Two novel transcriptional reporter systems for monitoring Helicobacter pylori stress responses

Highlights

• Screening of H. pylori cryptic plasmids allowed for the identification of candidates to construct novel vectors.

• Two E. coli/H. pylori shuttle vectors carrying promoterless gfp gene have been constructed to create transcriptional fusions.

• Creating various transcriptional fusions enabled monitoring the H. pylori response to diverse environmental changes.

Abstract

Helicobacter pylori, a human pathogen linked to many stomach diseases, is well adapted to colonize aggressive gastric environments, and its virulence factors contribute this adaptation. Here, we report the construction of two novel H. pylori vectors, pSv2 and pSv4, carrying a reporter gene fused to the promoters of virulence factor genes for monitoring the response of single H. pylori cells to various stresses. H. pylori cryptic plasmids were modified by the introduction of the Escherichia coli origin of replication, chloramphenicol resistance cassette, and promoterless gfp gene to produce E. coli/H. pylori shuttle vectors. The promoter regions of vacA and ureA genes encoding well-characterized H. pylori virulence factors were fused to the promoterless gfp gene. Recording the GFP fluorescence signal from the genetically modified H. pylori cells immobilized in specifically designed microfluidic devices revealed the response of transcriptional reporter systems to osmotic stress, acidic stress, elevated Ni²⁺ concentration or iron chelation. Our observations validate the utility of the pSv2 and pSv4 vectors to monitor the regulation of virulence factor genes in diverse strains and clinical isolates of H. pylori.

Introduction

Helicobacter pylori is a human pathogen that primarily colonizes stomach (Marshall and Warren, 1984). More than half of the world's population is infected by this pathogenic bacterium (British Society of Gastroenterologists, 2019). Development of chronic gastritis, peptic ulcers, gastric cancer, and some other stomach diseases has been shown to be associated with H. pylori colonization (Marshall and Warren, 1984; Seo et al., 2013). H. pylori is a gram-negative spiral-shaped bacterium with four to six polar flagella (Marshall and Warren, 1984). It localizes primarily on the surface of the gastric epithelium. H. pylori cells are 2.5–5.0 μm long with a diameter of 0.5–1.0 μm. The ability to colonize the gastric environment makes this bacterium unique. A number of genetic mechanisms support survival of H. pylori in the aggressive environment of the host stomach. The orchestrated regulation of gene expression in H. pylori cells facilitates its survival in the dynamic gastric environment.

Elucidating how H. pylori regulates virulence factor genes in response to environmental changes is crucial for understanding the pathogenesis of the diseases associated with this bacterium. Genome sequencing revealed a limited number of gene regulation mechanisms in H. pylori. (Alm et al., 1999a). Another common approach to studying gene regulation is the use of reporter systems. However, molecular tools for delivering reporter systems into H. pylori cells are still limited because of the restriction barrier of transformation. Cryptic plasmids seem to be promising vehicles for the delivery of reporter systems into H. pylori cells. Cryptic plasmids are assumed to significantly contribute to the genetic heterogeneity of H. pylori strains. Approximately a half of known H. pylori strains have been shown to contain cryptic plasmids which vary in size from 2 to approximately 100 kb (Penfold et al., 1988). However, the functions of these plasmids are still largely unknown. Recently, several small (<6 kb) plasmids of H. pylori were investigated in detail (Heuermann and Haas, 1995; Kleanthous et al., 1991; Minnis et al., 1995). The analysis of their sequences has allowed for dividing these plasmids into two distinct groups: (i) plasmids that are similar to pHPK255 (Heuermann and Haas, 1995) and reveal homology to plasmids of gram-positive bacteria with “rolling circle” replicating mechanism, and (ii) the iteron-containing plasmids (pHPM180, pHel1, and pHPS1) replicating via mechanism of the theta structure formation (De Ungria et al., 1999).

Modification of the cryptic plasmid pHP489 (Kleanthous et al., 1991) to construct an H. pylori-Escherichia coli shuttle vector was first used to obtain a vector for complementation in H. pylori cells. Later, the pHel1 cryptic plasmid and its derivatives pHel2 and pHel3 were used for genetic modification of H. pylori cells to create fluorescent “marker strains” by transcriptional fusions with the reporter gene gfp (Heuermann and Haas, 1998). Reporter genes lacZ and gfp have also been integrated into the bacterial chromosome using transposon mutagenesis (Bijlsma et al., 1999) to monitor gene expression in H. pylori. However, this technique is complex and provides bacterial cell transformation with low efficiency.

Therefore, in this study, we report the development and examination of two novel transcriptional reporter systems based on cryptic plasmids for monitoring the H. pylori response to various stress types. We modified two cryptic plasmids obtained from H. pylori clinical isolates to construct E. coli/H. pylori shuttle vectors for the expression of the gfp gene fused with H. pylori promoter regions sensitive to different stress types. To verify the utility of our reporter systems, we designed a microfluidic chip, which has previously been described (Belova et al., 2018). The fluorescent signal of GFP was recorded from single H. pylori cells attached to the surface of the channel in a microfluidic device.