The bacterial tyrosine kinase system CpsBCD governs the length of capsule polymers

Many pathogenic bacteria are encased in a layer of capsular polysaccharide (CPS). This layer is important for virulence by masking surface antigens, preventing opsonophagocytosis, and avoiding mucus entrapment. The bacterial tyrosine kinase (BY-kinase) regulates capsule synthesis and helps bacterial pathogens to survive different host niches. BY-kinases autophosphorylate at the C-terminal tyrosine residues upon external stimuli, but the role of phosphorylation is still unclear. Here, we report that the BY-kinase CpsCD is required for growth in Streptococcus pneumoniae. Cells lacking a functional cpsC or cpsD accumulated low molecular weight CPS and lysed because of the lethal sequestration of the lipid carrier undecaprenyl phosphate, resulting in inhibition of peptidoglycan (PG) synthesis. CpsC interacts with CpsD and the polymerase CpsH. CpsD phosphorylation reduces the length of CPS polymers presumably by controlling the activity of CpsC. Finally, pulse–chase experiments reveal the spatiotemporal coordination between CPS and PG synthesis. This coordination is dependent on CpsC and CpsD. Together, our study provides evidence that BY-kinases regulate capsule polymer length by fine-tuning CpsC activity through autophosphorylation.

Amoxicillin-resistant Streptococcus pneumoniae can be resensitized by targeting the mevalonate pathway as identified by sCRilecs-seq

Antibiotic resistance in the important opportunistic human pathogen Streptococcus pneumoniae is on the rise. This is particularly problematic in the case of the β-lactam antibiotic amoxicillin, which is the first intention therapy. It is therefore crucial to uncover targets that would kill or resensitize amoxicillin- resistant pneumococci. To do so, we developed a genome-wide, single-cell based, gene silencing screen using CRISPR interference called sCRilecs-seq (subsets of CRISPR interference libraries extracted by fluorescence activated cell sorting coupled to next generation sequencing). Since amoxicillin affects growth and division, sCRilecs-seq was used to identify targets that are responsible for maintaining proper cell size. Our screen revealed that downregulation of the mevalonate pathway leads to extensive cell elongation. We show that this phenotype is caused by insufficient transport of cell wall precursors across the cell membrane due to a limitation in the production of undecaprenyl phosphate (Und-P), the lipid carrier responsible for this process. The data suggest that septal peptidoglycan synthesis is more sensitive to reduced Und-P levels than peripheral peptidoglycan synthesis. We successfully exploited this knowledge to create a combination treatment strategy where the FDA-approved drug clomiphene, an inhibitor of Und-P synthesis, is paired up with amoxicillin. Our results show that clomiphene potentiates the antimicrobial activity of amoxicillin and that combination therapy resensitizes amoxicillin-resistant S. pneumoniae. These findings could provide a starting point to develop a solution for the increasing amount of hard-to-treat amoxicillin-resistant pneumococcal infections.

Identification of Homologous Polyprenols from Thermophilic Bacteria

Sixteen strains of five genera of thermophilic bacteria, i.e., Alicyclobacillus, Brevibacillus, Geobacillus, Meiothermus, and Thermus, were cultivated at a temperature from 42 to 70 °C. Twelve strains were obtained from the Czech Collection of Microorganisms, while four were directly isolated and identified by 16S rRNA gene sequencing from the hot springs of the world-famous Carlsbad spa (Czech Republic). Polyprenol homologs from C40 to C65 as well as free undecaprenol (C55), undecaprenyl phosphate, and undecaprenyl diphosphate were identified by shotgun analysis and RP-HPLC/MS-ESI+ (reverse phase high-performance liquid chromatography–high-resolution positive electrospray ionization mass spectrometry). The limit of detection (50 pM) was determined for individual homologs and free polyprenols and their phosphates. Thus, it has been shown that at least some thermophilic bacteria produce not just the major C55 polyprenol as previously described, but a mixture of homologs.

CbrA Mediates Colicin M Resistance in Escherichia coli through Modification of Undecaprenyl-Phosphate-Linked Peptidoglycan Precursors

ABSTRACT Colicin M is an enzymatic bacteriocin produced by some Escherichia coli strains which provokes cell lysis of competitor strains by hydrolysis of the cell wall peptidoglycan undecaprenyl-PP-MurNAc(-pentapeptide)-GlcNAc (lipid II) precursor. The overexpression of a gene, cbrA (formerly yidS), was shown to protect E. coli cells from the deleterious effects of this colicin, but the underlying resistance mechanism was not established. We report here that a major structural modification of the undecaprenyl-phosphate carrier lipid and of its derivatives occurred in membranes of CbrA-overexpressing cells, which explains the acquisition of resistance toward this bacteriocin. Indeed, a main fraction of these lipids, including the lipid II peptidoglycan precursor, now displayed a saturated isoprene unit at the α-position, i.e., the unit closest to the colicin M cleavage site. Only unsaturated forms of these lipids were normally detectable in wild-type cells. In vitro and in vivo assays showed that colicin M did not hydrolyze α-saturated lipid II, clearly identifying this substrate modification as the resistance mechanism. These saturated forms of undecaprenyl-phosphate and lipid II remained substrates of the different enzymes participating in peptidoglycan biosynthesis and carrier lipid recycling, allowing this colicin M-resistance mechanism to occur without affecting this essential pathway. IMPORTANCE Overexpression of the chromosomal cbrA gene allows E. coli to resist colicin M (ColM), a bacteriocin specifically hydrolyzing the undecaprenyl-PP-MurNAc(-pentapeptide)-GlcNAc (lipid II) peptidoglycan precursor of targeted cells. This resistance results from a CbrA-dependent modification of the precursor structure, i.e., reduction of the α-isoprenyl bond of C55-carrier lipid moiety that is proximal to ColM cleavage site. This modification, observed here for the first time in eubacteria, annihilates the ColM activity without affecting peptidoglycan biogenesis. These data, which further increase our knowledge of the substrate specificity of this colicin, highlight the capability of E. coli to generate reduced forms of C55-carrier lipid and its derivatives. Whether the function of this modification is only relevant with respect to ColM resistance is now questioned.

Membrane linkage of a Streptococcus pneumoniae Wzy capsular polysaccharide occurs through an acylglycerol

ABSTRACTCapsular polysaccharides (capsules) protect bacteria from environmental insults and can contribute to virulence in pathogenic bacteria. Their appropriate display on the bacterial surface is critical to their functions. In Gram-positive bacteria, most capsules are synthesized by the Wzy polymerase-dependent pathway, which is also utilized in the synthesis of many capsules and O-antigens of Gram-negative bacteria. Synthesis of capsule repeat units initiates on undecaprenyl-phosphate on the inner face of the cytoplasmic membrane, with polymerization occurring on the outer face of the membrane. In Gram-positive bacteria, the capsule can be transferred to peptidoglycan, as in Streptococcus pneumoniae where a direct glycosidic bond to the peptidoglycan N-acetylglucosamine occurs. In S. pneumoniae, capsule can also be detected on the membrane, and this has generally been assumed to reflect polysaccharide that is linked to undecaprenyl-phosphate and in the process of synthesis. We provide evidence here, however, that final membrane linkage occurs through an acylglycerol, and essentially all of the polysaccharide is transferred from the initial undecaprenyl-phosphate acceptor to an alternate acceptor. This step allows for recycling of undecaprenyl-phosphate and represents an additional terminal step in capsule synthesis. In this regard, capsule synthesis resembles that of the wall- and lipoteichoic acids of S. pneumoniae, wherein a common repeat unit and polymer structure are synthesized by the Wzy pathway with divergence at the terminal step that results in linkages to peptidoglycan and a membrane acylglycerol anchor.IMPORTANCELinkage of capsular polysaccharides to the bacterial cell surface is a critical step in assuring the ability of these polymers to fulfill their functions, such as the resistance to complement-mediated phagocytosis that can be essential for pathogenic organisms to survive in host environments. Knowledge of the mechanisms by which these linkages occur is incomplete. In this study, we provide evidence for linkage of an S. pneumoniae Wzy capsule to an acylglycerol, the most abundant class of lipids in the membrane. This linkage provides a terminal acceptor for capsule that occurs in addition to that of peptidoglycan. Transfer to these terminal receptors is an essential step in CPS synthesis, as failure to do so can be lethal for the cell.

A genetic screen to identify factors affected by undecaprenyl phosphate recycling uncovers novel connections to morphogenesis in Escherichia coli

Undecaprenyl phosphate (Und-P) is an essential lipid carrier that ferries cell wall intermediates across the cytoplasmic membrane in bacteria. Und-P is generated by dephosphorylating undecaprenyl diphosphate (Und-PP). In Escherichia coli, BacA, PgpB, YbjG, and LpxT dephosphorylate Und-PP and are conditionally essential. To identify vulnerabilities that arise when Und-P metabolism is defective, we developed a genetic screen for synthetic mutations in combination with ΔybjG ΔlpxT ΔbacA. The screen uncovered system-wide connections, including novel connections to cell division, DNA replication and repair, signal transduction, and glutathione metabolism. Further analysis revealed several new morphogenes; loss of one of these, qseC, caused cells to enlarge and lyse. QseC is the sensor kinase component of the QseBC two-component system. In the absence of QseC, the QseB response regulator is overactivated by PmrB cross-phosphorylation. Here, we show that deleting qseB completely reverses the shape defect of ΔqseC cells, as does overexpressing rprA (a small RNA). Surprisingly, deleting pmrB only partially suppressed qseC-related shape defects. Thus, QseB is activated by multiple factors in the absence of QseC and functions ascribed to QseBC may be related to cell wall defects. Altogether, our findings provide a framework for identifying new determinants of cell integrity that could be targeted in future therapies.

Distinct Pathways Carry Out α and β Galactosylation of Secondary Cell Wall Polysaccharide in Bacillus anthracis

ABSTRACT Bacillus anthracis, the causative agent of anthrax disease, elaborates a secondary cell wall polysaccharide (SCWP) that is required for the retention of surface layer (S-layer) and S-layer homology (SLH) domain proteins. Genetic disruption of the SCWP biosynthetic pathway impairs growth and cell division. B. anthracis SCWP is comprised of trisaccharide repeats composed of one ManNAc and two GlcNAc residues with O-3–α-Gal and O-4–β-Gal substitutions. UDP-Gal, synthesized by GalE1, is the substrate of galactosyltransferases that modify the SCWP repeat. Here, we show that the gtsE gene, which encodes a predicted glycosyltransferase with a GT-A fold, is required for O-4–β-Gal modification of trisaccharide repeats. We identify a DXD motif critical for GtsE activity. Three distinct genes, gtsA, gtsB, and gtsC, are required for O-3–α-Gal modification of trisaccharide repeats. Based on the similarity with other three-component glycosyltransferase systems, we propose that GtsA transfers Gal from cytosolic UDP-Gal to undecaprenyl phosphate (C55-P), GtsB flips the C55-P-Gal intermediate to the trans side of the membrane, and GtsC transfers Gal onto trisaccharide repeats. The deletion of galE1 does not affect growth in vitro, suggesting that galactosyl modifications are dispensable for the function of SCWP. The deletion of gtsA, gtsB, or gtsC leads to a loss of viability, yet gtsA and gtsC can be deleted in strains lacking galE1 or gtsE. We propose that the loss of viability is caused by the accumulation of undecaprenol-bound precursors and present an updated model for SCWP assembly in B. anthracis to account for the galactosylation of repeat units. IMPORTANCE Peptidoglycan is a conserved extracellular macromolecule that protects bacterial cells from turgor pressure. Peptidoglycan of Gram-positive bacteria serves as a scaffold for the attachment of polymers that provide defined bacterial interactions with their environment. One such polymer, B. anthracis SCWP, is pyruvylated at its distal end to serve as a receptor for secreted proteins bearing the S-layer homology domain. Repeat units of SCWP carry three galactoses in B. anthracis. Glycosylation is a recurring theme in nature and often represents a means to mask or alter conserved molecular signatures from intruders such as bacteriophages. Several glycosyltransferase families have been described based on bioinformatics prediction, but few have been studied. Here, we describe the glycosyltransferases that mediate the galactosylation of B. anthracis SCWP.

Three distinct glycosylation pathways are involved in the decoration of Lactococcus lactis cell wall glycopolymers

Extracytoplasmic sugar decoration of glycopolymer components of the bacterial cell wall contributes to their structural diversity. Typically, the molecular mechanism that underpins such a decoration process involves a three-component glycosylation system (TGS) represented by an undecaprenyl-phosphate (Und-P) sugar-activating glycosyltransferase (Und-P GT), a flippase, and a polytopic glycosyltransferase (PolM GT) dedicated to attaching sugar residues to a specific glycopolymer. Here, using bioinformatic analyses, CRISPR-assisted recombineering, structural analysis of cell wall–associated polysaccharides (CWPS) through MALDI-TOF MS and methylation analysis, we report on three such systems in the bacterium Lactococcus lactis. On the basis of sequence similarities, we first identified three gene pairs, csdAB, csdCD, and csdEF, each encoding an Und-P GT and a PolM GT, as potential TGS component candidates. Our experimental results show that csdAB and csdCD are involved in Glc side-chain addition on the CWPS components rhamnan and polysaccharide pellicle (PSP), respectively, whereas csdEF plays a role in galactosylation of lipoteichoic acid (LTA). We also identified a potential flippase encoded in the L. lactis genome (llnz_02975, cflA) and confirmed that it participates in the glycosylation of the three cell wall glycopolymers rhamnan, PSP, and LTA, thus indicating that its function is shared by the three TGSs. Finally, we observed that glucosylation of both rhamnan and PSP can increase resistance to bacteriophage predation and that LTA galactosylation alters L. lactis resistance to bacteriocin.