Two Novel Point Mutations in Clinical Staphylococcus aureus Reduce Linezolid Susceptibility and Switch on the Stringent Response to Promote Persistent Infection

Abstract

Staphylococcus aureus frequently invades the human bloodstream, leading to life threatening bacteremia and often secondary foci of infection. Failure of antibiotic therapy to eradicate infection is frequently described; in some cases associated with altered S. aureus antimicrobial resistance or the small colony variant (SCV) phenotype. Newer antimicrobials, such as linezolid, remain the last available therapy for some patients with multi-resistant S. aureus infections. Using comparative and functional genomics we investigated the molecular determinants of resistance and SCV formation in sequential S. aureus isolates from a patient who had a persistent and recurrent S. aureus infection, after failed therapy with multiple antimicrobials, including linezolid. Two point mutations in key staphylococcal genes dramatically affected clinical behaviour of the bacterium, altering virulence and antimicrobial resistance. Most strikingly, a single nucleotide substitution in relA (SACOL1689) reduced RelA hydrolase activity and caused accumulation of the intracellular signalling molecule guanosine 3′, 5′-bis(diphosphate) (ppGpp) and permanent activation of the stringent response, which has not previously been reported in S. aureus. Using the clinical isolate and a defined mutant with an identical relA mutation, we demonstrate for the first time the impact of an active stringent response in S. aureus, which was associated with reduced growth, and attenuated virulence in the Galleria mellonella model. In addition, a mutation in rlmN (SACOL1230), encoding a ribosomal methyltransferase that methylates 23S rRNA at position A2503, caused a reduction in linezolid susceptibility. These results reinforce the exquisite adaptability of S. aureus and show how subtle molecular changes cause major alterations in bacterial behaviour, as well as highlighting potential weaknesses of current antibiotic treatment regimens.

The Insect <em>Galleria mellonella</em> as a Powerful Infection Model to Investigate Bacterial Pathogenesis

“…Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model [8][9][10][11] . Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.…”

The Escherichia coli RlmN methyltransferase is a dual-specificity enzyme that modifies both rRNA and tRNA and controls translational accuracy

“…m 2 A2503 is located at the entrance of the nascent peptide exit tunnel and has been involved in the ribosomal mechanism that promotes translation arrest in response to specific peptide sequences (Vazquez-Laslop et al 2010;Ramu et al 2011). Lack of methylation of A2503 has been reported to produce a subtle yet significant effect on cell fitness and to increase linezolid resistance (Toh et al 2008;Gao et al 2010;Lamarre et al 2011). RlmN and its evolutionarily related resistance enzyme Cfr belong to the radical S-adenosyl-L-methionine (SAM) enzyme superfamily, and both use protein-free rRNA as a substrate (Atta et al 2010;Kaminska et al 2010;Yan et al 2010;Grove et al 2011;Yan and Fujimori 2011).…”

ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

“…The genome of S. aureus also encodes two other monofunctional synthetases, RelP and RelQ, and transcription of these genes increases when cells are exposed to cell wall-targeting antimicrobials (20,21). Recent work on S. aureus has shown that the ability to switch on the stringent response is essential for its virulence and is required for the organism to cause chronic infections (22)(23)(24)(25).…”