CHAPTER 3 Analysis of L. interrogans selective gene transcripts in the presence of
3.4 RESULTS
3.4.6 Analysis of the gene LA4185/LIC13341 transcription at physiological
As mentioned in section 3.4.3, LA4185/LIC13341 transcripts could not be detected from the cDNA of L. interrogans, although it could be amplified from the genomic DNA of L.
interrogans. To check for the transcription of LA4185/LIC13341 in the other pathogenic serovars of Leptospira, a PCR using the genomic DNA of L. interrogans serovar Copenhageni strain Fiocruz L1-130, L. interrogans serovar Lai strain Lai, L. interrogans serovar Canicola strain Hond Utrecht IV, L. interrogans serovar Pomona, and L. interrogans serovar Bataviae was set up. An amplified fragment of 1,065 bp indicated the presence of LA4185/LIC13341 in all the L. interrogans serovars examined (Fig. 3.15A). In contrast, no amplification of LIC13341 was observed in the non-pathogenic L. biflexa serovar Patoc strain Patoc 1. However, flaB, a constitutive gene of 849 bp amplified in all the tested pathogenic and non-pathogenic serovars of Leptospira. A reverse transcription-polymerase chain reaction (RT-PCR) was performed for amplifying LIC13341 from the cDNA
synthesized from the various serovars of L. interrogans. The amplification of flaB gene could be observed but no transcription of LA4185/LIC13341 could be validated in the pathogenic Leptospira serovars (Fig. 3.15B).
Figure 3.15. Molecular analysis of LA4185/LIC13341. Using PCR and RT-PCR, the presence of LA4185/LIC13341 and its transcription was evaluated in pathogenic and non-pathogenic serovars of Leptospira, respectively. (A) PCR of LA4185/LIC13341 using genomic DNA as the template of pathogenic and non-pathogenic serovars of Leptospira. An amplicon size of 1,065 bp confirmed the existence of LA4185/LIC13341 in pathogenic Leptospira serovars Copenhageni, Lai, Canicola, Pomona and Bataviae (Lane 2, 4, 6, 8 & 10). The constitutive gene, flaB (849 bp) was used as a positive control for determining the quality of DNA (Lane 1, 3, 5, 7, 9 & 11). No amplification of LA4185/LIC13341 was observed in the non-pathogenic serovar (Patoc) of Leptospira (Lane 12). (B) RT-PCR was performed using the cDNA synthesized from the mid-log phase culture of pathogenic Leptospira serovars to investigate transcripts of LA4185/LIC13341 grown in EMJH medium at 29°C. No transcription of LA4185/LIC13341 were detected in the pathogenic serovars of Leptospira grown in EMJH medium at 29°C (Lanes with the even number) whereas flaB gene transcripts could be detected (Lanes with the odd number).
It is now well documented that environmental factors such as temperature and osmolarity can influence the transcription of many genes of L. interrogans (Atzingen et al., 2008; Oliveira et al., 2010). To investigate if host factors such as osmolarity and temperature could induce the transcription of LA4185/LIC13341, L. interrogans was grown in the presence of physiological osmolarity and temperature. Induction of LA4185/LIC13341 transcription
under physiological osmolarity was assessed by growing Leptospira cultures at 29°C in EMJH medium supplemented with 10% rabbit serum and re-suspending mid-log phase culture in fresh EMJH medium (control) or in EMJH medium containing 120 mM NaCl for an overnight period. The addition of 120 mM NaCl (ionic osmolarity) to the medium mimics physiological conditions (~300 mOsm) encountered by leptospires upon entry into the host (Matsunaga et al., 2007a; Oliveira et al., 2011). We also evaluated gene transcripts of LA4185/LIC13341 from in vitro mid-log phase culture subjected to temperature upshifts from 29ºC to 37ºC of culture during an overnight period to simulate conditions experienced by leptospires in the early stages of infection and during febrile stage as described previously (Oliveira et al., 2011). Transcript analysis of LA4185/LIC13341 of L. interrogans grown in vitro at physiological osmolarity or temperature (37ºC) did not lead to detection of LA4185/LIC13341 transcripts. To validate the quality of the cDNA synthesized in our study, some genes previously shown to respond to physiological osmolarity or temperature were selected. We were able to reproduce the differential gene transcription of previously reported genes LIC10464 (ligB) (Matsunaga et al., 2007a), LIC11335 (groEL) (Nally et al., 2001c) and LIC10314 (lsa63) (Vieira et al., 2010c) under physiological osmolarity or temperature (37°C) using qRT-PCR (Fig. 3.16A and Fig. 3.16B). Thus, we were unable to switch on the transcription of LA4185/LIC13341 in the presence of physiological osmolarity or temperature used in our present study.
Figure 3.16. Validation of gene transcription from the cDNA of L. interrogans grown at physiological osmolarity or temperature using previously reported genes responding to osmolarity or temperature. (A) The qRT-PCR was used to quantify LIC10464 (ligB) and LIC10314 (lsa63) transcripts which are known to be differentially expressed at physiological osmolarity (120 mM NaCl) at 29°C. Transcription of target genes were quantified and normalized with 16S rRNA using 2-∆∆Ct method. At physiological osmolarity, there was an upregulation in the transcription of LIC10464 (ligB) whereas, a downregulation of the transcription was observed for LIC10314 (lsa63). (B) Evaluation of gene transcription of LIC11335 (groEL) and LIC10314 (lsa63) under physiological temperature (37 C) culture condition of Leptospira. The shift in temperature from 29°C to 37°C under in vitro culture condition leads to upregulation of groEL and downregulation of lsa63 transcription. The gene transcription is depicted as number of copies of the gene transcribed per 1000 copies of 16S rRNA. Bars denote the mean standard deviation from 2 independent qRT-PCR analyses.
3.4.7 Analysis of the gene LA4185/LIC13341 transcription using low passage L.
interrogans
It had been previously reported that in L. interrogans, there can be a loss in the virulence of the bacteria on continuous in vitro passaging. To check if this may be the reason for not detecting the transcription of LA4185/LIC13341, we procured low passaged strains of
Leptospira. Interestingly, as is evident from Fig. 3.17, we were able to demonstrate the transcription of LA4185/LIC13341 in all the low passage pathogenic serovars of L.
interrogans tested.
Figure 3.17. Molecular analysis of LA4185/LIC13341 in low passage L. interrogans. (A) PCR was performed for LA4185/LIC13341 gene using genomic DNA as the template of low passage pathogenic and non-pathogenic serovars of Leptospira. An amplicon size of 1,065 bp confirmed the existence of LA4185/LIC13341 in pathogenic Leptospira serovars Copenhageni, Lai, Canicola, Pomona and Bataviae (Lane 2, 4, 6, 8 & 10). The constitutive gene, flaB (849 bp) was used as a positive control for determining the quality of DNA (Lane 1, 3, 5, 7, 9 & 11). No amplification of LA4185/LIC13341 was observed in the non-pathogenic serovar (Patoc) of Leptospira (Lane 12). (B) RT-PCR was performed using the total RNA isolated from the mid-log phase culture of low passage pathogenic Leptospira serovars to investigate transcripts of LA4185/LIC13341 grown in EMJH medium at 29°C (Lanes with the even number) whereas flaB gene transcripts could be detected (Lanes with the odd number).
3.4.8 Analysis of the LA1939/LIC11966 transcription at physiological osmolarity