Katharina Peters

Regulation of bacterial cell wall growth

During growth and propagation, a bacterial cell enlarges and subsequently divides its peptidoglycan (PG) sacculus, a continuous mesh‐like layer that encases the cell membrane to confer mechanical strength and morphological robustness. The mechanism of sacculus growth, how it is regulated and how it is coordinated with other cellular processes is poorly understood. In this article, we will discuss briefly the current knowledge of how cell wall synthesis is regulated, on multiple levels, from both sides of the cytoplasmic membrane. According to the current knowledge, cytosolic scaffolding proteins connect PG synthases with cytoskeletal elements, and protein phosphorylation regulates cell wall growth in Gram‐positive species. PG‐active enzymes engage in multiple protein–protein interactions within PG synthesis multienzyme complexes, and some of the interactions modulate activities. PG synthesis is also regulated by central metabolism, and by PG maturation through the action of PG hydrolytic enzymes. Only now are we beginning to appreciate how these multiple levels of regulating PG synthesis enable the cell to propagate robustly with a defined cell shape under different and variable growth conditions.

A penicillin-binding protein inhibits selection of colistin-resistant, lipooligosaccharide-deficient Acinetobacter baumannii

Significance Antimicrobial drug resistance is a major threat to public health. Gram-negative bacteria are exceptionally resistant to antibiotics because of their outer-membrane barrier. Glycolipids called lipopolysaccharide (LPS) or lipooligosaccharide (LOS) fortify the outer membrane from many antimicrobials and biocides and were thought to be essential for Gram-negative bacterial survival. The last-resort treatment for multidrug-resistant Gram-negative infections is colistin, which targets the lipid A domain of LPS/LOS to disrupt the membrane, but the emerging pathogen Acinetobacter baumannii can develop colistin resistance by inactivating lipid A biosynthesis. This analysis advances our understanding of lipid A/LOS essentiality in A. baumannii and identifies antimicrobial targets. The Gram-negative bacterial outer membrane fortifies the cell against environmental toxins including antibiotics. Unique glycolipids called lipopolysaccharide/lipooligosaccharide (LPS/LOS) are enriched in the cell-surface monolayer of the outer membrane and promote antimicrobial resistance. Colistin, which targets the lipid A domain of LPS/LOS to lyse the cell, is the last-line treatment for multidrug-resistant Gram-negative infections. Lipid A is essential for the survival of most Gram-negative bacteria, but colistin-resistant Acinetobacter baumannii lacking lipid A were isolated after colistin exposure. Previously, strain ATCC 19606 was the only A. baumannii strain demonstrated to subsist without lipid A. Here, we show that other A. baumannii strains can also survive without lipid A, but some cannot, affording a unique model to study endotoxin essentiality. We assessed the capacity of 15 clinical A. baumannii isolates including 9 recent clinical isolates to develop colistin resistance through inactivation of the lipid A biosynthetic pathway, the products of which assemble the LOS precursor. Our investigation determined that expression of the well-conserved penicillin-binding protein (PBP) 1A, prevented LOS-deficient colony isolation. The glycosyltransferase activity of PBP1A, which aids in the polymerization of the peptidoglycan cell wall, was lethal to LOS-deficient A. baumannii. Global transcriptomic analysis of a PBP1A-deficient mutant and four LOS-deficient A. baumannii strains showed a concomitant increase in transcription of lipoproteins and their transporters. Examination of the LOS-deficient A. baumannii cell surface demonstrated that specific lipoproteins were overexpressed and decorated the cell surface, potentially compensating for LOS removal. This work expands our knowledge of lipid A essentiality and elucidates a drug resistance mechanism.

The Redundancy of Peptidoglycan Carboxypeptidases Ensures Robust Cell Shape Maintenance in Escherichia coli

ABSTRACT Peptidoglycan (PG) is an essential structural component of the bacterial cell wall and maintains the integrity and shape of the cell by forming a continuous layer around the cytoplasmic membrane. The thin PG layer of Escherichia coli resides in the periplasm, a unique compartment whose composition and pH can vary depending on the local environment of the cell. Hence, the growth of the PG layer must be sufficiently robust to allow cell growth and division under different conditions. We have analyzed the PG composition of 28 mutants lacking multiple PG enzymes (penicillin-binding proteins [PBPs]) after growth in acidic or near-neutral-pH media. Statistical analysis of the muropeptide profiles identified dd-carboxypeptidases (DD-CPases) that were more active in cells grown at acidic pH. In particular, the absence of the DD-CPase PBP6b caused a significant increase in the pentapeptide content of PG as well as morphological defects when the cells were grown at acidic pH. Other DD-CPases (PBP4, PBP4b, PBP5, PBP6a, PBP7, and AmpH) and the PG synthase PBP1B made a smaller or null contribution to the pentapeptide-trimming activity at acidic pH. We solved the crystal structure of PBP6b and also demonstrated that the enzyme is more stable and has a lower Km at acidic pH, explaining why PBP6b is more active at low pH. Hence, PBP6b is a specialized DD-CPase that contributes to cell shape maintenance at low pH, and E. coli appears to utilize redundant DD-CPases for normal growth under different conditions. IMPORTANCE Escherichia coli requires peptidoglycan dd-carboxypeptidases to maintain cell shape by controlling the amount of pentapeptide substrates available to the peptidoglycan synthetic transpeptidases. Why E. coli has eight, seemingly redundant dd-carboxypeptidases has remained unknown. We now show that one of these dd-carboxypeptidases, PBP6b, is important for cell shape maintenance in acidic growth medium, consistent with the higher activity and stability of the enzyme at low pH. Hence, the presence of multiple dd-carboxypeptidases with different enzymatic properties may allow E. coli to maintain a normal cell shape under various growth conditions. Escherichia coli requires peptidoglycan dd-carboxypeptidases to maintain cell shape by controlling the amount of pentapeptide substrates available to the peptidoglycan synthetic transpeptidases. Why E. coli has eight, seemingly redundant dd-carboxypeptidases has remained unknown. We now show that one of these dd-carboxypeptidases, PBP6b, is important for cell shape maintenance in acidic growth medium, consistent with the higher activity and stability of the enzyme at low pH. Hence, the presence of multiple dd-carboxypeptidases with different enzymatic properties may allow E. coli to maintain a normal cell shape under various growth conditions.

Robust peptidoglycan growth by dynamic and variable multi-protein complexes

In Gram-negative bacteria such as Escherichia coli the peptidoglycan sacculus resides in the periplasm, a compartment that experiences changes in pH value, osmolality, ion strength and other parameters depending on the cells environment. Hence, the cell needs robust peptidoglycan growth mechanisms to grow and divide under different conditions. Here we propose a model according to which the cell achieves robust peptidoglycan growth by employing dynamic multi-protein complexes, which assemble with variable composition from freely diffusing sets of peptidoglycan synthases, hydrolases and their regulators, whereby the composition of the active complexes depends on the cell cycle state - cell elongation or division - and the periplasmic growth conditions.

The Pneumococcal Cell Wall

Streptococcus pneumoniae has a highly complex cell wall made of a mesh-like peptidoglycan (PG) with covalently linked wall teichoic acid (WTA) and capsular polysaccharides. The glycan chains in PG are modified by deacetylation of N-acetylglucosamine residues and O-acetylation of N-acetylmuramic acid residues. Unlike many other species, pneumococci have the same, unusually complex repeating unit structure in WTA and lipoteichoic acid, which has recently been revised. Depending on the strain, the teichoic acids have a mean length of 7–8 repeats and contain 1–2 phosphorylcholine residues per repeat. The cell wall is synthesized at a central growth zone, presumably by large multiprotein complexes which alternate between peripheral and septal modes of synthesis, resulting in the characteristic oval cell shape. Cell wall components released from the bacterial cell during growth and cell lysis are recognized by components of the host, contributing to the multitude of interactions between this pathogen and the human organism.

New Aspects of the Interplay between Penicillin Binding Proteins, murM, and the Two-Component System CiaRH of Penicillin-Resistant Streptococcus pneumoniae Serotype 19A Isolates from Hungary

ABSTRACT The Streptococcus pneumoniae clone Hungary19A-6 expresses unusually high levels of β-lactam resistance, which is in part due to mutations in the MurM gene, encoding a transferase involved in the synthesis of branched peptidoglycan. Moreover, it contains the allele ciaH232, encoding the histidine kinase CiaH (M. Müller, P. Marx, R. Hakenbeck, and R. Brückner, Microbiology 157:3104–3112, 2011, https://doi.org/10.1099/mic.0.053157-0 ). High-level penicillin resistance primarily requires the presence of low-affinity (mosaic) penicillin binding protein (PBP) genes, as, for example, in strain Hu17, a closely related member of the Hungary19A-6 lineage. Interestingly, strain Hu15 is β-lactam sensitive due to the absence of mosaic PBPs. This unique situation prompted us to investigate the development of cefotaxime resistance in transformation experiments with genes known to play a role in this phenotype, pbp2x, pbp1a, murM, and ciaH, and penicillin-sensitive recipient strains R6 and Hu15. Characterization of phenotypes, peptidoglycan composition, and CiaR-mediated gene expression revealed several novel aspects of penicillin resistance. The murM gene of strain Hu17 (murMHu17), which is highly similar to murM of Streptococcus mitis, induced morphological changes which were partly reversed by ciaH232. murMHu17 conferred cefotaxime resistance only in the presence of the pbp2x of strain Hu17 (pbp2xHu17). The ciaH232 allele contributed to a remarkable increase in cefotaxime resistance in combination with pbp2xHu17 and pbp1a of strain Hu17 (pbp1aHu17), accompanied by higher levels of expression of CiaR-regulated genes, documenting that ciaH232 responds to PBP1aHu17-mediated changes in cell wall synthesis. Most importantly, the proportion of branched peptides relative to the proportion of linear muropeptides increased in cells containing mosaic PBPs, suggesting an altered enzymatic activity of these proteins.

The cell wall hydrolase Pmp23 is important for assembly and stability of the division ring in Streptococcus pneumoniae

Bacterial division is intimately linked to synthesis and remodeling of the peptidoglycan, a cage-like polymer that surrounds the bacterial cell, providing shape and mechanical resistance. The bacterial division machinery, which is scaffolded by the cytoskeleton protein FtsZ, includes proteins with enzymatic, structural or regulatory functions. These proteins establish a complex network of transient functional and/or physical interactions which preserve cell shape and cell integrity. Cell wall hydrolases required for peptidoglycan remodeling are major contributors to this mechanism. Consistent with this, their deletion or depletion often results in morphological and/or division defects. However, the exact function of most of them remains elusive. In this work, we show that the putative lysozyme activity of the cell wall hydrolase Pmp23 is important for proper morphology and cell division in the opportunistic human pathogen Streptococcus pneumoniae. Our data indicate that active Pmp23 is required for proper localization of the Z-ring and the FtsZ-positioning protein MapZ. In addition, Pmp23 localizes to the division site and interacts directly with the essential peptidoglycan synthase PBP2x. Altogether, our data reveal a new regulatory function for peptidoglycan hydrolases.

Copper inhibits peptidoglycan LD-transpeptidases suppressing β-lactam resistance due to bypass of penicillin-binding proteins

Significance The antimicrobial effect of copper has been known since ancient times and it is well known that copper alloy surfaces kill various disease-causing bacteria, fungi, and viruses, preventing the spread of antibiotic resistance through horizontal gene transfer between pathogens. However, there is little knowledge about specific targets of copper toxicity in bacteria. Here we show that copper inhibits peptidoglycan LD-transpeptidases, causing higher permeability in the outermembrane and precluding strains of Escherichia coli and Enterococcus faecium to utilize these enzymes in a bypass mechanism to achieve β-lactam resistance. Hence, copper affects bacterial cell envelope biogenesis at low concentration that does not affect growth rate. The peptidoglycan (PG) layer stabilizes the bacterial cell envelope to maintain the integrity and shape of the cell. Penicillin-binding proteins (PBPs) synthesize essential 4–3 cross-links in PG and are inhibited by β-lactam antibiotics. Some clinical isolates and laboratory strains of Enterococcus faecium and Escherichia coli achieve high-level β-lactam resistance by utilizing β-lactam–insensitive LD-transpeptidases (LDTs) to produce exclusively 3–3 cross-links in PG, bypassing the PBPs. In E. coli, other LDTs covalently attach the lipoprotein Lpp to PG to stabilize the envelope and maintain the permeability barrier function of the outermembrane. Here we show that subminimal inhibitory concentration of copper chloride sensitizes E. coli cells to sodium dodecyl sulfate and impair survival upon LPS transport stress, indicating reduced cell envelope robustness. Cells grown in the presence of copper chloride lacked 3–3 cross-links in PG and displayed reduced covalent attachment of Braun’s lipoprotein and reduced incorporation of a fluorescent d-amino acid, suggesting inhibition of LDTs. Copper dramatically decreased the minimal inhibitory concentration of ampicillin in E. coli and E. faecium strains with a resistance mechanism relying on LDTs and inhibited purified LDTs at submillimolar concentrations. Hence, our work reveals how copper affects bacterial cell envelope stability and counteracts LDT-mediated β-lactam resistance.

Structure of the essential peptidoglycan amidotransferase MurT/GatD complex from Streptococcus pneumoniae

The universality of peptidoglycan in bacteria underlies the broad spectrum of many successful antibiotics. However, in our times of widespread resistance, the diversity of peptidoglycan modifications offers a variety of new antibacterials targets. In some Gram-positive species such as Streptococcus pneumoniae, Staphylococcus aureus, or Mycobacterium tuberculosis, the second residue of the peptidoglycan precursor, D-glutamate, is amidated into iso-D-glutamine by the essential amidotransferase MurT/GatD complex. Here, we present the structure of this complex at 3.0 Å resolution. MurT has central and C-terminal domains similar to Mur ligases with a cysteine-rich insertion, which probably binds zinc, contributing to the interface with GatD. The mechanism of amidation by MurT is likely similar to the condensation catalyzed by Mur ligases. GatD is a glutaminase providing ammonia that is likely channeled to the MurT active site through a cavity network. The structure and assay presented here constitute a knowledge base for future drug development studies.The amidotransferase MurT/GatD complex catalyzes peptidoglycan precursor amidation in some Gram-positive bacteria. Here the authors present the crystal structure of the Streptococcus pneumoniae MurT/GatD complex and provide mechanistic insights, which are of interest for drug development.

The lytic transglycosylase MltB connects membrane homeostasis and in vivo fitness of Acinetobacter baumannii : The A. baumannii mltB gene encodes a crucial fitness factor

Acinetobacter baumannii has emerged as a leading nosocomial pathogen, infecting a wide range of anatomic sites including the respiratory tract and the bloodstream. In addition to being multi‐drug resistant, little is known about the molecular basis of A. baumannii pathogenesis. To better understand A. baumannii virulence, a combination of a transposon‐sequencing (TraDIS) screen and the neutropenic mouse model of bacteremia was used to identify the full set of fitness genes required during bloodstream infection. The lytic transglycosylase MltB was identified as a critical fitness factor. MltB cleaves the MurNAc‐GlcNAc bond of peptidoglycan, which leads to cell wall remodeling. Here we show that MltB is part of a complex network connecting resistance to stresses, membrane homeostasis, biogenesis of pili and in vivo fitness. Indeed, inactivation of mltB not only impaired resistance to serum complement, cationic antimicrobial peptides and oxygen species, but also altered the cell envelope integrity, activated the envelope stress response, drastically reduced the number of pili at the cell surface and finally, significantly decreased colonization of both the bloodstream and the respiratory tract.