Cyclic-di-adenosine monophosphate (c-di-AMP) is required for osmotic regulation in Staphylococcus aureus but dispensable for viability in anaerobic conditions

Cyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered signaling molecule important for the survival of Firmicutes, a large bacterial group that includes notable pathogens such as Staphylococcus aureus. However, the exact role of this molecule has not been identified. dacA, the S. aureus gene encoding the diadenylate cyclase enzyme required for c-di-AMP production, cannot be deleted when bacterial cells are grown in rich medium, indicating that c-di-AMP is required for growth in this condition. Here, we report that an S. aureus dacA mutant can be generated in chemically defined medium. Consistent with previous findings, this mutant had a severe growth defect when cultured in rich medium. Using this growth defect in rich medium, we selected for suppressor strains with improved growth to identify c-di-AMP-requiring pathways. Mutations bypassing the essentiality of dacA were identified in alsT and opuD, encoding a predicted amino acid and osmolyte transporter, the latter of which we show here to be the main glycine betaine-uptake system in S. aureus. Inactivation of these transporters likely prevents the excessive osmolyte and amino acid accumulation in the cell, providing further evidence for a key role of c-di-AMP in osmotic regulation. Suppressor mutations were also obtained in hepS, hemB, ctaA and qoxB, coding for proteins required for respiration. Furthermore, we show that dacA is dispensable for growth in anaerobic conditions. Together, these finding reveal an essential role for the c-di-AMP signaling network in aerobic, but not anaerobic, respiration in S. aureus

The immune evasion protein Sbi of Staphylococcus aureus occurs both extracellularly and anchored to the cell envelope by binding lipoteichoic acid

The Sbi protein of Staphylococcus aureus comprises two IgG-binding domains similar to those of protein A and a region that triggers the activation of complement C3. Sbi is expressed on the cell surface but its C-terminal domain lacks motifs associated with wall or membrane anchoring of proteins in Gram-positive bacteria. Cell-associated Sbi fractionates with the cytoplasmic membrane and is not solubilized during protoplast formation. S. aureus expressing Sbi truncates of the C-terminal Y domain allowed identification of residues that are required for association of Sbi with the membrane. Recombinant Sbi bound to purified cytoplasmic membrane material in vitro and to purified lipoteichoic acid. This explains how Sbi partitions with the membrane in fractionation experiments yet is partially exposed on the cell surface. An LTA-defective mutant of S. aureus had reduced levels of Sbi in the cytoplasmic membrane

The Cell Wall Polymer Lipoteichoic Acid Becomes Nonessential in Staphylococcus aureus Cells Lacking the ClpX Chaperone

Lipoteichoic acid (LTA) is an important cell wall component of Gram-positive bacteria and a promising target for the development of vaccines and antimicrobial compounds against Staphylococcus aureus. Here we demonstrate that mutations in the conditionally essential ltaS (LTA synthase) gene arise spontaneously in an S. aureus mutant lacking the ClpX chaperone. A wide variety of ltaS mutations were selected, and among these, a substantial portion resulted in premature stop codons and other changes predicted to abolish LtaS synthesis. Consistent with this assumption, the clpX ltaS double mutants did not produce LTA, and genetic analyses confirmed that LTA becomes nonessential in the absence of the ClpX chaperone. In fact, inactivation of ltaS alleviated the severe growth defect conferred by the clpX deletion. Microscopic analyses showed that the absence of ClpX partly alleviates the septum placement defects of an LTA-depleted strain, while other phenotypes typical of LTA-negative S. aureus mutants, including increased cell size and decreased autolytic activity, are retained. In conclusion, our results indicate that LTA has an essential role in septum placement that can be bypassed by inactivating the ClpX chaperone. IMPORTANCE Lipoteichoic acid is an essential component of the Staphylococcus aureus cell envelope and an attractive target for the development of vaccines and antimicrobials directed against antibiotic-resistant Gram-positive bacteria such as methicillin-resistant S. aureus and vancomycin-resistant enterococci. In this study, we showed that the lipoteichoic acid polymer is essential for growth of S. aureus only as long as the ClpX chaperone is present in the cell. Our results indicate that lipoteichoic acid and ClpX play opposite roles in a pathway that controls two key cell division processes in S. aureus, namely, septum formation and autolytic activity. The discovery of a novel functional connection in the genetic network that controls cell division in S. aureus may expand the repertoire of possible strategies to identify compounds or compound combinations that kill antibiotic-resistant S. aureus.Peer reviewe

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

Published versio

Cross-talk between two nucleotide-signaling pathways in Staphylococcus aureus.

Nucleotide-signaling pathways are found in all kingdoms of life and are utilized to coordinate a rapid response to external stimuli. The stringent response alarmones guanosine tetra- (ppGpp) and pentaphosphate (pppGpp) control a global response allowing cells to adapt to starvation conditions such as amino acid depletion. One more recently discovered signaling nucleotide is the secondary messenger cyclic diadenosine monophosphate (c-di-AMP). Here, we demonstrate that this signaling nucleotide is essential for the growth of Staphylococcus aureus, and its increased production during late growth phases indicates that c-di-AMP controls processes that are important for the survival of cells in stationary phase. By examining the transcriptional profile of cells with high levels of c-di-AMP, we reveal a significant overlap with a stringent response transcription signature. Examination of the intracellular nucleotide levels under stress conditions provides further evidence that high levels of c-di-AMP lead to an activation of the stringent response through a RelA/SpoT homologue (RSH) enzyme-dependent increase in the (p)ppGpp levels. This activation is shown to be indirect as c-di-AMP does not interact directly with the RSH protein. Our data extend this interconnection further by showing that the S. aureus c-di-AMP phosphodiesterase enzyme GdpP is inhibited in a dose-dependent manner by ppGpp, which itself is not a substrate for this enzyme. Altogether, these findings add a new layer of complexity to our understanding of nucleotide signaling in bacteria as they highlight intricate interconnections between different nucleotide-signaling networks

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

Published versio

The second messenger c-di-AMP regulates osmotic and pH adaptation in Staphylococcus aureus under aerobic and anaerobic conditions

Cyclic nucleotides that act as second messenger molecules play key roles in signaling pathways involved in rapid responses to changing environments. Cyclic di-adenosine monophosphate (c-di-AMP) is a secondary messenger that is mainly produced by Gram-positive bacteria, including many human pathogens. Changes in the intracellular concentration of this signaling nucleotide has been implicated in diverse physiological roles including potassium ion transport, cell wall biosynthesis, biofilm formation and resistance to some antibiotics. In Staphylococcus aureus, c-di-AMP is synthesized from two molecules of ATP by a single diadenylate cyclase enzyme, namely DacA, and is degraded by two phosphodiesterase enzymes, called GdpP and Pde2, to linear 5’-phosphadenylyl-adenosine (pApA). Pde2 can further degrade pApA to two molecules of AMP. Recently, it was shown that DacA is required for the growth of S. aureus in rich media and under standard laboratory conditions, but it is dispensable for viability under anaerobiosis. Therefore, in this work we investigated the role of c-di-AMP in the aerobic and anaerobic growth of S. aureus under salt and pH stresses. The growth of the MRSA USA300 strain LAC* (WT) and several isogenic mutants for the dacA, gdpP and pde2 genes, was assessed in media containing increasing concentrations of either NaCl or KCl and pH raging from 4.5 to 10.5. We found that DacA is likely to be important for S. aureus viability under both aerobic and anaerobic conditions when growing in media with high salt concentrations (>2 M), particularly in NaCl. Unexpectedly, high levels of c-di-AMP seemed to be detrimental under anaerobic conditions in presence of elevated salt concentrations (>1.5 M), as both phosphodiesterase GdpP and Pde2 are essential for S. aureus viability under these conditions. Furthermore, GdpP and Pde2 are required for anaerobic growth when exposed to low pH. To investigate in more detail de contribution of c-di-AMP to the osmotic adaptation of S. aureus growing under aerobiosis or anaerobiosis, suppressor strains for LAC* dacA and LAC* gdpPpde2 were obtained with improved growth in 2M KCl under aerobic conditions and in 2M NaCl under anaerobiosis, respectively. Currently, a whole-genome-sequencing approach is being employed to further identify the mutations restoring the viability of the suppressor strains. Taken together, these findings indicate that changes in the concentration of the c-di-AMP, mediated by DacA, GdpP and Pde2 enzymes, are critical for osmotic and pH adaptation in S. aureus under both aerobic and anaerobic conditions

Editorial overview: "All in all, it is not just another brick in the wall": new concepts and mechanisms on how bacteria build their wall.

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Genes Required for Glycolipid Synthesis and Lipoteichoic Acid Anchoring in Staphylococcus aureus▿

Staphylococcus aureus lipoteichoic acid (LTA) is composed of a linear 1,3-linked polyglycerolphosphate chain and is tethered to the bacterial membrane by a glycolipid (diglucosyl-diacylglycerol [Glc2-DAG]). Glc2-DAG is synthesized in the bacterial cytoplasm by YpfP, a processive enzyme that transfers glucose to diacylglycerol (DAG), using UDP-glucose as its substrate. Here we present evidence that the S. aureus α-phosphoglucomutase (PgcA) and UTP:α-glucose 1-phosphate uridyltransferase (GtaB) homologs are required for the synthesis of Glc2-DAG. LtaA (lipoteichoic acid protein A), a predicted membrane permease whose structural gene is located in an operon with ypfP, is not involved in Glc2-DAG synthesis but is required for synthesis of glycolipid-anchored LTA. Our data suggest a model in which LtaA facilitates the transport of Glc2-DAG from the inner (cytoplasmic) leaflet to the outer leaflet of the plasma membrane, delivering Glc2-DAG as a substrate for LTA synthesis, thereby generating glycolipid-anchored LTA. Glycolipid anchoring of LTA appears to play an important role during infection, as S. aureus variants lacking ltaA display defects in the pathogenesis of animal infections

In vivo and in vitro characterization of Staphylococcus aureus and Bacillus subtilis polyglycerolphosphate lipoteichoic acid synthases

Staphylococcus aureus lipoteichoic acid (LTA) consists of a 1,3-linked polyglycerolphosphate chain retained in the bacterial membrane by a glycolipid anchor. The LTA backbone is produced by the lipoteichoic acid synthase LtaS, a membrane protein with five transmembrane helices and a large extracellular enzymatic domain (eLtaS). Proteomic studies revealed that LtaS is efficiently cleaved, and here it was demonstrated that the eLtaS domain is released into the culture supernatant as well as partially retained within the cell wall fraction. However, using an in vivo LtaS activity assay, it was shown that only the full-length LtaS enzyme is able to synthesize LTA. Neither expression of a secreted eLtaS variant, created by replacing the N-terminal membrane domain with a conventional signal sequence, nor expression of eLtaS fused to a single or multi-transmembrane domains of other staphylococcal proteins resulted in the production of LTA. These data indicate that the transmembrane domain of LtaS play an essential, yet unknown, role in LtaS enzyme function. In addition, the protease responsible for LtaS cleavage was identified. It was found that a S. aureus strain in which the gene encoding for the essential signal peptidase SpsB was cloned under inducible expression control showed an accumulation of the full-length LtaS enzyme in the absence of the inducer. These data suggest that SpsB is involved in LtaS cleavage. Four LtaS orthologues, YflE, YfnI, YqgS and YvgJ, are present in Bacillus subtilis. Using an in vitro enzyme assay and purified protein, it was determined that all four B. subtilis proteins are Mn2+-dependent metal enzymes that use the lipid phosphatidylglycerol as substrate. It was shown that YflE, YfnI and YqgS are bonafide LTA synthases capable of producing polyglycerolphosphate chains, while YvgJ appears to function as an LTA primase, as indicated by the accumulation of a glycolipid with the expected chromatographic mobility of GroP-Glc2-DAG. Taken together, experimental evidence for the enzyme function of all four B. subtilis LtaStype proteins is provided in this work and it was shown that all four enzymes are involved in the LTA synthesis process.EThOS - Electronic Theses Online ServiceGBUnited Kingdo