PHASE AND ANTIGENIC VARIATION OF NEISSERIA GONORRHOEAE OPA GENES

淋病病原体淋病奈瑟氏菌(Ng)在发病过程中表现出高频期和抗原变异。导致表面成分(即偶发基因)表达变化的主要遗传机制涉及串联DNA重复序列的缺失或插入。淋病奈瑟氏菌的OPA基因家族是一组结构上相关的偶发基因的最著名的例子。NeisSeries OPA基因编码一个外膜蛋白家族，在实验室和体内表现出阶段可变的表达(正如在自然和男性志愿者感染研究中所显示的那样)。时相变化是由于在编码处理后的信号肽的区域中，每个OPA基因内五聚体([CTTCT])重复元件的数量发生变化。给定基因内的重复次数会导致起始密码子与编码成熟的OpA蛋白的序列对齐(框内)或错位(框外)。我们的目标是通过确定遗传和环境对相变的影响来理解非法重组的机制。报告系统已经被用来测量相位变化的频率。a.遗传对相位变化的影响。以前的研究表明，细菌细胞的DNA修复途径在利用串联DNA重复序列的高度可变基因中发生的遗传切换中是重要的。我们已经突变了淋病奈瑟菌的基因，这些基因参与了主要的DNA修复途径。此时发现和突变的基因涉及错配修复(MutS/HexA、MutL/HexB)、转录偶联修复(TcrF)、核苷酸切除修复(uvrA、uvrB和uvrC)和氧化损伤控制和修复(fpG、oxR和Katf)。将这些突变引入Ng应该会增加OPA基因报告基因的相变频率，但同时也会增加所有利用非法重组的基因的相变率，导致菌株同时表达所有可变成分。这些菌株将帮助我们了解变异在细菌发病中的作用，并可能有助于产生具有广泛免疫反应性的减毒菌株。B.环境对相变的影响。Ng OPA：：PhoA报告菌株被用来测量在一些环境条件下的相变频率。我们通过制作OPA：：BLA报告菌株来扩展我们的报告构建库，使我们能够直接选择特定的基因配置。在生长阶段的过程中，通过简单地比较相位变量的数量(开和关？)来测量频率或者？从关闭到打开？)出现在种群中。环境因素包括：1)生长期和饥饿，2)真核细胞接触，3)DNA转化和4)DNA损伤。-淋病奈瑟氏菌、淋病、性传播疾病、阶段变异、抗原变异、OPA、非法重组

Neisseria gonorrhoeae (Ng), the causative agent of gonorrhea, exhibits high frequency phase and antigenic variation during the course of disease. The predominant genetic mechanism responsible for variable expression of surface constituents (i.e. contingency genes) involves the deletion or insertion of tandem DNA repeats. The opa gene family of N. gonorrhoeae is the best known example of a structurally related group of contingency genes. Neisserial opa genes encode a family of outer-membrane proteins that show phase variable expression in the laboratory and in vivo (as shown in natural and male-volunteer infection studies). Phase variation results from changes in the number of pentameric ([CTTCT]) repeat elements within each opa gene in the region encoding the processed signal-peptide. The number of repeats within a given gene serves to either align (in frame) or misalign (out- of-frame) the start codon with the sequence encoding the mature Opa protein. Our goal is to understand the mechanisms for illegitimate recombination by determining the genetic and environmental influences on phase variation. Reporter systems have been used to measure phase variation frequencies.A. Genetic Influences on Phase Variation.Previous studies have shown that the DNA repair pathways of the bacterial cell are important in the genetic switching that occurs in hypermutable genes that utilize tandem DNA repeats. We have mutated genes in N. gonorrhoeae involved in the major DNA repair pathways. The genes identified and mutated at this time are involved in Mismatch Repair (mutS/hexA, mutL/hexB), Transcription Coupled Repair (tcrF), Nucleotide Excision Repair (uvrA, uvrB and uvrC) and Oxidative Damage Control and Repair (fpg, oxyR and katF). Introduction of these mutations into Ng should increase the phase variation frequency of the opa gene reporter but, at the same time, increase the phase change rates of all of the genes that utilize illegitimate recombination, resulting in strains that express all of the variable components simultaneously. These strains will help us understand the role of variation in bacterial pathogenesis and may also be useful in the generation of attenuated strains with broad immunological reactivity.B. Environmental Influences on Phase Variation.The Ng opa::phoA reporter strain was used to measure phase variation frequencies under a number of environmental conditions. We have expanded our repertoire of reporter costructions by making opa::bla reporter strains, allowing us to directly select for specific gene configurations. Frequencies were measured during the course of the growth phase by simply comparing the number of phase variants (either ?on to off? or ?off to on?) present in the population. Environmental factors which increased the frequency of phase variation included i) growth phase and starvation, ii) eukaryotic cell contact, iii) DNA transformation and iv) DNA damage. - Neisseria gonorrhoeae, gonorrhea, Sexually Transmitted Diseases, Phase Variation, Antigenic Variation, Opa, Illegitimate Recombination