Global analysis of the impact of linezolid onto virulence factor production in S. aureus USA300
Introduction
Methicillin resistant strains of the nosocomial pathogen Staphylococcus aureus (MRSA) are a major problem in global healthcare. Healthcare-associated (HA)-MRSA strains, for whose acquisition prior illness is a predisposing risk factor, infect primarily immune-suppressed, frail and elderly people. In contrast, community-associated (CA)-MRSA also infect healthy individuals, suggesting a greater virulence of CA-MRSA strains (DeLeo et al., 2010). Linezolid (LZD) is one of few remaining treatment options for infections caused by these important pathogens. LZD was the first antibiotic of the oxazolidinone translation inhibitor class approved for clinical use in the year 2000. Oxazolidinone translation inhibitors represented the first truly new class of antibiotics after nearly 20 years that act via a unique mechanism of action. Oxazolidinones prevent protein biosynthesis by binding to the peptidyl transferase center at the ribosomal A site. LZD stabilizes a distinct conformation of the conserved 23S rRNA nucleotide U2585. In this conformation the second tRNA cannot reach the correct position for formation of the first peptide bond. After GTP hydrolysis and release of EF-Tu the second tRNA dislocates from the ribosome, which remains locked (Wilson et al., 2008).
As with all antibiotics, development of bacterial resistance against LZD is of greatest concern. More than 99.5% of all S. aureus isolates tested in several studies were susceptible to LZD and all resistant Staphylococci were obtained from patients who had previously been treated with LZD (Gu et al., 2012). Resistance phenotypes identified so far are based on mutations in the 23S rRNA gene (Besier et al., 2008) and the genes coding for ribosomal proteins L3 and L4 (Locke et al., 2010). Other mechanisms of resistance in S. aureus include methylation of 23S rRNA by the plasmid-coded chloramphenicol florfenicol resistance (cfr) gene product (Morales et al., 2010). In addition, a mutation in the relA gene switching on the stringent response and reducing LZD susceptibility has been reported (Gao et al., 2010).
The effects of LZD on bacterial cells have been studied by several research groups. In a first study a decreased amount of staphylococcal and streptococcal exotoxins was detected after the cells were grown in presence of subinhibitory concentrations of LZD (Gemmell and Ford, 2002). A series of similar studies examining the expression and/or amount of certain exotoxins in presence of LZD and other antibiotics has been performed since then (Coyle et al., 2003, Dumitrescu et al., 2007, Otto et al., 2013). These conclusively show that LZD might inhibit toxin production (Diep et al., 2012). However, these studies focused only on specific virulence factors and the general impact of LZD onto bacterial physiology was not studied. Functional genomics studies can provide a detailed picture of the physiological adaptation responses of S. aureus to antibiotic treatment (Wecke and Mascher, 2011, Wenzel and Bandow, 2011). For instance, Bernardo et al. analyzed the exoproteome of LZD stressed S. aureus and detected a specific reduction in amount of extracellular virulence factors (Bernardo et al., 2004). Furthermore LZD was shown to inhibit accumulation but not the transcription of the exotoxin Panton-Valentine leukocidin, when added to exponentially growing cells at a concentration of five times the MIC (Stevens et al., 2007). However, none of these studies revealed the mechanism of the initially observed inhibition of virulence factors, which was also observed in in vivo studies with animal models (Yoshizawa et al., 2012, Diep et al., 2013).
To resolve the question if (and how) LZD reduces the synthesis of virulence factors, we designed an approach integrating complementing omics approaches with microscopy. An S. aureus USA300 derivative was used as a biological model. We performed absolute, label-free quantification of cytosolic and extracellular proteins to monitor the effect of translation inhibition on protein levels. For exact protein quantification we performed a metabolic labeling and analyzed the cytosolic and membrane proteome. In addition, we performed a DNA microarray analysis and microscopy experiments. Using this integrated approach we provide a global view of the physiological effects of LZD onto a clinically highly relevant pathogen and reveal that diminished protein translocation potential might result in decreased levels of extracellular toxins and reduced virulence observed after LZD treatment.
Section snippets
Bacterial growth conditions
Cells were grown aerobically at 37 °C in LB medium (Invitrogen), or for creation of the labeled standard in 15N-enriched BioExpress 1000 medium (Cambridge Isotopes). All transcriptomics and proteomics experiments were performed in triplicates. The minimal inhibitory concentration (MIC) of LZD in S. aureus USA300 was determined by broth microdilution. To analyze the LZD response of growing cells LZD was added at different concentrations (0.5–8× MIC) at OD540 of 0.5 (Fig. 1). For proteomic,
Bacterial growth
By broth microdilution a MIC of 2.5 mg/ml LZD could be determined for S. aureus USA300 in LB. Growing cells of S. aureus USA300 were stressed in the mid-exponential phase with sub-inhibitory and inhibitory concentrations of LZD, as in antibiotic stress experiments inhibitory concentrations that reduce the growth rate can be necessary (Bandow et al., 2003). As shown in Fig. 1, immediately after addition of LZD to cells growing exponentially at OD540 0.5 the growth rate decreased. At an OD540 of 2
Protein biosynthesis
Several functional genomics studies characterized the effects of translation inhibitors on bacteria. Protein synthesis inhibition can be caused on several levels for example by stopping peptide bond formation, premature chain termination and inhibition of the activity of aminoacyl tRNA synthetases. Each class of translation inhibitors has an individual signature on proteome level (Wenzel and Bandow, 2011). To obtain a straight-forward analysis we focused on studies of translation inhibitors
Conclusions
In the present study, several known effects of LZD and translation inhibitors in general could be interpreted in a new, comprehensive way by a combination of several orthogonal and complementing techniques. Based on the observed defects in membrane protein biogenesis and general protein export, we hypothesize that suppression of extracellular virulence factors by LZD could be based on the disintegration of transertion complexes. The observation that S. aureus cells resumed growth after a
Acknowledgements
The research leading to these results has received support from the Deutsche Forschungsgemeinschaft within the SFB/TRR 34 framework.
The authors like to thank Jürgen Bartel, Sebastian Grund, Daniela Ulbrich and Annette Meuche for excellent technical assistance. We further like to thank Sandra Maass and Holger Kock for critical revising the manuscript. Finally, we like to thank the ProteomeXchange consortium and the Gene Expression Omnibus team for their support.
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Present address: Nosocomial Pathogens and Antibiotic Resistance, Robert Koch Institute, 38855 Wernigerode, Germany.