HeLa cells were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) at 37°C in 5% CO₂. C. trachomatis serovar D at an MOI of 1 was added and incubated with the cells by centrifugation at 3400×g at 37°C for 1 hour. The samples were then incubated for another hour at 37°C in 5% CO₂. Extracellular bacteria were removed by aspirating the supernatant followed by the addition of fresh medium supplemented with 10% FBS and 0.5% glucose, and the cells were maintained under the same conditions until they were harvested at different time points.

For persistent infection, the procedures were the same as mentioned above except for the addition of 100 U/ml penicillin (Lukang Pharmaceutical Co., Ltd., Shandong, China) to the medium after centrifugation (Skilton et al., 2009). Uninfected cells cultured in medium with/without 100U/ml penicillin were used as controls.

In this study, we used the following antibodies: goat polyclonal to Chlamydia trachomatis MOMP coupled to FITC (Abcam, ab30951, USA); rabbit polyclonal anti-RAB14 (Abcam, ab28639, USA); rabbit monoclonal anti-GM130 (Abcam, ab52649, USA); rabbit polyclonal anti- GOLGA5/Golgin-84 (Abcam, ab224040, USA); rabbit polyclonal anti-Akt (pan) (Cell Signaling, 4685, USA); rabbit polyclonal anti-phosphorylated Akt (Ser-473) (Cell Signaling, 4060, USA); rabbit polyclonal anti-AS160 (Affinity, AF7630, USA); rabbit polyclonal anti-phosphorylated AS160 (Ser-318) (Affinity, AF2317, USA); rabbit polyclonal anti-GAPDH (Cell Signaling, 2118, USA); goat anti-rabbit HRP-conjugated IgG (ABclonal, AS007, China), and goat anti-rabbit Cy3-labeled IgG (ABclonal, AS007, China). To activate Akt, cells were pretreated with SC79 (Beyotime, SF2730, China) for 30 min. To inhibit Akt, Akt Inhibitor VIII (iAkt) (Beyotime, SF2730, China) was added to the culture medium at the indicated post-infection (p.i.) time. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, France) was used as a control for SC79 and iAkt.

For immunofluorescence staining, cells were fixed with 4% paraformaldehyde, permeabilized in 0.5% (v/v) Triton X-100 (Sigma–Aldrich, USA), and then blocked with 1% (w/v) BSA (Thermo Scientific, USA). Cells were then incubated with the primary antibody for 1.5h, washed in PBS, and incubated with Cy3-coupled secondary antibodies. Finally, the samples were counterstained with DAPI (Beyotime, China). Immunofluorescence images were acquired using a fluorescence microscope (Olympus, Japan).

Uninfected and infected HeLa cells were lysed in RIPA buffer (Thermo Scientific, USA) supplemented with protease and phosphatase inhibitors (KeyGEN, China) at the indicated time points, and the protein concentrations were determined using the Pierce BCA kit (Thermo Scientific, USA). Total proteins samples (15 g/lane) were separated on a reducing SDS-PAGE gel and then transferred onto a PVDF membrane. The membrane was incubated overnight with a primary antibody to detect the desired proteins. Protein loading was assessed using rabbit polyclonal anti-GAPDH (1:1000). The membranes were then washed and incubated with goat anti-rabbit HRP-conjugated IgG (1:5000) and developed using the Immobilon ECL Ultra Western HRP Substrate (Millipore, WBKLS0100, USA) in an ImageQuant LAS4000 (GE Healthcare Life Sciences, Japan).

Total RNA was extracted from infected HeLa cells with or without penicillin at the indicated time points using TRIzol reagent (Invitrogen, USA). The yield of RNA was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). Reverse transcription reactions were performed using Evo M-MLV RT Premix for qPCR (Accurate Biotechnology Co., Ltd., China) according to the manufacturer’s instructions. Quantitative PCR was performed with SYBR^® Green Premix Pro Taq HS qPCR Kit II (Accurate Biotechnology Co., Ltd., China) targeting the chlamydial 16s rRNA gene. PCR cycling conditions were as follows: initial denaturation for 2 min at 95°C, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s. Expression levels of the target gene were calculated using 2^−ΔΔCt, relative to the reference gene (GAPDH), allowing for the calculation of fold change relative to the control. The primers for 16s rRNA used were (Eszik et al., 2016): forward: 5’-CACAAGCAG TGGAGCATGTGGTTT-3’, reverse: 5’-ACTAACGATAAGGGTTGCGCTCGT-3’.

Infected cells were lysed at 48h p.i. The cell lysate was centrifuged for 10 minutes at 500×g to remove cell debris, and diluted 10-fold. The lysates were titrated with fresh HeLa cells. After 48h, the number of inclusions formed by chlamydial progeny was assessed by microscopic analysis at ×200 magnification. The average number of inclusion forming units/mL (IFUs/mL) was calculated for each sample and experimental condition.

HeLa cells were grown on either 6-well plates or 24-well plates until they reached 30-50% confluence, and transfected with siRNA-Rab14 or control-siRNA (100nM final concentration) (RiboBio, China) using riboFECT™ CP Reagent (RiboBio, China). At 72h post-transfection, cells were infected with C. trachomatis, as described above, and incubated for the indicated periods for further manipulation. We used immunoblotting to confirm the decrease in Rab14 protein expression at 72h post-transfection.

Cell viability was assessed using the Cell Counting kit-8 (CCK-8) (Servicebio, G4103, China). Briefly, 5000 HeLa cells per well were seeded into 96well plates. The cells were treated with different chemicals and incubated for different times to evaluate the toxicity of the chemicals. CCK-8 reagent was added to the wells and incubated at 37°C for 2 h. Absorbance was measured at 450 nm using a microplate reader (BioTek 800TS, USA).

GraphPad Prism 9 (GraphPad Software, La Jolla, CA, USA) was used for graphing and data analysis was performed using SPSS 25.0 (SPSS Inc, Chicago, IL, USA). Quantitative data were presented as means ± standard deviation (SD). The quantitative data were tested for normality using the Shapiro-Wilk test. Kruskal-Wallis followed by Dunn’s multiple comparisons test was used for evaluating of inclusion area. One-way ANOVA with Bonferroni’s multiple comparisons test was used for evaluation of infectivity assay and CCK-8 assay. Differences were considered significant at *P<0.05, ** P<0.01, *** P<0.001, ****P<0.0001.

It has been described that Akt is phosphorylated during the entire chlamydial developmental cycle (Capmany et al., 2019). Here, we detected the phosphorylation level of Akt at different post-infection times of acutely or persistently infected cells using immunoblotting analysis. Due to the induction of Akt phosphorylation by a component of FBS (Watton and Downward, 1999), we first examined the level of Akt phosphorylation treated with FBS deprivation for different time periods, and the data are shown in Figure S1 . Based on these results, we decided to perform the experiment after 4 h of FBS starvation. Phosphorylated Akt (pAkt) and total Akt expression levels in uninfected cells treated without/with penicillin remained steady. Figure 1A shows the typical immunoblotting results for the infected and uninfected cells. Figure 1B shows the ratio of pAkt in acutely or persistently infected cells, relative to uninfected cells, at different post-infection times based on three independent experiments. In acutely infected cells, phosphorylated Akt levels were elevated progressively since 36h p.i. Moreover, there were two peaks of phosphorylation in persistently infected cells: at the middle (8 h p.i.), and later stages (36 h p.i.) of the bacterial developmental cycle.

The level of pAkt in persistently infected cells was lower than that in acutely infected cells, at the same post-infection time except at 4h p.i. and 36h p.i. The dominance of pAkt in acutely infected cells was observed in the later stages of the developmental cycle (48h p.i. to 72h p.i.). Total Akt expression levels in two infection states remained unaltered with the time of infection. In addition, we detected the phosphorylation level of Akt Substrate of 160 kDa (AS160), and compared the level of phosphorylated AS160 (pAS160) between infected cells and uninfected cells, at different post-infection times respectively ( Figure S2 ). pAS160 level were observed to vary in a time-dependent manner, similar to those of pAkt. Taken together, our findings revealed that Akt phosphorylation is dynamic during different stages of chlamydial infection, and the level had a robust decrease in persistent infection.

To determine whether there was a difference in Rab14 recruitment between persistent infection and acute infection during the developmental cycle, we detected the intracellular distribution of endogenous Rab14 in cells of two infection states, at different post-infection times. Uninfected cells cultured without/with penicillin were used as control groups (control and control P).

Exposure to penicillin did not alter the Rab14 distribution in uninfected cells. However, although Rab14 presented dispersedly at the early stage (4h p.i.) of both infection states, Rab14 recruitment became associated to the inclusions and delimited the border of inclusions as the infection was processed. In the acute state, Rab14 tended to surround the inclusions since the 24h p.i. and gradually showed a rim-like staining pattern ( Figure 2A ). This indicates that Rab14 is closely associated with inclusions in acutely infected cells. In contrast, the chlamydial inclusions became smaller in the cells with persistent chlamydial infection induced by penicillin. In addition, the rim-like shape pattern of Rab14 was observed in the later stages of development (40h p.i to 72h p.i), but to a lesser extent ( Figure 2B ). However, our experiments might imply the potential pattern of Rab14 recruitment in acutely and persistently infected cells because we did not show the GTP-loading Rab14.

As Akt phosphorylation is associated with chlamydial infection, we next aimed to confirm the effect of Akt phosphorylation on the bacterial development and infectivity in the presence of SC79, an Akt activator, and Akt Inhibitor VIII (iAkt). First, we confirmed the non-toxicity of SC79, and the dose-dependent toxicity of iAkt to HeLa cells by CCK-8 assay ( Figures S3A, B ). Next, we detected the effects of SC79 and iAkt on Akt phosphorylation by immunoblotting and found that they both exert dose-dependent effects ( Figures S3C, D ). Consequently, we used 4μg/mL SC79 or 5μM iAkt in subsequent experimental procedures. DMSO was used as the control.

HeLa cells were pretreated with either DMSO or 4μg/mL SC79 for 30 minutes, and then infected with C. trachomatis (MOI 1) and cultured for 48h without/with penicillin. The inclusion size was enlarged in cells with SC79 treatment, for both infection states ( Figures 3A, B ). The infectivity of bacterial progeny, and the expression of the chlamydial 16s rRNA gene in SC79-pretreated cells with acute infection was increased, whereas that in cells with the same treatment in persistent infection was not changed ( Figures 3C, D ). These data indicate that activation of Akt phosphorylation could promote the growth of inclusions even under the suppression by penicillin, but not the developmental cycle.

A previous report demonstrated a reduction of bacterial internalization caused by Akt inhibition (Capmany et al., 2019). Thus, we added iAkt at 2 h p.i. to avoid interference with internalization. HeLa cells were infected with C. trachomatis (MOI 1) and then exposed to either DMSO or 5μM iAkt and cultured for 48h without/with penicillin. We observed that the inclusions became smaller in cells exposed to iAkt in both two infection states ( Figures 3E, F ). The infectivity and the expression of the chlamydial 16s rRNA gene decreased significantly with either iAkt treatment or penicillin alone, or iAkt + penicillin treatment ( Figures 3G, H ). These findings indicate that iAkt had an effect similar to that of penicillin, and the combination of the two treatments exerted synergistic repressive effects in chlamydial infection.

To determine the effect of Rab14 in acute and persistent chlamydial infection, we knocked down the expression of Rab14 in the HeLa cells using short interfering RNA (siRNA) transfection. HeLa cells were transfected with 100nM of chemically synthesized siRNA targeting Rab14, or a negative control siRNA. We used immunoblotting to confirm the efficacy of gene knockdown to Rab14 (reaching 70%) ( Figure S4A ), and used the CCK-8 assay to confirm the non-toxicity of siRNA to the HeLa cells ( Figure S4B ). At 72h post-transfection, cells were infected with C. trachomatis and cultured for 48h without/with penicillin. The cells were fixed and the chlamydial inclusions were detected using a goat polyclonal antibody targeting C. trachomatis MOMP coupled to FITC. In acutely infected cells, knockdown of Rab14 reduced the enlargement of chlamydial inclusions. In contrast, no changes occurred in the persistently infected cells, suggesting that Rab14 had a limited effect on the development of the inclusion in persistent infection ( Figures 4A, B ). We next collected the infectious particles from the cells and then titrated them on fresh HeLa cells by counting IFUs. The infectivity of bacterial progeny was reduced in acutely infected cells after Rab14-silencing. Nevertheless, knockdown of Rab14 did not change the bacterial infectivity, or the expression of the chlamydial 16s rRNA gene in persistent infection ( Figures 4C, D ). Taken together, these results indicate that Rab14 exerted limited effects on persistent chlamydial infection. Therefore, we suspect that the effects of Akt phosphorylation on Chlamydia development may not be mediated by Rab14 in persistent chlamydial infection.

We have previously discovered that persistent chlamydial infection causes little Golgi fragmentation (Zhu et al., 2014). Our current results showed that Akt phosphorylation has an impact on persistent chlamydial infection, and we wondered whether alteration of Akt phosphorylation level interferes with Chlamydia-induced Golgi fragmentation. We first examined Golgi morphology using immunofluorescence. Exposure to SC79, iAkt, or penicillin did not change Golgi apparatus structure in uninfected cells. The Golgi element showed a strip-like dispersed distribution surrounding inclusions in acutely infected cells, and this phenomenon was further decreased with SC79 pretreatment. In persistently infected cells, the Golgi apparatus is closely associated with the inclusions and the cell nucleus, and have a compact structure, but were observed in low amounts in SC79-pretreated cells ( Figure 5A ). However, when acutely infected cells were treated with iAkt, the Golgi apparatus remained a condensed structure similar to the persistently infected cells. Moreover, the combination of iAkt and penicillin caused an obvious reduction in chlamydial inclusion size with little Golgi fragmentation ( Figure 5B ).

We next assessed the amount and size of golgin-84, a Golgi matrix protein, in infected and uninfected cells treated with either SC79 or iAkt by immunoblotting. Golgin-84 was processed into two distinct fragments (78 kDa and 65 kDa) in the lysates of acutely infected cells, but the smaller fragment could hardly be seen in persistently infected cells. Strikingly, golgin-84 could be further processed in the infected cells with SC79 treatment but less in cells inhibited by iAkt ( Figures 5C, D ).

Taken together, our results suggests that Chlamydia-induced Golgi fragmentation could be regulated by Akt phosphorylation and further strengthens the hypothesis that Golgi fragmentation can influence C. trachomatis infections.