Research Groups

[86] RNA metabolism and antibiotic resistance mechanisms in Staphylococcus aureus.

Team Patrick LINDER: RNA metabolism and virulence factor expression in Staphylococcus aureus

Bacteria are everywhere and life without them is not possible. Most bacteria are harmless to human being, some are obligatory pathogens where their presence is equivalent to disease, others may not necessarily cause disease. Amongst this later category is the opportunistic pathogen Staphylococcus aureus. This Gram-positive bacterium forms golden grapes and is present in the nose of 30-50% of the people. In most of the healthy carriers, the presence of this bacterium is not noticeable, but in certain cases it can cause diseases ranging from furuncles to life threatening systemic infections. S. aureus is regularly in the Headlines because of its multiple resistances to antibiotics, including methicilin (MRSA, methicilin resistant S. aureus). Persistent infections of S. aureus are at least in part due to its extraordinary capability to form biofilms and to survive intracellularly.

Our laboratory has a long-standing interest in RNA helicases of the DEAD-box protein family. In eukaryotic cells, DEAD-box RNA helicases are involved in processes as divers as transcription, pre-mRNA splicing, ribosome biogenesis, RNA export, translation initiation, NMD (non-sense mediated decay), and organelle gene expression. All these processes are dynamic ribo-nucleo-protein (RNP) particles that need to be assembled, undergo conformational changes, and eventually be disassembled. Indeed a wonderful playground for RNA helicases!

The DEAD-box proteins are characterised by the presence of 12 highly conserved amino acid motifs that are involved in the binding of the substrates, ATP and RNA, and in intramolecular interactions. Unwinding of double stranded RNA or the dissociation of proteins from RNA does require ATP binding and ultimately ATP hydrolysis. Although not as abundant as in eukaryotes, most bacteria encode RNA helicases of the DEAD-box protein family. They have been identified by genetic screens or by bio-informatic analyses. In contrast to most eukaryotic DEAD-box protein genes, they are not essential under 'normal' (laboratory) growth conditions. However, even subtle differences in growth rate may result in huge differences in population density because of the short generation time of bacterial growth. Moreover, bacteria encounter various growth conditions that may require DEAD-box proteins. Indeed, early in the discovery of DEAD-box proteins, they were found to be associated with cold-shock in Escherichia coli.

The S. aureus genome encodes two DEAD-box RNA helicases. One of them was identified in a screen for biofilm deficient mutants in a clinical strain. Although growth of this mutant strain at temperatures of 37°C and above is normal, this strain is highly deficient in biofilm formation. At present we are analysing the role of this RNA helicase in gene expression to elucidate its role in biofilm formation (collaboration with the laboratory of Jacques Schrenzel, Genomic Research Laboratory, University Hospital, Geneva).

In a parallel project we are currently analysing the second RNA helicase encoded by the S. aureus genome. Disruption of this gene does confer a cold sensitive phenotype, but the strain grows normally at 37°C. Other projects in the laboratory are related to RNA metabolism in general.
Last but not least, we are trying to develop new strategies to make clinical S. aureus strains amenable to molecular genetic methods. Indeed, many of the clinical strains used in our studies cannot be transformed with plasmid DNA, even if the plasmids were prepared in the permissive laboratory strain RN4220. In this work we inactivate the host mediated restriction by the insertion of group II introns.

Team William KELLEY: Antibiotic resistance mechanisms and environmental sensing systems, primarily in the major human pathogen Staphylococcus aureus

The last steps of peptidoglycan biosynthesis are performed topologically outside the cell membrane by enzymes, termed penicillin binding proteins (PBPs). b-lactam and glycopeptide antibiotics (vancomycin and teicoplanin) target these enzymes, or their substrates, and disrupt peptidoglycan polymerization and crosslinking, leading ultimately to cell death.

The development of resistance to glycopeptide antibiotics is surprisingly complex and involves multiple transmembrane sensors triggering downstream gene expression and poorly understood changes in cell wall assembly pathways and metabolism. The resistance to all penicillin classes in staphylococcus except the latest 5th generation cephalosporins (ie, ceftaroline) occurs in MRSA strains by the horizontal acquistion of the SCCmec element encoding an alterative PBP (PBP2A) with poor affinity for penicillins. This is the fundamental underlying genetic change associated with the worldwide health problem associated with MRSA : loss of sensitivity to nearly all penicillins and multiple resistance to other drug classes leads to very limited treatment options. Glycopeptide antibiotics are considered first line defense for MRSA, but resistance, or reduced sensitivity, is associated with these drugs also, together with evidence  for the development of cross-resistance to other therapeutic alternatives currently available such as daptomycin.

Our work seeks to understand how resistance occurs and to develop strategies to restore sensitivity to antibiotics, or discover new insights that will guide future therapeutic development. Our recent published work has uncovered strategies to : i) block the emergence of glycopeptide resistance, ii) identified key extracellular protein folding proteins that facilitate the maturation of secreted proteins including PBPs, iii) identified novel signaling pathways that exploit an essential redox sensor Spx and antibiotic resistance, iv) discovered new genes involved in drug resistance, and v) identified novel mutations in PBP2A in certain clonotypes of archived clinical strains showing reduced sensitivity  to ceftaroline, but intriguingly, existing more than a decade before the commerical introduction of this drug in 2010. This entirely novel mechanism of resistance has lead us to initiate investigations to discover what genetic selections could possibly exist that can drive the formation of  PBP2A variants years before a 5th generation cephalosporin drug was even introduced.

In addition to antibiotics, we also have a longstanding interest in understanding the transcriptional regulation of toxins such as toxic shock superantigen (TSST-1).  Using both aerobic and anaerobic environmental conditions, we have been systematically identifying multiple trans-acting factors that control expression of the toxin. Many of the factors we have uncovered exert negative regulation over the tst promoter, providing a model framework to understand sporadic disease arising from spurious mutation in these regulators.

Our research goals and interests include :

  • Understanding  molecular details of antibiotic resistance
  • Develop strategies to restore sensitivity to b-lactams in MRSA strains
  • Understand the interplay of signaling systems that detect cell wall damage
  • Understand the molecular basis of cross-resistance
  • Dissect the pathways involved in secretion and maturation of PBPs
  • Dissect the role of reactive oxygen species elicted by cell-wall active antibiotics
  • Understand the essential redox stress sensor Spx and the repertoire of genes under its control
  • Understand why the gene encoding toxic shock superantigen is widespread, yet disease incidence is low by examining the trancriptional regulation of the tst promoter.