Daptomycin avoids drug resistance mediated by the BceAB transporter in Streptococcus pneumoniae

Abstract

Drug-resistant bacteria are a serious threat to human health as antibiotics are gradually losing their clinical efficacy. Comprehending the mechanism of action of antimicrobials and their resistance mechanisms plays a key role in developing new agents to fight antimicrobial resistance. The lipopeptide daptomycin is an antibiotic that selectively disrupts Gram-positive bacterial membranes, thereby showing slower resistance development than many classical drugs. Consequently, it is often used as a last resort antibiotic to preserve its use as one of the least potent antibiotics at our disposal. The mode of action of daptomycin has been debated but was recently found to involve the formation of a tripartite complex between undecaprenyl precursors of cell wall biosynthesis and the anionic phospholipid phosphatidylglycerol. BceAB-type ABC transporters are known to confer resistance to antimicrobial peptides that sequester some precursors of the peptidoglycan, such as the undecaprenyl pyrophosphate or lipid II. The expression of these transporters is upregulated by dedicated two-component regulatory systems in the presence of antimicrobial peptides that are recognized by the system. Here, we investigated whether daptomycin evades resistance mediated by the BceAB transporter from the bacterial pathogen Streptococcus pneumoniae. Although daptomycin can bind to the transporter, our data showed that the BceAB transporter does not mediate resistance to the drug and its expression is not induced in its presence. These findings show that the pioneering membrane-active daptomycin has the potential to escape the resistance mechanism mediated by BceAB-type transporters and confirm that the development of this class of compounds has promising clinical applications.IMPORTANCEAntibiotic resistance is rising in all parts of the world. New resistance mechanisms are emerging and dangerously spreading, threatening our ability to treat common infectious diseases. Daptomycin is an antimicrobial peptide that is one of the last antibiotics approved for clinical use. Understanding the resistance mechanisms toward last-resort antibiotics such as daptomycin is critical for the success of future antimicrobial therapies. BceAB-type ABC transporters confer resistance to antimicrobial peptides that target precursors of cell-wall synthesis. In this study, we showed that the BceAB transporter from the human pathogen Streptococcus pneumoniae does not confer resistance to daptomycin, suggesting that this drug and other calcium-dependent lipopeptide antibiotics have the potential to evade the action of this type of ABC transporters in other bacterial pathogens.

Keywords: ABC transporters; Streptococcus pneumoniae; antibiotic resistance; antibiotics; antimicrobial peptides; two-component regulatory systems.

Fig 1

Functional module involving TCS01 and BceAB in Streptococcus pneumoniae. In the absence of antimicrobial peptides, our current understanding is mostly based on work performed on the homologous system in Bacillus subtilis. BceAB and the histidine kinase likely form a relatively stable complex (31), and the transporter maintains its cognate kinase in an inactive state in the absence of bacitracin (32). Recognition of antimicrobial peptides and ATP binding/hydrolysis by BceAB are necessary to trigger phosphorelay signaling through BceS (33) (HK01 in S. pneumoniae). This signaling involves the autophosphorylation of the histidine kinase, and the phosphoryl group is then passed on to a conserved aspartate residue of the response regulator. Our previous work showed that the BceAB/TCS01 from the S. pneumoniae module can recognize the presence of antimicrobial peptides targeting undecaprenylpyrophosphate (UPP) or lipid II. In the presence of antimicrobial peptides, the phosphorylated RR01 upregulates the expression of the operon containing the bceAB genes but does not regulate the tcs01 operon (18). The overexpression of BceAB mediates antimicrobial peptide resistance. Figure created with Biorender.com.

Fig 2

Growth of wild-type and mutant R6 strains in the absence or presence of various concentrations of daptomycin. The daptomycin concentrations are indicated on top of the graphs. Cultures were performed in microplates, in the presence of 50 µg/mL of CaCl₂. Data shown here are the average of technical duplicates. These experiments were confirmed with biological replicates shown in Table 1.

Fig 3

Growth of wild-type and mutant R800 strains in the absence or presence of various concentrations of daptomycin. The daptomycin concentrations are indicated on top of the graphs. Cultures were performed in microplates, in the presence of 50 µg/mL of CaCl₂. Data shown here are the average of technical duplicates. These experiments were confirmed with biological replicates shown in Table 1.

Fig 4

Visualization of BceA-GFP fluorescence on the gel. The culture of the R800 bceA-gfp strain was performed until OD_600nm = 0.3. Bacteria were incubated for 30 min in the presence of different concentrations of daptomycin (in the presence of CaCl₂). Incubation in the presence of bacitracin (4 µg/mL) or the presence of actagardin (8 µg/mL) was used as positive control. Once treated, the bacterial pellets were loaded onto a 15% SDS-PAGE gel. In-gel fluorescence of BceA-GFP was scanned with a typhoon imager.

Fig 5

Analysis of BceAB-GFP expression in S. pneumoniae upon daptomycin treatment. These experiments were conducted in the R800 bceAB-gfp strains. Bacitracin (1 µg/mL) and actagardin (8 µg/mL) are used here as positive controls. Three daptomycin concentrations were tested: 2 µg/mL, 4 µg/mL, and 8 µg/mL. (A) Fluorescence microscopy of S. pneumoniae cells analyzing the drug-dependent expression of BceAB-GFP. Phase contrast (left panel), GFP fluorescent signal (middle panel), and overlays between phase contrast and GFP images (right panel) are shown. Enlargement is shown on the upper right corners of each panel. Scale bar, 1 µm. (B) Violin plots showing the distribution of cellular fluorescence mean intensities in individual cells corresponding to panel A. The boxes in the violin plots indicate the 25th to the 75th percentiles and the whiskers indicate the minimum and maximum value. The mean and the median are indicated with a dot and a line in the box, respectively. Significance was determined using Kruskal–Wallis and Dunn’s multiple comparison tests. A total of 11,185 cells were analyzed: actagardin n = 2,153; bacitracin n = 2,024; no drug n = 2,023; daptomycin 2 µg/mL n = 2,013, 4 µg/mL n = 1,673, 8 µg/mL n = 1,299.

Fig 6

ATPase activities of the BceAB transporter reconstituted in proteoliposomes. Data are the average of three replicates and error bars indicate the standard deviation of the replicates. Statistical significance was calculated by Student´s t-test with Welch’s correction between the conditions indicated with brackets. Statistically significant differences are indicated with ** (P ≤ 0.01).