Research interests

The focus of my research is the biogenesis of the cell wall envelope in Gram-positive bacterial pathogens. I am particularly interested in the assembly of proteins and wall polymers within the envelope of Staphylococcus aureus and other human pathogens such as Listeria monocytogenes.

Staphylococcus aureus is a microbial inhabitant of the human skin and nares. Breaches in epithelial barriers as well as defects in host immunity allow this normally commensal organism to cause a wide range of human infections. Among these are localised skin and soft tissue abscesses, pneumonia, endocarditis, osteomylitis, and septicemia. The cell wall envelope of S. aureus and other Gram-positive bacteria is a complex protective surface organelle, composed of peptidoglycan, proteins, polysaccharides and secondary wall polymers (Fig. 1). Many anti-staphylococcal therapies inhibit cell wall biosynthesis or destroy the physiological functions of the envelope in maintaining bacterial integrity and mediating evasion of host immune responses. These therapies include small molecules such as ß-lactams (i.e. methicillin) and glycopeptides (i.e. vancomycin) as well as lysostaphin, a bacteriocin that degrades the bacterial peptidoglycan layer.

I. Studies on lipoteichoic acid biosynthesis

A major focus of my research, is the biosynthesis pathway and function of the secondary wall polymer named lipotecihoic acid or short LTA. Along with proteins, secondary wall polymers fulfil important functions within the envelope of Gram-positive bacteria. Functions ascribed to LTA include scavenging Mg2+ ions required for the proper function of membrane associated enzymes, regulation of autolysis and protection against the action of antibacterial peptides produced by animals and humans as part of the innate immune system. Our own studies revealed that LTA is required in addition to these functions for proper cell division and essential for the assembly of a functional envelope, however the molecular mechanisms underlying these observations is currently unknown and are the subject of our research programme.

S. aureus LTA is composed of linear 1,3-linked polyglycerolphosphate chains linked to Glc₂-DAG, which retains the molecule in the bacterial membrane (Fig. 2). A similar polyglycerolphosphate LTA structure is found in many other Gram-positive pathogens including Bacillus cereus, B. anthracis, Enterococcus faecalis, Listeria sp., and Group A & B streptococci. The ubiquitous occurrence and the continued synthesis of LTA under conditions of phosphate limitation led to the idea that LTA plays an essential part in the bacterial cell physiology; indeed, very recently we provided experimental evidence for this notion (see below).

While the chemical structure of LTA has been known for some time, genes involved in its synthesis have been described only recently. Proteins encoded in the dlt operon are required for the addition of D-alanine residues, which play an important role in interaction of pathogenic S. aureus, Group A Streptococci, L. monocytogenes and B. anthraciswith their host. YpfP, PgcA and GtaB are necessary for glycolipid biosynthesis (Fig. 3). In addition, we revealed that LtaA (lipoteichoic acid protein A), a predicted membrane permease, is required for the synthesis of glycolipid-anchored LTA but dispensable for the synthesis of free membrane glycolipids. LtaA seems to facilitate the transport of Glc2-DAG from the inner (cytoplasmic) to the outer leaflet of the plasma membrane where it is used as lipid anchor for LTA (Fig. 3).

A S. aureus mutant unable to properly anchor LTA to the glycolipid has a defect in colonising liver and spleens in a murine model of infection. More importantly, we have identified the enzyme that is required for polyglycerolphosphate LTA synthesis. Depletion of a previously uncharacterised enzyme now named LtaS, for lipoteichoic acid synthase, leads to a halt in bacterial growth and to an absence of the polyglycerolphosphate LTA polymer on the surface of S. aureus. Electron microscopic analysis revealed severe morphological changes upon LTA depletion including a misplacement of cell division sites and thickening of the cell wall (Fig. 4). Based on additional work from our lab and the work from other groups it has now been established that LTA synthesis is also important for cell growth and morphology in other Gram-positive bacteria such as Bacillus subtilis and Listeria monocytogenes (Fig. 4).

Major research questions, which we are interested in pursuing:

• Why is LTA required for bacterial growth?
• Is there a direct link and interaction between LTA and cell division proteins?
• What is the enzyme mechanism of the lipoteichoic acid synthase LtaS?
• Can we develop LtaS-specific inhibitor as alternative antimicrobials?

Selected papers

Reichmann NT; Gründling A.* (2011). Location, synthesis and function of glycolipids and polyglycerolphosphate lipoteichoic acid in Gram-positive bacteria of the phylum Firmicutes. FEMS Microbiol Lett. 319:97-105. DOI Open Access copy. *corresponding author

Wörman n ME; Corrigan RM; Simpson PJ; Matthews SJ; Gründling A.* (2011). Enzymatic activities and functional interdependencies of Bacillus subtilis lipoteichoic acid synthesis enzymes. Mol Microbiol. 79:566-583. DOI Open Access copy. *corresponding author

Karatsa-Dodgson M; Wörmann ME; Gründling A.* (2010). In vitro analysis of the Staphylococcus aureus lipoteichoic acid synthase enzyme using fluorescently labeled lipids. J Bacteriol. 192:5341-5349. DOI. *corresponding author

Webb AJ; Karatsa-Dodgson M; Grundling A.* (2009). Two-enzyme systems for glycolipid and polyglycerolphosphate lipoteichoic acid synthesis in Listeria monocytogenes. MOL MICROBIOL. 74:299-314. DOI. *corresponding author

Lu D, Wörmann ME, Zhang X, Schneewind O, Gründling A* and Freemont PS* (2009). Structure-based mechanism of lipoteichoic acid synthesis by Staphylococcus aureus LtaS. Proc. Natl. Acad. Sci.: 106(5): 1584-1589; *co-corresponding authors.

Gründling A; Schneewind O. (2007). Synthesis of glycerol phosphate lipoteichoic acid in Staphylococcus aureus. Proc Natl Acad Sci U S A. 104:8478-8483. DOI.

Gründling A; Schneewind O. (Mar 2007). Genes required for glycolipid synthesis and lipoteichoic acid anchoring in Staphylococcus aureus. J Bacteriol. 189:2521-2530. DOI. 

II. Investigate the c-di-AMP signaling pathway in Staphylococcus aureus

Many complex physiological processes in bacterial cells, including the switch from planktonic to sedentary life styles and virulence gene expression, are regulated by small secondary messenger molecules such as c-di-GMP. It is now well documented that c-di-GMP plays an important role in controlling biofilm formation in a range of bacteria including important human pathogens such as Pseudomonas aeruginosa and a number of its target effectors have been described. Recently, it has also been suggested that this signalling molecule, which is widespread in bacterial species but apparently not found in higher eukaryotes, can act as a danger signal in eukaryotic cells prompting studies on the immunomodulatory and immunostimulatory properties of c-di-GMP. While Staphylococcus aureus does not possess c-di-GMP, we have recently identified c-di-AMP as an important signalling molecule in this bacterium using the screen described below.

To learn more about the function of LTA, we performed a genetic screen to obtain S. aureus suppressor stains that can grow in the absence of LTA. The compensatory mutations were identified using a whole genome sequencing approach and we found that mutations in gdpP, which encodes a c-di-AMP-specific phosphodiesterase allows S. aureus to grow in the absence of LTA. Only in 2008, c-di-AMP has been recognised as a naturally occurring molecule in living organisms. Up to now this molecule has been detected in the supernatant of Listeria monocytogenes cultures and in cytoplasmic extracts from Streptococcus pyogenes and from Bacillus subtilis, as well as from S. aureus as demonstrated by our own work. But it is expected that many more bacteria produce this signalling molecule. Furthermore, we have shown that c-di-AMP is produced in S. aureus by the protein DacA, which has diadenylate cyclase activity, while GdpP functions in vivo as a c-di-AMP-specific phosphodiesterase, as intracellular c-di-AMP levels increase drastically in gdpP deletion strains (Figure 5).

There is now growing evidence that c-di-AMP is essential for the growth of Gram-positive bacteria including S. aureus and L. monoytogenes since dacA mutants cannot be obtained. However nothing is known about the effector molecules to which c-di-AMP binds and through which it exerts its essential function. Furthermore, the molecular details linking the c-di-AMP signalling pathway and cell wall assembly and LTA synthesis in S. aureus are currently not understood (Figure 5).

Major research questions we are interested in pursuing:

• What genes are controlled by c-di-AMP?
• What is the molecular mechanism linking c-di-AMP and cell wall synthesis?
• Why is c-di-AMP essential for bacterial growth?

Selected paper

Corrigan, RM; Abbott, JC; Burhenne, H., Kaever, V; and Gründling, A.* (2011). Increased Intracellular c-di-AMP levels allow Staphylococcus aureus growth in the absence of lipoteichoic acid. PLoS Pathogens: 7(9)  e1002217. DOI. *corresponding author

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The Grundling Lab (November 2015)

Back row: Freja Kirsebom, Sophie Howard, Tommaso Tosi, Eleni Karinou, Chris Schuster, Angelika Grundling

Front row: Lisa Bowman, Merve Zeden, Charlotte Millership