The Innate Immune Glycoprotein Lactoferrin Represses the Helicobacter pylori cag Type IV Secretion System

Abstract

Chronic infection with Helicobacter pylori increases risk of gastric diseases including gastric cancer. Despite development of a robust immune response, H. pylori persists in the gastric niche. Progression of gastric inflammation to serious disease outcomes is associated with infection with H. pylori strains which encode the cag Type IV Secretion System (cag T4SS). The cag T4SS is responsible for translocating the oncogenic protein CagA into host cells and inducing pro-inflammatory and carcinogenic signaling cascades. Our previous work demonstrated that nutrient iron modulates the activity of the T4SS and biogenesis of T4SS pili. In response to H. pylori infection, the host produces a variety of antimicrobial molecules, including the iron-binding glycoprotein, lactoferrin. Our work shows that apo-lactoferrin exerts antimicrobial activity against H. pylori under iron-limited conditions, while holo-lactoferrin enhances bacterial growth. Culturing H. pylori in the presence of holo-lactoferrin prior to co-culture with gastric epithelial cells, results in repression of the cag T4SS activity. Concomitantly, a decrease in biogenesis of cag T4SS pili at the host-pathogen interface was observed under these culture conditions by high-resolution electron microscopy analyses. Taken together, these results indicate that acquisition of alternate sources of nutrient iron plays a role in regulating the pro-inflammatory activity of a bacterial secretion system and present novel therapeutic targets for the treatment of H. pylori-related disease.

Keywords: Helicobacter pylori; antimicrobial; bacterial pathogenesis; glycobiology; host-pathogen interactions; innate immunity; iron homeostasis; lactoferrin; toxin secretion; virulence.

Figure 1.

Lactoferrin is elevated in H. pylori-infected gastric tissues compared to uninfected gastric tissues. Immunohistochemical (IHC) and microscopical evaluation of gerbil gastric tissue specimens reveals elevated lactoferrin levels (indicated by the dark brown stain) associated with H. pylori chronic infection (B and D) compared to uninfected negative controls (A and C). Micrographs were collected at 100X (A and B) and 400X magnification (magnification bars indicate 100 μm) and representative micrographs are shown (N=3–4). Healthy, uninfected gastric tissue produces lower levels of lactoferrin, largely associated with gastric epithelia proximal to the lumen of the stomach. Infected tissues produce lactoferrin at higher levels and deeper into tissues towards gastric pits (white arrows) and muscularis (black arrows). H-DAB stain quantification (panel E) reveals that lactoferrin levels are significantly higher in H. pylori-infected gastric tissues compared to uninfected control tissues (*P=0.0148, Student’s t test with Welch’s correction).

Figure 2.

H. pylori can utilize holo-lactoferrin (holo-Lf) as an alternate source of nutrient iron under iron-limiting conditions. H. pylori growth in the presence of the synthetic iron chelator, dipyridyl (DIP), is attenuated. However, supplementation with an exogenous source of iron, such as ferric chloride (Fe) rescues growth. Similarly, supplementation with lactoferrin saturated with iron or holo-Lf enhances H. pylori growth in conditions where iron availability is low. Conversely, supplementation with lactoferrin lacking bound iron or apo-lactoferrin (apo-Lf) does not restore H. pylori growth under these conditions. (N=3, a, b, c = P<0.0001, comparing medium alone to medium supplemented with DIP + apo-Lf, and d, e, f = P<0.0001, comparing medium alone to medium supplemented with DIP, two-way ANOVA).

Figure 3.

High resolution scanning electron microscopy analysis of H. pylori cag T4SS. Bacteria were grown in culture medium alone or supplemented with either ferric chloride, apo-, or holo-lactoferrin prior to co-culture with human gastric epithelial cells. A) SEM analyses revealed that exposure to ferric iron (Fe) or holo-lactoferrin (holo-Lf) repressed the formation of cag T4SS pili at the bacterial-host interface, whereas bacterial exposure to medium alone (Medium Alone) or apo-lactoferrin (apo-Lf) results in pili biogenesis. B) Quantification of cag T4SS pili per cell and C) analysis of percentage of piliated cells indicates that exposure to ferric chloride or holo-lactoferrin results in inhibition of piliation compared to medium alone (P<0.05, one-way ANOVA). Magnification bar indicates 500 nm. Red arrows indicate cag T4SS pili. (N=3 biological replicates, *P<0.05, One-way ANOVA).

Figure 4.

Analysis of human gastric epithelial cell IL-8 response to the H. pylori cag T4SS. H. pylori was grown in culture medium alone or supplemented with either ferric chloride, apo-, or holo-lactoferrin prior to co-culture with human gastric epithelial cells. ELISA analysis of IL-8 secretion (a proxy for cag T4SS activity) revealed that exposure to ferric iron (Fe) or holo-lactoferrin (holo-Lf) repressed the activity of the cag T4SS, whereas bacterial exposure to medium alone (Medium Alone) or apo-lactoferrin (apo-Lf) results in robust IL-8 secretion, indicative of cag T4SS activity. (N=3–5 biological replicates, a,b,c=P<0.05, One-way ANOVA).

Figure 5.

Conceptual model of the interaction of H. pylori with lactoferrin. H. pylori infection of the gastric niche results in production of lactoferrin. Apo-lactoferrin lacks iron and, therefore, cannot be utilized as a source of nutrient iron under iron-limited conditions, resulting in the elaboration of the cag T4SS at the host interface. The H. pylori cag T4SS translocates proinflammatory substrates, including the oncogenic CagA cytotoxin, into host gastric epithelial cells. As a consequence of the activity of the H. pylori cag T4SS, host cells upregulate production of the proinflammatory cytokine and chemokine interleukin 8 (IL-8). Holo-lactoferrin can be utilized as a source of nutrient iron for H. pylori in conditions where iron availability is low. Holo-lactoferrin represses the elaboration of the H. pylori cag T4SS and represses the proinflammatory activity of this toxin secretion system, resulting in decreased IL-8 secretion by host cells.