Proteome profiling of evolved methicillin-resistant Staphylococcus aureus strains with distinct daptomycin tolerance and resistance phenotypes

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is a highly dangerous pathogen, and daptomycin has been increasingly used to treat its infections in clinics. Recently, several groups have shown that tolerance and resistance of microbes can evolve rapidly under cyclic antibiotic exposure. We have previously shown that the same tolerance and resistance development occurs in MRSA treated with daptomycin in an adaptive laboratory evolution (ALE) experiment. In the present study, we performed proteomic analysis to compare six daptomycin-tolerant and resistant MRSA strains that were evolved from the same ancestral strain. The strain with a higher tolerance level than the others had the most different proteome and response to antibiotic treatment, resembling those observed in persister cells, which are small subpopulations of bacteria that survive lethal antibiotics treatment. By comparing the proteome changes across strains with similar phenotypes, we identified the key proteins that play important roles in daptomycin tolerance and resistance in MRSA. We selected two candidates to be confirmed by gene overexpression analysis. Overexpression of EcsA1 and FabG, which were up-regulated in all of the tolerant evolved strains, led to increased daptomycin tolerance in wild-type MRSA. The proteomics data also suggested that cell wall modulations were implicated in both resistance and tolerance, but in different ways. While the resistant strains had peptidoglycan changes and a more positive surface charge to directly repel daptomycin, the tolerant strains possessed different cell wall changes that do not involve the peptidoglycan nor alterations of the surface charge. Overall, our study showed the differential proteome profiles among multiple tolerant and resistant strains, pinpointed the key proteins for the two phenotypes and revealed the differences in cell wall modulations between the daptomycin-tolerant/resistant strains.

Mutation in the Two-Component System Regulator YycH Leads to Daptomycin Tolerance in Methicillin-Resistant Staphylococcus aureus upon Evolution with a Population Bottleneck

“…Indeed, both our tolerant and resistant populations with yycH and yycI mutations displayed increased MICs of vancomycin (see Table S1). Since daptomycin is also known to indirectly affect the S. aureus cell wall as part of its activity and changes in cell wall properties commonly lead to daptomycin tolerance or resistance ( 13 , 37 , 51 – 54 ), these impaired functions of YycH and YycI, which downregulated WalKR activity, might influence the cells’ susceptibility to daptomycin, similar to results with vancomycin. Unlike YycI, the other mutated protein, ThrC, was still detected in the resistant population, although the expression was 3.5-fold lower than in the ancestral strain (see Table S7).…”

“…Interestingly, one of the downregulated proteins in the daptomycin-tolerant population, immunoglobulin-binding protein Sbi (reduced by 2.3-fold) (see Table S4), has also been found downregulated in 5 different daptomycin-tolerant strains in previous studies bearing single point mutations in different genes conferring different levels of tolerance (two strains in one study [ 13 ] and three strains in another [ 37 ]), but not in daptomycin-resistant strains. Sbi is anchored to the cell envelope through binding to the lipoteichoic acid (LTA) and it plays a role in S. aureus virulence by inhibiting both the innate and adaptive immune responses, although its actual function in antibiotic tolerance or resistance is still unknown.…”

“…Omics methodology has been recently applied to study the adaptation mechanisms of evolved tolerant and resistant strains from such in vitro evolution experiments ( 4 ), both in E. coli ( 12 , 34 ) and S. aureus ( 13 , 35 – 37 ). Here, we performed quantitative proteomics and compared the proteome profiles of the evolved populations to that of the ancestral strain to gain mechanistic insights into their adaptation.…”

Adaptive Laboratory Evolution of Staphylococcus aureus Resistance to Vancomycin and Daptomycin: Mutation Patterns and Cross-Resistance

“…The main mechanisms resulting in the decreased susceptibility of VISA/hVISA to vancomycin include thickening of the cell wall and accumulation of free D-Ala-Dla residues, decreased autolysis caused by a change in peptidoglycan biosynthesis, decreased activity of the staphylococcal global regulator decreased lysostaphin susceptibility, and changes in cell wall teichoic acids [8]. Mutations in two-component systems (TCS) regulating cell wall biosynthesis (WalKR, GraSR, and VraSR) and global regulators (Agr and RpoB) are involved in resistance development [9]. Recently, data on resistance were obtained for the cmk gene product, which participates in the pyrimidine synthesis pathway [10], the putative formate dehydrogenase Fdh2 [11], and the catabolite control protein E (CcpE), which is the first positive regulator of the tricarboxylic acid cycle [12].…”

“…Uncertainty exists regarding the correlation between changes in glycerol metabolism and the biosynthesis of membrane phospholipids. The involvement of numerous biosynthetic pathways in daptomycin resistance and tolerance development was demonstrated using a proteomic approach [9].…”