Genetics of Coxiella burnetii

伯氏柯克斯体是一种普遍存在的人畜共患细菌性病原体，也是人类急性Q热的病因，Q热是一种致残性流感样疾病。柯克斯体以前的专性细胞内特性显著阻碍了推测的毒力因子的遗传特征。然而，最近生物体的无宿主细胞(无菌)生长使穿梭载体、转座子和诱导基因表达技术的发展成为可能，靶向基因失活仍然是一个重要的挑战。为此，我们在Coxiella中开发了两种靶向基因缺失的方法，这两种方法利用基于PUC/ColE1Ori的编码SACB的自杀质粒进行突变的阳性选择。作为概念验证，选择编码IVB型分泌系统(T4BSS)结构成分的柯克斯氏菌doA和dotB进行缺失。第一种方法利用Cre-lox介导的重组。携带不同抗生素耐药标记和loxP位点的两个自杀质粒整合到DOTA的5‘和3’侧翼区域。用编码Cre重组酶的第三个自杀质粒转化该菌株，在蔗糖反选择下导致DOTA的缺失。第二种方法使用循环输入/循环输出策略来删除DOTA和DOTB。首次将单个自杀质粒整合到5‘或3’靶基因侧翼区。在蔗糖反选择的情况下，通过第二次交换事件来分解共整合质粒导致了基因的缺失，这一点得到了PCR和Southern印迹的证实。DoA和dotB突变体不能分泌T4BSS底物，也不能有效地感染宿主细胞。柯克斯体基因工具的全套工具现在允许使用传统的突变和互补策略来发现毒力因子。现在已经构建了30多株基因敲除菌株，包括那些额外的Dot/ICM基因缺失和编码已验证的T4BSS底物的基因。这些突变体将极大地帮助研究分泌器官和分泌的效应蛋白的功能。 柯克斯体T4BSS将具有效应功能的蛋白质直接分泌到宿主细胞胞浆中。柯克斯体似乎还参与了II型分泌，直接进入病原体占据的空泡，在那里分泌的蛋白质可能会改变管腔微环境，以促进病原体的复制。银染结合质谱学分析显示，从对数生长期培养得到的酸性柠檬酸半胱氨酸培养基(ACCM)中含有多个柯克斯体蛋白，其中大部分被注释为含有信号肽的假想蛋白。用编码C-末端3X-FLAG标记蛋白的质粒转化的细菌，证实了柯克斯体分泌一组蛋白质的活性。去除SEC依赖的信号序列后，野生型细菌的分泌被消除。 柯克斯体唯一确定的毒力因子是内毒素。在体外重复传代时，具有全长内毒素的毒力I相生物转变为严重截短内毒素的无毒II期生物。鉴于内毒素对柯克斯体毒力的重要作用，了解其相变的分子基础是很重要的。在我们的文库中，高传代的II期分离株不是克隆的，并且含有一小部分柯克斯氏菌亚群，仍然表达全长的I期内毒素。由此产生的混合基因型使INDELs(插入/缺失)的鉴定变得复杂，这些INDELs严格地与阶段变化有关。为了绕过这个问题，我们使用显微操作分离了九里岛、澳大利亚和加利福尼亚州高传代分离株的克隆II期群体。通过将他们的基因组DNA杂交到一个高密度微阵列中，该微阵列包含覆盖Nine Mile第一阶段分离株整个基因组的探针组，确定了第二阶段生物中可能导致内毒素生物合成缺陷的常见INDELs。 柯克斯体致病的核心是细胞内的两个阶段的发育周期，它产生两种不同的形态变异，可以通过超微结构和蛋白质组成来区分。小细胞变异体(SCV)是一种非复制形式，呈现浓缩的染色质，被认为是细胞外的生存形式。SCV分化为染色质分散的复制型大细胞变体(LCV)。LCV向SCV的转变与柯克斯体进入静止生长阶段相一致，随着感染细胞培养的延长孵育(2-4周)，几乎均一的SCV出现。作为一个服从模型，以帮助更好地理解柯克斯体的生物学相关性，分化，我们建立了SCV/LCV转变是由生长在无宿主细胞(无菌)ACCM中的微生物概括的。这一发现使柯克斯体发育生物学的研究不会遇到宿主细胞繁殖细菌的实验困难。

Coxiella burnetii is a ubiquitous zoonotic bacterial pathogen and the cause of human acute Q fever, a disabling influenza-like illness. Coxiella's former obligate intracellular nature significantly impeded genetic characterization of putative virulence factors. However, recent host cell-free (axenic) growth of the organism has enabled development of shuttle vector, transposon, and inducible gene expression technologies, with targeted gene inactivation remaining an important challenge. To this end, we developed two methods of targeted gene deletion in Coxiella that exploit pUC/ColE1 ori-based suicide plasmids encoding sacB for positive selection of mutants. As proof of concept, Coxiella dotA and dotB, encoding structural components of the type IVB secretion system (T4BSS), were selected for deletion. The first method exploited Cre-lox-mediated recombination. Two suicide plasmids carrying different antibiotic resistance markers and a loxP site were integrated into 5' and 3' flanking regions of dotA. Transformation of this strain with a third suicide plasmid encoding Cre recombinase resulted in deletion of dotA under sucrose counterselection. The second method utilized a loop-in/loop out strategy to delete dotA and dotB. A single suicide plasmid was first integrated into 5' or 3' target gene flanking regions. Resolution of the plasmid co-integrant by a second crossover event under sucrose counterselection resulted in gene deletion that was confirmed by PCR and Southern blot. dotA and dotB mutants failed to secrete T4BSS substrates and to productively infect host cells. The repertoire of Coxiella genetic tools now allows traditional mutation and complementation strategies for virulence factor discovery. Over 30 knockout strains have now been constructed, including those with deletions in additional Dot/Icm genes and genes encoding verified T4BSS substrates. These mutants will dramatically aid functional studies of both the secretion apparatus and secreted effector proteins. The Coxiella T4BSS secretes proteins with effector functions directly into the host cell cytosol. Coxiella also appears to engage in type II-like secretion directly into the pathogen-occupied vacuole where secreted proteins likely modify the lumenal microenvironment to promote pathogen replication. Sliver staining combined with mass spectrometry revealed multiple Coxiella proteins in acidified citrate cysteine medium (ACCM) harvested from log phase cultures, most of which are annotated as signal peptide-containing hypothetical proteins. Active secretion of a subset of proteins by Coxiella was confirmed using bacteria transformed with plasmids encoding C-terminal 3x-FLAG-tagged proteins expressed from an anhydrotetracycline-inducible promoter. Secretion by wild type bacteria was eliminated upon removal of the Sec-dependent signal sequence. The only defined virulence factor of Coxiella is LPS. Virulent phase I organisms with full-length LPS transition to avirulent phase II organisms with severely truncated LPS upon repeated in vitro passage. Given the critical importance of LPS to Coxiella virulence, it is important to understand the molecular basis of phase variation. The high passage phase II isolates in our stock collection are not clonal and contain a small subpopulation of Coxiella still expressing full-length phase I LPS. The resulting mixed genotype complicates identification of indels (insertions/deletions) strictly associated with phase variation. To circumvent this problem, we used micromanipulation to isolate clonal phase II populations of high passage Nine Mile, Australia and California isolates. By hybridizing their genomic DNA to a high-density microarray that contains probe sets encompassing the entire genome of the Nine Mile phase I isolate, common indels in phase II organisms were identified that may account for defective LPS biosynthesis. Central to Coxiella pathogenesis is an intracellular biphasic developmental cycle that generates two distinct morphological variants that can be distinguished by ultrastructure and protein composition. Small cell variants (SCV) are non-replicative forms that display condensed chromatin and are considered extracellular survival forms. SCVs differentiate into replicative large cell variants (LCVs) with dispersed chromatin. Transition of LCV back to SCV occurs coincident with entry of Coxiella into stationary growth phase with nearly homogeneous SCVs present with extended incubation (2 to 4 weeks) of infected cell cultures. As an amenable model to help better understand the biological relevance of Coxiella, differentiation, we established that SCV/LCV transitions are recapitulated by organisms growing in host cell-free (axenic) ACCM. This discovery enables studies of Coxiella developmental biology without experimental difficulties encountered with host cell-propagated bacteria.