Source data: FtsK is critical for the assembly of the unique divisome complex of the FtsZ-less Chlamydia trachomatis IF images

Chlamydia trachomatis serovar L2 (Ct), an obligate intracellular bacterium that does not encode FtsZ, divides by a polarized budding process. In the absence of FtsZ, we show that FtsK, a chromosomal translocase, is critical for divisome assembly in Ct. Chlamydial FtsK forms discrete foci at the septum and at the base of the progenitor mother cell, and our data indicate that FtsK foci at the base of the mother cell mark the location of nascent divisome complexes that form at the site where a daughter cell will emerge in the next round of division. The divisome in Ct has a hybrid composition, containing elements of the divisome and elongasome from other bacteria, and FtsK is recruited to nascent divisomes prior to the other chlamydial divisome proteins assayed, including the PBP2 and PBP3 transpeptidases, and MreB and MreC. Knocking down FtsK prevents divisome assembly in Ct and inhibits cell division and septal peptidoglycan synthesis. We further show that MreB does not function like FtsZ and serves as a scaffold for the assembly of the Ct divisome. Rather, MreB is one of the last proteins recruited to the chlamydial divisome, and it is necessary for the formation of septal peptidoglycan rings. Our studies illustrate the critical role of chlamydial FtsK in coordinating divisome assembly and peptidoglycan synthesis in this obligate intracellular bacterial pathogen.

Methods
Cell Culture
HeLa cells (ATCC, Manassas, VA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, Waltham, MA) containing 10% fetal bovine serum (FBS, Hyclone, Logan, UT) at 37°C in a humidified chamber with 5% CO2. HeLa cells were infected with Ct serovar L2 434/Bu in the same media. Infections of HeLa cells with chlamydial transformants were performed in DMEM containing 10% FBS and 0.36 U/mL penicillin G (Sigma-Aldrich).

Cloning
The plasmids and primers used for generating mCherry fusions of FtsK, PBP2, PBP3, and MreC are listed in Supp. Table S1. The chlamydial ftsK, pbp2, pbp3, and mreC genes were amplified by PCR with Phusion DNA polymerase (NEB, Ipswich, MA) using 10 ng C. trachomatis serovar L2 genomic DNA as a template. The PCR products were purified using a PCR purification kit (Qiagen) and inserted into the pBOMB4-Tet (-GFP) plasmid, which confers resistance to β-lactam antibiotics. The plasmid was cut at the NotI (FtsK-mCherry) or the KpnI (mCherry-PBP2, mCherry-PBP3, mCherry-MreC) site, and the chlamydial genes were inserted into the cut plasmid using the HiFi DNA Assembly kit (NEB) according to the manufacturer’s instructions. The products of the HiFi reaction were transformed into NEB-5αIq competent cells (NEB), and transformants were selected by growth on plates containing ampicillin. DNA from individual colonies was isolated using a mini-prep DNA isolation kit (Qiagen), and plasmids were initially characterized by restriction digestion to verify that the inserts were the correct size. Clones containing inserts of the correct size were DNA sequenced prior to use.

DNA and RNA purification and RT-qPCR
Total nucleic acids were extracted from HeLa cells infected with Ct plated in 6-well dishes as described previously (Ouellette 2015, Ouellette, Blay et al. 2021). For RNA isolation, cells were rinsed once with PBS, then lysed with 1 mL TRIzol (Invitrogen) per well. Total RNA was extracted from the aqueous layer after mixing with 200μL per sample of chloroform following the manufacturer’s instructions. Total RNA was precipitated with isopropanol and treated with DNase (Ambion) according to the manufacturer’s guidelines prior to cDNA synthesis using SuperScript III (Invitrogen). For DNA, infected cells were rinsed once with PBS, trypsinized, and pelleted before resuspending each pellet in 500μL of PBS. Each sample was split in half, and genomic DNA was isolated from each duplicate sample using the DNeasy extraction kit (Qiagen) according to the manufacturer’s guidelines. Quantitative PCR was used to measure C. trachomatis genomic DNA (gDNA) levels using an euo primer set. 150ng of each sample was used in 25μL reactions using standard amplification cycles on a QuantStudio3 thermal cycler (Applied Biosystems) followed by a melting curve analysis. ftsK, pbp2, euo, and omcB transcript levels were determined by RT-qPCR using SYBR Green as described previously (Ouellette SP 2021) (see Supp. Table S2 for primers used for measuring gDNA levels and RT-qPCR). Transcript levels were normalized to genomes and expressed as ng cDNA/gDNA.

Transformation of Ct

Ct was transformed as described previously (Wang 2011). Briefly, HeLa cells were plated in a 10cm plate at a density of 5 × 106 cells the day before beginning the transformation procedure. Ct lacking its endogenous plasmid (-pL2) was incubated with 10μg of plasmid DNA in Tris-CaCl2 buffer (10 mM Tris-Cl pH 7.5, 50 mM CaCl2) for 30 min at room temperature. HeLa cells were trypsinized, washed with 8 mL of 1x DPBS (Gibco), and pelleted. The pellet was resuspended in 300μL of the Tris-CaCl2 buffer. Ct was mixed with the HeLa cells and incubated at room temperature for an additional 20 min. The mixture was added to 10 mL of DMEM containing 10% FBS and 10 μg/mL gentamicin and transferred to a 10cm plate. At 48 hpi, the HeLa cells were harvested, and Ct in the population was used to infect a new HeLa cell monolayer in media containing 0.36 U/ml of penicillin G to select for transformants. The plate was incubated at 37°C for 48 hours. These harvest and re-infection steps were repeated every 48 hours until inclusions were observed.

Immunofluorescence Microscopy
HeLa cells were seeded in 10cm plates at a density of 5 × 106 cells per well the day before the infection. Ct L2 or chlamydial strains transformed with plasmids encoding FtsK-mCherry, mCherry-PBP2, mCherry-PBP3, or mCherry-MreC or with plasmids that direct the constitutive expression of the crRNAs targeting the pbp2 or ftsK promoters were used to infect HeLa cells in DMEM. For experiments with the transformants, aTc was added to the media of infected cells at the indicated concentration and time. At 21 hpi, cells were detached from the 10cm plate by scraping and pelleted by centrifugation for 30 seconds. The pellet was resuspended in 1 mL of 0.1x PBS (Gibco) and transferred to a 2 mL tube containing 0.5mm glass beads (ThermoFisher Scientific). Cells were vortexed for 3 mins, then centrifuged at 800 rpm for 2 mins. in a microfuge. 20μLs of the supernatant was mixed with 20μLs of 2x fixing solution (6.4% formaldehyde and 0.044% glutaraldehyde) and incubated on a glass slide for 10 min at room temperature. Cells were washed 3 times with PBS, and the cells were permeabilized by incubation with PBS containing 0.1% Triton X-100 for 1 min. Cells were washed with PBS twice. For experiments with Ct L2, the cells were incubated with a goat primary antibody against the major outer-membrane protein (MOMP; Meridian, Memphis, TN), and the mouse primary antibody that recognizes endogenous FtsK raised against recombinant CT739 protein (https://doi.org/10.1099/mic.0.047746-0), or with rabbit antibodies raised against peptides derived from PBP 2 or PBP3 (Ouellette SP 2012). Briefly, chlamydial antigens or peptides emulsified with Freund’s incomplete adjuvant were used to immunize animals via intramuscular injections three times with an interval of 2 weeks. Antisera were collected from the immunized animals 2 to 4 weeks after the final immunization as the primary antibodies. After the primary antibody labeling, the cells were then rinsed with PBS and incubated with donkey anti-goat IgG (Alexa 488) and donkey anti-mouse IgG (Alexa 594) or donkey-anti-rabbit IgG (Alexa 594) secondary antibodies (Invitrogen). Experiments in which we visualized the distribution of the various mCherry fusions, the localization of the mCherry fluorescence was compared to the distribution of MOMP. In some experiments, we determined the distribution of the MreB_6x His fusion by staining cells expressing the fusion with a rabbit anti-6x His antibody (Abcam, Cambridge, MA) and the goat anti-MOMP antibody, followed by the appropriate secondary antibodies. Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. During image acquisition, 0.3μm xy-slices were collected that extended above and below the cell. The images were collected such that the brightest spot in the image was saturated. The images were deconvolved using the nearest neighbor algorithm in the Zeiss Axiovision 4.7 software. Deconvolved images were viewed and assembled using Zeiss Zen-Blue software. For each experiment, three independent replicates were performed, and the values shown for localization are the average of the 3 experiments. In some instances, 3D projections of the acquired xy slices were generated using the Zeiss Zen-Blue software.

Peptidoglycan (PG) labeling
PG was labeled by incubating cells with 4mM ethylene-D-alanine-D-alanine (E-DA-DA) as described (Cox 2020). The incorporated E-DA-DA was fluorescently labeled using the Click & Go™ labeling kit (Vector Laboratories). The distribution of fluorescently labeled PG was compared to the distribution of MOMP and endogenous FtsK or the distribution of mCherry-PBP3. Three independent replicates were performed, and the values shown are the average of the 3 experiments.

Inclusion forming unit assay
HeLa cells were infected with Ct (-pL2) transformed with the pBOMB4 Tet (-GFP) plasmid encoding the indicated aTc-inducible gene. At 8 hpi, aTc was added to the culture media at the indicated concentration. Control cells were not induced. At 48 hpi, the HeLa cells were dislodged from the culture dishes by scraping and collected by centrifugation. The pellet was resuspended in 1 mL of 0.1x PBS (Gibco) and transferred to a 2 mL tube containing 0.5mm glass bead tubes (ThermoFisher Scientific). Cells were vortexed for 3 min. followed by centrifugation at 800 rpm for 2 min. The supernatants were mixed with an equal volume of a 2x sucrose-phosphate (2SP) solution (ref) and frozen at −80°C. At the time of the secondary infection, the chlamydiae were thawed on ice and vortexed. Cell debris was pelleted by centrifugation for 5 min at 1k x g at 4°C. The EBs in the resulting supernatant were serially diluted and used to infect a monolayer of HeLa cells in a 24-well plate. The secondary infections were allowed to grow at 37°C for 24 hrs before they were fixed and labeled for immunofluorescence microscopy by incubating with a goat anti-MOMP antibody followed by a secondary donkey anti-goat antibody (Alexa Fluor 594). The cells were rinsed in PBS, and inclusions were imaged using an EVOS imaging system (Invitrogen). The number of inclusions was counted in 5 fields of view and averaged. Three independent replicates were performed, and the values from the replicates were averaged to determine the number of inclusion forming units. Chi-squared analysis was used to compare IFUs in induced and uninduced samples.

Effect of A22 and mecillinam on the profile of division intermediates and on PG and divisome protein localization in Ct

HeLa cells were infected with Ct transformed with the pBOMB4-Tet (-GFP) plasmid encoding FtsK-mCherry, mCherry-PBP2, mCherry-PBP3, mCherry-MreC, or MreB-6xHis. The fusions were induced at 20 hpi with 10 nM aTc for 1 hr in the absence or presence of 75 μM A22. At 22 hpi, cells were harvested and prepared for staining as described above. Three independent replicates were performed, and the values shown for localization are the average of the 3 experiments.

HeLa cells were infected with Ct L2, and 20μM mecillinam (Sigma) was added to the media of infected cells at 17 hpi. Control cells were untreated. At 22 hpi, infected cells were harvested and RBs were prepared and stained with MOMP, FtsK, PBP2, or PBP3 antibodies as described above. Alternatively, cells were incubated with 4 mM EDA-DA at 17 hpi in the presence or absence of 20μM mecillinam. The cells were harvested at 22 hpi, and RBs were prepared, and PG was click-labeled, and its distribution was visualized in MOMP-stained cells as described above. Three independent replicates were performed, and the values shown for localization are the average of the 3 experiments.

Immunoblotting
HeLa cells infected with Ct L2 were harvested by scraping the infected cells from the plate at 24 hpi. Uninfected HeLa cells were included as a control. The HeLa cells were pelleted by centrifugation, resuspended in SDS sample buffer, and electrophoresed on a 10% SDS polyacrylamide gel. The gel was electrophoretically transferred to nitrocellulose (Schleicher and Schuell), and the filter was incubated with mouse polyclonal antibodies raised against chlamydial FtsK. The filter was rinsed and incubated with 800 donkey anti-mouse IgG secondary antibodies (LICOR, Lincoln, NE) and imaged using a LICOR Odyssey imaging system.

HeLa cells were infected with Ct transformed with plasmids that inducibly express FtsK-mCherry, mCherry-PBP2, mCherry-PBP3, or mCherry-MreC. The fusions were induced by the addition of 10nM aTc to the media of infected cells at 17 hpi. The cells were harvested and pelleted at 21 hpi. The cell pellet was resuspended in 1 mL of 0.1x PBS (Gibco) and transferred to a 2 mL tube containing 0.5mm glass beads (ThermoFisher Scientific). Cells were vortexed for 3 min. followed by centrifugation at 800 rpm for 2 min. The supernatant was collected and centrifuged for 3 min at 13,000 rpm, and the pellet containing Ct was resuspended in TBS containing 1% TX-100, 1X protease inhibitor cocktail (Sigma), and 5μM lactacystin. The suspension was sonicated 3 times on ice and centrifuged at 13,000 rpm for 3 mins. The supernatant was collected and mixed with SDS sample buffer. The samples were boiled and electrophoresed on a 10% SDS polyacrylamide gel, and the gel was electrophoretically transferred to nitrocellulose. The blots from these analyses were probed with a rabbit anti-mCherry primary antibody (Invitrogen) and an 800 donkey anti-rabbit IgG secondary antibody (LICOR, Lincoln, NE). The filters were imaged using a LICOR Odyssey imaging system.

HeLa cells were infected with Ct transformed with the pBOMB4-Tet (-GFP) plasmid encoding mCherry-PBP2 or mCherry-PBP3. The fusions were induced with 10nM aTc at 17 hpi. Uninduced cells were included as a control. The cells were harvested at 21 hpi, and samples were processed for immunoblotting as described above. The blots were probed with rabbit polyclonal antibodies raised against peptides derived from chlamydial PBP2 or PBP3 (Ouellette SP 2012). The blots were then rinsed and incubated with 800 donkey anti-rabbit IgG secondary antibodies (LICOR, Lincoln, NE). The filters were imaged using a LICOR Odyssey imaging system.

### 研究背景
沙眼衣原体血清型L2（*Chlamydia trachomatis serovar L2*，简称Ct）是一种不编码FtsZ蛋白（FtsZ）的专性胞内细菌，其通过极性出芽过程进行分裂。在缺失FtsZ的情况下，我们的研究表明，染色体移位酶FtsK（FtsK）对于Ct的分裂体（divisome）组装至关重要。沙眼衣原体FtsK会在分裂隔膜以及母细胞前体的基部形成离散的焦点；我们的数据显示，母细胞基部的FtsK焦点标记了新生分裂体复合物的位置，该复合物将在下一轮分裂中于子细胞形成位点组装。Ct的分裂体具有混合组成，包含其他细菌分裂体与延伸体（elongasome）的元件；且相较于其他已检测的沙眼衣原体分裂体蛋白（包括PBP2、PBP3转肽酶以及MreB和MreC），FtsK会被率先招募至新生分裂体。敲低FtsK会阻止Ct的分裂体组装，并抑制细胞分裂与隔膜肽聚糖合成。我们进一步发现，MreB的功能并不等同于FtsZ，而是作为Ct分裂体组装的支架蛋白。相反，MreB是最后被招募至沙眼衣原体分裂体的蛋白之一，且对于隔膜肽聚糖环的形成不可或缺。本研究阐明了沙眼衣原体FtsK在这种专性胞内致病菌的分裂体组装与肽聚糖合成过程中的关键调控作用。

## 实验方法
### 细胞培养
HeLa细胞（ATCC，弗吉尼亚州马纳萨斯）培养于含10%胎牛血清（FBS，Hyclone，犹他州洛根）的达尔伯克改良伊格尔培养基（DMEM；Invitrogen，马萨诸塞州沃尔瑟姆）中，培养条件为37℃、含5% CO₂的湿润培养箱。使用Ct血清型L2 434/Bu株感染同培养基培养的HeLa细胞。沙眼衣原体转化体感染HeLa细胞的实验采用含10% FBS与0.36 U/mL青霉素G（Sigma-Aldrich）的DMEM进行。

### 克隆构建
用于构建FtsK、PBP2、PBP3与MreC的mCherry融合蛋白的质粒及引物详见补充表S1。以10 ng沙眼衣原体血清型L2的基因组DNA为模板，使用Phusion DNA聚合酶（NEB，马萨诸塞州伊普斯维奇）通过PCR扩增沙眼衣原体ftsK、pbp2、pbp3与mreC基因。PCR产物经PCR纯化试剂盒（Qiagen）纯化后，插入至pBOMB4-Tet (-GFP)质粒（该质粒可赋予β-内酰胺类抗生素抗性）。分别使用NotI（针对FtsK-mCherry构建体）或KpnI（针对mCherry-PBP2、mCherry-PBP3、mCherry-MreC构建体）对质粒进行酶切，随后按照HiFi DNA组装试剂盒（NEB）的制造商说明书，将沙眼衣原体基因插入酶切后的质粒中。HiFi组装反应产物转化至NEB-5αIq感受态细胞（NEB），通过含氨苄青霉素的平板培养筛选转化子。使用微量制备DNA提取试剂盒（Qiagen）提取单个菌落的DNA，初始通过限制性酶切鉴定质粒插入片段的大小是否正确。插入片段大小符合要求的克隆在使用前进行DNA测序验证。

### DNA/RNA提取与实时定量逆转录PCR（RT-qPCR）
按照此前报道的方法（Ouellette 2015；Ouellette, Blay等，2021），从铺于6孔板的感染Ct的HeLa细胞中提取总核酸。对于RNA提取：细胞先用PBS漂洗一次，随后每孔加入1 mL TRIzol（Invitrogen）裂解。按照制造商说明书，每样本加入200 μL氯仿混匀后，从水相层提取总RNA。总RNA经异丙醇沉淀后，按照Ambion DNase的使用指南进行DNA酶处理，随后使用SuperScript III（Invitrogen）进行cDNA合成。对于DNA提取：感染细胞先用PBS漂洗一次，经胰酶消化后离心收集沉淀，将每管沉淀重悬于500 μL PBS中。每份样本均分为两份，使用DNeasy提取试剂盒（Qiagen）按照制造商指南从两份重复样本中分离基因组DNA。使用euo引物套装通过定量PCR检测沙眼衣原体基因组DNA（gDNA）水平。每份样本取150 ng用于25 μL反应体系，在QuantStudio3热循环仪（Applied Biosystems）上采用标准扩增循环程序进行反应，随后进行熔解曲线分析。通过RT-qPCR检测ftsK、pbp2、euo与omcB的转录水平，采用SYBR Green染料，方法同此前报道（Ouellette SP 2021）（用于检测gDNA水平与RT-qPCR的引物详见补充表S2）。转录水平以基因组为参照进行标准化，结果以ng cDNA/gDNA表示。

### Ct的转化
Ct的转化方法参照此前报道（Wang 2011）。简要步骤如下：转化实验前一天，将5×10^6个HeLa细胞铺于10 cm培养板中。将10 μg质粒DNA与缺失内源质粒的Ct（-pL2）在Tris-CaCl₂缓冲液（10 mM Tris-Cl pH 7.5，50 mM CaCl₂）中室温孵育30 min。胰酶消化HeLa细胞，用8 mL 1×DPBS（Gibco）洗涤后离心收集沉淀，将沉淀重悬于300 μL Tris-CaCl₂缓冲液中。将Ct与HeLa细胞混合后室温孵育20 min。将混合物加入至10 mL含10% FBS与10 μg/mL庆大霉素的DMEM中，转移至10 cm培养板。感染后48 h（48 hpi），收集HeLa细胞，使用培养体系中的Ct感染新的HeLa细胞单层，培养基添加0.36 U/mL青霉素G以筛选转化子。将培养板置于37℃孵育48 h。每48 h重复一次收集与重新感染的步骤，直至观察到包涵体。

### 免疫荧光显微镜成像
感染前一天，将5×10^6个HeLa细胞铺于10 cm培养板中。使用Ct L2株，或经编码FtsK-mCherry、mCherry-PBP2、mCherry-PBP3、mCherry-MreC的质粒转化的沙眼衣原体菌株，以及经可组成型表达靶向pbp2或ftsK启动子的crRNA的质粒转化的菌株，感染DMEM培养的HeLa细胞。对于转化体相关实验，按照指定浓度与时间向感染细胞的培养基中添加aTc。感染后21 h（21 hpi），通过刮取收集10 cm培养板中的细胞，经30秒离心沉淀细胞。将沉淀重悬于1 mL 0.1×PBS（Gibco）中，转移至含0.5 mm玻璃珠（ThermoFisher Scientific）的2 mL离心管中。涡旋振荡细胞3 min，随后以800 rpm离心2 min。取20 μL上清液与20 μL 2×固定液（6.4%甲醛与0.044%戊二醛）混合，滴加至载玻片上室温孵育10 min。细胞用PBS漂洗3次，随后用含0.1% Triton X-100的PBS孵育1 min进行透化处理，再用PBS漂洗2次。对于Ct L2相关实验，细胞分别用抗主要外膜蛋白（MOMP；Meridian，田纳西州孟菲斯）的山羊源一抗，以及抗重组CT739蛋白制备的识别内源性FtsK的小鼠源一抗（https://doi.org/10.1099/mic.0.047746-0），或抗PBP2/PBP3肽段制备的兔源一抗（Ouellette SP 2012）进行孵育。一抗制备简要步骤：将沙眼衣原体抗原或肽段与弗氏不完全佐剂乳化，通过肌内注射免疫动物，每2周免疫一次，共免疫3次；末次免疫后2~4周收集免疫动物的抗血清作为一抗。一抗孵育完成后，用PBS漂洗细胞，随后分别用驴抗山羊IgG（Alexa 488）、驴抗小鼠IgG（Alexa 594）或驴抗兔IgG（Alexa 594）二抗（Invitrogen）孵育。在可视化各类mCherry融合蛋白的实验中，将mCherry荧光的定位与MOMP的分布进行对比。在部分实验中，通过用兔抗6×His抗体（Abcam，马萨诸塞州剑桥）与山羊抗MOMP抗体染色表达MreB_6x His融合蛋白的细胞，再使用对应二抗，确定MreB_6x His融合蛋白的分布。使用配备100×油浸PlanApochromat物镜与CCD相机的Zeiss AxioImager2显微镜进行成像。图像采集时，以0.3 μm的间距采集细胞上下区域的xy层面切片；成像时确保图像中最亮的位点处于饱和状态。使用Zeiss Axiovision 4.7软件中的最近邻算法对图像进行反卷积处理，随后使用Zeiss Zen-Blue软件查看与拼接反卷积后的图像。每个实验均设置3次独立重复，定位相关结果为3次实验的平均值。部分实例中，使用Zeiss Zen-Blue软件对采集的xy层面切片生成三维投影。

### 肽聚糖（PG）标记
按照此前报道的方法（Cox 2020），通过将细胞与4 mM乙烯-D-丙氨酸-D-丙氨酸（E-DA-DA）孵育实现PG标记。使用Click & Go™标记试剂盒（Vector Laboratories）对掺入的E-DA-DA进行荧光标记。将荧光标记的PG的分布与MOMP和内源性FtsK的分布，或与mCherry-PBP3的分布进行对比。每个实验均设置3次独立重复，结果为3次实验的平均值。

### 包涵体形成单位（IFU）检测
使用编码指定aTc诱导型基因的pBOMB4 Tet (-GFP)质粒转化的Ct（-pL2）感染HeLa细胞。感染后8 h（8 hpi），按照指定浓度向培养基中添加aTc，未诱导组作为对照。感染后48 h（48 hpi），通过刮取收集培养皿中的HeLa细胞，经离心收集沉淀。将沉淀重悬于1 mL 0.1×PBS（Gibco）中，转移至含0.5 mm玻璃珠的2 mL离心管（ThermoFisher Scientific）中。涡旋振荡细胞3 min，随后以800 rpm离心2 min。上清液与等体积的2×蔗糖-磷酸盐（2SP）溶液（ref）混合后，置于-80℃冻存。二次感染时，将衣原体在冰上解冻并涡旋振荡，以1000×g、4℃离心5 min沉淀细胞碎片。将上清液中的原体（EBs）进行梯度稀释，用于感染24孔板中的HeLa细胞单层。二次感染后于37℃培养24 h，随后通过与山羊抗MOMP一抗、驴抗山羊IgG二抗（Alexa Fluor 594）孵育进行固定与免疫荧光标记。用PBS漂洗细胞后，使用EVOS成像系统（Invitrogen）成像包涵体。计数5个视野中的包涵体数量并取平均值。每个实验设置3次独立重复，取重复实验的平均值计算包涵体形成单位数量。采用卡方检验比较诱导组与未诱导组的IFU水平。

### A22与mecillinam对Ct分裂中间体谱及PG、分裂体蛋白定位的影响
使用编码FtsK-mCherry、mCherry-PBP2、mCherry-PBP3、mCherry-MreC或MreB-6xHis的pBOMB4-Tet (-GFP)质粒转化的Ct感染HeLa细胞。在添加或不添加75 μM A22的条件下，于感染后20 h（20 hpi）用10 nM aTc诱导融合蛋白表达1 h。感染后22 h（22 hpi）收集细胞，按照前述方法制备样本进行染色。每个实验设置3次独立重复，定位相关结果为3次实验的平均值。

使用Ct L2株感染HeLa细胞，于感染后17 h（17 hpi）向培养基中添加20 μM mecillinam（Sigma），未处理组作为对照。感染后22 h（22 hpi）收集细胞，制备网状体（RBs），并用MOMP、FtsK、PBP2或PBP3抗体进行染色，操作同前述方法。另一组实验中，于感染后17 h（17 hpi）将细胞与4 mM EDA-DA孵育，同时添加或不添加20 μM mecillinam；感染后22 h（22 hpi）收集细胞并制备RBs，按照前述方法对PG进行Click标记，并在MOMP染色的细胞中可视化PG的分布。每个实验设置3次独立重复，定位相关结果为3次实验的平均值。

### 免疫印迹实验
使用Ct L2株感染的HeLa细胞于感染后24 h（24 hpi）通过刮取收集，以未感染的HeLa细胞作为对照。离心收集HeLa细胞，重悬于SDS上样缓冲液后，经10% SDS-聚丙烯酰胺凝胶电泳分离。将凝胶中的蛋白电转至硝酸纤维素膜（Schleicher and Schuell），用抗沙眼衣原体FtsK的小鼠多克隆一抗孵育膜片。漂洗后用800标记的驴抗小鼠IgG二抗（LICOR，内布拉斯加州林肯）孵育，使用LICOR Odyssey成像系统进行成像。

使用经可诱导表达FtsK-mCherry、mCherry-PBP2、mCherry-PBP3或mCherry-MreC的质粒转化的Ct感染HeLa细胞。于感染后17 h（17 hpi）向培养基中添加10 nM aTc诱导融合蛋白表达。感染后21 h（21 hpi）收集细胞并离心沉淀。将沉淀重悬于1 mL 0.1×PBS（Gibco）中，转移至含0.5 mm玻璃珠的2 mL离心管（ThermoFisher Scientific）中。涡旋振荡细胞3 min，随后以800 rpm离心2 min。收集上清液并以13000 rpm离心3 min，将含Ct的沉淀重悬于含1% TX-100、1×蛋白酶抑制剂混合物（Sigma）与5 μM lactacystin的TBS中。将悬浮液冰上超声处理3次，随后以13000 rpm离心3 min。收集上清液并加入SDS上样缓冲液，煮沸后经10% SDS-聚丙烯酰胺凝胶电泳分离，再电转至硝酸纤维素膜。用兔抗mCherry一抗（Invitrogen）与800标记的驴抗兔IgG二抗（LICOR，内布拉斯加州林肯）孵育膜片，使用LICOR Odyssey成像系统成像。

使用编码mCherry-PBP2或mCherry-PBP3的pBOMB4-Tet (-GFP)质粒转化的Ct感染HeLa细胞。于感染后17 h（17 hpi）用10 nM aTc诱导融合蛋白表达，未诱导组作为对照。感染后21 h（21 hpi）收集细胞，按照前述方法处理样本进行免疫印迹。用抗沙眼衣原体PBP2或PBP3肽段的兔多克隆一抗（Ouellette SP 2012）孵育膜片，漂洗后用800标记的驴抗兔IgG二抗（LICOR，内布拉斯加州林肯）孵育，使用LICOR Odyssey成像系统成像。