Role for cis -Acting RNA Sequences in the Temperature-Dependent Expression of the Multiadhesive Lig Proteins in Leptospira interrogans

Abstract

fThe spirochete Leptospira interrogans causes a systemic infection that provokes a febrile illness. The putative lipoproteins LigA and LigB promote adhesion of Leptospira to host proteins, interfere with coagulation, and capture complement regulators. In this study, we demonstrate that the expression level of the LigA and LigB proteins was substantially higher when L. interrogans proliferated at 37°C instead of the standard culture temperature of 30°C. The RNA comprising the 175-nucleotide 5= untranslated region (UTR) and first six lig codons, whose sequence is identical in ligA and ligB, is predicted to fold into two distinct stem-loop structures separated by a single-stranded region. The ribosome-binding site is partially sequestered in doublestranded RNA within the second structure. Toeprint analysis revealed that in vitro formation of a 30S-tRNA fMet -mRNA ternary complex was inhibited unless a 5= deletion mutation disrupted the second stem-loop structure. To determine whether the lig sequence could mediate temperature-regulated gene expression in vivo, the 5= UTR and the first six codons were inserted between the Escherichia coli L-arabinose promoter and bgaB (␤-galactosidase from Bacillus stearothermophilus) to create a translational fusion. The lig fragment successfully conferred thermoregulation upon the ␤-galactosidase reporter in E. coli. The second stem-loop structure was sufficient to confer thermoregulation on the reporter, while sequences further upstream in the 5= UTR slightly diminished expression at each temperature tested. Finally, the expression level of ␤-galactosidase was significantly higher when point mutations predicted to disrupt base pairs in the second structure were introduced into the stem. Compensatory mutations that maintained base pairing of the stem without restoring the wild-type sequence reinstated the inhibitory effect of the 5= UTR on expression. These results indicate that ligA and ligB expression is limited by double-stranded RNA that occludes the ribosome-binding site. At elevated temperatures, the ribosome-binding site is exposed to promote translation initiation.

Control of Gene Expression in Leptospira spp. by Transcription Activator-Like Effectors Demonstrates a Potential Role for LigA and LigB in Leptospira interrogans Virulence

“…Previous research showed that LigA and LigB are surface-exposed proteins (22) and that they play a role in the adhesion of spirochetes to extracellular matrix proteins, complement regulators, and animal cells (15,23,24). Furthermore, single-gene mutants of either ligA in L. interrogans serovar Manilae strain L495 or ligB in L. interrogans serovar Copenhageni strain Fiocruz L1-130 did not reduce virulence in standard animal models and remained recoverable from animal tissue (25,26).…”

“…A TALE gene specific for the leptospiral immunoglobulinlike (lig) genes ligA and ligB was designed to anneal to the sequence 5=-TCCAATAAATCTTAAGAGA-3=. This sequence corresponds to the Ϫ10 promoter region, which is a homologous region 170 bp upstream of the ϩ1 sites of ligA (lmanv2_30028) and ligB (lmanv2_350013) (15). Three biologically distinct TALE lig transformants were characterized: TALE lig1 , TALE lig2 , and TALE lig3 .…”

“…The DNA fragment containing the transcriptional fusions was released by PvuII digestion and cloned into the SmaI site of the E. coli-L. biflexa shuttle vector pMAT (14) to generate plasmid ptale ␤gal , which replicates in L. biflexa. Another TALE construct, named the TALE lig gene, was designed to anneal to the sequence 5=-TCCAATAAATCTTAAGAGA-3=, which is located in homologous promoter regions of ligA and ligB in L. interrogans 170 bp upstream of position ϩ1 (15). The TALE lig gene was inserted downstream of the B. burgdorferi flgB promoter (as described above), and the DNA fragment containing the fusion was released by ApaI and KpnI digestions (Thermo Scientific, Waltham, MA), gel purified (Qiagen, Venlo, Netherlands), and subcloned into the corresponding sites of pAL614 (16).…”

Oral Immunization with Escherichia coli Expressing a Lipidated Form of LigA Protects Hamsters against Challenge with Leptospira interrogans Serovar Copenhageni

“…LigA is an outer membrane protein exposed on and released from the leptospiral surface (22,23). Several lines of evidence suggest that ligA is upregulated during infection of the mammalian host, including induction of expression by physiologic osmolarity (23) and temperature (24) and an early humoral immune response to the Lig proteins during infection (25). Several groups have reported that immunization with the LigA unique region, consisting of domains 7 to 13 (LigA7-13), confers protection from intraperitoneal (i.p.)…”

Identificação e avaliação de novas adesinas em <i>Leptospira interrogans</i> por <i>shotgun phage display</i>

“…indicam uma origem comum e revelaram uma grande divergência nos membros de Bartonella (LEHMANN et al, 2013) A família DUF_61 também está presente em bactérias patogênicas responsáveis por doenças gástricas e enterohepáticas em animais e/ou humanos, como espécies não pilóricas do gênero Helicobacter, espécies de Campylobacter e a espécie Piscirickettsia salmonis (O"ROURKE et al, 2001;FRYER & HEDRICK, 2003;KERSULYTE et al, 2013;MÉNARD et al, 2014;KAAKOUSH et al, 2015).…”