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Abstract：
Spontaneous mutants with defects in the primary glucose phosphotransferase permease (manLMNO) of Streptococcus sanguinis SK36 showed enhanced fitness at low pH. Transcriptomics and metabolomics with a manL deletion mutant (SK36/manL) revealed redirection of pyruvate to production of acetate and formate, rather than lactate. These observations were consistent with measurements of decreased lactic acid accumulation and increased excretion of acetate, formate, pyruvate, and H2O2. Genes showing increased expression in SK36/manL included those encoding carbohydrate transporters, extracellular glycosidases, intracellular polysaccharide metabolism, and arginine deiminase and pathways for metabolism of acetoin, ethanolamine, ascorbate, and formate, along with genes required for membrane biosynthesis and adhesion. Streptococcus mutans UA159 persisted much better in biofilm cocultures with SK36/manL than with SK36, an effect that was further enhanced by culturing the biofilms anaerobically but dampened by adding arginine to the medium. We posited that the enhanced persistence of S. mutans with SK36/manL was in part due to excess excretion of pyruvate by the latter, as addition of pyruvate to S. mutans-S. sanguinis cocultures increased the proportions of UA159 in the biofilms. Reducing the buffer capacity or increasing the concentration of glucose benefited UA159 when cocultured with SK36, but not with SK36/manL, likely due to the altered metabolism and enhanced acid tolerance of the mutant. When manL was deleted in S. mutans or Streptococcus gordonii, the mutants presented altered fitness characteristics. Our study demonstrated that phosphotransferase system (PTS)-dependent modulation of central metabolism can profoundly affect streptococcal fitness and metabolic interactions, revealing another dimension in commensal-pathogen relationships influencing dental caries development. IMPORTANCE Dental caries is underpinned by a dysbiotic microbiome and increased acid production. As beneficial bacteria that can antagonize oral pathobionts, oral streptococci such as S. sanguinis and S. gordonii can ferment many carbohydrates, despite their relative sensitivity to low pH. We characterized the molecular basis for why mutants of glucose transporter ManLMNO of S. sanguinis showed enhanced production of hydrogen peroxide and ammonia and improved persistence under acidic conditions. A metabolic shift involving more than 300 genes required for carbohydrate transport, energy production, and envelope biogenesis was observed. Significantly, manL mutants engineered in three different oral streptococci displayed altered capacities for acid production and interspecies antagonism, highlighting the potential for targeting the glucose-PTS to modulate the pathogenicity of oral biofilms.
摘要：
血链球菌SK36的原发性葡萄糖磷酸转移酶通透酶（manLMNO）缺陷的自发突变体在低pH下显示出增强的适应性。具有manL缺失突变体（SK36/manL）的转录组学和代谢组学揭示了丙酮酸盐的重定向，以产生乙酸盐和甲酸盐。而不是乳酸。这些观察结果与乳酸积累减少和乙酸盐排泄增加的测量结果一致，甲酸盐,丙酮酸,和H2O2。在SK36/manL中显示表达增加的基因包括编码碳水化合物转运蛋白的基因，胞外糖苷酶，胞内多糖代谢，精氨酸脱亚胺酶和乙酰丙酮的代谢途径，乙醇胺,抗坏血酸，和甲酸盐，以及膜生物合成和粘附所需的基因。变形链球菌UA159在与SK36/manL的生物膜共培养物中的持久性要比与SK36的更好，这种作用通过厌氧培养生物膜而得到进一步增强，但通过向培养基中添加精氨酸而得到抑制。我们认为，变异链球菌与SK36/manL的持久性增强部分是由于后者的丙酮酸的过量排泄，作为向S.mutans-S中添加丙酮酸盐血共培养物增加了生物膜中UA159的比例。当与SK36共培养时，降低缓冲能力或增加葡萄糖浓度有益于UA159，但不与SK36/manL共培养，可能是由于突变体的代谢改变和酸耐受性增强。当变形链球菌或戈顿链球菌中的manL被删除时，突变体表现出改变的适应度特征。我们的研究表明，磷酸转移酶系统（PTS）依赖性调节中枢代谢可以深刻地影响链球菌的适应性和代谢相互作用，揭示了共生-病原体关系中影响龋齿发展的另一个维度。重要性龋齿是由生态失调的微生物组和增加的酸产生支撑的。作为可以拮抗口腔疾病的有益细菌，口腔链球菌如血链球菌和戈顿链球菌可以发酵许多碳水化合物，尽管它们对低pH值相对敏感。我们表征了为什么血链球菌的葡萄糖转运蛋白ManLMNO突变体在酸性条件下显示过氧化氢和氨的产生增强和持久性改善的分子基础。涉及碳水化合物运输所需的300多个基因的代谢转变，能源生产，并观察到包膜生物发生。重要的是,在三种不同的口腔链球菌中工程化的manL突变体显示出酸产生和种间拮抗作用的能力改变，突出了靶向葡萄糖-PTS调节口腔生物膜致病性的潜力。
