Role of urease in megasome formation and Helicobacter pylori survival in macrophages

Abstract

Previous studies have demonstrated that Helicobacter pylori (Hp) delays its entry into macrophages and persists inside megasomes, which are poorly acidified and accumulate early endosome autoantigen 1. Herein, we explored the role of Hp urease in bacterial survival in murine peritoneal macrophages and J774 cells. Plasmid-free mutagenesis was used to replace ureA and ureB with cat in Hp Strains 11637 and 11916. ureAB null Hp lacked detectable urease activity and did not express UreA or UreB as judged by immunoblotting. Deletion of ureAB had no effect on Hp binding to macrophages or the rate or extent of phagocytosis. However, intracellular survival of mutant organisms was impaired significantly. Immunofluorescence microscopy demonstrated that (in contrast to parental organisms) mutant Hp resided in single phagosomes, which were acidic and accumulated the lysosome marker lysosome-associated membrane protein-1 but not early endosome autoantigen 1. A similar phenotype was observed for spontaneous urease mutants derived from Hp Strain 60190. Treatment of macrophages with bafilomycin A1, NH 4 Cl, or chloroquine prevented acidification of phagosomes containing mutant Hp. However, only ammonium chloride enhanced bacterial viability significantly. Rescue of ureAB null organisms was also achieved by surface adsorption of active urease. Altogether, our data indicate a role for urease and urease-derived ammonia in megasome formation and Hp survival.

Helicobacter pylori as a trigger and aggravating factor in rheumatoid arthritis and other rheumatic diseases

“…Но H. pylori имеет мощный механизм защиты от су-пероксид-анионов, в частности, связанный с экспрессией ферментов каталазы и супероксиддисмутазы. Кроме того, установлено, что уреаза изменяет рН в фагосомах, что спо-собствует выживанию H. pylori в макрофагах [33]. По неко-торым данным, эта бактерия не только выживает, но и раз-множается в макрофагах, эпителиальных и дендритных клетках [34].…”

A role for altered phagosome maturation in the long-term persistence ofHelicobacter pyloriinfection

“…Despite this engulfment, H. pylori appears to be able to avoid intracellular killing. For example, after phagocytizing H. pylori, neutrophils become activated, but the ingested bacteria are not always eliminated (10,123,124), and H. pylori has been shown to survive for up to 48 h within monocytes and macrophages (14,29,110,126,130,153). There is increasing evidence that H. pylori facilitates this survival by altering phagosome maturation to attenuate the destructive effects of the phagosome.…”

“…The appearance of megasomes was reported to be characteristic of type 1 H. pylori strains (14,153), whereas LCVs were observed in macrophages, regardless of the H. pylori strain's vacA or cag status (30,126). The study of isogenic mutant strains of H. pylori has demonstrated that VacA and urease are essential for megasome formation (130,153). Megasomes and LCVs appear to arise 2-4 h after internalization and persist for Ն24 h (14,30,126,130,153).…”

“…During normal phagosome maturation, phagosomes progressively recruit vacuolar-type H ϩ -ATPase (vATPase) to the phagosomal membrane, which decreases pH from 6.1-6.5 in the early phagosome to 4.5 in the phagolysosome (56). It is likely that H. pylori resists the phagosome's increasing proton concentration in the same way that it tolerates the high acid conditions encountered in the stomach lumen: with ureasemediated production of bicarbonate and ammonium ions (130) (Fig. 3).…”

“…3). However, H. pylori appears to generate a hybrid compartment with reduced degradative capacity that retains characteristics of early-stage phagosomes and phagolysosomes (30,75,130,153) (Fig. 3).…”