Antigenic variation by multiple promoter inversions in Mycoplasma penetrans

描述（由申请方提供）：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，M。在HIV感染者中主要鉴定出M.反式支原体衍生的脂蛋白刺激HIV-1的复制，增加了这种支原体可能是艾滋病进展中的共同病原体的可能性。对M. Appletrans公开了50种脂蛋白基因的存在，其中38种构成包括丰富的脂蛋白P35和P42的大旁系同源家族（命名为mpl）。虽然mpl家族的确切功能尚不清楚，但其单个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体繁殖群体内产生表面多样性的适应性策略。P42的相位变化是由含有脂蛋白基因启动子的135-bp DNA盒的分子倒位引起的。这个可逆位点的重新定向既排除了P42转录，又产生了一个假定的终止子。值得注意的是，类似的盒位于33个mpl基因的近端，这表明每个偶然性基因经历相位变异和组合表达谱的随机分类，在群体内产生十亿个潜在的抗原变体。尽管在支原体中已经表征了许多启动子倒位系统，但是在适当的启动子并置之后，仅存在单个可逆启动子来介导家族内单个基因的转录，即单个细胞主要表达基因家族的仅一个成员。相比之下，具有允许组合表达的多个密切相关的可逆片段的扩展mpl库在支原体中是前所未有的，并且大大超过了脆弱拟杆菌中报道的最大的13个片段的原核系统。此外，可逆启动子的大小与已知的最小单位（119 bp）相似，可能接近与对位相容的极限。这个提议的关键是，介导mpl分子体操的重组酶还没有被鉴定出来。因此，为了理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与同源DNA结合位点的相互作用。特定目的整合了基因组、分子、生物化学和微生物学方法，以i）通过在两种大肠杆菌中的转化测定中的功能表达来鉴定重组酶。大肠杆菌和最近开发的支原体宿主-载体系统中，ii）描述允许启动子区倒位的序列要求。这一建议解决了一个独特的相位变化的情况下，在一个神秘的支原体，并将提供新的见解抗原多样性产生系统。通过完成特定目标产生的数据可以提供一个平台，从中可以设计和实施免疫或化疗干预策略。公共卫生相关性：这项拟议中的研究有望揭示一种与艾滋病相关的细菌--反式支原体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，并为未来制定治疗和预防策略提供平台。

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.