The Small RNA Teg41 Regulates Expression of the Alpha Phenol-Soluble Modulins and Is Required for Virulence in Staphylococcus aureus

Overproduction of Teg41 leads to increased hemolytic activity in S. aureus. (A) Hemolytic activity of S. aureus cells in human blood is increased upon Teg41 overproduction. Human blood samples were inoculated with S. aureus containing a vector control (wild-type [WT] pMK4) or Teg41-overproducing plasmid (WT pMK4_Teg41). At the time points indicated, the degree of hemolytic activity was determined. No difference in hemolysis was observed at early time points; however, a significant difference was observed at 4 h and 5 h postinoculation. No difference in bacterial numbers was detected at any of the time points. The strain background was TCH1516. (B) Hemolytic activity of cell-free S. aureus 15-h culture supernatants is increased upon Teg41 overproduction. The strain background was TCH1516. (C) Overproduction of Teg41 results in an increase in hemolytic activity in cell-free 15-h culture supernatants in various wild-type S. aureus backgrounds. Hemolytic activity in the wild-type strain containing the vector control (WT pMK4) is set at 100%, and the relative hemolytic activity of the Teg41-overproducing strain (WT pMK4_Teg41) is indicated as a percentage. All statistical analyses were performed using Student’s t test. Statistical significance is indicated by bars and asterisks as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.005.

Teg41 does not encode a hemolytic peptide. (A) The Teg41 DNA sequence is shown with the potential ORF highlighted in pink. (B) Translation of Teg41 ORF. A 24-amino-acid peptide is potentially encoded. (C) Expression of Teg41 in S. epidermidis does not result in increased hemolytic activity. Hemolytic activity in S. aureus strain JE2 is shown as a positive control (set at 100%), and the relative hemolytic activity of S. epidermidis strains is indicated.

Teg41 overproduction leads to an increase in αPSM-dependent hemolysis. (A) Overproduction of Teg41 leads to a significant increase in hemolysis in the wild-type background (WT pMK4 compared to WT pMK4_Teg41); however, overproduction of Teg41 in an αPSM mutant (psmα pMK4_Teg41) does not result in increased hemolytic activity compared to the αPSM mutant with the vector control (psmα pMK4). The strain background was JE2. (B) Overproduction of Teg41 leads to a significant increase in hemolysis in the wild-type background (WT pMK4 compared to WT pMK4_Teg41). A similar increase in hemolytic activity is observed upon overproduction of Teg41 in an hla mutant (hla pMK4 compared to hla pMK4_Teg41). The strain background was AH1263. Hemolytic activity in the wild-type strain containing the vector control (WT pMK4) is set at 100%, and the relative hemolytic activity of all other strains is indicated as a percentage. Statistical analyses were performed using Student’s t test. Statistical significance is indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.005; ns, not significant.

Overproduction of Teg41 leads to increased αPSM levels. (A) Hemolytic activity of S. aureus culture supernatants prior to butanol extraction. No difference in hemolytic activity was observed for strains with and without the vector plasmid. Overproduction of Teg41 leads to a significant increase in hemolysis in the wild-type background (WT pMK4 compared to WT pMK4_Teg41) but not in the αPSM mutant (psmα pMK4 compared to psmα pMK4_Teg41). (B) Coomassie blue-stained SDS-PAGE analysis of butanol extracts. A band corresponding in size to the PSMs was observed in all strains. Bands of similar intensities were observed in the wild-type strain with and without the empty vector. A band with increased intensity was observed in the wild-type strain overproducing Teg41. An overall decrease in PSM levels was observed in the αPSM mutant, which did not vary upon overproduction of Teg41. (C) Hemolytic activity of butanol-extracted peptides. Butanol-extracted samples were dissolved in water and used in human blood hemolysis assays. A similar result was obtained to that shown in panel A (using culture supernatants), indicating that the hemolysin responsible was purified during the butanol extraction process. Hemolytic activity in the wild-type strain containing the vector control (WT pMK4) is set at 100%, and the relative hemolytic activity of all other strains is indicated as a percentage. Statistical analyses were performed using Student’s t test. Statistical significance is indicated as follows: *, P < 0.05; ns, not significant. The strain background was JE2.

The 3′ end of Teg41 is required for hemolytic activity. (A and B) In silico analysis predicts an interaction between the 3′ end of Teg41 (nucleotides 183 to 194; colored purple) and a region of the αPSM transcript downstream of the PSMα4 translation start site (nucleotides 376 to 388; colored red). (A) The αPSM-Teg41 locus. The regions of each transcript predicted to interact are boxed. (B) Predicted interaction between the αPSM transcript (red) and Teg41 (purple). Canonical RNA-RNA base pairs are indicated by vertical lines; noncanonical base pairing is indicated by colons. (C) Hemolytic activity of S. aureus culture supernatants is significantly reduced in the Teg41Δ3′ strain. A 10-fold reduction in hemolytic activity was observed in culture supernatants from the Teg41Δ3′ strain. Hemolysis was restored to wild-type levels by introducing full-length Teg41 on a plasmid (Teg41Δ3′ pMK4_Teg41). Hemolytic activity in the wild-type strain is set at 100%, and the relative hemolytic activity of the other strains is indicated as a percentage. Statistical analyses were performed using Student’s t test (**, P < 0.01; ns, not significant). (D) PSM levels are significantly reduced in the Teg41Δ3′ strain. Culture supernatants from panel C were butanol extracted and analyzed by SDS-PAGE. A reduction in PSM peptide levels was observed in the Teg41Δ3′ strain. Expressing full-length Teg41 from a plasmid in the Teg41Δ3′ strain (Teg41Δ3′ pMK4_Teg41) resulted in an increase in PSM production. The strain background was AH1263.

Teg41 contributes to virulence in S. aureus. Wild-type S. aureus and the Teg41Δ3′ strain were inoculated subcutaneously into groups of 12 mice. (A and B) Abscess size (A) and the number of bacteria present in abscesses (CFU/gram) (B) was determined following a 7-day infection period. A 4.2-fold reduction in abscess size and a 302-fold reduction in bacterial numbers was observed in the abscesses of mice infected with Teg41Δ3′ compared to those infected with the wild-type strain. (C) After histopathological staining, the abscesses of mice infected with the wild-type strain displayed a large amount of infiltrating inflammation-associated leukocytes (stained purple) and necrotic muscle tissue (stained pink). (D) In contrast, mice infected with Teg41Δ3′ displayed abscesses with a low population of leukocytes and intact muscle tissue with no necrotic areas. All pictures were taken at a magnification of ×20. Statistical significance was determined using Student’s t test (***, P < 0.005; ****, P < 0.001). The strain background was AH1263.

The Teg41Δ3′ strain demonstrates decreased rabbit erythrocyte lysis. Compared to the wild type, Teg41Δ3′ displays decreased lysis of rabbit erythrocytes after a 5-min incubation at 37°C. Although significant, the reduction in hemolytic activity was not as pronounced as in an hla mutant strain. Hemolytic activity in the wild-type strain (WT) is set at 100%, and the relative hemolytic activity of all other strains is indicated as a percentage. Statistical analyses were performed using Student’s t test. Statistical significance is indicated as follows: ***, P < 0.005. The strain background was AH1263.

The 3′ end of Teg41 is necessary and sufficient for S. aureus hemolytic activity. (A) Expression of truncated Teg41 (pMK4_Teg41_5) does not result in an increase in hemolytic activity in either the wild-type or Teg41Δ3′ strain compared to empty vector (pMK4) controls or strains without plasmids. (B) Overexpression of full-length Teg41 (pCN51_Teg41) or the Teg41 3′ end (pCN51_Teg41_3’) results in an increase in hemolytic activity in both wild-type S. aureus and the Teg41Δ3′ strain. In panels A and B, hemolytic activity in the wild-type strain without plasmid was set at 100%, and the relative hemolytic activity of all other strains was indicated as a percentage. Statistical analyses were performed using Student’s t test. Statistical significance is indicated as follows: **, P < 0.01; ns = not significant. The strain background was AH1263.

Analysis of Teg41 and αPSM transcripts. (A) Northern blot of Teg41 levels in wild-type S. aureus and the Teg41Δ3′ strain. RNA was extracted from cells growing for 6 h in TSB and probed with a radiolabeled Teg41 probe. (B) RT-qPCR analysis of the 5′ end of Teg41. RT-qPCR was performed using primers that anneal to the 5′ end of Teg41. Data shown are the averages of two technical replicates from three biological replicates. Expression levels are normalized using gyrB, and the wild-type sample is set at 1. Error bars represent standard deviations. Statistical analyses were performed using Student’s t test. Statistical significance is indicated as follows: **, P < 0.01. (C) Northern blot analysis of αPSM levels in wild-type S. aureus and the Teg41Δ3′ strain. RNA was extracted from cells growing for 3 h and 6 h in TSB and probed with a radiolabeled αPSM probe. (D) RT-qPCR analysis of αPSM transcript levels. RT-qPCR was performed on RNA samples identical to those used in panel C (i.e., wild-type S. aureus and the Teg41Δ3′ strain at 3 h and 6 h). Data shown are the averages of two technical replicates from three biological replicates. Expression levels are normalized using gyrB, and the wild-type sample is set at 1. Error bars represent standard deviations. Statistical analyses were performed using Student’s t test. Statistical significance is indicated as follows: *, P < 0.05. The strain background was AH1263.