Proteolytic Regulation of Toxin-Antitoxin Systems by ClpPC in Staphylococcus aureus

Abstract

Bacterial toxin-antitoxin (TA) systems typically consist of a small, labile antitoxin that inactivates a specific longer-lived toxin. In Escherichia coli, such antitoxins are proteolytically regulated by the ATP-dependent proteases Lon and ClpP. Under normal conditions, antitoxin synthesis is sufficient to replace this loss from proteolysis, and the bacterium remains protected from the toxin. However, if TA production is interrupted, antitoxin levels decrease, and the cognate toxin is free to inhibit the specific cellular component, such as mRNA, DnaB, or gyrase. To date, antitoxin degradation has been studied only in E. coli, so it remains unclear whether similar mechanisms of regulation exist in other organisms. To address this, we followed antitoxin levels over time for the three known TA systems of the major human pathogen Staphylococcus aureus, mazEF, axe1-txe1, and axe2-txe2. We observed that the antitoxins of these systems, MazE sa , Axe1, and Axe2, respectively, were all degraded rapidly (half-life [t 1/2 ], ϳ18 min) at rates notably higher than those of their E. coli counterparts, such as MazE (t 1/2 , ϳ30 to 60 min). Furthermore, when S. aureus strains deficient for various proteolytic systems were examined for changes in the half-lives of these antitoxins, only strains with clpC or clpP deletions showed increased stability of the molecules. From these studies, we concluded that ClpPC serves as the functional unit for the degradation of all known antitoxins in S. aureus.Staphylococcus aureus is a versatile human pathogen responsible for an increasing number of hospital-and community-acquired infections (33, 41) ranging from superficial skin lesions to life-threatening sepsis, endocarditis, and toxic shock (29). S. aureus' capacity to cause illness is enhanced by its robust stress response, which allows it to endure adverse conditions, such as heat, antibiotics, and nutritional deprivation. This is mediated in part by transcriptional regulators, like CtsR (11), CodY (31), and the alternative sigma factor B (24), that allow the bacteria to rapidly adjust to challenging environments.The roles of chromosomal toxin-antitoxin (TA) modules in environmental and antibiotic stress response have been documented for a variety of organisms, especially Escherichia coli, but only recently have they been investigated in S. aureus (12,18,43). These systems typically consist of a pair of cotranscribed stress-inducible genes (19) that encode a stable toxin and a more labile antitoxin. Depletion of the antitoxin allows activation of its cognate toxin, which is then free to interfere with a specific cellular target, such as mRNA, DNA gyrase, or DNA helicase. Depending on the species and the TA system, this activation results in a variety of phenotypes, but those related to growth, stress response, starvation, and persistence are often seen (12,19,30). For example, Streptococcus mutans devoid of its TA systems is more susceptible to changes in nutrient availability and pH than its counterpart wild-type strains (26). Further...

The ClpXP Protease Is Responsible for the Degradation of the Epsilon Antidote to the Zeta Toxin of the Streptococcal pSM19035 Plasmid

“…In Gram-positive bacteria, the chaperones ClpX, ClpC, ClpE, and ClpY interact with the ClpP protease core. In a previous paper, the ClpC chaperone was shown to be essential for the ClpP-mediated degradation of the MazEsa, Axe1, and Axe2 antitoxins in S. aureus, a Gram-positive human pathogen (16). Increased stability of these antitoxins was observed only in S. aureus strains that were deficient for clpC or clpP.…”

“…One reason for this could be a different proteolytic regulation of this TA system. It was demonstrated for the mazEF system that homologous antidotes were degraded by various proteases in the host strains (12,16), which were also dependent on environmental conditions (13).…”

“…In contrast to the E. coli antitoxins, knowledge of the proteolysis of antitoxins from other bacterial species is scarce. Until now, only Donegan et al (16) had described this process in Gram-positive bacteria. These authors demonstrated that the MazEsa, Axe1, and Axe2 antitoxins from the three known TA systems in Staphylococcus aureus are rapidly degraded in vivo by the ClpCP protease.…”

Regulation of Host Hemoglobin Binding by the Staphylococcus aureus Clp Proteolytic System

“…S. aureus expresses three intracellular, ATP-dependent proteases, known as ClpP, FtsH, and HslV (ClpQ), that are important for managing protein levels and directing stress responses. The ClpP protease is believed to be the principal degradation machinery in S. aureus, while FtsH and HslV play less critical roles in the bacterial stress response (16)(17)(18)(19). Experiments using strains inactivated for clpP, ftsH, or hslV demonstrated that none of these proteases are responsible for ensuring the heme-dependent stability of IsdG (15).…”

A regulatory role for Staphylococcus aureus toxin–antitoxin system PemIKSa

“…Under favourable growth conditions genes of most TA systems are coexpressed and the deleterious activity of the toxin is prevented by the antitoxin 7 . However, various stress stimuli alter the toxin/antitoxin balance, usually by inducing increased proteolytic degradation of the labile antitoxin [8][9][10] . The activated toxin induces a dormant state or other adaptations that enable the bacteria to survive environmentally unfavourable conditions [11][12][13] .…”