Angelika Gründling

c-di-AMP Is a New Second Messenger in Staphylococcus aureus with a Role in Controlling Cell Size and Envelope Stress

The cell wall is a vital and multi-functional part of bacterial cells. For Staphylococcus aureus, an important human bacterial pathogen, surface proteins and cell wall polymers are essential for adhesion, colonization and during the infection process. One such cell wall polymer, lipoteichoic acid (LTA), is crucial for normal bacterial growth and cell division. Upon depletion of this polymer bacteria increase in size and a misplacement of division septa and eventual cell lysis is observed. In this work, we describe the isolation and characterization of LTA-deficient S. aureus suppressor strains that regained the ability to grow almost normally in the absence of this cell wall polymer. Using a whole genome sequencing approach, compensatory mutations were identified and revealed that mutations within one gene, gdpP (GGDEF domain protein containing phosphodiesterase), allow both laboratory and clinical isolates of S. aureus to grow without LTA. It was determined that GdpP has phosphodiesterase activity in vitro and uses the cyclic dinucleotide c-di-AMP as a substrate. Furthermore, we show for the first time that c-di-AMP is produced in S. aureus presumably by the S. aureus DacA protein, which has diadenylate cyclase activity. We also demonstrate that GdpP functions in vivo as a c-di-AMP-specific phosphodiesterase, as intracellular c-di-AMP levels increase drastically in gdpP deletion strains and in an LTA-deficient suppressor strain. An increased amount of cross-linked peptidoglycan was observed in the gdpP mutant strain, a cell wall alteration that could help bacteria compensate for the lack of LTA. Lastly, microscopic analysis of wild-type and gdpP mutant strains revealed a 13–22% reduction in the cell size of bacteria with increased c-di-AMP levels. Taken together, these data suggest a function for this novel secondary messenger in controlling cell size of S. aureus and in helping bacteria to cope with extreme membrane and cell wall stress.

Synthesis of glycerol phosphate lipoteichoic acid in Staphylococcus aureus

Lipoteichoic acid (LTA), a glycerol phosphate surface polymer, is a component of the envelope of Gram-positive bacteria. However, the molecular basis for its synthesis or function is not known. Here we report that Staphylococcus aureus LtaS synthesizes glycerol phosphate LTA. Construction of a mutant S. aureus strain with inducible ltaS expression revealed that LTA synthesis is required for bacterial growth and cell division. An ltaS homologue of Bacillus subtilis restored LTA synthesis and the growth of ltaS mutant staphylococci. Thus, LtaS inhibition can be used as a target to treat human infections caused by antibiotic-resistant S. aureus or other bacterial pathogens.

Cyclic di-AMP: another second messenger enters the fray

Nucleotide signalling molecules contribute to the regulation of cellular pathways in all forms of life. In recent years, the discovery of new signalling molecules in bacteria and archaea, as well as the elucidation of the pathways they regulate, has brought insights into signalling mechanisms not only in bacterial and archaeal cells but also in eukaryotic host cells. Here, we provide an overview of the synthesis and regulation of cyclic di-AMP (c-di-AMP), one of the latest cyclic nucleotide second messengers to be discovered in bacteria. We also discuss the currently known receptor proteins and pathways that are directly or indirectly controlled by c-di-AMP, the domain structure of the enzymes involved in its production and degradation, and the recognition of c-di-AMP by the eukaryotic host.

Holins kill without warning

Holins comprise the most diverse functional group of proteins known. They are small bacteriophage-encoded proteins that accumulate during the period of late-protein synthesis after infection and cause lysis of the host cell at a precise genetically programmed time. It is unknown how holins achieve temporal precision, but a conserved feature of their function is that energy poisons subvert the normal scheduling mechanism and instantly trigger membrane disruption. On this basis, timing has been proposed to involve a progressive decrease in the energized state of the membrane until a critical triggering level is reached. Here, we report that membrane integrity is not compromised after the induction of holin synthesis until seconds before lysis. The proton motive force was monitored by the rotation of individual cells tethered by a single flagellum. The results suggest an alternative explanation for the lysis “clock,” in which holin concentrations build to a critical level that leads to formation of an oligomeric complex that disrupts the membrane.

Systematic identification of conserved bacterial c-di-AMP receptor proteins.

Nucleotide signaling molecules are important messengers in key pathways that allow cellular responses to changing environments. Canonical secondary signaling molecules act through specific receptor proteins by direct binding to alter their activity. Cyclic diadenosine monophosphate (c-di-AMP) is an essential signaling molecule in bacteria that has only recently been discovered. Here we report on the identification of four Staphylococcus aureus c-di-AMP receptor proteins that are also widely distributed among other bacteria. Using an affinity pull-down assay we identified the potassium transporter-gating component KtrA as a c-di-AMP receptor protein, and it was further shown that this protein, together with c-di-AMP, enables S. aureus to grow in low potassium conditions. We defined the c-di-AMP binding activity within KtrA to the RCK_C (regulator of conductance of K+) domain. This domain is also found in a second S. aureus protein, a predicted cation/proton antiporter, CpaA, which as we show here also directly binds c-di-AMP. Because RCK_C domains are found in proteinaceous channels, transporters, and antiporters from all kingdoms of life, these findings have broad implications for the regulation of different pathways through nucleotide-dependent signaling. Using a genome-wide nucleotide protein interaction screen we further identified the histidine kinase protein KdpD that in many bacteria is also involved in the regulation of potassium transport and a PII-like signal transduction protein, which we renamed PstA, as c-di-AMP binding proteins. With the identification of these widely distributed c-di-AMP receptor proteins we link the c-di-AMP signaling network to a central metabolic process in bacteria.

Requirement of the Listeria monocytogenes Broad-Range Phospholipase PC-PLC during Infection of Human Epithelial Cells

In this study, we investigated the requirement of the Listeria monocytogenes broad-range phospholipase C (PC-PLC) during infection of human epithelial cells. L. monocytogenes is a facultative intracellular bacterial pathogen of humans and a variety of animal species. After entering a host cell, L. monocytogenes is initially surrounded by a membrane-bound vacuole. Bacteria promote their escape from this vacuole, grow within the host cell cytosol, and spread from cell to cell via actin-based motility. Most infection studies with L. monocytogenes have been performed with mouse cells or an in vivo mouse model of infection. In all mouse-derived cells tested, the pore-forming cytolysin listeriolysin O (LLO) is absolutely required for lysis of primary vacuoles formed during host cell entry. However, L. monocytogenes can escape from primary vacuoles in the absence of LLO during infection of human epithelial cell lines Henle 407, HEp-2, and HeLa. Previous studies have shown that the broad-range phospholipase C, PC-PLC, promotes lysis of Henle 407 cell primary vacuoles in the absence of LLO. Here, we have shown that PC-PLC is also required for lysis of HEp-2 and HeLa cell primary vacuoles in the absence of LLO expression. Furthermore, our results indicated that the amount of PC-PLC activity is critical for the efficiency of vacuolar lysis. In an LLO-negative derivative of L. monocytogenes strain 10403S, expression of PC-PLC has to increase before or upon entry into human epithelial cells, compared to expression in broth culture, to allow bacterial escape from primary vacuoles. Using a system for inducible PC-PLC expression in L. monocytogenes, we provide evidence that phospholipase activity can be increased by elevated expression of PC-PLC or Mpl, the enzyme required for proteolytic activation of PC-PLC. Lastly, by using the inducible PC-PLC expression system, we demonstrate that, in the absence of LLO, PC-PLC activity is not only required for lysis of primary vacuoles in human epithelial cells but is also necessary for efficient cell-to-cell spread. We speculate that the additional requirement for PC-PLC activity is for lysis of secondary double-membrane vacuoles formed during cell-to-cell spread.

Cross-Linked Peptidoglycan Mediates Lysostaphin Binding to the Cell Wall Envelope of Staphylococcus aureus

Staphylococcus simulans bv. staphylolyticus secretes lysostaphin, a bacteriocin that cleaves pentaglycine cross bridges in the cell wall of Staphylococcus aureus. The C-terminal cell wall-targeting domain (CWT) of lysostaphin is required for selective binding of this bacteriocin to S. aureus cells; however, the molecular target for this was unknown. We used purified green fluorescent protein fused to CWT (GFP-CWT) to reveal species-specific association of the reporter with staphylococci. GFP-CWT bound S. aureus cells as well as purified peptidoglycan sacculi. The addition of cross-linked murein, disaccharides linked to interconnected wall peptides, blocked GFP-CWT binding to staphylococci, whereas murein monomers or lysostaphin-solubilized cell wall fragments did not. S. aureus strain Newman variants lacking the capacity for synthesizing polysaccharide capsule (capFO), poly-N-acetylglucosamine (icaAC), lipoprotein (lgt), cell wall-anchored proteins (srtA), or the glycolipid anchor of lipoteichoic acid (ypfP) bound GFP-CWT similar to wild-type staphylococci. A tagO mutant strain, defective in the synthesis of polyribitol wall teichoic acid attached to the cell wall envelope, displayed increased GFP-CWT binding. In contrast, a femAB mutation, reducing both the amount and the length of peptidoglycan cross-linking (monoglycine cross bridges), showed a dramatic reduction in GFP-CWT binding. Thus, the CWT domain of lysostaphin directs the bacteriocin to cross-linked peptidoglycan, which also serves as the substrate for its glycyl-glycine endopeptidase domain.

Lipoteichoic Acid Synthesis and Function in Gram-Positive Bacteria

Lipoteichoic acid (LTA) is an important cell wall polymer found in gram-positive bacteria. Although the exact role of LTA is unknown, mutants display significant growth and physiological defects. Additionally, modification of the LTA backbone structure can provide protection against cationic antimicrobial peptides. This review provides an overview of the different LTA types and their chemical structures and synthesis pathways. The occurrence and mechanisms of LTA modifications with D-alanyl, glycosyl, and phosphocholine residues will be discussed along with their functions. Similarities between the production of type I LTA and osmoregulated periplasmic glucans in gram-negative bacteria are highlighted, indicating that LTA should perhaps be compared to these polymers rather than lipopolysaccharide, as is presently the case. Lastly, current efforts to use LTAs as vaccine candidates, synthesis proteins as novel antimicrobial targets, and LTA mutant strains as improved probiotics are highlighted.

Location, synthesis and function of glycolipids and polyglycerolphosphate lipoteichoic acid in Gram-positive bacteria of the phylum Firmicutes.

Lipoteichoic acid (LTA) is a zwitterionic polymer found in the cell wall of many Gram-positive bacteria. A widespread and one of the best-studied forms of LTA consists of a polyglycerolphosphate (PGP) chain that is tethered to the membrane via a glycolipid anchor. In this review, we will summarize our current understanding of the enzymes involved in glycolipid and PGP backbone synthesis in a variety of different Gram-positive bacteria. The recent identification of key LTA synthesis proteins allowed the construction and analysis of mutant strains with defined defects in glycolipid or backbone synthesis. Using these strains, new information on the functions of LTA for bacterial growth, physiology and during developmental processes was gained and will be discussed. Furthermore, we will reintroduce the idea that LTA remains in close proximity to the bacterial membrane for its function during bacterial growth rather than as a surface-exposed structure.

Effects of ribosomal proteins S1, S2 and the DeaD/CsdA DEAD-box helicase on translation of leaderless and canonical mRNAs in Escherichia coli

Leaderless mRNAs beginning with the AUG initiating codon occur in all kingdoms of life. It has been previously reported that translation of the leaderless λcI mRNA is stimulated in an Escherichia coli rpsB mutant deficient in ribosomal protein S2. Here, we have studied this phenomenon at the molecular level by making use of an E. coli rpsBts mutant. The analysis of the ribosomes isolated under the non‐permissive conditions revealed that in addition to ribosomal protein S2, ribosomal protein S1 was absent, demonstrating that S2 is essential for binding of S1 to the 30S ribosomal subunit. In vitro translation assays and the selective translation of a leaderless mRNA in vivo at the non‐permissive temperature corroborate and extend previous in vitro ribosome binding studies in that S1 is indeed dispensable for translation of leaderless mRNAs. The deaD/csdA gene, encoding the ‘DeaD/CsdA’ DEAD‐box helicase, has been isolated as a multicopy suppressor of rpsBts mutations. Here, we show that expression of a plasmid borne deaD/csdA gene restores both S1 and S2 on the ribosome at the non‐permissive temperature in the rpsBts strain, which in turn leads to suppression of the translational defect affecting canonical mRNAs. These data are discussed in terms of a model, wherein DeaD/CsdA is involved in ribosome biogenesis rather than acting directly on mRNA.