Rickettsia typhi possesses phospholipase A2 enzymes that are involved in infection of host cells.
Rickettsia typhi possesses phospholipase A2 enzymes that are involved in infection of host cells.
复制标题
DOI：
10.1371/journal.ppat.1003399
复制
发表时间：
2013
期刊：
影响因子：
6.7
通讯作者：
Azad AF
中科院分区：
文献类型：
--
作者：
Rahman MS;Gillespie JJ;Kaur SJ;Sears KT;Ceraul SM;Beier-Sexton M;Azad AF
磷脂酶A2（PLA 2）酶参与立克次体感染宿主细胞的长期假设已得到最近来自病原体普氏立克次体和立克次体的具有PLA 2活性的patatin磷脂酶（Pat 2）的表征的支持。伤寒。然而，pat 2并不是在所有的立克次体基因组中编码的;另一种未表征的patatin（Pat 1）确实是普遍存在的。在此，对46个立克次体基因组中的两种patatins的进化分析显示：1）pat 1和pat 2基因座在所有基因组中是同线的，2）Pat 1和Pat 2都不包含预测的Sec依赖性信号序列，3）pat 2在立克次体进化中被多次假基因化，和4）普遍存在的pat 1形成两个不同的组（pat 1A和pat 1B），有强有力的证据表明pat 1B和质粒编码的同源物之间的重组。根据这些发现，我们扩展了R.伤寒Pat 1和Pat 2蛋白，并确定其在感染过程中的作用。如前所述，我们确定了1）Pat 1在R.伤寒杆菌感染，2）重组Pat 1的表达对酵母细胞具有细胞毒性，3）重组Pat 1具有需要宿主辅因子的PLA 2活性，和4）PLA 2抑制剂降低Pat 1的细胞毒性和PLA 2活性，并通过催化性Ser/Asp残基的定点诱变消除。为了明确Pat 1和Pat 2在R.伤寒感染时，使用针对两种蛋白质的抗体来预处理立克次体。随后的侵袭和空斑检测均表明R.伤寒感染与免疫前IgG相比。此外，抗体预处理的R.伤寒阻断/延迟吞噬体逃逸。总之，这些数据表明这两种酶都参与了感染过程的早期。总体而言，我们的研究表明，R。伤寒杆菌利用两种进化上不同的马铃薯糖蛋白磷脂酶来维持其细胞内生命周期，这是一种使其与其它立克次体物种区别开来的机制。伤寒群立克次体包括伤寒立克次体和R. prowazekii，分别是鼠斑疹伤寒和流行性斑疹伤寒的病原体。立克次氏体专性细胞内生命周期使遗传操作的常规方法复杂化，阻碍了致病机制分子的表征。磷脂酶A2（PLA 2）酶是可能介导宿主进入、吞噬体逃逸和宿主裂解以进行细胞间传播的过程的因子。在这里，我们证明了46个立克次体基因组中编码两种PLA 2酶（pat 1和pat 2）的基因具有不同的进化历史和不一致的选择性限制，pat 2从大多数其他非TG立克次体基因组中删除。如前所确定的帕特2，我们在这里表明，R。伤寒杆菌Pat 1在感染过程中分泌到宿主细胞的细胞质中，并且需要宿主辅因子来进行酶活性。与Pat 2一样，重组Pat 1蛋白对酵母细胞具有细胞毒性。R.抗Pat 1或抗Pat 2抗体的伤寒杆菌感染立克次体的数量显著减少，提示Pat 1和Pat 2在立克次体早期阶段均起作用。伤寒宿主细胞感染。总的来说，我们的工作表明，R。伤寒在其细胞内生命周期中利用两种进化趋异的磷脂酶，这是一种将TG立克次体相关细胞生物学和发病机制与其他立克次体病，特别是与斑点热群病原体相关的立克次体病区分开来的机制。
The long-standing proposal that phospholipase A2 (PLA2) enzymes are involved in rickettsial infection of host cells has been given support by the recent characterization of a patatin phospholipase (Pat2) with PLA2 activity from the pathogens Rickettsia prowazekii and R. typhi. However, pat2 is not encoded in all Rickettsia genomes; yet another uncharacterized patatin (Pat1) is indeed ubiquitous. Here, evolutionary analysis of both patatins across 46 Rickettsia genomes revealed 1) pat1 and pat2 loci are syntenic across all genomes, 2) both Pat1 and Pat2 do not contain predicted Sec-dependent signal sequences, 3) pat2 has been pseudogenized multiple times in rickettsial evolution, and 4) ubiquitous pat1 forms two divergent groups (pat1A and pat1B) with strong evidence for recombination between pat1B and plasmid-encoded homologs. In light of these findings, we extended the characterization of R. typhi Pat1 and Pat2 proteins and determined their role in the infection process. As previously demonstrated for Pat2, we determined that 1) Pat1 is expressed and secreted into the host cytoplasm during R. typhi infection, 2) expression of recombinant Pat1 is cytotoxic to yeast cells, 3) recombinant Pat1 possesses PLA2 activity that requires a host cofactor, and 4) both Pat1 cytotoxicity and PLA2 activity were reduced by PLA2 inhibitors and abolished by site-directed mutagenesis of catalytic Ser/Asp residues. To ascertain the role of Pat1 and Pat2 in R. typhi infection, antibodies to both proteins were used to pretreat rickettsiae. Subsequent invasion and plaque assays both indicated a significant decrease in R. typhi infection compared to that by pre-immune IgG. Furthermore, antibody-pretreatment of R. typhi blocked/delayed phagosomal escapes. Together, these data suggest both enzymes are involved early in the infection process. Collectively, our study suggests that R. typhi utilizes two evolutionary divergent patatin phospholipases to support its intracellular life cycle, a mechanism distinguishing it from other rickettsial species. Typhus Group (TG) rickettsiae include Rickettsia typhi and R. prowazekii, the etiological agents of murine and epidemic typhus, respectively. The rickettsial obligate intracellular life cycle complicates conventional approaches to genetic manipulation, impeding characterization of molecules underlying pathogenesis. Phospholipase A2 (PLA2) enzymes are factors that likely mediate such processes as host entry, phagosomal escape, and host lysis for cell-to-cell spread. Here, we demonstrate across 46 Rickettsia genomes that genes encoding two PLA2 enzymes (pat1 and pat2) have divergent evolutionary histories and discordant selective constraints, with pat2 deleted from most other non-TG rickettsiae genomes. As previously determined for Pat2, we show here that R. typhi Pat1 is secreted into the host cell cytoplasm during infection and requires a host cofactor for enzymatic activity. Like Pat2, recombinant Pat1 protein is cytotoxic to yeast cells. Pretreatment of R. typhi with anti-Pat1 or anti-Pat2 antibody results in a significant decrease in rickettsial infection, implicating roles for both Pat1 and Pat2 during the early stage of R. typhi host cell infection. Collectively, our work suggests that R. typhi utilizes two evolutionary divergent phospholipases during its intracellular life cycle, a mechanism distinguishing TG rickettsiae-associated cell biology and pathogenesis from other rickettsioses, particularly those associated with Spotted Fever Group pathogens.
登录
查看更多内容
影响因子：
10.7
作者：
Castresana, J
通讯作者：
Castresana, J
影响因子：
3.7
作者：
Gillespie, Joseph J.;Williams, Kelly;Shukla, Maulik;Snyder, Eric E.;Nordberg, Eric K.;Ceraul, Shane M.;Dharmanolla, Chitti;Rainey, Daphne;Soneja, Jeetendra;Shallom, Joshua M.;Vishnubhat, Nataraj Dongre;Wattam, Rebecca;Purkayastha, Anjan;Czar, Michael;Crasta, Oswald;Setubal, Joao C.;Azad, Abdu F.;Sobral, Bruno S.
通讯作者：
Sobral, Bruno S.
影响因子：
3.7
作者：
Gillespie JJ;Ammerman NC;Dreher-Lesnick SM;Rahman MS;Worley MJ;Setubal JC;Sobral BS;Azad AF
通讯作者：
Azad AF
影响因子：
3.6
作者：
Atmakuri, K;Ding, ZY;Christie, PJ
通讯作者：
Christie, PJ
影响因子：
11.8
作者：
Azad, AF;Radulovic, S;Troyer, JM
通讯作者：
Troyer, JM