Vergauwen Bjorn, Herbert Mark, Van Beeumen Jozef J
Laboratory of Protein Biochemistry and Protein Engineering, Ghent University, K.L. Ledeganckstraat 35, 9000 Gent, Belgium.
BMC Microbiol. 2006 Jan 23;6:3. doi: 10.1186/1471-2180-6-3.
A potentially lethal flux of hydrogen peroxide (H2O2) is continuously generated during aerobic metabolism. It follows that aerobic organisms have equipped themselves with specific H2O2 dismutases and H2O2 reductases, of which catalase and the alkyl hydroperoxide reductase (AhpR) are the best-studied prokaryotic members. The sequenced Haemophilus influenzae Rd genome reveals one catalase, designated HktE, and no AhpR. However, Haemophilus influenzae type b strain Eagan (Hib), a causative agent of bacterial sepsis and meningitis in young children, disrupted in its hktE gene is not attenuated in virulence, and retains the ability to rapidly scavenge H2O2. This redundancy in H2O2-scavenging is accounted for by peroxidatic activity which specifically uses glutathione as the reducing substrate.
We show here that inside acatalasaemic H. influenzae all of the residual peroxidatic activity is catalyzed by PGdx, a hybrid peroxiredoxin-glutaredoxin glutathione-dependent peroxidase. In vitro kinetic assays on crude hktE- pgdx- H. influenzae Rd extracts revealed the presence of NAD(P)H:peroxide oxidoreductase activity, which, however, appears to be physiologically insignificant because of its low affinity for H2O2 (Km = 1.1 mM). Hydroperoxidase-deficient hktE- pgdx- H. influenzae Rd showed a slightly affected aerobic growth phenotype in rich broth, while, in chemically defined medium, growth was completely inhibited by aerobic conditions, unless the medium contained an amino acid/vitamin supplement. To study the role of PGdx in virulence and to assess the requirement of H2O2-scavenging during the course of infection, both a pgdx single mutant and a pgdx/hktE double mutant of Hib were assayed for virulence in an infant rat model. The ability of both mutant strains to cause bacteremia was unaffected.
Catalase (HktE) and a sole peroxidase (PGdx) account for the majority of scavenging of metabolically generated H2O2 in the H. influenzae cytoplasm. Growth experiments with hydroperoxidase-deficient hktE- pgdx- H. influenzae Rd suggest that the cytotoxicity inflicted by the continuous accumulation of H2O2 during aerobic growth brings about bacteriostasis rather than bacterial killing. Finally, H2O2-scavenging is not a determinant of Hib virulence in the infant rat model of infection.
在有氧代谢过程中会持续产生具有潜在致死性的过氧化氢（H₂O₂）通量。因此，需氧生物自身配备了特定的H₂O₂歧化酶和H₂O₂还原酶，其中过氧化氢酶和烷基过氧化氢还原酶（AhpR）是研究得最为深入的原核生物成员。已测序的流感嗜血杆菌Rd基因组显示有一个过氧化氢酶，命名为HktE，但没有AhpR。然而，b型流感嗜血杆菌伊根株（Hib）是幼儿细菌性败血症和脑膜炎的病原体，其hktE基因被破坏后毒力并未减弱，并且仍保留快速清除H₂O₂的能力。这种H₂O₂清除的冗余性是由特异性以谷胱甘肽作为还原底物的过氧化物酶活性所导致的。
我们在此表明，在无过氧化氢酶的流感嗜血杆菌中，所有剩余的过氧化物酶活性均由PGdx催化，PGdx是一种混合的过氧化物还原酶-谷氧还蛋白谷胱甘肽依赖性过氧化物酶。对hktE - pgdx - 流感嗜血杆菌Rd粗提物进行的体外动力学分析显示存在NAD(P)H：过氧化物氧化还原酶活性，然而，由于其对H₂O₂的亲和力较低（Km = 1.1 mM），该活性在生理上似乎并不重要。缺乏氢过氧化物酶的hktE - pgdx - 流感嗜血杆菌Rd在丰富肉汤中显示出有氧生长表型略有受影响，而在化学成分确定的培养基中，有氧条件下生长完全受到抑制，除非培养基含有氨基酸/维生素补充剂。为了研究PGdx在毒力中的作用并评估感染过程中清除H₂O₂的必要性，对Hib的pgdx单突变体和pgdx/hktE双突变体在幼鼠模型中进行了毒力测定。两种突变株引起菌血症的能力均未受影响。
过氧化氢酶（HktE）和唯一的过氧化物酶（PGdx）在流感嗜血杆菌细胞质中代谢产生的H₂O₂清除中占大部分。对缺乏氢过氧化物酶的hktE - pgdx - 流感嗜血杆菌Rd进行的生长实验表明，有氧生长过程中H₂O₂持续积累所造成的细胞毒性导致抑菌而非杀菌。最后，在幼鼠感染模型中，清除H₂O₂不是Hib毒力的决定因素。
