lok-To Sham

Simons Foundation (Fellow of the Life Sciences Research Foundation Award)

  • Hoong C Lim

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (310030_156945)

  • John D McKinney

Seventh Framework Programme (FP7/2007-2013/ERC Grant agreement No. 307338 (NaMic))

  • Georg E Fantner

European Molecular Biology Organization (191-2014)

  • Haig A Eskandarian

National Science Foundation (DGE1144152)

  • Karen J Kieser

National Institutes of Health (F32AI104287)

  • E Hesper Rego

National Institutes of Health (R01 GM76710)

  • Suzanne Walker

National Institutes of Health (R01AI083365)

  • Thomas G Bernhardt

National Institutes of Health (F32GM123579)

  • Michael A Welsh

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (205321_134786)

  • Georg E Fantner

Innovative Medicines Initiative (115337)

  • John D McKinney

EU-FP7/Eurostars (E!8213)

  • Georg E Fantner

European Molecular Biology Organization (750-2016)

  • Haig A Eskandarian

National Science Foundation (DGE0946799)

  • Karen J Kieser

National Institutes of Health (U19AI109764)

  • Thomas G Bernhardt

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (205320_152675)

  • Georg E Fantner

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Cara Boutte and Erkin Kuru for discussion and thorough manuscript review. We are grateful to the Rubin and Fortune laboratory for discussion and input. We also thank the Microbiology and Immunobiology department at Harvard Medical School for sharing equipment and reagents, as well as the BSL3 staff at the Harvard School of Public health for tremendous support. This work was supported by the National Institutes of Health U19 AI107774 to EJR and TRI, F32AI104287 to EHR (who is also supported by a Career Award at the Scientific Interface from BWF), R01 GM76710 to SW, R01AI083365 and U19AI109764 to TGB. MAW is supported by an F32 GM123579. LTS was supported by an American Heart Association Postdoctoral fellowship (14POST18480014). HCL is funded by a Simons Foundation Fellow of the Life Sciences Research Foundation award. This work was also supported in part by the Swiss National Science Foundation (310030_156945) and the Innovative Medicines Initiative (115337) to JDM, and from the Swiss National Science Foundation (205321_134786 and 205320_152675), the European Union FP7/2007-2013/ERC Grant Agreement No. 307338 (NaMic), and EU-FP7/Eurostars E!8213 to GEF Support for HAE comes from a European Molecular Biology Organization Long Term Fellowships (191–2014 and 750–2016). KJK was supported by the National Science Foundation Graduate Research Fellowship (DGE1144152, DGE0946799).

Senior Editor

  1. Wendy S Garrett, Harvard TH Chan School of Public Health, United States

Reviewing Editor

  1. Bavesh D Kana, University of the Witwatersrand, South Africa

Reviewer

  1. Michel Arthur, INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, France

Publication history

  1. Received: April 12, 2018
  2. Accepted: October 11, 2018
  3. Accepted Manuscript published: October 16, 2018 (version 1)
  4. Version of Record published: November 12, 2018 (version 2)
  5. Version of Record updated: September 5, 2019 (version 3)

Copyright

© 2018, Baranowski et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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