Abstract
Modification of bacterial surface structures, such as the lipid A portion of lipopolysaccharide (LPS), is used by many pathogenic bacteria to help evade the host innate immune response. Helicobacter pylori, a gram-negative bacterium capable of chronic colonization of the human stomach, modifies its lipid A by removal of phosphate groups from the 1- and 4′-positions of the lipid A backbone. In this study, we identify the enzyme responsible for dephosphorylation of the lipid A 4′-phosphate group in H. pylori, Jhp1487 (LpxF). To ascertain the role these modifications play in the pathogenesis of H. pylori, we created mutants in lpxE (1-phosphatase), lpxF (4′-phosphatase) and a double lpxE/F mutant. Analysis of lipid A isolated from lpxE and lpxF mutants revealed lipid A species with a 1 or 4′-phosphate group, respectively while the double lpxE/F mutant revealed a bis-phosphorylated lipid A. Mutants lacking lpxE, lpxF, or lpxE/F show a 16, 360 and 1020 fold increase in sensitivity to the cationic antimicrobial peptide polymyxin B, respectively. Moreover, a similar loss of resistance is seen against a variety of CAMPs found in the human body including LL37, β-defensin 2, and P-113. Using a fluorescent derivative of polymyxin we demonstrate that, unlike wild type bacteria, polymyxin readily associates with the lpxE/F mutant. Presumably, the increase in the negative charge of H. pylori LPS allows for binding of the peptide to the bacterial surface. Interestingly, the action of LpxE and LpxF was shown to decrease recognition of Helicobacter LPS by the innate immune receptor, Toll-like Receptor 4. Furthermore, lpxE/F mutants were unable to colonize the gastric mucosa of C57BL/6J and C57BL/6J tlr4 -/- mice when compared to wild type H. pylori. Our results demonstrate that dephosphorylation of the lipid A domain of H. pylori LPS by LpxE and LpxF is key to its ability to colonize a mammalian host.
Highlights
Helicobacter pylori, a gram-negative bacterium with only one welldefined niche, the human stomach, can persist for several years without manifestation of symptoms
Previous work in our laboratory identified and characterized the Kdo2-lipid A modification machinery in H. pylori, including LpxE (1-phosphatase), EptA, KdoH1/2 (Kdo Hydrolase) and LpxR (39-O-deacylase), which all act in an ordered efficient manner to produce a single lipid A species found on the bacterial surface (Figure 2) [19,20,22]
H. pylori is a great example, producing a highly modified form of lipid A that aids in camouflaging the bacterium from the innate immune response of its host allowing it to set up a life long chronic infection of the gastric mucosa
Summary
Helicobacter pylori, a gram-negative bacterium with only one welldefined niche, the human stomach, can persist for several years without manifestation of symptoms. Similar to most gram-negative bacteria the outer membrane of H. pylori is composed of lipopolysaccharide (LPS), a surface exposed glycolipid. LPS is anchored to the bacterial membrane by its hydrophobic lipid A domain. The first sugar of the core, Kdo (3-deoxy-D-manno-octulosonic acid), serves as a bridge to link the lipid anchor to the carbohydrate domains of LPS. H. pylori shows great diversity in expression of its O-antigen [3], achieving a form of molecular mimicry by assembling surface polysaccharides resembling human blood group antigens, contributing to its ability to evade immune detection [4]. MIC are reported as mg/ml and are the average of three experiments using Polymyxin B E-Test strips (Biomerieux) on TSA supplemented with 5% blood. MIC are reported as mg/ml and are the average of three experiments. 1Helicobacter pylori (2–20); 2 human neutrophil peptide-2 (a-defensin); 3human beta defensin-2 (b-defensin); 4 human cathelicidin LL-37; 5 P-113 fragment of histatin-5.
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