Browsing by Author "Li, Dengfeng"

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Function of non-heme-binding domains of the Staphylococcus aureus IsdB protein in heme assimilation from methemoglobin
(Montana State University - Bozeman, College of Agriculture, 2014) Li, Dengfeng; Chairperson, Graduate Committee: Benfang Lei
As a hemoglobin acceptor, IsdB rapidly and efficiently acquires heme from methemoglobin (metHb) in the heme acquisition pathway of Staphylococcus aureus. The pathway of heme assimilation in S. aureus involving IsdB has been established; however, the mechanism of rapid and efficient heme assimilation of metHb heme by IsdB remains unclear. IsdB consists of five major domains: the N-terminal (ND), NEAr Transporter 1 (N1), middle (MD), heme binding NEAr Transporter 2 (N2), and C-terminal (CD) domains. The goal of this study is to elucidate the roles of these IsdB domains in the metHb-to-IsdB heme transfer reaction. Deletion of the CD region does not alter the kinetics and equilibrium of the reaction. Sequential deletions of ND and N1 of ND-N1-MD-N2 progressively reduce heme transfer rates but have no effect on the reaction equilibrium. Further deletion of MD decreases the efficiency of heme transfer from metHb to N2. The MD domain reduces heme dissociation from holo-N2 and drives the metHb/N2 reaction to the formation of holo-N2. ND-N1-MD and N2 fragments, but not ND-N1, MD, and N2, reconstitute the rapid metHb/IsdB reaction, indicating an MD/N2 interaction. Analyses of MD, N2, and MD-N2 mixture by size exclusion chromatography support an interaction between MD and N2. These results indicate that ND-N1 and MD domains critically contribute to the kinetics and equilibrium of the metHb-to-IsdB heme transfer reaction, respectively. The results also suggest that CD functions as a spacer to position IsdB in the cell wall envelope for heme relay through the cell wall. These findings support a mechanism of direct extraction of metHb heme by IsdB that involves the four structural domains of IsdB.
Non-Heme-Binding Domains and Segments of the Staphylococcus aureus IsdB Protein Critically Contribute to the Kinetics and Equilibriumof Heme Acquisition from Methemoglobin
(2014-06) Zhu, Hui; Li, Dengfeng; Liu, Mengyao; Copie, Valerie; Lei, Benfang
The hemoglobin receptor IsdB rapidly acquires heme from methemoglobin (metHb) in the heme acquisition pathway of Staphylococcus aureus. IsdB consists of N-terminal segment (NS), NEAT1 (N1), middle (MD), and heme binding NEAT2 (N2) domains, and C-terminal segment (CS). This study aims to elucidate the roles of these domains or segments in the metHb/IsdB reaction. Deletion of CS does not alter the kinetics and equilibrium of the reaction. Sequential deletions of NS and N1 in NS-N1-MD-N2 progressively reduce heme transfer rates and change the kinetic pattern from one to two phases, but have no effect on the equilibrium of the heme transfer reaction, whereas further deletion of MD reduces the percentage of transferred metHb heme. MD-N2 has higher affinity for heme than N2. MD in trans reduces rates of heme dissociation from holo-N2 and increases the percentage of metHb heme captured by N2 by 4.5 fold. NS-N1-MD and N2, but not NS-N1, MD, and N2, reconstitute the rapid metHb/IsdB reaction. NS-N1-MD-NIsdC, a fusion protein of NS-N1-MD and the NEAT domain of IsdC, slowly acquires heme from metHb by itself but together with N2 results in rapid heme loss from metHb. Thus, NS-N1 and MD domains specifically and critically contribute to the kinetics and equilibrium of the metHb/IsdB reaction, respectively. These findings support a mechanism of direct heme acquisition by IsdB in which MD enhances the affinity of N2 for heme to thermodynamically drive heme transfer from metHb to IsdB and in which NS is required for the rapid and single phase kinetics of the metHb/IsdB reaction.
Serotype M3 and M28 Group A Streptococci Have Distinct Capacities to Evade Neutrophil and TNF-α Responses and to Invade Soft Tissues
(2015-06-05) Stetzner, Zachary W.; Li, Dengfeng; Feng, Wenchao; Liu, Mengyao; Liu, Guanghui; Wiley, James A.; Lei, Benfang
The M3 Serotype of Group A Streptococcus (GAS) is one of the three most frequent serotypes associated with severe invasive GAS infections, such as necrotizing fasciitis, in the United States and other industrialized countries. The basis for this association and hypervirulence of invasive serotype M3 GAS is not fully understood. In this study, the sequenced serotype M3 strain, MGAS315, and serotype M28 strain, MGAS6180, were characterized in parallel to determine whether contemporary M3 GAS has a higher capacity to invade soft tissues than M28 GAS. In subcutaneous infection, MGAS315 invaded almost the whole skin, inhibited neutrophil recruitment and TNF-α production, and was lethal in subcutaneous infection of mice, whereas MGAS6180 did not invade skin, induced robust neutrophil infiltration and TNF-α production, and failed to kill mice. In contrast to MGAS6180, MGAS315 had covS G1370T mutation. Either replacement of the covS1370T gene with wild-type covS in MGAS315 chromosome or in trans expression of wild-type covS in MGAS315 reduced expression of CovRS-controlled virulence genes hasA, spyCEP, and sse by >10 fold. MGAS315 covSwt lost the capacity to extensively invade skin and to inhibit neutrophil recruitment and had attenuated virulence, indicating that the covS G1370T mutation critically contribute to the hypervirulence of MGAS315. Under the background of functional CovRS, MGAS315 covSwt still caused greater lesions than MGAS6180, and, consistently under the background of covS deletion, MGAS6180 ΔcovS caused smaller lesions than MGAS315 ΔcovS. Thus, contemporary invasive M3 GAS has a higher capacity to evade neutrophil and TNF-α responses and to invade soft tissue than M28 GAS and that this skin-invading capacity of M3 GAS is maximized by natural CovRS mutations. These findings enhance our understanding of the basis for the frequent association of M3 GAS with necrotizing fasciitis.
Serotype M3 and M28 Group A Streptococci Have Distinct Capacities to Evade Neutrophil and TNF-α Responses and to Invade Soft Tissues
(2015-06) Stetzner, Zachary W.; Li, Dengfeng; Feng, Wenchao; Liu, Mengyao; Liu, Guanghui; Wiley, James A.; Lei, Benfang
The M3 Serotype of Group A Streptococcus (GAS) is one of the three most frequent serotypes associated with severe invasive GAS infections, such as necrotizing fasciitis, in the United States and other industrialized countries. The basis for this association and hypervirulence of invasive serotype M3 GAS is not fully understood. In this study, the sequenced serotype M3 strain, MGAS315, and serotype M28 strain, MGAS6180, were characterized in parallel to determine whether contemporary M3 GAS has a higher capacity to invade soft tissues than M28 GAS. In subcutaneous infection, MGAS315 invaded almost the whole skin, inhibited neutrophil recruitment and TNF-α production, and was lethal in subcutaneous infection of mice, whereas MGAS6180 did not invade skin, induced robust neutrophil infiltration and TNF-α production, and failed to kill mice. In contrast to MGAS6180, MGAS315 had covS G1370T mutation. Either replacement of the covS1370T gene with wild-type covS in MGAS315 chromosome or in trans expression of wild-type covS in MGAS315 reduced expression of CovRS-controlled virulence genes hasA, spyCEP, and sse by >10 fold. MGAS315 covSwt lost the capacity to extensively invade skin and to inhibit neutrophil recruitment and had attenuated virulence, indicating that the covS G1370T mutation critically contribute to the hypervirulence of MGAS315. Under the background of functional CovRS, MGAS315 covSwt still caused greater lesions than MGAS6180, and, consistently under the background of covS deletion, MGAS6180 ΔcovS caused smaller lesions than MGAS315 ΔcovS. Thus, contemporary invasive M3 GAS has a higher capacity to evade neutrophil and TNF-α responses and to invade soft tissue than M28 GAS and that this skin-invading capacity of M3 GAS is maximized by natural CovRS mutations. These findings enhance our understanding of the basis for the frequent association of M3 GAS with necrotizing fasciitis.