Acyl acceptor recognition by Enterococcus faecium l,d‐transpeptidase Ldtfm

In Mycobacterium tuberculosis and ampicillin‐resistant mutants of Enterococcus faecium, the classical target of β‐lactam antibiotics is bypassed by l ,d ‐transpeptidases that form unusual 3 → 3 peptidoglycan cross‐links. β‐lactams of the carbapenem class, such as ertapenem, are mimics of the acyl donor substrate and inactivate l ,d ‐transpeptidases by acylation of their catalytic cysteine. We have blocked the acyl donor site of E. faecium l ,d ‐transpeptidase Ldt_fm by ertapenem and identified the acyl acceptor site based on analyses of chemical shift perturbations induced by binding of peptidoglycan fragments to the resulting acylenzyme. An nuclear magnetic resonance (NMR)‐driven docking structure of the complex revealed key hydrogen interactions between the acyl acceptor and Ldt_fm that were evaluated by site‐directed mutagenesis and development of a cross‐linking assay. Three residues are reported as critical for stabilisation of the acceptor in the Ldt_fm active site and proper orientation of the nucleophilic nitrogen for the attack of the acylenzyme carbonyl. Identification of the catalytic pocket dedicated to the acceptor substrate opens new perspectives for the design of inhibitors with an original mode of action that could act alone or in synergy with β‐lactams.     

Discrete and overlapping functions of peptidoglycan synthases in growth,cell division and virulence of Listeria monocytogenes

Upon ingestion of contaminated food, Listeria monocytogenes can cause serious infections in humans that are normally treated with β‐lactam antibiotics. These target Listeria's five high molecular weight penicillin‐binding proteins (HMW PBPs), which are required for peptidoglycan biosynthesis. The two bi‐functional class A HMW PBPs PBP A1 and PBP A2 have transglycosylase and transpeptidase domains catalyzing glycan chain polymerization and peptide cross‐linking, respectively, whereas the three class B HMW PBPs B1, B2 and B3 are monofunctional transpeptidases. The precise roles of these PBPs in the cell cycle are unknown. Here we show that green fluorescent protein (GFP)‐PBP fusions localized either at the septum, the lateral wall or both, suggesting distinct and overlapping functions. Genetic data confirmed this view: PBP A1 and PBP A2 could not be inactivated simultaneously, and a conditional double mutant strain is largely inducer dependent. PBP B1 is required for rod‐shape and PBP B2 for cross‐wall biosynthesis and viability, whereas PBP B3 is dispensable for growth and cell division. PBP B1 depletion dramatically increased β‐lactam susceptibilities and stimulated spontaneous autolysis but had no effect on peptidoglycan cross‐linkage. Our in vitro virulence assays indicated that the complete set of all HMW PBPs is required for maximal virulence.     

Activation of the l,d‐transpeptidation peptidoglycan cross‐linking pathway by a metallo‐d,d‐carboxypeptidase in Enterococcus faecium

Bypass of the penicillin‐binding proteins by an l ,d ‐transpeptidase (Ldt_fm) confers cross‐resistance to β‐lactam and glycopeptide antibiotics in mutants of Enterococcus faecium selected in vitro. Ldt_fm is produced by the parental strain D344S although it insignificantly contributes to peptidoglycan cross‐linking as pentapeptide stems cannot be used as acyl donors by this enzyme. Here we show that production of the tetrapeptide substrate of Ldt_fm is controlled by a two‐component regulatory system (DdcRS) and a metallo‐d ,d ‐carboxypeptidase (DdcY). The locus was silent in D344S and its activation was due to amino acid substitutions in DdcS or DdcR that led to production of DdcY and hydrolysis of the C‐terminal d ‐Ala residue of the cytoplasmic peptidoglycan precursor UDP‐MurNAc‐pentapeptide. The T¹⁶¹A and T¹⁶¹M substitutions affected a position of DdcS known to be essential for the phosphatase activity of related sensor kinases. Complete elimination of UDP‐MurNAc‐pentapeptide, which was required specifically for resistance to glycopeptides, involved substitutions in DdcY that increased the catalytic efficiency of the enzyme (E¹²⁷K) and affected its interaction with the cell envelope (I¹⁴N). The ddc locus displays striking similarities with portions of the van vancomycin resistance gene clusters, suggesting possible routes of emergence of cross‐resistance to glycopeptides and β‐lactams in natural conditions.     

Distinct mechanisms contribute to immunity in the lantibiotic NAI‐107 producer strain Microbispora ATCC PTA‐5024

The investigation of self‐resistance in antibiotic producers is important to understand the emergence of antibiotic resistance in pathogens and to improve antibiotic production. Lantibiotics are ribosomally synthesized antibiotics that mostly target peptidoglycan biosynthesis. The actinomycete Microbispora ATCC PTA‐5024 produces the lantibiotic NAI‐107, which interferes with peptidoglycan biosynthesis by binding bactoprenol‐pyrophosphate‐coupled peptidoglycan precursors. In order to understand how Microbispora counteracts the action of its own antibiotic, its peptidoglycan composition was analysed in detail. Microbispora peptidoglycan consists of muropeptides with D‐Ala and Gly in similar proportion at the fourth position of the peptide stems and alternative 3‐3 cross‐links besides the classical 4‐3 cross‐links. In addition, the NAI‐107 biosynthetic gene cluster (mlb) was analysed for the expression of immunity proteins. We show that distinct immunity determinants are encoded in the mlb cluster: the ABC transporter MlbYZ acting cooperatively with the transmembrane protein MlbJ and the lipoprotein MlbQ. NMR structural analysis of MlbQ revealed a hydrophobic surface patch, which is proposed to bind the cognate lantibiotic. This study demonstrates that immunity in Microbispora is not only based on one determinant but on the action of the distinct immunity proteins MlbQ, MlbYZ and MlbJ.     

Differential modes of crosslinking establish spatially distinct regions of peptidoglycan in Caulobacter crescentus

The diversity of cell shapes across the bacterial kingdom reflects evolutionary pressures that have produced physiologically important morphologies. While efforts have been made to understand the regulation of some prototypical cell morphologies such as that of rod‐shaped Escherichia coli, little is known about most cell shapes. For Caulobacter crescentus, polar stalk synthesis is tied to its dimorphic life cycle, and stalk elongation is regulated by phosphate availability. Based on the previous observation that C. crescentus stalks are lysozyme‐resistant, we compared the composition of the peptidoglycan cell wall of stalks and cell bodies and identified key differences in peptidoglycan crosslinking. Cell body peptidoglycan contained primarily DD‐crosslinks between meso‐diaminopimelic acid and D‐alanine residues, whereas stalk peptidoglycan had more LD‐transpeptidation (meso‐diaminopimelic acid‐meso‐diaminopimelic acid), mediated by LdtD. We determined that ldtD is dispensable for stalk elongation; rather, stalk LD‐transpeptidation reflects an aging process associated with low peptidoglycan turnover in the stalk. We also found that lysozyme resistance is a structural consequence of LD‐crosslinking. Despite no obvious selection pressure for LD‐crosslinking or lysozyme resistance in C. crescentus, the correlation between these two properties was maintained in other organisms, suggesting that DAP‐DAP crosslinking may be a general mechanism for regulating bacterial sensitivity to lysozyme.     

Insight into a strategy for attenuating AmpC‐mediated β‐lactam resistance: Structural basis for selective inhibition of the glycoside hydrolase NagZ

NagZ is an exo‐N‐acetyl‐β‐glucosaminidase, found within Gram‐negative bacteria, that acts in the peptidoglycan recycling pathway to cleave N‐acetylglucosamine residues off peptidoglycan fragments. This activity is required for resistance to cephalosporins mediated by inducible AmpC β‐lactamase. NagZ uses a catalytic mechanism involving a covalent glycosyl enzyme intermediate, unlike that of the human exo‐N‐acetyl‐β‐glucosaminidases: O‐GlcNAcase and the β‐hexosaminidase isoenzymes. These latter enzymes, which remove GlcNAc from glycoconjugates, use a neighboring‐group catalytic mechanism that proceeds through an oxazoline intermediate. Exploiting these mechanistic differences we previously developed 2‐N‐acyl derivatives of O‐(2‐acetamido‐2‐deoxy‐D ‐glucopyranosylidene)amino‐N‐phenylcarbamate (PUGNAc), which selectively inhibits NagZ over the functionally related human enzymes and attenuate antibiotic resistance in Gram‐negatives that harbor inducible AmpC. To understand the structural basis for the selectivity of these inhibitors for NagZ, we have determined its crystallographic structure in complex with N‐valeryl‐PUGNAc, the most selective known inhibitor of NagZ over both the human β‐hexosaminidases and O‐GlcNAcase. The selectivity stems from the five‐carbon acyl chain of N‐valeryl‐PUGNAc, which we found ordered within the enzyme active site. In contrast, a structure determination of a human O‐GlcNAcase homologue bound to a related inhibitor N‐butyryl‐PUGNAc, which bears a four‐carbon chain and is selective for both NagZ and O‐GlcNAcase over the human β‐hexosamnidases, reveals that this inhibitor induces several conformational changes in the active site of this O‐GlcNAcase homologue. A comparison of these complexes, and with the human β‐hexosaminidases, reveals how selectivity for NagZ can be engineered by altering the 2‐N‐acyl substituent of PUGNAc to develop inhibitors that repress AmpC mediated β‐lactam resistance.     

FtsEX is required for CwlO peptidoglycan hydrolase activity during cell wall elongation in Bacillus subtilis

The peptidoglycan (PG) sacculus, a meshwork of polysaccharide strands cross‐linked by short peptides, protects bacterial cells against osmotic lysis. To enlarge this covalently closed macromolecule, PG hydrolases must break peptide cross‐links in the meshwork to allow insertion of new glycan strands between the existing ones. In the rod‐shaped bacterium Bacillus subtilis, cell wall elongation requires two redundant endopeptidases, CwlO and LytE. However, it is not known how these potentially autolytic enzymes are regulated to prevent lethal breaches in the cell wall. Here, we show that the ATP‐binding cassette transporter‐like FtsEX complex is required for CwlO activity. In Escherichia coli, FtsEX is thought to harness ATP hydrolysis to activate unrelated PG hydrolases during cell division. Consistent with this regulatory scheme, B. subtilis FtsE mutants that are unable to bind or hydrolyse ATP cannot activate CwlO. Finally, we show that in cells depleted of both CwlO and LytE, the PG synthetic machinery continues moving circumferentially until cell lysis, suggesting that cross‐link cleavage is not required for glycan strand polymerization. Overall, our data support a model in which the FtsEX complex is a remarkably flexible regulatory module capable of controlling a diverse set of PG hydrolases during growth and division in different organisms.     

BMP‐2 modulates β‐catenin signaling through stimulation of Lrp5 expression and inhibition of β‐TrCP expression in osteoblasts

Canonical BMP and Wnt signaling pathways play critical roles in regulation of osteoblast function and bone formation. Recent studies demonstrate that BMP‐2 acts synergistically with β‐catenin to promote osteoblast differentiation. To determine the molecular mechanisms of the signaling cross‐talk between canonical BMP and Wnt signaling pathways, we have used primary osteoblasts and osteoblast precursor cell lines 2T3 and MC3T3‐E1 cells to investigate the effect of BMP‐2 on β‐catenin signaling. We found that BMP‐2 stimulates Lrp5 expression and inhibits the expression of β‐TrCP, the F‐box E3 ligase responsible for β‐catenin degradation and subsequently increases β‐catenin protein levels in osteoblasts. In vitro deletion of the β‐catenin gene inhibits osteoblast proliferation and alters osteoblast differentiation and reduces the responsiveness of osteoblasts to the BMP‐2 treatment. These findings suggest that BMP‐2 may regulate osteoblast function in part through modulation of the β‐catenin signaling. J. Cell. Biochem. 108: 896–905, 2009. © 2009 Wiley‐Liss, Inc.     

Synthesis and Immunostimulating Properties of Novel Adamant‐1‐yl Tripeptides

The aim of this work was to prepare L ‐ and D ‐(adamant‐1‐yl)‐Gly‐L ‐Ala‐D ‐isoGln peptides in order to study their adjuvant (immunostimulating) activities. Adjuvant activity of adamant‐1‐yl tripeptides was tested in the mouse model using ovalbumin as an antigen and in comparison to the peptidoglycan monomer (PGM; β‐D ‐GlcNAc‐(1→4)‐D ‐MurNAc‐L ‐Ala‐D ‐isoGln‐mesoDAP(εNH₂)‐D ‐Ala‐D ‐Ala) and structurally related adamant‐2‐yl tripeptides.     

Bacterial growth does require peptidoglycan hydrolases

Most bacteria surround their cytoplasmic membrane with a net‐like, elastic heteropolymer, the peptidoglycan sacculus, to protect themselves from bursting due to the turgor and to maintain cell shape. It has been assumed that growing bacteria require peptidoglycan hydrolases to open meshes in the peptidoglycan net allowing the insertion of the newly synthesized material for surface expansion. However, peptidoglycan hydrolases essential for bacterial growth have long remained elusive. In this issue of Molecular Microbiology Singh et al. ( 2012 ) report the identification in Escherichia coli of three new DD‐endopeptidases (Spr, YdhO and YebA) which are collectively required for peptidoglycan growth. Cells depleted of the three enzymes fail to incorporate new peptidoglycan, indicating that the cleavage of cross‐links by the new endopeptidases is needed for surface growth of the sacculus. These results are corroborated by recent data showing that Bacillus subtilis cells require the DL‐endopeptidase activity of CwlO or LytE for growth.     

Colocalization and interaction between elongasome and divisome during a preparative cell division phase in Escherichia coli

The rod‐shaped bacterium Escherichia coli grows by insertion of peptidoglycan into the lateral wall during cell elongation and synthesis of new poles during cell division. The monofunctional transpeptidases PBP2 and PBP3 are part of specialized protein complexes called elongasome and divisome, respectively, which catalyse peptidoglycan extension and maturation. Endogenous immunolabelled PBP2 localized in the cylindrical part of the cell as well as transiently at midcell. Using the novel image analysis tool Coli‐Inspector to analyse protein localization as function of the bacterial cell age, we compared PBP2 localization with that of other E. coli cell elongation and division proteins including PBP3. Interestingly, the midcell localization of the two transpeptidases overlaps in time during the early period of divisome maturation. Försters Resonance Energy Transfer (FRET) experiments revealed an interaction between PBP2 and PBP3 when both are present at midcell. A decrease in the midcell diameter is visible after 40% of the division cycle indicating that the onset of new cell pole synthesis starts much earlier than previously identified by visual inspection. The data support a new model of the division cycle in which the elongasome and divisome interact to prepare for cell division.     

Three redundant murein endopeptidases catalyse an essential cleavage step in peptidoglycan synthesis of Escherichia coli K12

Bacterial peptidoglycan (PG or murein) is a single, large, covalently cross‐linked macromolecule and forms a mesh‐like sacculus that completely encases the cytoplasmic membrane. Hence, growth of a bacterial cell is intimately coupled to expansion of murein sacculus and requires cleavage of pre‐existing cross‐links for incorporation of new murein material. Although, conceptualized nearly five decades ago, the mechanism of such essential murein cleavage activity has not been studied so far. Here, we identify three new murein hydrolytic enzymes in Escherichia coli, two (Spr and YdhO) belonging to the NlpC/P60 peptidase superfamily and the third (YebA) to the lysostaphin family of proteins that cleave peptide cross‐bridges between glycan chains. We show that these hydrolases are redundantly essential for bacterial growth and viability as a conditional mutant lacking all the three enzymes is unable to incorporate new murein and undergoes rapid lysis upon shift to restrictive conditions. Our results indicate the step of cross‐link cleavage as essential for enlargement of the murein sacculus, rendering it a novel target for development of antibacterial therapeutic agents.     

Mycobacterium tuberculosis proteasomal ATPase Mpa has a β‐grasp domain that hinders docking with the proteasome core protease

Mycobacterium tuberculosis (Mtb) has a proteasome system that is essential for its ability to cause lethal infections in mice. A key component of the system is the proteasomal adenosine triphosphatase (ATPase) Mpa, which captures, unfolds, and translocates protein substrates into the Mtb proteasome core particle for degradation. Here, we report the crystal structures of near full‐length hexameric Mtb Mpa in apo and ADP‐bound forms. Surprisingly, the structures revealed a ubiquitin‐like β‐grasp domain that precedes the proteasome‐activating carboxyl terminus. This domain, which was only found in bacterial proteasomal ATPases, buries the carboxyl terminus of each protomer in the central channel of the hexamer and hinders the interaction of Mpa with the proteasome core protease. Thus, our work reveals the structure of a bacterial proteasomal ATPase in the hexameric form, and the structure finally explains why Mpa is unable to stimulate robust protein degradation in vitro in the absence of other, yet‐to‐be‐identified co‐factors.     

Mycobacterium tuberculosis RpfE crystal structure reveals a positively charged catalytic cleft

Resuscitation promoting factor (Rpf) proteins, which hydrolyze the sugar chains in cell‐wall peptidoglycan (PG), play key roles in prokaryotic cell elongation, division, and escape from dormancy to vegetative growth. Like other bacteria, Mycobacterium tuberculosis (Mtb) expresses multiple Rpfs, none of which is individually essential. This redundancy has left unclear the distinct functions of the different Rpfs. To explore the distinguishing characteristics of the five Mtb Rpfs, we determined the crystal structure of the RpfE catalytic domain. The protein adopts the characteristic Rpf fold, but the catalytic cleft is narrower compared to Mtb RpfB. Also in contrast to RpfB, in which the substrate‐binding surfaces are negatively charged, the corresponding RpfE catalytic pocket and predicted peptide‐binding sites are more positively charged at neutral pH. The complete reversal of the electrostatic potential of the substrate‐binding site suggests that the different Rpfs function optimally at different pHs or most efficiently hydrolyze different micro‐domains of PG. These studies provide insights into the molecular determinants of the evolution of functional specialization in Rpfs.     

Substrate specificity of an elongation‐specific peptidoglycan endopeptidase and its implications for cell wall architecture and growth of Vibrio cholerae

The bacterial cell wall consists of peptidoglycan (PG), a sturdy mesh of glycan strands cross‐linked by short peptides. This rigid structure constrains cell shape and size, yet is sufficiently dynamic to accommodate insertion of newly synthesized PG, which was long hypothesized, and recently demonstrated, to require cleavage of the covalent peptide cross‐links that couple previously inserted material. Here, we identify several genes in Vibrio cholerae that collectively are required for growth – particularly elongation – of this pathogen. V. cholerae encodes three putative periplasmic proteins, here denoted ShyA, ShyB, and ShyC, that contain both PG binding and M23 family peptidase domains. While none is essential individually, the absence of both ShyA and ShyC results in synthetic lethality, while the absence of ShyA and ShyB causes a significant growth deficiency. ShyA is a D,d ‐endopeptidase able to cleave most peptide chain cross‐links in V. cholerae's PG. PG from a ?shyA mutant has decreased average chain length, suggesting that ShyA may promote removal of short PG strands. Unexpectedly, ShyA has little activity against muropeptides containing pentapeptides, which typically characterize newly synthesized material. ShyA's substrate‐dependent activity may contribute to selection of cleavage sites in PG, whose implications for the process of side‐wall growth are discussed.     

A single‐stranded DNA aptamer against mannose‐capped lipoarabinomannan enhances anti‐tuberculosis activity of macrophages through downregulation of lipid‐sensing nuclear receptor peroxisome proliferator‐activated receptor γ expression

Mannose‐capped lipoarabinomannan (ManLAM) is an immunomodulatory epitope of Mycobacterium tuberculosis (Mtb). An aptamer (ZXL1) that specifically binds to ManLAM from the virulent Mtb H37Rv strain was previously generated and it was found that ZXL1 functions as an antagonist, inhibiting the ManLAM‐induced immunosuppression of DCs. In the present study, it was found that ZXL1 inhibits Mtb entry into murine macrophages and that ZXL1 enhances IL‐1β and IL‐12 mRNA expression and cytokine production in ManLAM‐treated macrophages but decreases IL‐10 production. Inducible nitric oxide synthase expression in macrophages was upregulated in the presence of ZXL1 after stimulation with ManLAM. ZXL1 was also found to inhibit expression of lipid‐sensing nuclear receptor peroxisome proliferator‐activated receptor γ (PPAR‐γ). These results suggest that ZXL1 promotes anti‐tuberculosis activity through downregulation of PPAR‐γ expression, which may contribute to M1 macrophage polarization and Mtb killing by macrophages.     

FadD3 is an acyl‐CoA synthetase that initiates catabolism of cholesterol rings C and D in actinobacteria

The cholesterol catabolic pathway occurs in most mycolic acid‐containing actinobacteria, such as Rhodococcus jostii RHA1, and is critical for Mycobacterium tuberculosis (Mtb) during infection. FadD3 is one of four predicted acyl‐CoA synthetases potentially involved in cholesterol catabolism. A ΔfadD3 mutant of RHA1 grew on cholesterol to half the yield of wild‐type and accumulated 3aα‐H‐4α(3′‐propanoate)‐7aβ‐methylhexahydro‐1,5‐indanedione (HIP), consistent with the catabolism of half the steroid molecule. This phenotype was rescued by fadD3 of Mtb. Moreover, RHA1 but not ΔfadD3 grew on HIP. Purified FadD3_Mtb catalysed the ATP‐dependent CoA thioesterification of HIP and its hydroxylated analogues, 5α‐OH HIP and 1β‐OH HIP. The apparent specificity constant (k_cat/K_m) of FadD3_Mtb for HIP was 7.3 ± 0.3 × 10⁵ M^?1 s^?1, 165 times higher than for 5α‐OH HIP, while the apparent K_m for CoASH was 110 ± 10 μM. In contrast to enzymes involved in the catabolism of rings A and B, FadD3_Mtb did not detectably transform a metabolite with a partially degraded C17 side‐chain. Overall, these results indicate that FadD3 is a HIP‐CoA synthetase that initiates catabolism of steroid rings C and D after side‐chain degradation is complete. These findings are consistent with the actinobacterial kstR2 regulon encoding ring C/D degradation enzymes.     

Influence of Mannosylation on Immunostimulating Activity of Adamant‐1‐yl Tripeptide

The mannosylated derivative of adamant‐1‐yl tripeptide (D ‐(Ad‐1‐yl)Gly‐L ‐Ala‐D ‐isoGln) was prepared to study the effects of mannosylation on adjuvant (immunostimulating) activity. Mannosylated adamant‐1‐yl tripeptide (Man‐OCH₂CH(Me)CO‐D ‐(Ad‐1‐yl)Gly‐L ‐Ala‐D ‐isoGln) is a non‐pyrogenic, H₂O‐soluble, and non‐toxic compound. Adjuvant activity of mannosylated adamantyl tripeptide was tested in the mouse model with ovalbumin as an antigen and in comparison to the parent tripeptide and peptidoglycan monomer (PGM, β‐D ‐GlcNAc‐(1→4)‐D ‐MurNAc‐L ‐Ala‐D ‐isoGln‐mesoDAP(εNH₂)‐D ‐Ala‐D ‐Ala), a well‐known effective adjuvant. The mannosylation of adamantyl tripeptide caused the amplification of its immunostimulating activity in such a way that it was comparable to that of PGM.     

Septal and lateral wall localization of PBP5, the major D,D‐carboxypeptidase of Escherichia coli,requires substrate recognition and membrane attachment

The distribution of PBP5, the major D,D‐carboxypeptidase in Escherichia coli, was mapped by immunolabelling and by visualization of GFP fusion proteins in wild‐type cells and in mutants lacking one or more D,D‐carboxypeptidases. In addition to being scattered around the lateral envelope, PBP5 was also concentrated at nascent division sites prior to visible constriction. Inhibiting PBP2 activity (which eliminates wall elongation) shifted PBP5 to midcell, whereas inhibiting PBP3 (which aborts divisome invagination) led to the creation of PBP5 rings at positions of preseptal wall formation, implying that PBP5 localizes to areas of ongoing peptidoglycan synthesis. A PBP5(S44G) active site mutant was more evenly dispersed, indicating that localization required enzyme activity and the availability of pentapeptide substrates. Both the membrane bound and soluble forms of PBP5 converted pentapeptides to tetrapeptides in vitro and in vivo, and the enzymes accepted the same range of substrates, including sacculi, Lipid II, muropeptides and artificial substrates. However, only the membrane‐bound form localized to the developing septum and restored wild‐type rod morphology to shape defective mutants, suggesting that the two events are related. The results indicate that PBP5 localization to sites of ongoing peptidoglycan synthesis is substrate dependent and requires membrane attachment.