FICHA DE OBSERVACIÓN APLICADO A LOS ESTUDIANTES DE INICIAL

H. pylori has been studied extensively, however, its ability to persist and interact

with the host is still not fully understood. The discovery of SLR motifs within HcpE

suggests they are interacting with host cells and/or proteins (39). However, the process or

cells with which they interact has not been studied. Thus, to determine the effect of HcpE

on the host, in terms of both colonization and recruitment of inflammatory mediators, H.

pylori wild-type as well as hcpE and dsbHP knockout mutants need to be studied in vivo.

H. pylori Sydney strain (SS1), is a mouse adapted strain which was repeatedly orally

passaged through C57BL/6 mice until it was capable of efficiently colonizing mice for long

periods of infection (≤ 8 months) (Lee et al, 1997). Colonization results in inflammation of the gastric mucosa within 4-5 weeks, increased neutrophil recruitment, and expression

of Major Histocompatibility Complex II (MHC II) class antigens. However, peptic

ulceration and gastric cancers have not been observed (Fan et al, 1998). As in humans, the

Figure 31: HcpE produced and secreted by H. pylori isolated from infected patients.

The presence and abundance of HcpE was analyzed in clinical isolates obtained from

patients diagnosed with various gastric disorders.

PanelA: Ponceau S red staining and Western blotting for the presence of HcpE using anti-

HcpE antibodies in total cell lysate of 16 clinical isolates. HcpE is present in the cell lysate

of all clinical strains tested, with the strains obtained from ulcer patients displaying the

highest abundance of HcpE.

Panel B: Ponceau S red staining and Western blotting for the presence of HcpE using anti-

HcpE antibodies in culture supernatants of 12 clinical isolates. HcpE is present in the

supernatant of all clinical strains tested, with the strains obtained from ulcer patients

displaying the highest abundance of HcpE.

WT: wild-type, KO: hcpE::kan knockout mutant, GU: Gastric ulcer, DU: Duodenal ulcer,

A. B. WT KO 1 2 3 4 5 6 7 8 9 10 11 Strain WT KO 12 13 14 15 16 Strain WT KO 1 2 3 4 5 6 7 Strain WT KO 8 9 10 11 12 Strain GU GU GU DU DU GU A A G G A Patient Disease G A A G G Patient Disease GU GU DU DU GU A A Patient Disease G G A G G Patient Disease

H. pylori. Thus, this mouse model has been successfully developed to study infections in

vivo which show similar characteristics to infection in humans.

Hcps are highly conserved within H. pylori strains, thus must be beneficial to their

perseverance. Other Hcp proteins such as HcpA and HcpB have been implicated in the

production of pro-inflammatory cytokines, suggesting they may play a role in H. pylori

induced gastritis. To determine whether HcpE impacts colonization or inflammation,

C57BL/6 mice were intra-gastrically inoculated with strain SS1 wild-type and knockout

mutants as described by Ferrero et al. Briefly, mice were orally gavaged with 109 bacteria

3 times, once every other day for the course of a week. Infection was allowed to proceed

for 4 and 6 weeks post gavage before mice were sacrificed. To assess infection, plating of

stomach homogenate for cfu counting was conducted. After being harvested and divided

into 2 halves, the murine stomach tissue was placed into PBS buffer with antibiotics and

homogenized with an electric homogenizer. After homogenization, the gastric homogenate

was diluted 10 and 100 fold, and each dilution was plated onto Columbia blood agar. Plates

were incubated in micro-aerobic conditions for 5-7 days. Colonization was also assessed

via histological sections of stomach tissue that were prepared, and observed using light

microscopy. The dsbHP and hcpE knockout mutants in SS1 strain had not been previously

established, thus knockout mutants were constructed using the same constructs and

technique described in section 2.10.

The presence of H. pylori within the gastric mucosa of mice gavaged with either

wild-type, dsbHP::kan, or hcpE::kan knockout mutants was not detected via plating of the

gastric homogenate. No colonies from mice gavaged with either strain were recovered.

detect spirochetes, established the presence of H. pylori within the gastric mucosa of

infected mice (Figure 32). Both dsbHP and hcpE knockout mutants had impaired

colonization compared to wild-type based on total cells visualized and counted within the

processed tissue, with the dsbHP knockout mutant being the most attenuated (Figure 33).

This trend was observed for mice sacrificed at both 4 and 6 weeks post gavage, indicating

that H. pylori is still capable of colonization over long periods when devoid of either DsbHP

or HcpE, but colonization is significantly impaired compared to wild-type. We currently

do not fully understand why this defect in colonization is occurring, but we hypothesize

that motility may be a factor due to the fact that both knockout mutants have impaired

motility compared to wild-type (Figure 34). The dsbHP knockout mutant is less capable of

colonizing than the hcpE mutant, however retains more motility. Thus there is another

factor(s) that is contributing to the decreased colonization observed in the dsbHP knockout

mutant.