Amino Acid Substitutions of MagA in <em>Klebsiella pneumoniae</em> Affect the Biosynthesis of the Capsular Polysaccharide

Mucoviscosity-associated gene A (magA) of Klebsiella pneumoniae contributes to K1 capsular polysaccharide (CPS) biosynthesis. Based on sequence homology and gene alignment, the magA gene has been predicted to encode a Wzy-type CPS polymerase. Sequence alignment with the Wzy_C and RfaL protein families (which catalyze CPS or lipopolysaccharide (LPS) biosynthesis) and topological analysis has suggested that eight highly conserved residues, including G308, G310, G334, G337, R290, P305, H323, and N324, were located in a hypothetical loop region. Therefore, we used site-directed mutagenesis to study the role of these residues in CPS production, and to observe the consequent phenotypes such as mucoviscosity, serum and phagocytosis resistance, and virulence (as assessed in mice) in pyogenic liver abscess strain NTUH-K2044. Alanine substitutions at R290 or H323 abolished all of these properties. The G308A mutant was severely impaired for these functions. The G334A mutant remained mucoid with decreased CPS production, but its virulence was significantly reduced in vivo. No phenotypic change was observed for strains harboring magA G310A, G337A, P305A, or N324A mutations. Therefore, R290, G308, H323, and G334 are functionally important residues of the MagA (Wzy) protein of K. pneumoniae NTUH-K2044, capsular type K1. These amino acids are also likely to be important for the function of Wzy in other capsular types in K. pneumoniae and other species bearing Wzy_C family proteins.肺炎克雷伯菌（Klebsiella pneumoniae）的黏液黏度相关基因A（Mucoviscosity-associated gene A，magA）参与K1荚膜多糖（capsular polysaccharide，CPS）的生物合成。基于序列同源性与基因比对结果，magA基因被预测编码一类Wzy型荚膜多糖聚合酶。通过与催化荚膜多糖或脂多糖（lipopolysaccharide，LPS）生物合成的Wzy_C、RfaL蛋白家族进行序列比对，并结合拓扑结构分析，研究人员推测包含G308、G310、G334、G337、R290、P305、H323及N324在内的8个高度保守残基，均位于一处假想环区域中。为此，本研究采用定点突变技术，探究上述残基在荚膜多糖合成中的功能作用，并观察化脓性肝脓肿菌株NTUH-K2044的相关表型变化，包括黏液黏稠度、血清抗性、吞噬抗性以及小鼠感染模型中评估的毒力。实验结果显示：R290或H323位点的丙氨酸替换可完全废除上述所有特性；G308A突变体的相关功能严重受损；G334A突变体仍保持黏液样表型，但荚膜多糖合成量下降，且其体内毒力显著降低；携带magA G310A、G337A、P305A或N324A突变的菌株未观察到任何表型变化。综上，对于K1荚膜型肺炎克雷伯菌NTUH-K2044的MagA（Wzy）蛋白而言，R290、G308、H323及G334为功能关键残基。这些氨基酸残基对于肺炎克雷伯菌其他荚膜型菌株，以及携带Wzy_C家族蛋白的其他物种中的Wzy蛋白功能，可能同样具有重要意义。