scholarly journals The alkaliphilic side of Staphylococcus aureus

2019 ◽  
Author(s):  
Manisha Vaish ◽  
Amyeo Jereen ◽  
Amall Ali ◽  
Terry Ann Krulwich
Keyword(s):  
Staphylococcus Aureus ◽  
Salt Tolerance ◽  
Significant Role ◽  
Catalytic Properties ◽  
Stress Conditions ◽  
Infection Model ◽  
Alkaline Stress ◽  
Alkaline Ph ◽  
Ph Homeostasis ◽  
Synergistic Relationship

AbstractThe 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.

scholarly journals The Lysine 299 Residue Endows the Multisubunit Mrp1 Antiporter with Dominant Roles in Na+Resistance and pH Homeostasis inCorynebacterium glutamicum

2018 ◽  
Vol 84 (10) ◽  
Author(s):  
Ning Xu ◽  
Yingying Zheng ◽  
Xiaochen Wang ◽  
Terry A. Krulwich ◽  
Yanhe Ma ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Expression Patterns ◽  
Alkaline Stress ◽  
Alkaline Ph ◽  
Ph Homeostasis ◽  
Conserved Residues ◽  
Content Type ◽  
Ion Translocation ◽  
Alkaline Tolerance ◽  
Alkaline Conditions

ABSTRACTCorynebacterium glutamicumis generally regarded as a moderately salt- and alkali-tolerant industrial organism. However, relatively little is known about the molecular mechanisms underlying these specific adaptations. Here, we found that the Mrp1 antiporter played crucial roles in conferring both environmental Na+resistance and alkali tolerance whereas the Mrp2 antiporter was necessary in coping with high-KCl stress at alkaline pH. Furthermore, the Δmrp1Δmrp2double mutant showed the most-severe growth retardation and failed to grow under high-salt or alkaline conditions. Consistent with growth properties, the Na+/H+antiporters ofC. glutamicumwere differentially expressed in response to specific salt or alkaline stress, and an alkaline stimulus particularly induced transcript levels of the Mrp-type antiporters. When the major Mrp1 antiporter was overwhelmed,C. glutamicummight employ alternative coordinate strategies to regulate antiport activities. Site-directed mutagenesis demonstrated that several conserved residues were required for optimal Na+resistance, such as Mrp1A K299, Mrp1C I76, Mrp1A H230, and Mrp1D E136. Moreover, the chromosomal replacement of lysine 299 in the Mrp1A subunit resulted in a higher intracellular Na+level and a more alkaline intracellular pH value, thereby causing a remarkable growth attenuation. Homology modeling of the Mrp1 subcomplex suggested two possible ion translocation pathways, and lysine 299 might exert its effect by affecting the stability and flexibility of the cytoplasm-facing channel in the Mrp1A subunit. Overall, these findings will provide new clues to the understanding of salt-alkali adaptation duringC. glutamicumstress acclimatization.IMPORTANCEThe capacity to adapt to harsh environments is crucial for bacterial survival and product yields, including industrially usefulCorynebacterium glutamicum. AlthoughC. glutamicumexhibits a marked resistance to salt-alkaline stress, the possible mechanism for these adaptations is still unclear. Here, we present the physiological functions and expression patterns ofC. glutamicumputative Na+/H+antiporters and conserved residues of Mrp1 subunits, which respond to different salt and alkaline stresses. We found that the Mrp-type antiporters, particularly the Mrp1 antiporter, played a predominant role in maintaining intracellular nontoxic Na+levels and alkaline pH homeostasis. Loss of the major Mrp1 antiporter had a profound effect on gene expression of other antiporters under salt or alkaline conditions. The lysine 299 residue may play its essential roles in conferring salt and alkaline tolerance by affecting the ion translocation channel of the Mrp1A subunit. These findings will contribute to a better understanding of Na+/H+antiporters in sodium antiport and pH regulation.

scholarly journals Catalytic Properties and Specificity of the Extracellular Nuclease of Staphylococcus aureus

1967 ◽  
Vol 242 (7) ◽  
pp. 1541-1547 ◽  
Author(s):  
Pedro Cuatrecasas ◽  
Sara Fuchs ◽  
Christian B. Anfinsen
Keyword(s):  
Staphylococcus Aureus ◽  
Catalytic Properties ◽  
Extracellular Nuclease

scholarly journals Decreased Vancomycin Susceptibility in Staphylococcus aureus Caused by IS256Tempering of WalKR Expression

2013 ◽  
Vol 57 (7) ◽  
pp. 3240-3249 ◽  
Author(s):  
Christopher R. E. McEvoy ◽  
Brian Tsuji ◽  
Wei Gao ◽  
Torsten Seemann ◽  
Jessica L. Porter ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Fluorescent Protein ◽  
Site Directed Mutagenesis ◽  
Infection Model ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Phenotype Analysis ◽  
Dosing Regimens ◽  
Mrsa Strains ◽  
Green Fluorescent

ABSTRACTVancomycin-intermediateStaphylococcus aureus(VISA) strains often arise by mutations in the essential two-component regulatorwalKR; however their impact onwalKRfunction has not been definitively established. Here, we investigated 10 MRSA strains recovered serially after exposure of vancomycin-susceptibleS. aureus(VSSA) JKD6009 to simulated human vancomycin dosing regimens (500 mg to 4,000 mg every 12 h) using a 10-day hollow fiber infection model. After continued exposure to the vancomycin regimens, two isolates displayed reduced susceptibility to both vancomycin and daptomycin, developing independent IS256insertions in thewalKR5′ untranslated region (5′ UTR). Quantitative reverse transcription-PCR (RT-PCR) revealed a 50% reduction inwalKRgene expression in the IS256mutants compared to the VSSA parent. Green fluorescent protein (GFP) reporter analysis, promoter mapping, and site-directed mutagenesis confirmed these findings and showed that the IS256insertions had replaced two SigA-likewalKRpromoters with weaker, hybrid promoters. Removal of IS256reverted the phenotype to VSSA, showing that reduced expression of WalKR did induce the VISA phenotype. Analysis of selected WalKR-regulated autolysins revealed upregulation ofssaAbut no change in expression ofsakandsceDin both IS256mutants. Whole-genome sequencing of the two mutants revealed an additional IS256insertion withinagrCfor one mutant, and we confirmed that this mutation abolishedagrfunction. These data provide the first substantial analysis ofwalKRpromoter function and show that prolonged vancomycin exposure can result in VISA through an IS256-mediated reduction inwalKRexpression; however, the mechanisms by which this occurs remain to be determined.

scholarly journals Physiological and Biochemical Responses of Jerusalem Artichoke Seedlings to Mixed Salt-Alkali Stress Conditions

2015 ◽  
Vol 43 (2) ◽  
pp. 473-478 ◽  
Author(s):  
Shuai SHAO ◽  
Mingming QI ◽  
Shuang TAO ◽  
Jixiang LIN ◽  
Yingnan WANG ◽  
...  
Keyword(s):  
Seedling Growth ◽  
Jerusalem Artichoke ◽  
Soil Salinization ◽  
Stress Conditions ◽  
Energy Crop ◽  
Alkaline Stress ◽  
Alkali Stress ◽  
Mixed Salt ◽  
Effective Utilization ◽  
Alkaline Conditions

Soil salinization and alkalization frequently co-occur in the grassland, but little information exists concerning the mixed effects of salt-alkaline stress on plant. Jerusalem artichoke is an economically and ecologically important energy crop and also considered as a salt-tolerant species. In this study, we investigated the effects of 12 mixed salt-alkaline conditions on the seedling growth and responses of Jerusalem artichoke to such conditions. The results showed that the seedling growth decreased with the increasing salinity and pH, and the destructive effects were more markedly under the interactions of highest salinity and pH. The Na+, Mg2+ and Ca2+ concentrations were all increased with the increasing salinity and pH, but the K+ kept stable. The Cl- concentration increased when the treatment without alkali salts, and the NO3– and H2PO4- concentrations were decreased with the increasing salinity. Jerusalem artichoke seedlings enhanced organic acids and proline to supply the shortage of inorganic anions and cope with osmotic stress from the high Na+ concentration. Above results show that the toxicity effects of the interactions of salt stress and alkali stress on plant is much greater than that only salt or alkali stress. A better understanding of the seedlings of Jerusalem artichoke under mixed salt-alkali stress conditions should facilitate the effective utilization of this species under such complex environment in Northeast China.

scholarly journals Role of BrnQ1 and BrnQ2 in Branched-Chain Amino Acid Transport and Virulence in Staphylococcus aureus

2014 ◽  
Vol 83 (3) ◽  
pp. 1019-1029 ◽  
Author(s):  
Julienne C. Kaiser ◽  
Sameha Omer ◽  
Jessica R. Sheldon ◽  
Ian Welch ◽  
David E. Heinrichs
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Gene ◽  
Defined Medium ◽  
Infection Model ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Branched Chain Amino Acid ◽  
Branched Chain

The branched-chain amino acids (BCAAs; Ile, Leu, and Val) not only are important nutrients for the growth ofStaphylococcus aureusbut also are corepressors for CodY, which regulates virulence gene expression, implicating BCAAs as an important link between the metabolic state of the cell and virulence. BCAAs are either synthesized intracellularly or acquired from the environment.S. aureusencodes three putative BCAA transporters, designated BrnQ1, BrnQ2, and BrnQ3; their functions have not yet been formally tested. In this study, we mutated all threebrnQparalogs so as to characterize their substrate specificities and their roles in growthin vitroandin vivo. We demonstrated that in the community-associated, methicillin-resistantS. aureus(CA-MRSA) strain USA300, BrnQ1 is involved in uptake of all three BCAAs, BrnQ2 transports Ile, and BrnQ3 does not have a significant role in BCAA transport under the conditions tested. Of the three, only BrnQ1 is essential for USA300 to grow in a chemically defined medium that is limited for Leu or Val. Interestingly, we observed that abrnQ2mutant grew better than USA300 in media limited for Leu and Val, owing to the fact that this mutation leads to overexpression ofbrnQ1. In a murine infection model, thebrnQ1mutant was attenuated, but in contrast,brnQ2mutants had significantly increased virulence compared to that of USA300, a phenotype we suggest is at least partially linked to enhancedin vivoscavenging of Leu and Val through BrnQ1. These data uncover a hitherto-undiscovered connection between nutrient acquisition and virulence in CA-MRSA.

scholarly journals Galleria mellonella Larvae as an Infection Model to Investigate sRNA-Mediated Pathogenesis in Staphylococcus aureus

2021 ◽  
Vol 11 ◽  
Author(s):  
Guillaume Ménard ◽  
Astrid Rouillon ◽  
Gevorg Ghukasyan ◽  
Mathieu Emily ◽  
Brice Felden ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Galleria Mellonella ◽  
Inflammatory Responses ◽  
Expression Profiles ◽  
Bacterial Pathogens ◽  
Infection Model ◽  
Animal Testing ◽  
Easy Method ◽  
Larval Infection ◽  
Over Time

Small regulatory RNAs (sRNAs) are key players in bacterial regulatory networks. Monitoring their expression inside living colonized or infected organisms is essential for identifying sRNA functions, but few studies have looked at sRNA expression during host infection with bacterial pathogens. Insufficient in vivo studies monitoring sRNA expression attest to the difficulties in collecting such data, we therefore developed a non-mammalian infection model using larval Galleria mellonella to analyze the roles of Staphylococcus aureus sRNAs during larval infection and to quickly determine possible sRNA involvement in staphylococcal virulence before proceeding to more complicated animal testing. We began by using the model to test infected larvae for immunohistochemical evidence of infection as well as host inflammatory responses over time. To monitor sRNA expression during infection, total RNAs were extracted from the larvae and invading bacteria at different time points. The expression profiles of the tested sRNAs were distinct and they fluctuated over time, with expression of both sprD and sprC increased during infection and associated with mortality, while rnaIII expression remained barely detectable over time. A strong correlation was observed between sprD expression and the mortality. To confirm these results, we used sRNA-knockout mutants to investigate sRNA involvement in Staphylococcus aureus pathogenesis, finding that the decrease in death rates is delayed when either sprD or sprC was lacking. These results demonstrate the relevance of this G. mellonella model for investigating the role of sRNAs as transcriptional regulators involved in staphylococcal virulence. This insect model provides a fast and easy method for monitoring sRNA (and mRNA) participation in S. aureus pathogenesis, and can also be used for other human bacterial pathogens.

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