Department of Microbiology and Immunology, University of Mississippi Medical Center, Jackson, Mississippi, USA.
Department of Biology, Georgia State University, Atlanta, Georgia, USA.
mSphere. 2020 Dec 9;5(6):e01117-20. doi: 10.1128/mSphere.01117-20.
and other streptococci produce a greenish halo on blood agar plates referred to as alpha-hemolysis. This phenotype is utilized by clinical microbiology laboratories to report culture findings of alpha-hemolytic streptococci, including , and other bacteria. The alpha-hemolysis halo on blood agar plates has been related to the hemolytic activity of pneumococcal pneumolysin (Ply) or, to a lesser extent, to lysis of erythrocytes by -produced hydrogen peroxide. We investigated the molecular basis of the alpha-hemolysis halo produced by Wild-type strains TIGR4, D39, R6, and EF3030 and isogenic derivative Δ mutants produced similar alpha-hemolytic halos on blood agar plates, while cultures of hydrogen peroxide knockout Δ Δ mutants lacked this characteristic halo. Moreover, in the presence of catalase, the alpha-hemolysis halo was absent in cultures of the wild-type (wt) and Δ mutant strains. Spectroscopic studies demonstrated that culture supernatants of TIGR4 released hemoglobin-bound heme (heme-hemoglobin) from erythrocytes and oxidized oxy-hemoglobin to met-hemoglobin within 30 min of incubation. As expected, given Ply hemolytic activity and that hydrogen peroxide contributes to the release of Ply, TIGR4Δ and Δ Δ isogenic mutants had significantly decreased release of heme-hemoglobin from erythrocytes. However, TIGR4Δ that produces hydrogen peroxide oxidized oxy-hemoglobin to met-hemoglobin, whereas TIGR4Δ Δ failed to produce oxidation of oxy-hemoglobin. Studies conducted with all other wt strains and isogenic mutants resulted in similar findings. We demonstrated that the so-called alpha-hemolysis halo is caused by the oxidation of oxy-hemoglobin (Fe) to a non-oxygen-binding met-hemoglobin (Fe) by -produced hydrogen peroxide. There is a misconception that alpha-hemolysis observed on blood agar plate cultures of and other alpha-hemolytic streptococci is produced by a hemolysin or, alternatively, by lysis of erythrocytes caused by hydrogen peroxide. We noticed in the course of our investigations that wild-type strains and hemolysin (e.g., pneumolysin) knockout mutants produced the alpha-hemolytic halo on blood agar plates. In contrast, hydrogen peroxide-defective mutants prepared in four different strains lacked the characteristic alpha-hemolysis halo. We also demonstrated that wild-type strains and pneumolysin mutants oxidized oxy-hemoglobin to met-hemoglobin. Hydrogen peroxide knockout mutants, however, failed to oxidize oxy-hemoglobin. Therefore, the greenish halo formed on cultures of and other so-called alpha-hemolytic streptococci is caused by the oxidation of oxy-hemoglobin produced by hydrogen peroxide. Oxidation of oxy-hemoglobin to the nonbinding oxygen form, met-hemoglobin, might occur in the lungs during pneumococcal pneumonia.
和其他链球菌在血琼脂平板上产生一个被称为α-溶血的绿色晕环。这种表型被临床微生物学实验室用来报告α-溶血链球菌的培养结果，包括、和其他细菌。血琼脂平板上的α-溶血晕环与肺炎球菌肺炎溶素（Ply）的溶血活性有关，或者与产生的过氧化氢对红细胞的裂解作用有关。我们研究了野生型菌株 TIGR4、D39、R6 和 EF3030 产生的α-溶血晕环的分子基础，以及同源缺失突变体 Δ产生的类似α-溶血晕环，而过氧化氢缺失突变体 Δ Δ 的培养物缺乏这种特征性的晕环。此外，在过氧化氢酶存在的情况下，野生型（wt）和 Δ 突变株的培养物中不存在α-溶血晕环。光谱研究表明，TIGR4 的培养上清液从红细胞中释放出与血红蛋白结合的血红素（血红素-血红蛋白），并在孵育 30 分钟内将氧合血红蛋白氧化为高铁血红蛋白。正如预期的那样，鉴于 Ply 的溶血活性以及过氧化氢有助于 Ply 的释放，TIGR4Δ 和 Δ Δ 同源缺失突变体从红细胞中释放的血红素-血红蛋白明显减少。然而，产生过氧化氢的 TIGR4Δ 将氧合血红蛋白氧化为高铁血红蛋白，而 TIGR4Δ Δ 则未能产生氧合血红蛋白的氧化。对所有其他 wt 菌株和同源缺失突变体进行的研究得出了类似的结果。我们证明，所谓的α-溶血晕环是由 -产生的过氧化氢将氧合血红蛋白（Fe）氧化为非氧结合的高铁血红蛋白（Fe）引起的。有一种误解认为，在血琼脂平板培养的 和其他α-溶血链球菌中观察到的α-溶血是由一种溶血素或由过氧化氢引起的红细胞裂解引起的。在我们的研究过程中，我们注意到野生型 菌株和溶血素（如肺炎球菌溶血素）缺失突变体在血琼脂平板上产生α-溶血晕环。相比之下，在四个不同菌株中制备的过氧化氢缺陷突变体缺乏特征性的α-溶血晕环。我们还证明，野生型菌株和肺炎球菌溶血素突变体将氧合血红蛋白氧化为高铁血红蛋白。然而，过氧化氢缺失突变体未能氧化氧合血红蛋白。因此，在 和其他所谓的α-溶血链球菌的培养物上形成的绿色晕环是由过氧化氢产生的氧合血红蛋白的氧化引起的。在肺炎球菌肺炎期间，可能会发生氧合血红蛋白向非结合氧形式高铁血红蛋白的氧化。
