Mutation in the C-di-AMP cyclase dacA affects fitness and resistance of methicillin resistant Staphylococcus aureus

Abstract

Faster growing and more virulent strains of methicillin resistant Staphylococcus aureus (MRSA) are increasingly displacing highly resistant MRSA. Elevated fitness in these MRSA is often accompanied by decreased and heterogeneous levels of methicillin resistance; however, the mechanisms for this phenomenon are not yet fully understood. Whole genome sequencing was used to investigate the genetic basis of this apparent correlation, in an isogenic MRSA strain pair that differed in methicillin resistance levels and fitness, with respect to growth rate. Sequencing revealed only one single nucleotide polymorphism (SNP) in the diadenylate cyclase gene dacA in the faster growing but less resistant strain. Diadenylate cyclases were recently discovered to synthesize the new second messenger cyclic diadenosine monophosphate (c-di-AMP). Introduction of this mutation into the highly resistant but slower growing strain reduced resistance and increased its growth rate, suggesting a direct connection between the dacA mutation and the phenotypic differences of these strains. Quantification of cellular c-di-AMP revealed that the dacA mutation decreased c-di-AMP levels resulting in reduced autolysis, increased salt tolerance and a reduction in the basal expression of the cell wall stress stimulon. These results indicate that c-di-AMP affects cell envelope-related signalling in S. aureus. The influence of c-di-AMP on growth rate and methicillin resistance in MRSA indicate that altering c-di-AMP levels could be a mechanism by which MRSA strains can increase their fitness levels by reducing their methicillin resistance levels.

Figure 1. Oxacillin resistance and fitness of RA120, ME51 and RA120::dacA-SNP.

A, oxacillin population analysis profiles and B, growth curves of the strain RA120 (black squares) and the dacA-SNP strains ME51 (white diamonds) and RA120::dacA-SNP (white squares). Representative data from three independent experiments are shown.

Figure 2. Cellular levels of c-di-AMP and PBPs of RA120, ME51 and RA120::dacA-SNP.

A, cellular levels of c-di-AMP quantified by LC-MS in ng per mg cellular dry weight (cdw). Average values from five biological replicates with standard deviation are shown. Asterisks indicate significant differences compared to RA120 (** p<0.01). B, PBP2a (76 kDa) Western blot analysis, C, visualisation of PBP1 (83 kDa), PBP2 (80 kDa) and PBP3 (77 kDa) using the fluorescent penicillin analogue Bocillin-FL and D, PBP4 (48 kDa) Western blot analysis of membrane fractions of the strains RA120 (1), ME51 (2) and RA120::dacA-SNP (3) sampled at indicated ODs. Representative results from three independent protein extractions are shown.

Figure 3. Analysis of phenotypes associated with cell envelope properties in RA120, ME51 and RA120::dacA-SNP.

A, spontaneous autolysis of RA120 (black squares) and the dacA-SNP strains ME51 (white diamonds) and RA120::dacA-SNP (white squares) in phosphate buffer and B, induced autolysis of strains with 0.01% Triton X-100 in phosphate buffer. Average values with standard deviation from three independent experiments are shown. Asterisks indicate measurement time points with significant difference between both RA120/ME51 and RA120/RA120::dacA-SNP (* p<0.05, ** p<0.01) using Student’s t-test. Two-way ANOVA analysis considering all time points resulted in significant differences (p<0.01) for stain pairs RA120/ME51 and RA120/RA120::dacA-SNP for both spontaneous and induced autolysis. C, NaCl tolerance of RA120, ME51 and RA120::dacA-SNP. Overnight cultures were serially diluted 1∶5 and plated on plain LB agar or LB agar with 2.2 M NaCl. Representative results from three independent experiments are shown. D, left, relative light units (RLU) measured from cell wall stress stimulon reporter construct psas016 _p -luc+ in RA120 (black squares) and the dacA-SNP strains ME51 (white diamonds) and RA120::dacA-SNP (white squares) and right, the corresponding OD values of the cultures at each sampling point. Average values with standard deviation from three independent experiments are shown. Asterisks indicate measurement time points with significant differences in RLU between both RA120/ME51 and RA120/RA120::dacA-SNP strain pairs. If the strain pairs had different categories of p-values these were indicated separately (* p<0.05, ** p<0.01). Two-way ANOVA analysis considering RLU values from all time points resulted in significant differences (p<0.01) for both strain pairs RA120/ME51 and RA120/RA120::dacA-SNP.

Figure 4. Effect of dacA-SNP on oxacillin resistance, fitness and the c-di-AMP levels in MSSA strain BB255 and MRSA strain COL.

A, oxacillin population analysis profile of wild type strains BB255 (black triangle) and COL (black circle) and their corresponding dacA-SNP strains BB255::dacA-SNP (white triangle) and COL::dacA-SNP (white circle). Representative data from three independent experiments are shown. B, growth curves of BB255 and BB255::dacA-SNP and C, growth curves of COL and COL::dacA-SNP. Representative data from three independent experiments are shown. D, cellular levels of c-di-AMP quantified by LC-MS in ng per mg cellular dry weight (cdw). Average values from five biological replicates with standard deviation are shown. Grey bar with asterisk (*) indicates c-di-AMP concentration was blow detection limit and the given value represents maximal concentration which was estimated by dividing the limit of detection (32 nM) by the samples biomass concentrations. Two asterisks (**) indicate a significant difference using Student’s t-test (p<0.01) and the dashed bracket indicates statistical analysis was performed using the estimated maximal value for BB255::dacA-SNP.