A putative cro-like repressor contributes to arylomycin resistance in Staphylococcus aureus

Abstract

Antibiotic-resistant bacteria are a significant public health concern and motivate efforts to develop new classes of antibiotics. One such class of antibiotics is the arylomycins, which target type I signal peptidase (SPase), the enzyme responsible for the release of secreted proteins from their N-terminal leader sequences. Despite the essentiality, conservation, and relative accessibility of SPase, the activity of the arylomycins is limited against some bacteria, including the important human pathogen Staphylococcus aureus. To understand the origins of the limited activity against S. aureus, we characterized the susceptibility of a panel of strains to two arylomycin derivatives, arylomycin A-C16 and its more potent analog arylomycin M131. We observed a wide range of susceptibilities to the two arylomycins and found that resistant strains were sensitized by cotreatment with tunicamycin, which inhibits the first step of wall teichoic acid synthesis. To further understand how S. aureus responds to the arylomycins, we profiled the transcriptional response of S. aureus NCTC 8325 to growth-inhibitory concentrations of arylomycin M131 and found that it upregulates the cell wall stress stimulon (CWSS) and an operon consisting of a putative transcriptional regulator and three hypothetical proteins. Interestingly, we found that mutations in the putative transcriptional regulator are correlated with resistance, and selection for resistance ex vivo demonstrated that mutations in this gene are sufficient for resistance. The results begin to elucidate how S. aureus copes with secretion stress and how it evolves resistance to the inhibition of SPase.

FIG 1

Heterogeneous resistance to the arylomycins in S. aureus. (A) The MIC of arylomycin A-C₁₆ was determined for a panel of 117 S. aureus strains consisting of well-characterized research strains, clinical MSSA isolates, and MRSA isolates. (B) The MICs of arylomycin M131 for a subset of the isolates from panel A consisting of all 16 arylomycin A-C₁₆-resistant isolates and 16 randomly selected arylomycin-sensitive isolates were determined. The structures of arylomycin A-C₁₆ and arylomycin M131 are shown above the respective graphs for comparison. Open bars indicate the strains with arylomycin A-C₁₆ MICs of >128 μg/ml.

FIG 2

Growth inhibition of S. aureus NCTC 8325 by arylomycin M131. S. aureus was grown with shaking in TSB for 4 h at 37°C to an OD₆₀₀ of 2. The culture was split and treated with 4× MIC of arylomycin M131 or DMSO, and growth was monitored for an additional 6 h. Samples were taken for global transcriptional analysis 30 and 120 min after arylomycin M131 addition, corresponding to 4.5 and 6 h of total growth, respectively.

FIG 3

Induction of the CWSS in arylomycin-sensitive and -resistant strains by arylomycin M131. S. aureus strains were grown in TSB to an OD₆₀₀ of 1.0 and subjected to either DMSO or 4 μg/ml arylomycin M131 for 30 min. Extracted RNA was converted to cDNA and subjected to RT-PCR using primers to amplify genes involved in the CWSS (vraR, spsIB, and prsA). gmk was used as an external control, and gene expression was normalized to 16S rRNA. Fold changes in gene expression were calculated using the ΔΔC_T method; data shown are the average and standard error of the mean (SEM) for three independent samples.

FIG 4

Alleles of SAOUHSC_00331 in representative arylomycin-sensitive and -resistant S. aureus strains. The SAOUHSC_00331 gene was amplified using genomic DNA from various S. aureus strains, and the resulting PCR product was sequenced. Included are isolates of N315 in which resistance to arylomycin M131 evolved. Residue changes correlated with resistance are shown in red; those not correlated with resistance are shown in blue (✱, stop codon; •, deletion).