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 genome of Staphylococcus aureus has eight structurally distinct cation/proton antiporters (CPA) that play significant roles in maintaining cytoplasmic pH and ions in extreme conditions. These antiporters enable S. aureus to persist under conditions that are favorable to the bacterium but unfavorable to animal host including humans. In this study, we report physiological roles and catalytic properties of NhaC (NhaC1, NhaC2 and NhaC3), CPA1 (CPA1-1 and CPA1-2) and CPA2 family antiporters and how these antiporters crosstalk with Mnh1, a CPA3 family antiporter, recently shown to play important roles in virulence and pH tolerance. Catalytic properties of antiporters were determined by Na+/H+ and K+/H+ antiport assays using everted membrane vesicles of a CPA-deficient E. coli KNabC host. NhaC and CPA1 candidates exhibited Na+/H+ and K+/H+ antiporter activity in the pH range between pH 7 to 9.5 but did not show significant role in halotolerance and osmotolerance alone. Interestingly, NhaC3 exhibited significant antiporter activity at alkaline pH and play major roles in pH and salt tolerance. CPA2 neither exhibited Na+or K+/H+ exchange nor showed any active role in pH and salt tolerance. Double deletion of mnhA1 with nhaC1, nhaC3, cpa1-1 or cpa1-2 respectively, made S. aureus severely sensitive at pH 7.5 under stress conditions indicating synergistic relationship of Mnh1 with these antiporters. The functional loss study of these antiporters in in-vivo mouse infection model, nhaC3 deletion showed significant loss of S. aureus virulence. Altogether, the current study indicates NhaC3 as a potential target against S. aureus virulence under extreme pH and salt conditions.ImportanceIn this study, we established catalytic properties and physiological roles of S. aureus NhaC, CPA1 and CPA2 family antiporters and their importance under salt and alkaline stress conditions. Except CPA2, all five antiporters of both families were active for Na+/H+ and K+/H+ exchange. CPA1-1 showed significant role in pH homeostasis at pH 7.5 whereas CPA1-2 and NhaCs were major contributors to halotolerance and osmotolerance at alkaline pH. The severity of growth deficit in double knockouts of mnhA1 with each of nhaC1, nhaC2, nhaC3, cpa1-1 or cpa1-2 establishes their synergistic relationship in regulating pH and salt homeostasis. Deletion of cpa1-1, cpa1-2 and nhaC1, nhaC2, and nhaC3 were assessed in mice model and NhaC3 was shown to play a major role in S. aureus virulence.

Background: The etonogestrel implant is generally considered an effective, three-year, long-acting reversible contraceptive device. Previous research, such as the landmark CHOICE study, has reported a oneyear continuation rate of 72% to 84%, however, in a real-world setting these rates may be significantly lower.Objective: To study etonogestrel implant continuation rates and factors associated with early discontinuation in a specific clinical setting.Study design: Single-center, retrospective cohort study of patients who received the etonogestrel implant between January 1, 2015, and December 31, 2017, at several practices at an academic community hospital network. Records were reviewed up to three years after implant insertion to determine continuation rates (one to three years), early discontinuation rates (≤12 months), and reasons for early discontinuation. A sample size calculation was performed to guide a subanalysis of side effects.Results: A total of 774 patients underwent etonogestrel insertion during the study period. The one-year continuation rate was lower than that of the CHOICE study (62% vs. 83%, P <0.001). A subanalysis (n=216) revealed that a majority (82%, n=177) of patients reported side effects. Side effects were more common in patients with early discontinuation compared with patients who continued use longer than one year (93% vs. 71%, P <0.001). The most common side effect, abnormal uterine bleeding, was not significantly associated with early discontinuation. A significant association (P=0.02) was found between early discontinuation and neurologic/psychiatric complaints.Conclusions: The one-year continuation rate of the etonogestrel implant in our population is significantly lower than the value reported by CHOICE. Implant side effects are common and significantly affect rates of discontinuation. Our data suggest there is an opportunity for education and counseling for individuals opting for this method of long-acting contraception.

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