Characterisation of worldwide Helicobacter pylori strains reveals genetic conservation and essentiality of serine protease HtrA.

HtrA proteases and chaperones exhibit important roles in periplasmic protein quality control and stress responses. The genetic inactivation of htrA has been described for many bacterial pathogens. However, in some cases such as the gastric pathogen Helicobacter pylori, HtrA is secreted where it cleaves the tumour-suppressor E-cadherin interfering with gastric disease development, but the generation of htrA mutants is still lacking. Here, we show that the htrA gene locus is highly conserved in worldwide strains. HtrA presence was confirmed in 992 H. pylori isolates in gastric biopsy material from infected patients. Differential RNA-sequencing (dRNA-seq) indicated that htrA is encoded in an operon with two subsequent genes, HP1020 and HP1021. Genetic mutagenesis and complementation studies revealed that HP1020 and HP1021, but not htrA, can be mutated. In addition, we demonstrate that suppression of HtrA proteolytic activity with a newly developed inhibitor is sufficient to effectively kill H. pylori, but not other bacteria. We show that Helicobacter htrA is an essential bifunctional gene with crucial intracellular and extracellular functions. Thus, we describe here the first microbe in which htrA is an indispensable gene, a situation unique in the bacterial kingdom. HtrA can therefore be considered a promising new target for anti-bacterial therapy.

Morphologic, genetic, and biochemical characterization of Helicobacter magdeburgensis, a novel species isolated from the intestine of laboratory mice.

BACKGROUND: The presence of enterohepatic Helicobacter species (EHS) is commonly noted in mouse colonies. These infections often remain unrecognized but can cause severe health complications or more subtle host immune perturbations and therefore can confound the results of animal experiments. The aim of this study was to isolate and characterize a putative novel EHS that has previously been detected by PCR screening of specific-pathogen-free mice. MATERIALS AND METHODS: Biochemical analysis of enzyme activities (API campy), morphologic investigation (Gram-staining and electron microscopy) and genetic analyses (16SrRNA and 23SrRNA analyses, DNA fingerprinting, restriction fragment polymorphisms, and pulsed-field gel electrophoresis) were used to characterize isolated EHS. Genomic DNA fragments were sequenced to develop a species-specific PCR detection assay. RESULTS: Scanning electron microscopy revealed the presence of spiral-shaped EHS, which varied in length (2.5-6 µm) and contained single monopolar or single bipolar sheathed flagella. The bacteria were grown under anaerobic conditions, preferably on agar plates containing serum or blood. The 16SrRNA, genetic, and biochemical analyses indicated the identification of a novel EHS species, named Helicobacter magdeburgensis. We also examined the genome content using pulsed-field gel electrophoresis. Based on the pattern produced by two restriction enzymes, BamIII and KspI, the genome size was determined to be about 1.7-1.8 Mbp. CONCLUSION: We isolated and characterized a novel EHS species, H. magdeburgensis, morphologically, biochemically, and genetically. These results are important for future studies on the prevalence and pathophysiologic relevance of such infections. Our PCR assay can be used to detect and discriminate H. magdeburgensis from other Helicobacter species.