Bacterial strains and culture conditions
Yersinia pestis microtus strain 201 is isolated from Microtus brandti and is extremely lethal to mice but avirulent to humans and other large animals[31]. Y. pestis wild-type (WT) strain 201 is reserved in our lab, and used in this study. In general, Y. pestis is grown in lysogeny broth (LB) medium or agar plate at 26 °C or 37 °C, unless otherwise specified. Resuscitated Y. pestis microtus strain 201 was inoculated at a dilution of 1:20 into the HARV bioreactors. When HARVs were filled with ~58ml fresh LB medium and the bubbles were removed, the low shear force simulated microgravity (LSMMG) cultivation condition is generated by the HARVs’ axis of rotation perpendicular to gravity direction, in contrast, the normal gravity (NG) cultivation condition is generated by the axis parallel to gravity direction[4]. There is a silicified gas-permeable membrane on the back of the HARVs, which can provide oxygen supply and prevent the leakage of liquid during bacterial culture.
Under the condition of 26 °C and rotary speed of 25 rpm, Y. pestis was respectively grown to the middle exponential phase (OD620nm=1.0) for 24 hours under the SMG and NG conditions, then diluted to new HARV bioreactors for 40 consecutive passages of cultivation.
Crystal violet staining
The biofilm formation ability of Y. pestis is assessed by the crystal violet staining as follows. With the cultures being removed, the bioreactors were washed lightly with Phosphate Buffer Saline (PBS) and then were stained with 0.1% crystal violet solution for 20 min at indoor temperature.
In order to quantitatively analyze the difference of biofilm formation ability between SMG and NG group, the cultures were taken on a 24-well plate at a dilution of 1:100 and incubated for 24 hours on a shaker at 37 °C and 200 rpm. The optical density (OD) values were measured for each well at 620 nm. Removed the bacterial solutions thoroughly and put the 24-well plate at 80℃ for 15 min to fix the biofilm. Each well was dyed with 0.1% crystal violet solution for 15 min and washed three times with PBS. Each well was added with 2 mL dimethyl sulfoxide solution to dissolve the crystal violet, and measured the OD values at 570 nm. Finally, the ratios of OD570nm to OD620nm of each well were calculated.
RAW 264.7 cells intracellular survival assay
We intended to choose the RAW 264.7 cells evaluate the bacterial intracellular survival ability. The cultures grown to OD620nm = 1.0 under SMG and NG groups were added to 24-well plates that contains 70-80% single-layer adherent RAW 264.7 cells at a MOI of ~5. At 0.5 hour past infection (hpi), gentamycin was added to kill the extracellular bacteria and the infected cells were lysed by Triton-X solution. Then living bacteria counting of the lysate was performed on the agar plates in triplicate. The above operations were repeated at 4, 6, 8 hpi.
HeLa cells cytotoxicity/rounding assay
HeLa cells were selected to evaluate the cytotoxicity of Y. pestis. Following the published methods for the HeLa cell rounding/cytotoxicity assay[12], saturated cultures of the two groups were added to infect the HeLa cells in six-well plates for 2.5 hours at a MOI of ~50. The HeLa cells cytotoxic effects were evaluated by observing the cell rounding phenotype through the microscopes.
However, considering that HeLa cells may be rounded due to crowded growth and errors existed in observing cell morphological changes with naked eyes, we intended to utilize the xCELLigence real-time cell analysis (RTCA, ACEA Biosciences Inc) system to analyze the dynamic and quantitative response profile of HeLa cells in vitro, and this technology is non-invasive and label-free. Cellular changes including cell number (cell index, CI) are recorded and analyzed via the RTCA system [13, 32, 33]. The principle of the RTCA system is to read the values of electronic impedance from the gold-plated sensor electrodes located on the bottom of microplates(E-plate). Electronic readings changes as the HeLa cells adhere or detach to the surface of the electrodes, cell index (CI) values are drawn via the software calculation[34].
HeLa cells were added into the 8-well microplates (E-plate) and cultured at 37 °C with 5% CO2 for 12 hours. The bacteria grown under SMG and NG conditions were added into the E-plate for 24 hours at a MOI of ~5. The cell index data was collected and analyzed by the RTCA Data Analysis Software 1.0, and then the data was reanalyzed per 2 hours from 3-hour to 36-hour point using GraphPad Prism 5.0 software.
BALB/c mice infection assay
Saturated cultures grown under SMG and NG conditions were centrifuged at the rotary speed of 4500rpm, discarding the supernatant, and diluted to a concentration of one hundred microliters containing 100CFU or 10CFU with phosphate-buffered saline (PBS). The actual number of bacteria contained per 100 microliters is counted through dropping on the Hottinger’s Agar plates. We randomly divided 32 8-week-old BALB/c female mice into 4 groups, and the diluted bacteria of 40th passage were inoculated into each group with intraperitoneal route. The infected mice were monitored for 14 consecutive days and recorded the number of dead mice daily. The survival curves were drawn with GraphPad Prism 5.0 software, and the p-values less than 0.05 were considered statistically significant through log-rank (Mantel-Cox) test. The BABL/c mice used in this experiment were provided by Beijing Vital River Laboratory Animal Technology Co. Ltd [laboratory animal permit no. SCXK (Jing) 2016-0006]. All mice participating in this experiment were treated in accordance with the guidelines for the welfare and ethics of laboratory animals.
RNA-seq-based transcriptional analysis
The cultures in SMG and NG bioreactors were grown to saturation (OD620nm=1.0) as mentioned before. Each group was setting as three biological replicates. The mRNAs were obtained by removing the ribosomal RNA (rRNA) from the total RNA extracted by the PureLink™ RNA Mini Kit (Invitrogen, Thermo Fisher Scientific, USA), and then used for creating cDNA library and deep sequencing. According to the values of FPKM (Fragments Per Kilobase Million), the ratio of transcript levels between SMG and NG groups were used as the logarithm to the base of 2 (Two-Fold Change), for example, a two-fold change value of 1.0 indicates 2-fold greater expression of a certain gene. The differential values at least a 2-fold were applied to analyze the differential expression of genes according to the Y. pestis 91001 genome annotation.
Quantitative real-time PCR
The total RNA was extracted as described above, and purified by TURBO DNA-free ™ Kit (Invitrogen, Thermo Fisher Scientific, USA) upon the manufacturer’s instructions. The total RNAs were reverse transcribed into cDNA as templates by SuperSript™ Reverse Transcription Kit (Invitrogen, Thermo Fisher Scientific, USA). All primer pairs were designed to produce amplicons with expected sizes of 50-200 nt through the Primer Premier 5.0 software. Meanwhile, the highly expressed 16S ribosomal RNA gene was chose as the reference gene to draw the relative standard curve, in order to quantitatively compare and analyze differential expression levels of the target genes. The quantitative PCR (qPCR) was carried out in the Light Cycler R480 quantitative PCR instrument (Roche, CA, USA) and its software would calculate the relative fold change of target genes in the RNA samples of SMG and NG groups.