The Bifunctional Enzyme SpoT Is Involved in the Clarithromycin Tolerance of Helicobacter pylori by Upregulating the Transporters HP0939, HP1017, HP0497, and HP0471

Abstract

Clarithromycin (CLA) is a commonly recommended drug for Helicobacter pylori eradication. However, the prevalence of CLA-resistant H. pylori is increasing. Although point mutations in the 23S rRNA are key factors for CLA resistance, other factors, including efflux pumps and regulation genes, are also involved in the resistance of H. pylori to CLA. Guanosine 3′-diphosphate 5′-triphosphate and guanosine 3′,5′-bispyrophosphate [(p)ppGpp)], which are synthesized by the bifunctional enzyme SpoT in H. pylori, play an important role for some bacteria to adapt to antibiotic pressure. Nevertheless, no related research involving H. pylori has been reported. In addition, transporters have been found to be related to bacterial drug resistance. Therefore, this study investigated the function of SpoT in H. pylori resistance to CLA by examining the shifts in the expression of transporters and explored the role of transporters in the CLA resistance of H. pylori. A ΔspoT strain was constructed in this study, and it was shown that SpoT is involved in H. pylori tolerance of CLA by upregulating the transporters HP0939, HP1017, HP0497, and HP0471. This was assessed using a series of molecular and biochemical experiments and a cDNA microarray. Additionally, the knockout of genes hp0939, hp0471, and hp0497 in the resistant strains caused a reduction or loss (the latter in the Δhp0497 strain) of resistance to CLA. Furthermore, the average expression levels of these four transporters in clinical CLA-resistant strains were considerably higher than those in clinical CLA-sensitive strains. Taken together, our results revealed a novel molecular mechanism of H. pylori adaption to CLA stress.

Bifunctional Enzyme SpoT Is Involved in Biofilm Formation of Helicobacter pylori with Multidrug Resistance by Upregulating Efflux Pump Hp1174 ( gluP )

“…Studies have shown that (p)ppGpp is involved in bacterial tolerance to various antibiotics, such as penicillin ( 41 ), vancomycin ( 42 ), and ampicillin ( 43 ); in H. pylori , (p)ppGpp is synthesized from SpoT ( 21 ). Our previous studies discovered that (p)ppGpp modulates the expression of H. pylori efflux transporters ( 44 ), accompanied by the importance of the efflux pump to multidrug resistance ( 24 ); thus, we wondered whether (p)ppGpp participates in the multidrug resistance of H. pylori . This research confirmed that inference.…”

“…To determine bacterial shape and viability, planktonic H. pylori bacteria were stained with membrane-permeant and membrane-impermeant fluorescent dyes from LIVE/DEAD BacLight bacterial viability kits (Molecular Probes, Invitrogen, USA). Then, they were observed by confocal microscopy, which was performed as described in a previous study ( 44 ). For biofilm-forming cells, the NC membrane with the biofilm was removed from the agar plate and then gently washed three times with PBS.…”

“…(p)ppGpp production was assayed according to the method described in a previous study with slight modification ( 44 ). The treatment of the planktonic bacteria was the same as previously described ( 44 ).…”

“…(p)ppGpp production was assayed according to the method described in a previous study with slight modification ( 44 ). The treatment of the planktonic bacteria was the same as previously described ( 44 ). The biofilm bacteria were washed with PBS, diluted to an OD 600 of 0.2, and incubated for an additional 2 h. When all the strains reached an OD 600 of approximately 0.3, samples from each culture plate were centrifuged at 10,000 rpm for 5 min and resuspended in 250 μl of liquid culture medium.…”

“…In our previous studies, we successfully constructed an spoT mutant strain (the Δ spoT strain) ( 62 ) and an spoT -complemented strain (the spoT* strain) ( 44 ). The plasmids (pILL570 and pUC18K2) used to construct the mutant strain were kindly provided by Agnès Labigne (Unité de Pathogénie Bactérienne des Muqueuses, Institut Pasteur).…”