Department of Microbiology, Immunology and Molecular Genetics, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.
Department of Developmental Dentistry, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.
Anaerobe. 2020 Aug;64:102231. doi: 10.1016/j.anaerobe.2020.102231. Epub 2020 Jun 27.
Treponema denticola is a spirochete that is involved in causing periodontal diseases. This bacterium can produce HS from thiol compounds found in the gingival crevicular fluid. Determining how HS is made by oral bacteria is important since this molecule is present at high levels in periodontally-diseased pockets and the biological effects of HS can explain some of the pathologies seen in periodontitis. Thus, it is of interest to identify the enzyme, or enzymes, involved in the synthesis of HS by T. denticola. We, and others, have previously identified and characterized a T. denticola cystalysin, called HlyA, which hydrolyzes cysteine into HS (and pyruvate and ammonia). However, there have been no studies to show that HlyA is, or is not, the only pathway that T. denticola can use to make HS. To address this question, allelic replacement mutagenesis was used to make a deletion mutant (ΔhlyA) in the gene encoding HlyA. The mutant produces the same amount of HS from cysteine as do wild type spirochetes, indicating that T. denticola has at least one other enzyme that can generate HS from cysteine. To identify candidates for this other enzyme, a BLASTp search of T. denticola strain 33520 was done. There was one gene that encoded an HlyA homolog so we named it HlyB. Recombinant His-tagged HlyB was expressed in E. coli and partially purified. This enzyme was able to make HS from cysteine in vitro. To test the role of HlyB in vivo, an HlyB deletion mutant (ΔhlyB) was constructed in T. denticola. This mutant still made normal levels of HS from cysteine, but a strain mutated in both hly genes (ΔhlyA ΔhlyB) synthesizes significantly less HS from cysteine. We conclude that the HlyA and HlyB enzymes perform redundant functions in vivo and are the major contributors to HS production in T. denticola. However, at least one other enzyme can still convert cysteine to HS in the ΔhlyA ΔhlyB mutant. An in silico analysis that identifies candidate genes for this other enzyme is presented.
齿密螺旋体是一种参与引起牙周病的螺旋体。这种细菌可以从牙龈沟液中的硫醇化合物中产生 HS。确定口腔细菌如何产生 HS 很重要，因为这种分子在牙周病袋中含量很高，HS 的生物学效应可以解释牙周炎中观察到的一些病理学变化。因此，确定齿密螺旋体合成 HS 的酶或酶是很有意义的。我们和其他人之前已经鉴定并表征了一种齿密螺旋体晶体溶菌素，称为 HlyA，它将半胱氨酸水解成 HS（以及丙酮酸和氨）。然而，还没有研究表明 HlyA 是或不是齿密螺旋体产生 HS 的唯一途径。为了解决这个问题，我们使用等位基因替换诱变技术在编码 HlyA 的基因中制造了一个缺失突变体（ΔhlyA）。突变体从半胱氨酸产生的 HS 量与野生型螺旋体相同，这表明齿密螺旋体至少还有一种其他酶可以从半胱氨酸生成 HS。为了确定这种其他酶的候选物，对齿密螺旋体菌株 33520 进行了 BLASTp 搜索。有一个基因编码 HlyA 同源物，因此我们将其命名为 HlyB。重组 His 标记的 HlyB 在大肠杆菌中表达并部分纯化。该酶能够在体外从半胱氨酸生成 HS。为了在体内测试 HlyB 的作用，在齿密螺旋体中构建了 HlyB 缺失突变体（ΔhlyB）。该突变体仍能从半胱氨酸中产生正常水平的 HS，但在 hly 两个基因都突变的菌株（ΔhlyA ΔhlyB）中，从半胱氨酸合成的 HS 明显减少。我们得出结论，HlyA 和 HlyB 酶在体内具有冗余功能，是齿密螺旋体 HS 产生的主要贡献者。然而，在 ΔhlyA ΔhlyB 突变体中，至少还有一种其他酶仍能将半胱氨酸转化为 HS。提出了一种用于鉴定该其他酶的候选基因的计算机分析。
