Jonson G, Lebens M, Holmgren J
Department of Medical Microbiology and Immunology, Göteborg University, Sweden.
Mol Microbiol. 1994 Jul;13(1):109-18. doi: 10.1111/j.1365-2958.1994.tb00406.x.
The mannose-sensitive haemagglutinin (MSHA) pilus that is associated with Vibrio cholerae strains of El Tor biotype has been shown to be a potential colonization factor and protective antigen. The gene encoding the structural subunit of MSHA pili was cloned from size-fractionated SacI-cleaved chromosomal DNA in the expression phage vector lambda ZAPII. Positive clones carried a c. 5.3 kb SacI fragment and were identified on the basis of MSHA expression and hybridization with a synthetic oligonucleotide probe based upon the N-terminus of MshA, the structural subunit of MSHA. The mshA gene was localized to a 2.6 kb SalI-EcoRI fragment, which was subcloned and shown to express MshA from its own promoter in Escherichia coli. Nucleotide sequencing of the entire fragment revealed six open reading frames (ORFs) of which four were complete. The mshA gene encodes an 18,094 Da prepilin protein, which in its mature form has a size of 17,436 Da. MshA is a type 4 (N-MePhe) pilin protein that is more homologous to pilins produced by Pseudomonas aeruginosa and Neisseria gonorrhoeae than to TcpA, the structural subunit of the toxin-coregulated pilus of V. cholerae. The protein seems to be directly involved in receptor binding, as an in-frame mutation in the mshA gene was found to abolish both D-mannose-dependent haemagglutination and binding of V. cholerae bacteria to D-mannose-containing agarose beads. Three additional ORFs, all in the same transcriptional orientation as mshA, were found to encode type 4 pilin-like proteins. A potential promoter with a sequence homologous to that of cAMP-CRP-activated promoters in E. coli was identified upstream of ORF3, the gene preceding mshA.
与霍乱弧菌埃尔托生物型菌株相关的甘露糖敏感血凝素（MSHA）菌毛已被证明是一种潜在的定植因子和保护性抗原。编码MSHA菌毛结构亚基的基因是从经SacI切割的染色体DNA大小分级片段中克隆出来的，克隆到表达噬菌体载体λZAPII中。阳性克隆携带一个约5.3 kb的SacI片段，并根据MSHA表达以及与基于MSHA结构亚基MshA的N端合成寡核苷酸探针的杂交进行鉴定。mshA基因定位于一个2.6 kb的SalI - EcoRI片段，该片段被亚克隆并显示在大肠杆菌中从其自身启动子表达MshA。对整个片段进行核苷酸测序揭示了六个开放阅读框（ORF），其中四个是完整的。mshA基因编码一种18,094 Da的前菌毛蛋白，其成熟形式的大小为17,436 Da。MshA是一种4型（N - MePhe）菌毛蛋白，与铜绿假单胞菌和淋病奈瑟菌产生的菌毛蛋白的同源性高于与霍乱弧菌毒素共调节菌毛的结构亚基TcpA的同源性。该蛋白似乎直接参与受体结合，因为发现mshA基因中的一个框内突变消除了D - 甘露糖依赖性血凝以及霍乱弧菌细菌与含D - 甘露糖的琼脂糖珠的结合。另外三个ORF，均与mshA处于相同的转录方向，被发现编码4型菌毛样蛋白。在mshA之前的基因ORF3上游鉴定出一个与大肠杆菌中cAMP - CRP激活启动子序列同源的潜在启动子。
