In vitro studies indicate a high resistance potential for the lantibiotic nisin in Staphylococcus aureus and define a genetic basis for nisin resistance

Abstract

Lantibiotics such as nisin (NIS) are peptide antibiotics that may have a role in the chemotherapy of bacterial infections. A perceived benefit of lantibiotics for clinical use is their low propensity to select resistance, although detailed resistance studies with relevant bacterial pathogens are lacking. Here we examined the development of resistance to NIS in Staphylococcus aureus, establishing that mutants, including small-colony variants, exhibiting substantial (4- to 32-fold) reductions in NIS susceptibility could be selected readily. Comparative genome sequencing of a single NISr mutant exhibiting a 32-fold increase in NIS MIC revealed the presence of only two mutations, leading to the substitutions V229G in the purine operon repressor, PurR, and A208E in an uncharacterized protein encoded by SAOUHSC_02955. Independently selected NISr mutants also harbored mutations in the genes encoding these products. Reintroduction of these mutations into the S. aureus chromosome alone and in combination revealed that SAOUHSC_02955(A208E) made the primary contribution to the resistance phenotype, conferring up to a 16-fold decrease in NIS susceptibility. Bioinformatic analyses suggested that this gene encodes a sensor histidine kinase, leading us to designate it "nisin susceptibility-associated sensor (nsaS)." Doubling-time determinations and mixed-culture competition assays between NISr and NISs strains indicated that NIS resistance had little impact on bacterial fitness, and resistance was stable in the absence of selection. The apparent ease with which S. aureus can develop and maintain NIS resistance in vitro suggests that resistance to NIS and other lantibiotics with similar modes of action would arise in the clinic if these agents are employed as chemotherapeutic drugs.

Fig. 1.

Predicted functional domains and structure of the NsaS sensor histidine kinase and genetic context of the nsaS gene. (Top) Amino acid sequence of NsaS showing transmembrane domains (blue), the HisKA domain (green) containing the conserved histidine residue (bright red), and the HATPase_c domain (magenta), as predicted by InterProScan and SOSUI. (Middle left) Structural model of NsaS protein (orange) and X-ray crystal structure of the T. maritima sensor histidine kinase (gray) on which the model is based. Shown in blue are the transmembrane domains of NsaS predicted by SOSUI. (Middle right) Alternate view of the model on the left showing the ATP analogue ADPβN (yellow) bound into the T. maritima crystal structure and the residues with which it interacts (green). Also shown are NIS^r residues A₂₀₈ and A₁₀₅ in NsaS (red) and the conserved histidine residues in the T. maritima crystal structure (blue) and the NsaS model (black). (Bottom) Comparison of genetic architecture and protein products of the nsaRS and graRS operons of S. aureus SH1000. SHK, sensor histidine kinase; RR, response regulator; PERM, permease domain of ABC transporter; ATP, ATP-binding domain of ABC transporter. Gene sizes (bp) are indicated in bold, carets mark intergenic distances (bp), and % identity between paralogous protein products is shown.