A nonsense mutation in agrA accounts for the defect in agr expression and the avirulence of Staphylococcus aureus 8325-4 traP::kan

Abstract

TraP is a triply phosphorylated staphylococcal protein that has been hypothesized to be the mediator of a second Staphylococcus aureus quorum-sensing system, "SQS1," that controls expression of the agr system and therefore is essential for the organism's virulence. This hypothesis was based on the loss of agr expression and virulence by a traP mutant of strain 8325-4 and was supported by full complementation of both phenotypic defects by the cloned traP gene in strain NB8 (Y. Gov, I. Borovok, M. Korem, V. K. Singh, R. K. Jayaswal, B. J. Wilkinson, S. M. Rich, and N. Balaban, J. Biol. Chem. 279:14665-14672, 2004), in which the wild-type traP gene was expressed in trans in the 8325-4 traP mutant. We initiated a study of the mechanism by which TraP activates agr and found that the traP mutant strain used for this and other recently published studies has a second mutation, an adventitious stop codon in the middle of agrA, the agr response regulator. The traP mutation, once separated from the agrA defect by outcrossing, had no effect on agr expression or virulence, indicating that the agrA defect accounts fully for the lack of agr expression and for the loss of virulence attributed to the traP mutation. In addition, DNA sequencing showed that the agrA gene in strain NB8 (Gov et al., J. Biol. Chem., 2004), in contrast to that in the agr-defective 8325-4 traP mutant strain, had the wild-type sequence; further, the traP mutation in that strain, when outcrossed, also had no effect on agr expression.

FIG. 1.

Schematic illustration of hemolytic activities on SBA. Bacteria to be tested (horizontal black bars) are streaked at a right angle to RN4220 (vertical black bar) and the plate incubated overnight. β-Hemolysin forms a turbid zone of hemolysis surrounding the vertical streak of RN4220. δ-Hemolysin and β-hemolysin are synergistic, producing a zone of clear hemolysis where they intersect. Three such zones are shown. At the upper right is a strain producing only δ-hemolysin. Below that is a strain producing all three hemolysins. The α-hemolysin zone is more turbid than seen with α-hemolysin alone because of inhibition by β-hemolysin. In this case, δ-hemolysin produces a narrow clear zone surrounding the streak owing to its interaction with β-hemolysin. At the upper left is a nonhemolytic strain; next is a strain producing α-hemolysin and δ-hemolysin. The V-shaped zone is characteristic of the intersection of α-hemolysin and β-hemolysin zones, as α-hemolysin and β-hemolysin are mutually inhibitory. Within the region of intersection, β-hemolysin and δ-hemolysin interact to give a region of greater clearing than that seen with α-hemolysin alone. At the lower left is a strain producing only β-hemolysin.

FIG. 2.

Effects of traP-3 on hemolytic activity of S. aureus (experiments performed in New York). (A) Hemolytic patterns on SBA with RN4220 (black streak at top). (B) PCR products obtained with primers that would amplify either traP (0.3-kb band) or traP with the Km^r insert (1.8-kb band). Lanes: 1, 8325-4(NB) from N. Balaban; 2, 8325-4traP-3(NB), also from N. Balaban (note the faint turbid β-hemolysin zone surrounding this streak); 3, RN6734 (standard agr⁺ traP⁺, φ13 lysogen); 4, RN6734t3 (Km^r traP-3 transductant of RN6734); 5, 8325-4(RN); 6, 8325-4(RN)t3 [Km^r traP-3 transductant of 8325-4(RN)]. (C) Single-colony hemolytic patterns on SBA of the 8325-4(NB) culture provided by N. Balaban, containing at least 3 types of colonies: +, showing zones for β-hemolysin and δ-hemolysin [one of these was isolated and used in further studies and was designated 8325-4(NB)-s (α-hemolysin is produced but is difficult to identify in such single colonies)]; n, producing only β-hemolysin; i, possible intermediate type.

FIG. 3.

Effect of traP-3 on protease production by S. aureus (experiments performed in Stockholm). (A) PCR analysis of traP with (lanes 1 to 4) and without (lane 5) the kan insertion. In lane 4 is 8325-4traP-3(NB) (N. Balaban's traP mutant), and lanes 1 to 3 represent Km^r transductants of 8325-4(RN) with 8325-4traP-3(NB) as the donor. Lane M, size marker; lane C, negative control without bacterial DNA. (B) Protease indicator plate (casein agar) with stabs of the strains shown in panel A. RN6911 is an agr-null strain; note that strain 8325-4traP-3(NB) (N. Balaban's traP-3 mutant) (lane 4), like RN6911, shows no protease activity, whereas the transductants (lanes 1 to 3) are fully active, as is the 8325-4(RN) recipient (lane 5).

FIG. 4.

Effect of traP-3 on exoprotein profiles and agr-RNAIII production. (A) Six-hour culture supernatants were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis according to the method of Laemmli (9), stained with Coomassie brilliant blue, and photographed. (B) PCR analysis of chromosomal DNA as in Fig. 3. Lanes: 1, 8325-4(NB); 2, 8325-4traP-3(NB); 3, 8325-4(RN); 4, 8325-4(RN)t3 [Km^r transductant of 8325-4(RN)]; 5, RN6734; 6, RN6734t3 (Km^r transductant of RN6734). (C) Northern blot hybridization analysis of RNAIII. Lanes 7 and 8, 4-h culture samples; lanes 9 and 10, 6-h samples. The samples in lanes 7 and 9 are from an 8325-4(RN) culture; those in lanes 8 and 10 are from an 8325-4(RN)t3 culture. Results shown in lanes 1 to 6 are from experiments performed in New York; those in lanes 7 to 10 are from experiments performed in Stockholm.

FIG. 5.

Complementation of 8325-4traP-3(NB) by cloned agrA but not by cloned traP. (A and B) Hemolytic patterns (A) and PCR analysis (B) as in Fig. 2. (C) Northern blot hybridization patterns using 16S RNA and agr-RNAIII probes as indicated. The same 6-h culture samples used for PCR were also used for whole-cell RNA extraction. RNA samples were separated on agarose and Northern blotted with ³²P-labeled oligonucleotides specific for 16S RNA and agr-RNAIII, respectively. Blots were developed with a phosphorimager. Lanes: 1, 8325-4traP-3(NB); 2, 8325-4traP-3(NB) containing cloned agrA; 3, 8325-4traP-3(NB) containing cloned traP; 4, 8325-4(RN); 5, NB8; 6, 8325-4(RN)t4 [Km^r transductant of 8325-4(RN) with NB8 as the donor].

FIG. 6.

Sequencing of agrA. The electropherogram of the sequencing reaction for agrA in strain 8325-4traP-3(NB) is shown at the bottom, with the deduced nucleotide and amino acid sequences below. At the top is the agrA sequence for strain NB8 in comparison to the 8325-4(RN) agrA sequence from GenBank.

FIG. 7.

Effects of traP on virulence in the murine subcutaneous abscess model. See text for details. Odd-numbered mice were infected with traP⁺ bacteria, and even-numbered mice were infected with traP-3. Strains: 1 and 2, 8325-4(NB)-s; 3 and 4, RN6734; 5 and 6, RN7206.