Comparative Transcriptomic and Functional Assessments of Linezolid-Responsive Small RNA Genes in Staphylococcus aureus

Abstract

Staphylococcus aureus contains a repertoire of at least 50 and possibly 500 small RNAs (sRNAs). The functions of most sRNAs are not understood, although some are known to respond to environmental changes, including the presence of antibiotics. Here, in an effort to better understand the roles of sRNAs in the context of antibiotic exposure, we took a clinical methicillin-resistant S. aureus (MRSA) isolate and separately deleted eight sRNAs that were significantly upregulated in response to the last-line antibiotic linezolid as revealed by transcriptome sequencing (RNA-seq) comparisons. We also deleted an additional 10 sRNAs that were either highly expressed or previously found to respond to antibiotic exposure. There were no significant changes for any of the 18 mutants in a variety of phenotypic screens, including MIC screens, growth competition assays in the presence of linezolid, biofilm formation, and resistance to whole-blood killing. These data suggest sRNA functional redundancy, because despite their high expression levels upon antibiotic exposure, individual sRNA genes do not affect readily observable bacterial phenotypes. The sRNA transcriptional changes we measured during antibiotic exposure might also reflect sRNA “indifference,” that is, a general stress response not specifically related to sRNA function. These data underscore the need for sensitive assays and new approaches to try and decipher the functions of sRNA genes in S. aureus. IMPORTANCE Bacterial small RNAs (sRNAs) are RNA molecules that can have important regulatory roles across gene expression networks. There is a growing understanding of the scope and potential breadth of impact of sRNAs on global gene expression patterns in Staphylococcus aureus, a major human pathogen. Here, transcriptome comparisons were used to examine the roles of sRNA genes with a potential role in the response of S. aureus to antibiotic exposure. Although no measurable impact on key bacterial phenotypes was observed after deleting each of 18 sRNAs identified by these comparisons, this research is significant because it underscores the subtle modes of action of these sometimes abundant molecules within the bacterium.

Transcriptome Profiling of Staphylococcus aureus Associated Extracellular Vesicles Reveals Presence of Small RNA-Cargo

“…Although there are multiple studies in Gram-positive bacteria showing altered sRNA expression due to antibiotic treatment (Felden and Cattoir, 2018 ; Gao et al, 2020 ), few studies have evaluated RNA content associated with EVs upon antibiotics exposure. Exposure to antimicrobials such as ciprofloxacin, tetracycline and melittin treatment of Acholeplasma laidlawii resulted in very variable numbers of sequence reads of predominantly 14–60 nt RNAs associated with EVs.…”

Diversity and Versatility in Small RNA-Mediated Regulation in Bacterial Pathogens

“…In S. aureus , antibiotic exposure causes the specific expression of several sRNAs in the multidrug-resistant strain JKD6008 ( Howden et al, 2013 ). Recently, the same authors identified a set of 18 sRNAs whose expressions vary under linezolid treatment ( Gao et al, 2020 ). Although, no phenotypic variations were observed after genetic deletions of these sRNAs, which questions their actual roles in antibiotic resistance/adaptation, other studies have given direct evidence of direct sRNA involvement in antibiotic resistance.…”

Evaluation of the fitness benefit conferred by RNA cis-regulators toStreptococcus pneumoniaeduring infection

“…Phenotypes associated with the loss of any one RNA regulator are often not observed, or are very subtle 23,24 . This lack may be due to the relatively small dynamic range of many such regulators 25 , redundancy of function for trans-acting regulators 26,27 , or lack of assessment in the appropriate environment. Models have suggested that benefits of trans-acting sRNA networks include more graduated responses 28 , rapid adaptation to large input signal changes 29,30 , and noise reduction 31,32 , and the role of negative feedback in biological systems seems to be accelerating return to equilibrium 33 .…”