Phenotypic and proteomic approaches of the response to iron-limited condition in Staphylococcus lugdunensis

Abstract

Background Staphylococcus lugdunensis is a coagulase-negative Staphylococcus part of the commensal skin flora but emerge as an important opportunistic pathogen. Because iron limitation is a crucial stress during infectious process, we performed phenotypic study and compared proteomic profiles of this species incubated in absence and in presence of the iron chelator 2,2′-dipyridyl (DIP). Results No modification of cell morphology nor cell wall thickness were observed in presence of DIP. However iron-limitation condition promoted biofilm formation and reduced the ability to cope with oxidative stress (1 mM H2O2). In addition, S. lugdunensis N920143 cultured with DIP was significantly less virulent in the larvae of Galleria mellonella model of infection than that grown under standard conditions. We verified that these phenotypes were due to an iron limitation by complementation experiments with FeSO4. By mass spectrometry after trypsin digestion, we characterized the first iron-limitation stress proteome in S. lugdunensis. Among 1426 proteins identified, 349 polypeptides were differentially expressed. 222 were more and 127 less abundant in S. lugdunensis incubated in iron-limitation condition, and by RT-qPCR, some of the corresponding genes have been shown to be transcriptionally regulated. Our data revealed that proteins involved in iron metabolism and carriers were over-expressed, as well as several ABC transporters and polypeptides linked to cell wall metabolism. Conversely, enzymes playing a role in the oxidative stress response (especially catalase) were repressed. Conclusions This phenotypic and global proteomic study allowed characterization of the response of S. lugdunensis to iron-limitation. We showed that iron-limitation promoted biofilm formation, but decrease the oxidative stress resistance that may, at least in part, explained the reduced virulence of S. lugdunensis observed under low iron condition.

Inactivation of the Response Regulator AgrA Has a Pleiotropic Effect on Biofilm Formation, Pathogenesis and Stress Response in Staphylococcus lugdunensis

“…From the AgrA-regulated genes, it can be hypothesized that the downregulation of genes involved in iron homeostasis could lead to reduced biofilm formation. In S. lugdunensis , it has been clearly shown that the iron-limitation condition triggered biofilm production in parallel with overexpression of isd operon ( 16 , 52 ). Note that the isd operon is not present in the S. epidermidis genome and was not identified as an AgrA-regulated gene in S. aureus ( 4 ).…”

“…The oxidative killing assays of S. lugdunensis N920143 and Δ agrA mutant were performed as previously described ( 16 ). Briefly, bacteria were grown 24 h in BHI broth and subcultured in 10 ml broth at a starting density of OD 600 of 0.1.…”

“…The total protein extraction and the mass-spectrometry were done as described previously ( 16 ). Briefly, 200 ml of S. lugdunensis N920143 and Δ agrA mutant cell cultures were centrifuged (6000 g) for 15 min at 4°C, washed twice with recovery buffer (Tris HCl 50 mM, Na 2 SO 4 50 mM, glycerol 15%), and incubated for 12 h at −80°C.…”

“…RNAs were extracted from cells of S. lugdunensis N920143 and Δ agrA mutant cultured in BHI harvested in the late-exponential phase (OD 600 of 1) as previously described ( 16 ). Following incubation of the cell pellets for 12 h at −80°C, RNAs were extracted with the Direct-zol RNA miniprep kit (Zymo Research, Irvine, CA, USA) and residual DNA was removed using Turbo DNase (ThermoFisher) according to the manufacturer’s instructions.…”

“…It is probably the most aggressive CoNS species and several virulence factors have been characterized ( 15 ). Likewise, S. lugdunensis is well-equipped for the acquisition of nutritional iron, which has been shown to be crucial for biofilm formation, virulence, and resistance to oxidative stress ( 16 , 17 ). This a priori harmless commensal CoNS should therefore be regarded as an opportunistic pathogen.…”