Construction of Shine-Dalgarno and leaderless M. tuberculosis reporter strains
To study the role of leaderless translation in M. tuberculosis response to stress and infection, we first devised a system to easily quantify translation in M. tuberculosis by engineering a promoterless firefly luciferase (FFluc) encoding vector that could be used to generate suitable integrative translational reporters. As backbone we used the mycobacterial integrating vector pMV30622 with five copies of the transcriptional terminator trp23. We then cloned the ffluc coding region codon-optimized for M. tuberculosis from pMV306hsp+FFluc24 and removed the optimised Shine-Dalgarno sequence to create pTC1 (Supplementary figure 1A). Bioluminescence production in the resulting strain was not significantly different to that in the H37Rv wild-type strain with no ffluc gene, indicating that there was no background transcription and translation of the ffluc gene in the promoterless vector (Supplementary Figure 1B).
To compare translational efficiency between leaderless and Shine-Dalgarno transcripts, we selected the desA1 (Rv0824c) and desA2 (Rv1094) gene pair which encode homologous acyl-ACP desaturases sharing 30% primary sequence identity. Both genes are strongly expressed during exponential growth and have a typical TANNNT -10 promoter motif; however, while desA1 contains a 5’UTR with the Shine-Dalgarno sequence, desA2 is expressed as a leaderless transcript15. We obtained the Shine-Dalgarno reporter construct pTC2 by fusing the desA1 50 bp promoter region, the 5’UTR and the region encoding the first six amino acids of desA1 to ffluc in pTC1 (Figure 1A); similarly, we generated the leaderless reporter construct pTC3 by fusing the desA2 50 bp promoter region and the region encoding the first six amino acids of desA2 to ffluc in pTC1 (Figure 1A). To make sure that any differences observed between the Shine-Dalgarno and leaderless reporters were due to translational regulation and not to transcriptional regulation, we swapped the 50-bp desA1 and desA2 promoters in pTC2 and pTC3 to create the Shine-Dalgarno reporter pTC4 and the leaderless reporter pTC5, respectively (Figure 1A). All reporter sequences were verified by sequencing and plasmids were subsequently electroporated in M. tuberculosis. Successful integration was verified by polymerase chain reaction (PCR) followed by sequencing. Additionally, we verified that transcription of the reporters was driven by the desired promoter by determining the transcriptional start sites using 5’ rapid amplification of cDNA ends (RACE). Finally, we verified translation of the FFluc reporter by measuring luminescence of the reporter strains over a 5-day period of exponential growth as an indirect measurement of protein expression. All reporter strains showed comparable levels of luminescence suggesting they have comparable levels of luciferase production (Figure 1B). As a result, we selected the Shine-Dalgarno pTC2 and leaderless pTC5 reporter pair, for which pTC2 contains a naturally occurring canonical Shine-Dalgarno organisation for further experiments.
The leaderless reporter is robustly translated in M. tuberculosis during exponential and stationary in vitro growth
Our results so far suggested that the leaderless reporter was translated with similar efficiency to that of the Shine-Dalgarno reporter at least during exponential growth (Figure 1B). To identify possible differences in the translation efficiencies of the Shine-Dalgarno and leaderless reporters that could be associated with the growth status of the bacteria we used pTC2 and pTC5 to closely monitor luminescence production during the switch from exponential to stationary growth. As a control, we also measured translation of the reporters in M. smegmatis as experiments using fluorescent reporters have shown that leaderless translation in M. smegmatis, a close relative of M. tuberculosis that also encodes a high percentage of leaderless proteins in its genome15,16, has similar efficiency to that of a Shine-Dalgarno reporter21. Additionally, we measured translation of the reporters in E. coli, a bacterium with scarce leaderless transcripts that are translated at low levels by the 70S monosome25.
Introduction of the reporter vectors did not affect bacterial growth in any of the three bacterial models tested (Figure 2A-C). As predicted, during exponential growth luminescence production from the leaderless reporter in E. coli was 1 log lower than that from the Shine-Dalgarno reporter (one-way ANOVA, p <0.0001; Figure 2D and 2G). By contrast, in M. smegmatis luminescence from the leaderless reporter was significantly higher than that from the Shine-Dalgarno reporter (one-way ANOVA, p <0.001) (Figure 2E and 2H), whereas in M. tuberculosis luminescence production of the leaderless reporter was comparable to that of the Shine-Dalgarno reporter (Figure 2F and 2I). Upon entrance into stationary growth, median luminescence levels increased for both reporters in E. coli, but the increase was not statistically significant in the case of the leaderless reporter (Figure 2G). In M. smegmatis, the translation of the Shine-Dalgarno reporter significantly decreased whilst the median levels of leaderless translation were maintained (one-way ANOVA, p <0.0001) (Figure 2H). In the case of M. tuberculosis, Shine-Dalgarno translation was not significantly reduced upon entrance into stationary growth, while leaderless translation was significantly increased (one-way ANOVA p <0.0001) (Figure 2I).
In summary, our results show a robust translation of the leaderless reporter during exponential growth in M. tuberculosis, to a level comparable to that of the Shine-Dalgarno reporter, and a significant increase in leaderless translation upon entrance into stationary growth.
The preference for translation of the leaderless reporter varies during different in vitro stresses in M. tuberculosis
Our results indicated that the ratio of translation of leaderless and Shine-Dalgarno transcripts differed during exponential and stationary phase in M. tuberculosis. Next, we studied whether this was also the case during stress conditions. In particular, we determined the translation of leaderless and Shine-Dalgarno reporters in M. tuberculosis during nutrient starvation and following a transient stress with a sub-lethal concentration of NO. These two conditions are representative of conditions encountered by M. tuberculosis during non-replicating growth26 and active infection in humans27,28, respectively. In each condition tested, we addressed translation kinetics of the leaderless and Shine-Dalgarno reporters by measuring luminescence produced from the pTC2 and TC5 reporter vectors and quantified changes at the transcriptional level through quantitative real-time PCR to rule out changes due to differences in promoter activities.
For the nutrient starvation experiments, cells growing exponentially in nutrient-rich media were washed with PBS and used to inoculate (time 0). PBS (nutrient starvation) and nutrient-rich media (7H9 media, control) cultures were incubated for 28 days. The control cultures grew exponentially for 5 days before reaching stationary phase (Figure 3A). These cultures reached stationary phase 2 days earlier than the cultures in Figure 2C, likely because of the higher starting OD (see Material and methods). As expected, nutrient limitation affected the growth and translation efficiency of both the leaderless and Shine-Dalgarno M. tuberculosis reporter strains compared to those in the control cultures (Figures 3A and 3B). In both the control and the nutrient starvation cultures, the leaderless reporter was translated at a higher level than the Shine-Dalgarno reporter (multiple t-tests, p <0.03) (Figure 3B). When comparing changes in the translation of the reporters during nutrient starvation compared to that during control conditions, we found a significant decrease in the translation of the Shine-Dalgarno reporter compared to that of the leaderless reporter after 24 hours of nutrient starvation (multiple t-tests, p < 0.001) (Figure 3C). This difference was not driven by transcriptional changes, as transcription levels of the Shine-Dalgarno and leaderless reporters did not change significantly (Tukey’s multiple comparisons test, p >0.163) (Figure 3D). This more pronounced effect of the stress on Shine-Dalgarno-mediated translation was transient, as we observed comparable reduction of translation of both types of reporters at later timepoints (Figure 3C).
Next, we studied changes in translation during exposure to a sub-lethal concentration of NO by monitoring changes in luminescence in exponentially growing cultures of the M. tuberculosis leaderless and Shine-Dalgarno reporter strains exposed to 0.25 mM NO or sodium hydroxide (used to dissolve the NO adduct, see Material and methods) for 7 days. As expected, exposure to NO caused a rapid but transient growth arrest of both the Shine-Dalgarno and leaderless reporter strains that could be observed already at 24 hours post-NO exposure (Figure 4A). Throughout the experiment, the leaderless reporter was translated at a higher level than the Shine-Dalgarno reporter in both the stressed and the control cultures (multiple t-tests, p <0.03) (Figure 4B). Indeed, although the transient inhibition in growth resulted in a decrease in translation of both reporters compared with the corresponding controls, both reporters were affected to a similar degree and hence translation of the leaderless reporter was higher than that of the Shine-Dalgarno reporter (Figures 4B and 4C). Forty-eight hours after NO treatment started, both reporter strains resumed growth and luminescence suggesting that they had recovered from the stress (Figures 4A and 4B). At this point, the percentage of translation of the leaderless reporter in comparison with that in the corresponding control was significantly higher than that of the Shine-Dalgarno reporter (27% vs 14%, respectively, multiple t-tests, p <0.002), suggesting a quicker recovery of leaderless translation following exposure to NO stress (Figure 4C). These differences were not driven by transcriptional changes, as we found no significant differences in the transcription levels of Shine-Dalgarno and leaderless transcripts (Tukey’s multiple comparisons test, p >0.162) (Figure 4D).
Overall, the results show that leaderless translation is significantly less affected by nutrient starvation and recovers more quickly from nitrosative stress than Shine-Dalgarno translation.
Translation of the leaderless reporter is less affected during the early stages of macrophage infection
Finally, we studied translation efficiency of Shine-Dalgarno and leaderless transcripts during M. tuberculosis intracellular growth in macrophages. To this end we measured luminescence of the reporter strains during infection of PMA-activated THP-1 cells. We used an MOI of 10 to ensure enough luminescence from the reporters was detected.
Following infection of THP-1 cells, no increase in luminescence and growth was observed for the reporter strains during the first 24 hours (Figure 5A and 5B). At 48 hours post infection, both reporter strains resumed growth and translation, suggesting that they had adapted to the intracellular environment. Luminescence from the leaderless reporter was higher than that from the Shine-Dalgarno reporter and this difference was significant between 24 and 72 hours post infection (multiple t-tests, p <0.002) (Figure 5B). To quantify how translation of the leaderless and Shine-Dalgarno reporters responded during the different stages of macrophage infection, we normalised the luminescence levels of the reporters at different time points against the luminescence levels at time 0. This revealed that during the first 48 hours of infection, before bacterial growth resumed, translation of the leaderless reporter was less affected than that of the Shine-Dalgarno reporter, and this difference was statistically significant at 48 hours post infection (multiple t-test, p <0.05) (Figure 5C). As growth resumed following adaptation to the intracellular environment, the translation efficiency of both types of transcripts increased to comparable levels (Figure 5C). Our data show that during the early stages of macrophage infection, the translation efficiency of the leaderless reporter is significantly less affected by exposure to the intracellular host environment than that of the Shine-Dalgarno.