Academic literature on the topic 'Peptidoglycan remodeling'

In response to the presence of compatible rhizobium bacteria, legumes form symbiotic organs called nodules on their roots. These nodules house nitrogen-fixing bacteroids that are a differentiated form of the rhizobium bacteria. In some legumes, the bacteroid differentiation comprises a dramatic cell enlargement, polyploidization, and other morphological changes. Here, we demonstrate that a peptidoglycan-modifying enzyme in Bradyrhizobium strains, a DD-carboxypeptidase that contains a peptidoglycan-binding SPOR domain, is essential for normal bacteroid differentiation in Aeschynomene species. The corresponding mutants formed bacteroids that are malformed and hypertrophied. However, in soybean, a plant that does not induce morphological differentiation of its symbiont, the mutation does not affect the bacteroids. Remarkably, the mutation also leads to necrosis in a large fraction of the Aeschynomene nodules, indicating that a normally formed peptidoglycan layer is essential for avoiding the induction of plant immune responses by the invading bacteria. In addition to exopolysaccharides, capsular polysaccharides, and lipopolysaccharides, whose role during symbiosis is well defined, our work demonstrates an essential role in symbiosis for yet another rhizobial envelope component, the peptidoglycan layer.

The obligate intracellular Chlamydiaceae do not need to resist osmotic challenges and thus lost their cell wall in the course of evolution. Nevertheless, these pathogens maintain a rudimentary peptidoglycan machinery for cell division. They build a transient peptidoglycan ring, which is remodeled during the process of cell division and degraded afterwards. Uncontrolled degradation of peptidoglycan poses risks to the chlamydial cell, as essential building blocks might get lost or trigger host immune response upon release into the host cell. Here, we provide evidence that a primordial enzyme class prevents energy intensive de novo synthesis and uncontrolled release of immunogenic peptidoglycan subunits in Chlamydia trachomatis. Our data indicate that the homolog of a Bacillus NlpC/P60 protein is widely conserved among Chlamydiales. We show that the enzyme is tailored to hydrolyze peptidoglycan-derived peptides, does not interfere with peptidoglycan precursor biosynthesis, and is targeted by cysteine protease inhibitors in vitro and in cell culture. The peptidase plays a key role in the underexplored process of chlamydial peptidoglycan recycling. Our study suggests that chlamydiae orchestrate a closed-loop system of peptidoglycan ring biosynthesis, remodeling, and recycling to support cell division and maintain long-term residence inside the host. Operating at the intersection of energy recovery, cell division and immune evasion, the peptidoglycan recycling NlpC/P60 peptidase could be a promising target for the development of drugs that combine features of classical antibiotics and anti-virulence drugs.

ABSTRACT Members of a family of serine/threonine protein kinases (STPKs), unique to gram-positive bacteria, comprise an intracellular kinase domain and reiterated extracellular PASTA (for “penicillin-binding protein and serine/threonine kinase associated”) domains. PASTA domains exhibit low affinity for β-lactam antibiotics that are structurally similar to their likely normal ligands: stem peptides of unlinked peptidoglycan. The PASTA-domain STPKs are found in the actinobacteria and firmicutes and, as exemplified by PknB of Mycobacterium tuberculosis, they are functionally implicated in aspects of growth, cell division, and development. Whereas the kinase domains are well conserved, there is a wide divergence in the sequences of the multiple PASTA domains. Closer inspection reveals position-dependent evolution of individual PASTA domains: a domain at one position within a gene has a close phylogenetic relationship with a domain at a similar position in an orthologous gene, whereas neighboring domains have clearly diverged one from one another. A similar position-dependent relationship is demonstrated in the second family of proteins with multiple PASTA domains: the high-molecular-weight type II penicillin-binding protein (PBP2x) family. These transpeptidases are recruited to the division site by a localized pool of unlinked peptidoglycan. We infer that protein localization is guided by low-affinity interactions between structurally different unlinked peptidoglycan stem peptides and individual PASTA domains. The STPKs possess a greater multiplicity and diversity of PASTA domains, allowing interactions with a wider range of stem-peptide ligands. These interactions are believed to activate the intracellular kinase domain, allowing an STPK to coordinate peptidoglycan remodeling and reproduction of a complex cell wall structure.

ABSTRACTNeisseria gonorrhoeae(gonococci) andNeisseria meningitidis(meningococci) are human pathogens that cause gonorrhea and meningococcal meningitis, respectively. BothN. gonorrhoeaeandN. meningitidisrelease a number of small peptidoglycan (PG) fragments, including proinflammatory PG monomers, althoughN. meningitidisreleases fewer PG monomers. The PG fragments released byN. gonorrhoeaeandN. meningitidisare generated in the periplasm during cell wall remodeling, and a majority of these fragments are transported into the cytoplasm by an inner membrane permease, AmpG; however, a portion of the PG fragments are released into the extracellular environment through unknown mechanisms. We previously reported that the expression of meningococcalampGinN. gonorrhoeaereduced PG monomer release by gonococci. This finding suggested that the efficiency of AmpG-mediated PG fragment recycling regulates the amount of PG fragments released into the extracellular milieu. We determined that three AmpG residues near the C-terminal end of the protein modulate AmpG's efficiency. We also investigated the association between PG fragment recycling and release in two species of human-associated nonpathogenicNeisseria:N. siccaandN. mucosa. BothN. siccaandN. mucosarelease lower levels of PG fragments and are more efficient at recycling PG fragments thanN. gonorrhoeae. Our results suggest thatN. gonorrhoeaehas evolved to increase the amounts of toxic PG fragments released by reducing its PG recycling efficiency.IMPORTANCENeisseria gonorrhoeaeandNeisseria meningitidisare human pathogens that cause highly inflammatory diseases, althoughN. meningitidisis also frequently found as a normal member of the nasopharyngeal microbiota. NonpathogenicNeisseria, such asN. siccaandN. mucosa, also colonize the nasopharynx without causing disease. Although all four species release peptidoglycan fragments,N. gonorrhoeaeis the least efficient at recycling and releases the largest amount of proinflammatory peptidoglycan monomers, partly due to differences in the recycling permease AmpG. Studying the interplay between bacterial physiology (peptidoglycan metabolism) and pathogenesis (release of toxic monomers) leads to an increased understanding of how different bacterial species maintain asymptomatic colonization or cause disease and may contribute to efforts to mitigate disease.

Bacterial dormancy can take many forms, including formation of Bacillus endospores, Streptomyces exospores, and metabolically latent Mycobacterium cells. In the actinobacteria, including the streptomycetes and mycobacteria, the rapid resuscitation from a dormant state requires the activities of a family of cell-wall lytic enzymes called resuscitation-promoting factors (Rpfs). Whether Rpf activity promotes resuscitation by generating peptidoglycan fragments (muropeptides) that function as signaling molecules for spore germination or by simply remodeling the dormant cell wall has been the subject of much debate. Here, to address this question, we used mutagenesis and peptidoglycan binding and cleavage assays to first gain broader insight into the biochemical function of diverse Rpf enzymes. We show that their LysM and LytM domains enhance Rpf enzyme activity; their LytM domain and, in some cases their LysM domain, also promoted peptidoglycan binding. We further demonstrate that the Rpfs function as endo-acting lytic transglycosylases, cleaving within the peptidoglycan backbone. We also found that unlike in other systems, Rpf activity in the streptomycetes is not correlated with peptidoglycan-responsive Ser/Thr kinases for cell signaling, and the germination of rpf mutant strains could not be stimulated by the addition of known germinants. Collectively, these results suggest that in Streptomyces, Rpfs have a structural rather than signaling function during spore germination, and that in the actinobacteria, any signaling function associated with spore resuscitation requires the activity of additional yet to be identified enzymes.

Abstract Bacteria of the Planctomycetes phylum have many unique cellular features, such as extensive membrane invaginations and the ability to import macromolecules. These features raise intriguing questions about the composition of their cell envelopes. In this study, we have used microscopy, phylogenomics, and proteomics to examine the composition and evolution of cell envelope proteins in Tuwongella immobilis and other members of the Planctomycetes. Cryo-electron tomography data indicated a distance of 45 nm between the inner and outer membranes in T. immobilis. Consistent with the wide periplasmic space, our bioinformatics studies showed that the periplasmic segments of outer-membrane proteins in type II secretion systems are extended in bacteria of the order Planctomycetales. Homologs of two highly abundant cysteine-rich cell wall proteins in T. immobilis were identified in all members of the Planctomycetales, whereas genes for peptidoglycan biosynthesis and cell elongation have been lost in many members of this bacterial group. The cell wall proteins contain multiple copies of the YTV motif, which is the only domain that is conserved and unique to the Planctomycetales. Earlier diverging taxa in the Planctomycetes phylum contain genes for peptidoglycan biosynthesis but no homologs to the YTV cell wall proteins. The major remodeling of the cell envelope in the ancestor of the Planctomycetales coincided with the emergence of budding and other unique cellular phenotypes. The results have implications for hypotheses about the process whereby complex cellular features evolve in bacteria.

Il rivestimento dei batteri Gram negativi consiste in una membrana interna (IM) e una membrana esterna (OM) separate da uno spazio periplasmatico contenente un sottile strato di peptidoglicano (PG) ancorato alla OM tramite la lipoproteina di Braun (LPP). Mentre la IM è costituita da un doppio strato di fosfolipidi, la OM è una membrana lipidica asimmetrica, contenente fosfolipidi nel foglietto interno, e un lipide complesso il Lipopolisaccaride (LPS) nel foglietto esterno. Il LPS sintetizzato nel citoplasma, viene traslocato sul lato periplasmatico della IM e preso in carico dal complesso multiproteico Lpt (LPS transport), composto in Escherichia coli da sette proteine essenziali (Lpt ABCDEFG) che si occupano del suo trasporto fino al raggiungimento della sua sede finale, la OM. Analisi biochimiche hanno dimostrato che le sette proteine Lpt formano un complesso “transenvelope” che connette IM e OM e studi di tipo genetico suggeriscono che esse operino in concerto come un singolo macchinario. Infatti, la deplezione di un qualsiasi componente del complesso Lpt causa lo stesso fenotipo, ovvero l’accumulo del LPS nel versante periplasmatico della IM, la decorazione del LPS con acido colanico e la formazione di una IM anomala, con una densità intermedia tra la IM e la OM. Nel nostro laboratorio è stata condotta l’analisi differenziale del proteoma delle membrane totali di E. coli in deplezione di LptC, per studiare la risposta globale al blocco del trasporto del LPS. Tra le proteine il cui livello cambia nel confronto tra il ceppo non depleto e il depleto sono state trovate proteine coinvolte nella biogenesi e nel rimodellamento del PG. Lo scopo di questa tesi è stato lo studio della correlazione tra il blocco del LPS e il rimodellamento del PG. Inizialmente è stata analizzata la struttura del PG in deplezione di LptC. Questa analisi ha evidenziato che in questa condizione la struttura del PG varia sia per composizione che per tipo di legami crociati tra i filamenti glicanici adiacenti. Nei batteri Gram negativi il legame tra i filamenti glicanici è generalmente un legame diretto 3-4, che si forma tra il gruppo aminico del diaminoacido in posizione 3 di un tetrapeptide e il gruppo carbossilico della D-alanina in posizione 4 del tretrapeptide adiacente. Il legame 3-4 avviene ad opera delle D,D transpeptidasi PBP. Un altro tipo di legame crociato presente nel PG è quello tipo 3-3 che si forma tra il gruppo aminico del diaminoacido in posizione 3 ed il gruppo carbossilico del diaminoacido presente nel tetrapeptide del filamento glicanico adiacente ed è catalizzato da L,D-transpeptidasi . In E. coli, sono noti cinque enzimi con attività L,D-transpeptidasica, di cui tre (LdtA, LdtB, LdtC) ancorano la lipoproteina più abbondante della OM (lipoproteina di Braun) al PG e due (LdtD, LdtE) catalizzano il legame crociato 3-3. La delezione di tutti questi geni, singolarmente o in combinazione, non presenta nessun fenotipo, suggerendo che in condizioni normali questo legame è dispensabile. Per studiare la correlazione tra il legame crociato 3-3 e il blocco del trasporto del LPS abbiamo creato mutanti arabinosio dipendenti per alcuni dei componenti del sistema Lpt deleti contemporaneamente per i geni che esprimono le L,D-transpeptidasi LdtD e LdtE. In precedenza, nel nostro laboratorio è stato dimostrato che la deplezione di uno qualsiasi dei geni lpt causa la formazione di cellule filamentose e l’arresto della crescita ma non la lisi cellulare. Invece, nei mutanti ΔldtDΔldtE, in deplezione dei geni lpt, oltre alla formazione di cellule filamentose si osserva la formazione di un setto anomalo e la lisi cellulare. Questi dati suggeriscono che il rimodellamento del peptidoglicano a seguito della formazione di legami 3-3 potrebbe essere una forma di riposta al danno alla membrana esterna.
The cell envelope of Gram-negative bacteria is a complex multi-layered structure consisting of a cytoplasmic and an outer membrane (CM and OM), which delimit the periplasm containing a thin layer of peptidoglycan (PG) called the sacculus. The primary function of the OM is to establish a selective permeability barrier that enables the cell to maintain favourable intracellular conditions even in hash environments and the lipopolysaccharide (LPS) layer greatly contributes to this peculiar property. The integrity of the PG mesh is essential to protect the cell from bursting due to its turgor and maintain the shape of the cell. OM and PG are synthetized and assembled by multiprotein machineries that need to be finely coordinated as imbalanced growth of these layers may compromise structural integrity of the cell. In order to gain more insight in the mechanism by which the cells coordinate the growth of these two layers, we analysed the PG composition when the biogenesis of OM is compromised due to the block of LPS transport. In this work we shown that when OM is impaired, E. coli cells remodel PG architecture by increasing the non-canonical 3- 3 cross-linkage. We can assume that this is a salvage mechanism to prevent cell lysis when OM is damaged.

The three layered Gram-negative bacteria envelope consists of an inner membrane (IM), the periplasm‐containing peptidoglycan (PG), and an asymmetric outer membrane (OM) decorated with lipopolysaccharide (LPS) in the outer leaflet. Growth and assembly of cell envelope is orchestrated by action of dedicated protein machineries which span the entire envelope and whose coordinated activity guarantees proper envelope stiffness. Defects in biogenesis in any of these layers compromise the whole cell integrity and lead to cell death. In this thesis we show that Escherichia coli remodels the PG structure by increasing the level of 3-3 crosslinks produced by LD – Transpeptidases (LDTs), to avoid cell lysis when the LPS transport to the OM is disrupted. E. coli codes for six LDTs (LdtA-F): LdtA, LdtB, and LdtC covalently attach Lpp to PG while LdtD and LdtE introduce 3-3crosslinks. LdtF has no LD-Transpeptidase (LD-Tpase) activity but enhances the enzymatic activity of LdtD and LdtE. Our data outlines a major contribution of LdtD in PG remodelling and suggest that LdtD works in concert with the PG synthase PBP1B, its activator LpoB and the DD-CPase PBP6a to form a dedicated PG repair machine that runs a PG remodeling program to counteract damages to the OM. We also show that the lysis phenotype and morphological defects seen in mutants with an impaired LPS transport and lacking ldtF, are rescued and suppressed, respectively, by the loss of YgeR an uncharacterized lipoprotein predicted to be OM anchored. YgeR belongs to the family of LytM-domain factors which are hydrolases or hydrolase regulators implicated in PG remodeling/turnover. Important PG hydrolases are amidases which promote PG septal splitting and daughter cell separation. Our biochemical data reveal that YgeR is an amidase regulator able to activate AmiA, AmiB and AmiC the three amidases encoded by E. coli. We also show that YgeR binds purified PG and physically interacts with the amidase AmiC. Our biochemical analyses are complemented by in vivo data showing that YgeR preferentially activates AmiC and that it does it through its LytM domain. Altogether, our results point out an unexplored protective role of the 3-3 crosslinks in PG to overcome severe OM biogenesis defects and propose YgeR as a novel amidase activator whose action seems required upon envelope stress.

La paroi des bactéries à Gram positif se compose du peptidoglycane (PG) et des acides téichoïques (TA). Leur étude a révélé de nouveaux mécanismes de régulation chez le pathogène humain Streptococcus pneumoniae. Nous avons montré que la O-acétylation intervient précocement dans la biosynthèse du PG, participe à sa maturation et à la division cellulaire. Nous avons développé une approche innovante basée sur la chimie click pour le marquage in vivo des TAs, et révélé que leur synthèse est septale et corrélée à celle du PG. Le PG et les TAs contribuent aussi à réguler l'activité enzymatique de l'autolysine majeur du pneumocoque LytA: la O-acétylation du PG protège les cellules en division de l'autolyse par LytA et les TAs, sur lesquels elle se fixe, régulent sa localisation de surface. Pour conclure, ce travail souligne le rôle coopératif du PG et des TAs dans la synthèse de la paroi, la division cellulaire et la régulation de composants de la surface bactérienne
Gram-positive bacteria cell wall (CW) is composed by peptidoglycan (PG) and teichoic acids (TA). We studied both CW components and revealed new regulation mechanisms in the human pathogen Streptococcus pneumoniae. We showed that PG O-acetylation occurs in the early steps of PG biosynthesis, promotes the formation of mature PG and plays a role in cell division. We developed an innovative click chemistry-based approach to label TA in live cells, opening the way to explore mechanistic issues of pneumococcal TA biosynthesis. We revealed that TA synthesis occurs at the division site and is correlated with PG synthesis. Finally, we showed that both PG and TA polymers contribute to regulate the major autolysin LytA which binds TA and cleaves the PG: the O-acetylation of PG protects dividing cells from LytA-induced autolysis while TA finely regulates LytA surface localization. In conclusion, our work highlights the cooperative role of PG and TA in CW biosynthesis, cell division and regulation of surface components

There are so many people who I would like to thank. If it takes a village to raise a child, it certainly takes a city to train a scientist. Firstly, I would like to thank my supervisors, A/Prof Reto Guler, Prof Frank Brombacher and Dr Mumin Ozturk for allowing me to further my studies and allowing me to work on several interesting projects. Specifically, I would like to take this opportunity to thank A/Prof Reto Guler for his insightful patient advice, training and ever willingness to talk about my work. His brilliant example has made me a better scientist. Further, I would also like to thank Dr Mumin Ozturk for his constant, patient mentorship, help, advice and friendship. His influence, guidance and example have affected me more than he'll ever know. Lastly, but certainly not least, I would like Professor Bavesh Kana and for all his advice and support. I would also like to express my gratitude to my labmates from the Brombacher group. All the conversations, laughs, celebrations and commiserations have made this journey undeniably easier. In particular, I would like to thank Shelby-Sara Jones for her constant willingness to help with lab work whilst chatting about everything under the sun. To my friends and family, there are no words to express my unending gratitude. Without their love and support along the way, I would never have gotten to this stage in my life. To my parents and sister, I would like to say a huge thank you for their constant support and love during my academic career so far. You guys have been wonderful. A huge thank you to Daniela de Almeida and the French's for their support and love from afar– you guys have been great. I would like to say a special thank you to Dustin Fischer who has always been there for a beer and good old-fashioned rant. I can only hope that my friendship and advice have been even the smallest bit as helpful to him as he has been to me during our long trek through academia. To the Cunniffes, thank you for all your support down this long road and truly making me feel like one of the family. Last but by no means least, I would like to thank my partner, Teagan Cunniffe, whose effortless grace, wit, humour, friendship and constant love have been the single greatest gifts I have ever received. I look forward to our adventures to come. Thank you for being there every step of the way and keeping me sane - This dissertation is dedicated to you.

A dissertation submitted to the Faculty of Health Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science in Medicine. May 2018.
Mycobacterium tuberculosis (TB), the causative agent of tuberculosis, is responsible for over one million deaths per annum, a substantive proportion of these due to multidrug resistant or extensively drug resistant strains. The available antibiotics are rapidly becoming ineffective due to the development of resistance mechanisms by the pathogen. Considering this, there is an urgent need for novel and highly effective new TB drugs, with novel modes of action. The peptidogycan (PG) layer in the cell wall has emerged as a rich area for drug discovery. It undergoes constant reconstruction by penicillin binding proteins (PBPs) and other enzymes to allow for cell growth and division, while preventing lysis. In this study, we characterize the function of two Low Molecular Mass PBPs, known as D,D-carboxypeptidases (D,D-CPases, MSMEG_6113 and MSMEG_2433) in Mycobacterium smegmatis, a model organism for TB research. Using a bacterial two hybrid system, we demonstrate that MSMEG_2433 interacts with PonA1 (A High Molecular Mass PBP involved in PG synthesis) and FtsH (An AAA family protease and a member of the divisome complex). We also demonstrate that MSMEG_6113 forms a complex with FtsI (A High Molecular mass PBP and also part of the divisome complex) and a cell division control protein, Cdc48. We demonstrate that the two D,D-CPases associate with their partnering proteins via their C-terminal transpeptidase domain. The importance of these identified interactions for cell division and growth was tested through deletion of partnering proteins from the mycobacterial genome, particularly FtsI and Cdc48. FtsI is essential for mycobacterial growth in vitro as demonstrated by the inability to recover mutants through allelic exchange by homologous recombination, while Cdc48 is dispensable for growth. We noted morphological and cell division defects in the Cdc48 deletion mutant strain. The absence of Cdc48 results in bulging, kinking and chaining phenotypes, in addition to misplacement of the FtsZ ring. Collectively, our observations describe the presence of novel PG hydrolysing protein complexes that may mediate essential steps in PG synthesis and bacterial proliferation. Targeting these complexes may provide an attractive avenue for the development of novel TB therapeutics.
LG2018