Characterization of an acidic-pH-inducible stress protein (hsp70), a putative sulfatide binding adhesin, from Helicobacter pylori

Abstract

The in vitro glycolipid binding specificity of the gastric pathogen Helicobacter pylori is altered to include sulfated glycolipids (sulfatides) following brief exposure of the organism to acid pH typical of the stomach. This change is prevented by anti-hsp70 antibodies, suggesting that hsp70 may be a stress-induced surface adhesin, mediating sulfatide recognition. To facilitate investigation of the role of hsp70 in attachment, we have cloned and sequenced the H. pylori hsp70 gene (dnaK). The hsp70 gene was identified by probing a cosmid DNA library made from H. pylori 439 with a PCR amplicon generated with oligonucleotides synthesized to highly conserved regions of dnaK. The 1.9-kb H. pylori hsp70 gene encodes a product of 616 amino acids. Primer extension analysis revealed a single transcription start site, while Northern blot analysis established that hsp70 was preferentially induced by low pH rather than by heat shock. The ability of H. pylori to alter its glycolipid binding specificity following exposure to low pH by upregulating hsp70 and by expressing hsp70 on the bacterial surface may provide a survival advantage during periods of high acid stress.

FIG. 1

(A) DNA sequence of H. pylori hsp70. Indicated are the transcriptional start site (+1), a putative Shine-Dalgarno (SD) sequence, and −35 and −10 putative promoters. The 1,851-bp ORF starts at an ATG codon (underlined); a potential rho-independent transcription terminator sequence (doubly underlined) and a stop codon (underlined TA) are also shown. (B) Deduced amino acid sequence of H. pylori hsp70. Asterisks represent the positions of stop codons. (C) Amino acid sequence comparison between H. pylori hsp70 and homologs from Salmonella typhimurium (S.typh.), E. coli, and H. influenzae (H. flu). Amino acids 153 to 158 and 366 to 373 were used in the design of PCR primers. Identical amino acids are boxed, and the consensus sequence is shown in bold.

FIG. 2

Primer extension analysis of the transcriptional start site of the H. pylori hsp70 gene. A [γ-³²P]ATP-labeled oligonucleotide complementary to the 5′ end of H. pylori hsp70 was hybridized to 25 and 50 μg of total RNA (lanes 1 and 2, respectively). Lanes T, G, C, and A are products of sequencing reactions using the same oligonucleotide as the primer. The start site at position −62 (P) is indicated on the right. The upstream sequence of the antisense strand is shown in Fig. 1A.

FIG. 3

Northern blot analysis of hsp70 expression after stress. H. pylori mRNA was isolated before and after heat shock or acid pH shock. Northern hybridization was performed with 5 μg of total RNA per lane. A 50-mer synthetic oligonucleotide probe designed from the hsp70 gene was used as a probe. Lanes: 1, mRNA from H. pylori incubated at 37°C (control); 2, heat-shocked (at 42°C) organisms; 3, acid pH-shocked (at pH 2.0) organisms. The hybridization bands were quantitated by densitometry. The hsp mRNA was increased 5.9-fold following pH shock relative to heat-shocked organisms. Under the assay conditions, transcription in nonstressed organisms could not be detected.

FIG. 4

Comparison of ³⁵S metabolic labeling and Western blotting using anti-hsp antibodies to identify pH 2.5-induced stress proteins. Each lane contained 5 μg of total protein extracted with 1% SDS. (A) SDS-PAGE. Lane 1, prestained MW standards (from top to bottom, phosphorylase [111 kDa], bovine serum albumin [77 kDa], ovalbumin [48.2 kDa], carbonic anhydrase [33.8 kDa], soybean trypsin inhibitor [28.6 kDa], and lysozyme [20.5 kDa]); lanes 2 and 3, total protein stained with Coomassie blue; lanes 4 and 5, autoradiograms corresponding to lanes 2 and 3. Lanes 2 and 4, control H. pylori; lanes 3 and 5, acid pH-shocked H. pylori. (B) Western blotting using anti-hsp60 antibody. Lanes 1 and 2, immunostain; lanes 3 and 4, autoradiograms of lanes 1 and 2, respectively. Lanes 1 and 3, control H. pylori; lanes 2 and 4, pH-shocked H. pylori. (C) Western blotting using anti-hsp70 antibody. Lanes 1 and 2, immunostain; lanes 3 and 4, autoradiograms of lanes 1 and 2, respectively. Lanes 1 and 3, control H. pylori; lanes 2 and 4, pH-shocked H. pylori.