Roles of Staphylococcus aureus Mnh1 and Mnh2 Antiporters in Salt Tolerance, Alkali Tolerance, and Pathogenesis

Abstract

Staphylococcus aureus has three types of cation/proton antiporters. The type 3 family includes two multisubunit Na+/H+ (Mnh) antiporters, Mnh1 and Mnh2. These antiporters are clusters of seven hydrophobic membrane-bound protein subunits. Mnh antiporters play important roles in maintaining cytoplasmic pH in prokaryotes, enabling their survival under extreme environmental stress. In this study, we investigated the physiological roles and catalytic properties of Mnh1 and Mnh2 in S. aureus. Both Mnh1 and Mnh2 were cloned separately into a pGEM3Z+ vector in the antiporter-deficient KNabc Escherichia coli strain. The catalytic properties of the antiporters were measured in everted (inside out) vesicles. The Mnh1 antiporter exhibited a significant exchange of Na+/H+ cations at pH 7.5. Mnh2 showed a significant exchange of both Na+/H+ and K+/H+ cations, especially at pH 8.5. Under elevated salt conditions, deletion of the mnhA1 gene resulted in a significant reduction in the growth rate of S. aureus in the range of pH 7.5 to 9. Deletion of mnhA2 had similar effects but mainly in the range of pH 8.5 to 9.5. Double deletion of mnhA1 and mnhA2 led to a severe reduction in the S. aureus growth rate mainly at pH values above 8.5. The effects of functional losses of both antiporters in S. aureus were also assessed via their support of virulence in a mouse in vivo infection model. Deletion of the mnhA1 gene led to a major loss of S. aureus virulence in mice, while deletion of mnh2 led to no change in virulence.IMPORTANCE This study focuses on the catalytic properties and physiological roles of Mnh1 and Mnh2 cation/proton antiporters in S. aureus and their contributions under different stress conditions. The Mnh1 antiporter was found to have catalytic activity for Na+/H+ antiport, and it plays a significant role in maintaining halotolerance at pH 7.5 while the Mnh2 antiporter has catalytic antiporter activities for Na+/H+ and K+/H+ that have roles in both osmotolerance and halotolerance in S. aureus. Study of S. aureus with a single deletion of either mnhA1 or mnhA2 was assessed in an infection model of mice. The result shows that mnhA1, but not mnhA2, plays a major role in S. aureus virulence.

The alkaliphilic side ofStaphylococcus aureus

“…In the previous study (18) it was reported that Mnh1 significantly contributes in pH homeostasis at pH 7.5 and Mnh2 was active at higher pH range. However, it was still unclear that how rest of the antiporters in cohort regulates pH tolerance and salt tolerance in extreme conditions.…”

“…Strains with Δcpa1-1, Δcpa1-2, ΔnhaC1 and ΔnhaC2 deletions exhibited virulence similar to Newman wild type, whereas strain with ΔnhaC3 deletion showed marked attenuation of virulence (Fig 6A). To confirm the virulence defect observed in ΔnhaC3 strain is due to lack of functional nhaC3 , complemented strain of wild type nhaC3 in ΔnhaC3 mutant strain was constructed by using pOS1 plasmid as mentioned in previous studies (18, 19). The virulence defect was reversed with the restoration of wild type nhaC3 in ΔnhaC3 mutant strain (Fig 6B).…”

“…Growth experiments were done according to our previous study (18). Briefly, glycerol stocks of S. aureus wild type and mutant strain were inoculated in LB0 medium, pH 7.5, and grown for 16 h at 37°C with shaking at 225 rpm prior to growth experiments.…”

“…Recently, we found that a multi-drug resistant pathogen S. aureus seven-subunit cation/proton antiporter Mnh1 of CPA3 family plays important role in diminishing sodium toxicity and maintaining pH homeostasis at neutral pH. We found Mnh1 to be essential for virulence and pathogenesis of S. aureus in the in-vivo mice infection model, and therefore it could be a potential therapeutic target (18).…”

High‐throughput transposon sequencing highlights the cell wall as an important barrier for osmotic stress in methicillin resistant Staphylococcus aureus and underlines a tailored response to different osmotic stressors

“…Inactivating or reducing the expression of such genes is expected to help S. aureus survive under salt stress. Among these dispensable genes were genes coding for the sodium antiporter Mnh2, which has previously been shown to transport Na + /H + and K + /H + in high pH medium and to play an important role in cytoplasmic pH maintenance (Vaish et al, ). As noted earlier, a large number of genes involved in respiration, such as the cta/qox/men/hem genes as well as genes involved indirectly in respiration through quinone synthesis (shikimate pathway, aro operon) were also identified as dispensable during salt stress.…”