The Rho-Independent Transcription Terminator for the porA Gene Enhances Expression of the Major Outer Membrane Protein and Campylobacter jejuni Virulence in Abortion Induction

Abstract

Campylobacter jejuni is a leading cause of foodborne illnesses worldwide. Its porA gene encodes the major outer membrane protein (MOMP) that is abundantly expressed and has important physiological functions, including a key role in systemic infection and abortion induction in pregnant animals. Despite the importance of porA in C. jejuni pathogenesis, mechanisms modulating its expression levels remain elusive. At the 3′ end of the porA transcript, there is a Rho-independent transcription terminator (named TporA in this study). Whether TporA affects the expression and function of MOMP remains unknown and is investigated in this study. Green fluorescent protein (GFP) fusion constructs with the porA promoter at the 5′ end and an intact TporA or no TporA at the 3′ end of the gfp coding sequence revealed that both the transcript level of gfp and its fluorescence signals were more than 2-fold higher in the construct with TporA than in the one without TporA. Real-time quantitative PCR (qRT-PCR) analysis of the porA mRNA and immunoblot detection of MOMP in C. jejuni showed that disruption of TporA significantly reduced the porA transcript level and the expression of MOMP. An mRNA decay assay demonstrated that disruption of TporA resulted in a shortened transcript half-life of the upstream gfp or porA gene, indicating that TporA enhances mRNA stability. In the guinea pig model, the C. jejuni construct with an interrupted TporA was significantly attenuated in abortion induction. Together, these results indicate that TporA enhances the expression level of MOMP by stabilizing its mRNA and influences the virulence of C. jejuni.

Critical Role of 3′-Downstream Region of pmrB in Polymyxin Resistance in Escherichia coli BL21(DE3)

“…Given that the TOP10 derivatives, JL1371 and JL1381, which carry pmrB-pmrA ORF and pmrB ORF, respectively, displayed the same susceptibility to colistin (MIC = 0.5 µg/mL), the 176 bp downstream sequence of pmrB clearly played a critical role in PmrB-mediated colistin resistance in BL21. It was reported that the 3′-untranslated region (UTR) of a specific gene may form secondary structures, consequently affecting the expression and function of the preceding genes [ 30 , 38 ]. Analysis of 3′-UTR of pmrB using RNAfold WebServer [ 39 ] revealed multiple stem-loop structures ( Figure 1 B).…”

“…Since a significantly low level of pmrB BL21 mRNA was observed in JL1507 when compared to that in JL1509 ( Figure 2 A), the 3′-UTR length may modulate and determine the stability of pmrB BL21 mRNA, as observed in the regulation of other genes [ 30 , 38 ]. To test this hypothesis, the half-life of pmrB in JL1507 and JL1509 was further measured by rifampin arrest treatment followed by qRT-PCR.…”

“…Genomic DNA was removed with DNase I (Qiagen) digestion, and RNA was re-purified using an RNeasy mini kit (Qiagen). Subsequent qRT-PCR analysis was carried out as previously described [ 28 , 30 ]. 16S rRNA gene was targeted as an internal control using primers 16S-F and 16S-R ( Table 2 ).…”

“…TOP10 derivative strains JL1507 and JL1509, which contain pUS150-PmrB-DS86 and pUS150-PmrB-DS126, respectively, were used to determine the pmrB mRNA decay rate (half-life) using the rifampin arrest method as previously described [ 30 ] with some modifications. Briefly, E. coli overnight cultures were inoculated into fresh medium containing kanamycin (30 μg/mL), and grew to logarithm phase (OD 600nm = 0.5–1.0) in an orbital shaker (250 rpm) at 37°C.…”

“…The qRT-PCR data used to calculate the pmrB mRNA half-life were analyzed using GraphPad Prism software (GraphPad, San Diego, CA, USA). The mRNA half-life was determined by one-phase decay using a nonlinear regression model, and the Kolmogorov–Smirnov statistic was used to determine if differences in distributions were significant, as previously described [ 30 ]. Levels of significance for p value are 5% (0.05).…”