Antigenic variation by multiple promoter inversions in Mycoplasma penetrans

描述（由申请人提供）：人支原体引起慢性和机会性感染，也可能导致其他微生物病原体的发病。事实上，流行病学研究表明，穿透支原体主要在hiv感染者中发现，再加上观察到穿透支原体衍生的脂蛋白刺激HIV-1复制，提出了这种支原体可能是艾滋病进展的共同病原体的可能性。通过对M. penetrans全基因组序列的分析，发现存在50个脂蛋白基因，其中38个构成一个大的同源家族（称为mpl），其中包括丰富的脂蛋白P35和P42。虽然mpl家族的确切功能尚不清楚，但个体成员被认为是偶然性基因；高度可变的表面蛋白编码序列，具有表现出高频相位变化的倾向。脂蛋白表达的这种随机变化反映了在生物繁殖种群中产生表面多样性的适应性策略。P42的相位变化源于含有脂蛋白基因启动子的135 bp DNA盒的分子倒置。这个可逆位点的重新定位既阻止了P42的转录，又产生了一个假定的终止子。值得注意的是，类似的磁带位于33个mpl基因的附近，这表明每个偶然性基因都受到相位变化的影响，并且组合表达谱的随机分类在人群中产生了10亿个潜在的抗原变异。尽管在支原体中已经发现了许多启动子反转系统，但只有一个可逆启动子介导一个家族中单个基因的转录，并遵循适当的启动子并立，即单个细胞主要只表达一个基因家族的一个成员。相比之下，广泛的mpl库具有多个密切相关的可逆片段，允许组合表达，在支原体中是前所未有的，并且大大超过了在脆弱拟杆菌中报道的13个片段的最大原核系统。此外，可逆启动子的大小与已知最小的单位（119bp）相似，并且可能接近与对位相容的极限。对这一建议至关重要的是，介导mpl分子体操的重组酶尚未被确定。因此，为了了解控制这种病理适应策略的分子事件，建议鉴定重组酶并表征其与同源DNA结合位点的相互作用。具体目标整合了基因组学，分子，生化和微生物学方法，i)通过在大肠杆菌和最近开发的支原体宿主-载体系统中的倒置实验中的功能表达来鉴定重组酶，ii)描述允许反转启动子区域的序列要求。这一建议解决了神秘支原体中独特的阶段变化情况，并将为抗原多样性产生系统提供新的见解。完成特定目标所产生的数据可以为免疫或化疗干预策略的设计和实施提供一个平台。公共卫生相关性：拟议的研究有望揭示穿透支原体（一种与艾滋病相关的细菌）在人类感染期间适应和生存能力的关键组成部分。对这种生物体中抗原变异系统的研究将进一步加深我们对这一复杂过程的理解，并为未来治疗和预防策略的发展提供一个平台。

DESCRIPTION (provided by applicant): Human mycoplasmas cause chronic and opportunistic infections and may also contribute to the pathogenesis of other microbial agents. Indeed, epidemiological studies reveal that M. penetrans is predominantly identified in HIV-infected individuals, which coupled to the observation that M. penetrans-derived lipoproteins stimulate HIV-1 replication, raises the possibility that this Mycoplasma may be a contributory co-pathogen in AIDS progression. Analysis of the complete genome sequence of M. penetrans discloses the presence of 50 lipoprotein genes, 38 of which constitute a large paralogous family (designated mpl) that includes the abundant lipoproteins P35 and P42. Although the precise function of the mpl family is unknown, the individual members are considered to be contingency genes; highly mutable surface protein-encoding sequences with the demonstrated propensity to exhibit high frequency phase variation. Such stochastic changes in lipoprotein expression reflect an adaptive strategy to generate surface diversity within a propagating population of organisms. Phase variation of P42 results from the molecular inversion of a 135-bp DNA cassette that contains the promoter of the lipoprotein gene. Re-orientation of this invertible locus both precludes P42 transcription and creates a putative terminator. Remarkably, analogous cassettes are located proximal to 33 of the mpl genes, suggestive of each contingency gene being subject to phase variation and of the stochastic assortment of combinatorial expression profiles generating a billion potential antigenic variants within the population. Although a number of promoter inversion systems have been characterized in mycoplasmas, only a single invertible promoter is present to mediate transcription of a single gene within a family, following appropriate promoter juxtaposition i.e. a single cell predominantly expresses only one member of a gene family. In contrast, the expansive mpl repertoire with multiple, closely-related invertible segments allowing combinatorial expression, is unprecedented in mycoplasmas and greatly exceeds the largest reported prokaryotic system of 13 segments in Bacteroides fragilis. Furthermore, the invertible promoter is similar in size to the smallest known unit (119 bp) and may be approaching the limit compatible with contraposition. Critical to this proposal, the recombinase that mediates mpl molecular gymnastics has not been identified. Accordingly, to understand the molecular events that govern this strategy of pathoadaptation, it is proposed to identify the recombinase and characterize the interaction with the cognate DNA binding site. The Specific Aims integrate genomic, molecular, biochemical and microbiological approaches to i) identify the recombinase by functional expression in an inversion assay in both E. coli and in a recently developed Mycoplasma host-vector system, ii) delineate the sequence requirements that permit inversion of the promoter region. This proposal addresses a unique phase variation scenario in an enigmatic Mycoplasma and will provide new insight into antigenic diversity generating systems. Data generated by completion of the Specific Aims may provide a platform from which strategies for immune or chemotherapeutic intervention can be devised and implemented. PUBLIC HEALTH RELEVANCE: The proposed research is expected to reveal a critical component of the ability of Mycoplasma penetrans, an AIDS-associated bacterium, to adapt and survive during human infection. The study of the system of antigenic variation in this organism will both further our understanding of this complex process and provide a platform for the future development of strategies for treatment and prevention.