Exposure of Mycobacterium marinum to low-shear modeled microgravity: effect on growth, the transcriptome and survival under stress

Abstract

Waterborne pathogenic mycobacteria can form biofilms, and certain species can cause hard-to-treat human lung infections. Astronaut health could therefore be compromised if the spacecraft environment or water becomes contaminated with pathogenic mycobacteria. This work uses Mycobacterium marinum to determine the physiological changes in a pathogenic mycobacteria grown under low-shear modeled microgravity (LSMMG). M. marinum were grown in high aspect ratio vessels (HARVs) using a rotary cell culture system subjected to LSMMG or the control orientation (normal gravity, NG) and the cultures used to determine bacterial growth, bacterium size, transcriptome changes, and resistance to stress. Two exposure times to LSMMG and NG were examined: bacteria were grown for ~40 h (short), or 4 days followed by re-dilution and growth for ~35 h (long). M. marinum exposed to LSMMG transitioned from exponential phase earlier than the NG culture. They were more sensitive to hydrogen peroxide but showed no change in resistance to gamma radiation or pH 3.5. RNA-Seq detected significantly altered transcript levels for 562 and 328 genes under LSMMG after short and long exposure times, respectively. Results suggest that LSMMG induced a reduction in translation, a downregulation of metabolism, an increase in lipid degradation, and increased chaperone and mycobactin expression. Sigma factor H (sigH) was the only sigma factor transcript induced by LSMMG after both short and long exposure times. In summary, transcriptome studies suggest that LSMMG may simulate a nutrient-deprived environment similar to that found within macrophage during infection. SigH is also implicated in the M. marinum LSMMG transcriptome response.

Figure 1

Growth under LSMMG alters culture density of M. marinum. LHM4 and 1218R was grown in 10 ml HARVs under NG or LSMMG for 4 days continuously and the OD₆₀₀ determined. The data from 13 HARVs for LHM4 and 3 HARVs for 1218R for each condition was used to obtain the average and s.d. (a). OD₆₀₀ was monitored for LHM4 (b) and 1218R (c) in 50 ml HARVs. At each time point, a small culture volume (1–2 ml) was removed to measure OD₆₀₀ and the volume replaced with fresh medium (2–4% total volume). Strain 1218R was also grown in medium with catalase (d). Three independent experiments were performed for each strain and growth condition. As data were not gathered at the exact time point in each experiment, all the data points were pooled for a strain and condition and are shown graphically (b–d). A transition from exponential phase was detected at ~37 h in LSMMG cultures compared with NG cultures. The data between 37 and 70 h for the three experiments were plotted (e, f) and regression analysis performed, demonstrating that the increase in OD₆₀₀ during this time period was statistically different when the bacteria were grown under NG and LSMMG (P<0.0001).

Figure 2

Gene expression changes identified by RNA-Seq. Transcriptional profiles were compared between 1218R grown at LSMMG and NG for 40 h (a, LSMMG/NG) or 4 days ~35 h (b, LSMMG/NG) and between 1218R grown under LSMMG for the two time periods (c, 4 days ~35 h/40 h). The significant (P<0.05) changes in transcript levels were identified. The real fold change (RFC) is the ratio of the normalized mean count values for LSMMG divided by the normalized mean count values for NG. The genes that changed ⩾1.5-fold were assigned to categories (1–9) and the distribution of the RFC is shown graphically. No genes were significantly altered ⩾1.5-fold in the insertion sequences and phage category and so this category is not included. The mean value for the RFC for a category is marked by the black spot and the number of genes in the category is shown in parentheses.

Figure 3

Growth under LSMMG increases expression of genes involved in lipid degradation and mycobactin synthesis but decreases expression of genes involved in mycolic acid synthesis. Genes involved in the designated categories lipid degradation (a), mycolic acid synthesis (b) and mycobactin synthesis (c) were identified using the mycobrowser website (http://mycobrowser.epfl.ch/) and the literature. The FPKMs (fragments per kilobase of transcript per million reads) for each gene in the 12 RNA samples were analyzed using Genesifter. Only genes with ⩾1.5-fold change in expression (P<0.05) were considered. The data were log transformed. The heat maps were generated showing expression in short (growth for ~40 h) and long (growth for 4 days ~35 h) exposure samples. Genes colored brown are discussed in the manuscript text.

Figure 4

Changes in expression of SigH-regulated genes show a linkage to growth under LSMMG. Mehr et al. treated wild-type and sigH mutant M. tuberculosis with diamide and determined SigH-related gene expression changes after 0 and 30 min recovery time. Peak sigH transcript was at 30 min. M. marinum orthlogs of these genes were identified (http://mycobrowser.epfl.ch/). The FPKMs (fragments per kilobase of transcript per million reads) for each gene in the 12 RNA samples were analyzed for hierarchical clustering using GeneSifter with a threshold of 1.5-fold change and a statistical difference (P<0.05) determined using an ANOVA. The data were log transformed. The heat maps show expression changes during LSMMG and NG for the orthologs of the M. tuberculosis genes that changed after 0 (a) and 30 min (b). Genes colored brown are discussed in the text.

Figure 5

The effect of LSMMG on genes involved in the enduring hypoxic response (EHR) or nutrient starvation response. Genes involved in these two stress responses have been identified in M. tuberculosis.^, The M. marinum orthologs were identified (http://mycobrowser.epfl.ch/) for the EHR (a) and relA nutrient starvation (b) response (Supplemental Data Set 1 and section 3 in ref. 43). The FPKMs (fragments per kilobase of transcript per million reads) for each gene in the 12 RNA samples were analyzed for hierarchical clustering using GeneSifter with a threshold of 1.5-fold change and a statistical difference (P<0.05) determined using an ANOVA analysis. The data were log transformed. Genes colored brown are discussed in the text.

Figure 6

LHM4 grown under LSMMG are more sensitive to H₂O₂ but not gamma radiation or acid in comparison with NG cultures. Bacteria were grown in HARVs under LSMMG or NG for ~40 h (short exposure time) or 4 days ~35 h (long exposure time). Cultures were removed and treated with gamma radiation using a ¹³⁷Cesium source (a), H₂O₂ (b), or growth medium at pH 3.5 (c). Bacteria were grown on solid medium and colonies counted. The percent survival was calculated by dividing the colony number/ml by that obtained for a culture treated with no dose at the same time. At least three experiments were performed for each culture and treatment type, except for the short exposure time LSMMG and NG cultures treated with H₂O₂ where two experiments were performed. The average and s.d. are shown. Survival was compared between the LSMMG and NG samples using the area under the curve and the only statistically significant difference was between the long exposure LSMMG culture and the long exposure NG culture treated with H₂O₂ (*P=0.004).