helicobacter pylori commensal or pathogen? · pdf fileslide 5 gastric mucosa, silver stain,...

Helicobacter pyloriCommensal or Pathogen?

Vietnam 2004

Barry MarshallClinical Professor of Microbiology UWA

Marshall & Warren, Perth 1983Marshall & Warren, Perth 1983

It was very difficult for gastroenterologists and physicians to accept the

proposal of these two investigators, Marshall and Warren, who stated in 1983

that spiral bacteria in the human stomach were likely to be the cause of ulcers

and stomach cancer.

Established gastroenterologists and scientists found it hard to accept the

teachings of the two Australians! Instead, most gastroenterologists of the

time assumed that Helicobacter were commensals which merely colonized

people with ulcers or gastritis

Oct-04 © BJM

First Attempt at Publication: 1983

Score = bottom 20%

Dear Dr. Marshall,

I regret that your research paper was not accepted for presentation...

The number of abstracts we receive continues to increase and for this meeting 67 were submitted and we could only accept 56.

As shown here, our discovery was not “appreciated” by Australian

gastroenterologists (read text from slide). Our discovery was rated in the

lowest 20% and was not accepted even for a poster!

I only show this slide to remind young investigators that they should always

keep their rejection letters. Although it is too painful to read them now, you

may be proved correct in the future and then you can proudly display them.

Oct-04 © BJM

An attempt to Fulfill Koch’s Postulates for Campylobacter pyloridis

Med J. Aust 1984

Because of this rejection, in 1984 it was necessary for me to drink a culture of

Helicobacter pylori to prove to the skeptics that Hp could cause gastritis and

was not just a commensal present in people with ulcers or stomach cancer.

This was published in the Medical Journal of Australia and in an editorial for

The Lancet. Many gastroenterologists took notice but were determined to

prove me wrong! However, it stimulated much of the successful research

which we have seen presented since that time.

Gastric Mucosa, Silver Stain, Day 8

The self infection in 1984 fulfilled Koch’s postulates for H.pylori, proving that it

caused gastritis. This shows a silver stain of the gastric mucosa with the

acute infection. The black helical organisms are seen on damaged (brown)

epithelial cells. Note that the cells do not have the normal transparent mucus

content usually seen in the gastric epithelial layer.

Oct-04 © BJM

Repels waterdue to phospholipid

DamagedBy Hp

This high power view reminds us that the gastric wall has a mucus layer to

keep the acid from touching the mucosa and digesting it.

This diagram shows how the mucus barrier works. Here you see the mucus

above in green and the cells below. In the lumen of the stomach is the acid

represented by the H+. The surface of the mucus layer can actually repel

water and acid. This enables the cells below to remain healthy because even

though the stomach acid is strong, with pH of 2.0, the pH down on the cells

(below the mucus barrier) is about 6.0.

In addition, a greasy component of the mucus gel (hydrophobic phospholipid)

tends to repel water and acid from it.

H.Pylori damages this barrier in two ways. Directly by its phospholipase and

proteases which make the mucus layer less repellant to water and acid.

Indirectly by causing mucus cells to separate, become loosened from each

other, stimulating the inflammatory response causing cells from below the

mucosa to migrate through the epithelium.

Oct-04 © BJM

Epidemiology and Disease Associations of H.pylori

O ct-04 c BJM

H. pylori+H. pylori+

duodenal ulcerduodenal ulcer

gastric ulcergastric ulcer

gastric cancergastric cancer

gastric lymphomagastric lymphoma

Western (USA)30% Hp +

O ct-04 c BJM

H. pylori+H. pylori+

duodenal ulcerduodenal ulcer

gastric ulcergastric ulcer

gastric cancergastric cancer

gastric lymphomagastric lymphoma

Developing (Vietnam)60% Hp +

Epidemiology and Disease Associations of H.pylori Many studies have shown variants on the association with peptic ulcer, gastric

cancer, and gastric lymphoma. In any developed country, about 20-30% of

the population are infected with Helicobacter pylori (H. pylori). It is within this

group that peptic ulcer disease develops. The great majority of duodenal

ulcers and gastric ulcers are in the infected patients shown as the red inner

circle.

Although the majority of persons with H. pylori are asymptomatic at any point

in time, many of them eventually develop disease. In prospective studies, the

conversion rate to active peptic ulcer is about 1% per annum. Thus, during a

lifetime, about one third of infected persons develop symptomatic disease. In

addition, persons with H.pylori have a 1%-5% chance of developing gastric

cancer in their lifetime.

In Vietnam, with a population fo 80 million people, there are probably 40

million with Hp.

The fact that so many people with Hp never actually develop disease has

been reason for continuing controversy about Helicobacter.

Perhaps Hp is not always so bad. Actually there are several factors which

affect the ultimate outcome of Hp infection, some in the organism, some in the

host, and some in the environment.

Oct-04 © BJM

In many cases of H.pylori infection, the mucosa appears normal or only slightly irregular.

Antral “gooseflesh”also called “chicken skin”

The association between H.pylori and peptic ulcer is well known, but in many

persons the endoscopy is normal. Peptic ulcer disease is a condition

characterised by remissions and relapses so that the ulcer crater is not

always present at endoscopy. One visible lesion found to be the best

predictor of gastritis is the cobblestone, “gooseflesh” or “chicken skin”

appearance of the antral mucosa. Other appearances such as redness

remain for many years after treatment of the gastritis so they cannot be used

as indicators of active Helicobacter infection.

Oct-04 © BJM

All persons with hp have some degree of chronic gastritis and this becomes more severe and extensive in the stomach with time.

Chronic Gastritis.

Gastritis is always present in persons with H.pylori. Before discussing variations in the pathogenicity of Hp, I wish to emphasize

that Gastritis is always present in persons with Hp. Here is the histology characteristic of Hp showing infiltration of the gastric

mucosa with chronic inflammation – lymphocytes macrophages and plasma

cells. These cells are making IgG antibody which allows serological detection

to work quite well. Also there is acute inflammation present with scattered

neutrophils.

Even though some Hp are worse than others, ALL PERSONS with Hp HAVE

SOME DEGREE OF CHRONIC GASTRITIS AND THIS BECOMES MORE

SEVERE AND EXTENSIVE IN THE STOMACH WITH TIME.

Oct-04 © BJM

Mortality from Gastric Cancer by Country

Ferlay J, Bray F, Pisani P, Markin D. 2001. GLOBOCAN 2000: Cancer Incidence, Mortality and Prevalence Worldwide. V. 1.0. Lyon: IARCPress.

Cancer Incidence and Mortality The countries are listed sorted both by cancer mortality and by cancer

incidence. Note that in Japan, incidence is far greater than mortality. This is

because effective screening programs detect gastric cancer at an early stage

and cure is possible.

Although high gastric cancer usually correlates with H. pylori prevalence,

there are some notable exceptions. UAR and Kuwait are known to have a 50-

70% prevalence of H.pylori, but have rather low cancer mortality. Kenya is

the representative African country with a rather low gastric cancer rate but a

figh H. pylori prevalence. This paradox, referred to as the “African enigma” by

some, demonstrates that cancer causation from H. pylori must be modulated

by many factors. These could be dietary, ethnic and bacterial strain related.

Oct-04 © BJM

Variation in Pathogenicity of Hp

All Hp cause gastritisGastritis is worse if CagA pathogenicity island is presentCagA has various pathogenic potentials

• East Asian (Japan - severe)• Asian (Vietnam - mixed)• Western (mild)

But the pathogenicity of Hp varies according to the presence and type of

CagA toxin.

Oct-04 © BJM

Distribution of the Diversity of the CagA Protein in Japan

015354Gastric cancer

6117087Chronic gastritis

cagA (-)WesternEast Asian

n

Azuma T, Ohtani M, Yamazaki Y, Higashi H, Hatakeyama M. Meta-analysis of the relationship between CagA seropositivity and gastric cancer. Gastroenterology 2004;126(7):1926-7.

Distribution of the Diversity of the CagA Protein in Japan Note that in patients with gastric cancer in Japan, almost all will have the East

Asian type of CagA. (Azuma T, Ohtani M, Yamazaki Y, Higashi H,

Hatakeyama M. Meta-analysis of the relationship between CagA seropositivity

and gastric cancer. Gastroenterology 2004;126(7):1926-7.)

Oct-04 © BJM

36.7105Korea24.56020China12.83010VietnamAsia3.31913Thailand3.8330India

13.0730IrelandThe West14.1210Austria

27.8410Italy17.6610England6.1560USA8.6430Australia

CagA type Mortality rate of gastric cancer (/100,00males in 2000)WesternEast AsianCountry

58.3912123JapanEast Asia

The Distribution of CagA Protein Diversity Worldwide

Type of CagA and the Risk of Cancer Azuma and co-workers suggest that cancer rates are related to the CagA type

predominating in each country.

This table is preliminary data and hasnot been confirmed yet. It was just a

letter to a journal. The number of strains tested from many countries is quite

low.

However, the bad news is that strains from Vietnam appear to be very similar

to Japanese strains – East Asian Type in the majority. This means that they

have quite a strong cancer potential.

Contrast this with Thailand where Westen style CagA strains are more

common and cancer incidence is lower.

(Azuma T, Ohtani M, Yamazaki Y, Higashi H, Hatakeyama M. Meta-analysis

of the relationship between CagA seropositivity and gastric cancer.

Gastroenterology 2004;126(7):1926-7)

Oct-04 © BJM

Location of Location of H. pyloriH. pyloripH=2

pH=6

aerobic

micro-aerophilic

anaerobic

Oxygen +++Oxygen +++

No OxygenNo Oxygen

bacteria are stuck herecannot move away}

Microaerophilic metabolism directs H. H. pyloripylori away from the lumen, towards the

mucosa

Hp is unique – almost a commensal This cartoon emphasizes the location of Helicobacter beneath the gastric

mucus layer, attached to the stomach mucus cells. The bacteria actually

avoid the very acidic stomach contents (at top) where the pH is 2.0, instead,

they stay at the bottom of the mucus layer.

But Helicobacter do not survive where oxygen levels are high such as in the

tissues. Therefore they remain half way between the oxygenated tissue and

the low oxygen environment of the stomach contents. This means that

Helicobacter can never become invasive like other pathogens.

This location creates problems for the immune system which cannot eradicate

gastric bacteria. If the immune response is too aggressive, the mucosal

barrier will break and ulceration will occur.

There is some evidence that Hp can down-regulate the immune system, thus

allowing it to persist.

Oct-04 © BJM

Virulence FactorsFactor Function DistributionUrease Buffers stomach acid All strainsFlagella Motility All strainsNAP Neutrophil activation All strainsBabA Adhesion for Leb Prevalent on

type I strainsLPS Low toxicity All strainsLewisx,y antigens Molecular mimicry Some strainsIceA Homolog of Nla III restriction Some strains

endonucleaseVacA Cytotoxicity (two alleles) All strainscag PAI 31 genes coding for type IV Type I strains

secretion systemCagA Immunodominant antigen Type I strains (part of cag PAI)PicB Equivalent to CagE Type I

Covacci et al, Science 1999 284: 1328-1333

Virulence Factors of H.pylori In-vitro studies using cultured epithelial cells have identified many factors

secreted by H.pylori which cause pathologic changes. Most of these factors

are present in the majority of Hp strains, but they may vary in their

pathogenicity.

Urease is essential for colonization because it protects from acid allowing the

bacterium to survive long enough to reach the mucosa.

Nap increases inflammation by attracting neutrophils.

BabA, is an adhesin responsible for attaching Hp to the epithelial cells. If the

adhesion is strong, then the other toxins have far more effect.

Lewis antigens affect the ability of Hp to down-regulate the immune response

perhaps by mimicing carbohydrates in the mucosa.

VacA and CagA, I will discuss in detail below. CagA toxin is part of the “Cag

pathogenicity island” also refered to as the “CagPAI”.

PicB is a component of the CagPAI which is an ATPase responsible for

driving the CagPAI machinery. It causes release of IL-8 and attracts

neutrophils.

Oct-04 © BJM

Secretion Systems


CagPAIis Type IV

Secretion Systems CagA is a toxin associated with the Cag pathogenicity Island (CagPAI).

Bacteria have numerous secretion systems. Type IV secretion refers to the

ability of some bacteria to create a “molecular syringe”, which is a modified

flagellum or pilus structure, through which bacterial proteins and even genes

can be injected into a hoist cell.

The prototype of this mechanism is the Type IV system of Agrobacterium

which is extensively used to create new GM varieties of plants such as

Vitamin enriched rice etc.

Oct-04 © BJM

Type IV Secretion Homologues

S

B9B9

D4

NTPNTP

B4

NTPNTP

B6

B8

B11

NTPNTP

B10

B3

B 7

S

B 7

S

S

B5 B5


B2B2

Escherich

ia coli

Agrobacteriu tumefacie

ns

Brucella suis

Bordetella pertussi

s

Helicobacte

r pylo

ri

Rickettsi

a prowazekii

Legionella pneumophila

D4

G

F

O

N

D

Eex

KorA

M

L

C

B

A

D4 D4

DotBB11 292B11 525H

B10 291 IcME527B10 G

B9 B9 F 528 290

B8 B8E E 287

B7 IB7 CagT

B6 DB6

B5 B5

B4 B4 C CagE 103

B3 B3 B

B2 AB2

B1 B1

tra(pKM101)

vir vir ptl cag RP ORF

Dot/icm

B1

core

subu

nits

Transfer of secreted components across the bacterial envelope.

Genes present in Hp and various other Type IV systems

Oct-04 © BJM

urea

vacuoles

Action of vacA Toxin

VacA Toxin: VacA toxin is present in all Helicobacters. But when the CagA pathogenicity

island is present, then a more virulent form of VacA is present.

VacA is secreted from the Hp bacterium where, in the presence of low pH, it

polymerizes to form a membrane channel. It inserts into the epithelial cell

membrane and creates a pore through which anions can pass. The cell

becomes leaky, perhaps to the advantage of the attached Hp organism. The

channels also insert into intracellular organelles causing vacuoles to form.

Oct-04 © BJM

Redundancy in the Genetic Code

• CGTA in triplets• 64 combinations

for 20 AA’s• Mutations in third

codon may be “synonymous”

mRNA codes

The Molecular epidemiology of Hp The Molecular epidemiology of Hp can be studied as shown here. The

principle relies on the fact that there is redundency in the genetic code. This

slide shows the genetic codes which can form each amino acid. Notice that

changes in the third base of a codon often does not change the amino acid.

So, taking the case of Glycine, mutations affecting the third base, which can

be U, C, A or G, all code for Glycine. Thus mutations here do not affect the

Hp bacterium and go unnoticed in evolution. However, they can be counted

to measure the number of years since strains have diverged from one

another.

Oct-04 © BJM

Hp and Human Migrations


10 – 40 00012 000

40 000

60 – 70 00060 – 100 000

40 000

Human and Helicobacter Migrations If Hp was merely a pathogen, we might find that it was only recently acquired

by humans. However, if Hp was a commensal, then we might expect that Hp

has evolved with man for millions of years, as has the rest of the intestinal

flora.

By studying the sequence variations of CagA, VacA and various other

essential “housekeeping” genes (present in all Hp strains), several groups

have mapped the global migration of Hp in parallel to the known human

migrations.

This world map indicates the direction of human migrations (arrows) and time

range (years since migrations happened), as taken from Cavalli-Sforza (47)

and Diamond (48), The geographic centers of the major Hp genotypes known

today are indicated by concentric circles of different colors. It appears that Hp

followed man during the migrations indicated by the arrows, giving rise to the

present genotype distribution (indicated by circles).

Light green areas indicate the locations where the development of agriculture

and animal breeding was initially started, resulting in the expansion of the

initial human populations (46-48).

So far, it appears that Hp has followed humans for at least 50,000 years.

Thus, perhaps Hp resembles a commensal in some ways.

Oct-04 © BJM

Pathophysiology of Gastritis, Ulcers and Cancer

Saurbaum michetti

Natural History of Helicobacter pylori Infection.

H. pylori is usually acquired in childhood. Acute H. pylori infection causes transient

hypochlorhydria and is rarely diagnosed.

Chronic gastritis will develop in virtually all persistently colonized persons, but 80 to

90 percent will never have symptoms.

The further clinical course is highly variable and depends on bacterial and host

factors. Patients with higher acid output are likely to have antral predominant gastritis,

which predisposes them to duodenal ulcers. Patients with lower acid output are more

likely to have gastritis in the body of the stomach, which predisposes them to gastric

ulcer and can initiate a sequence of events that, in rare cases, leads to gastric

carcinoma.

H. pylori infection induces the formation of mucosa -associated lymphoid tissue

(MALT) in the gastric mucosa.

Malignant lymphoma arising from such acquired MALT is another rare complication

of H. pylori infection.

Oct-04 © BJM

No cancer in patients with DU

Helicobacter pylori infection and the development of gastric cancer.Uemura N, Okamoto S, Yamamoto S, et al. N Engl J Med 2001;345:784-789.

By itself, H.pylori rarely causes cancer. Cancer only occurs in long term infection where acid secretion is suppressed or removed by atrophic gastritis

HP+, >3% ca. in 12 yrs

Here is an important paradox about H.pylori and Cancer Perhaps there is some reason for a little optimism about Hp. Helicobacter is

said to be a risk factor for stomach cancer. But this important observation has

been largely overlooked. Stomach cancer rates for persons with a history of

duodenal ulcer are normal, or even lower than normal.

In this prospective study reported in the New England Journal of Medicine,

Uemura performed surveillance endoscopy on patients with Hp for up to 12

years. He observed that about 4% developed stomach cancer during that

time.

But there was no stomach cancer in patients who had duodenal ulcer, even

though duodenal ulcer is associated with the more harmful, toxin producing,

strains of Helicobacter.

Thus, by itself, Helicobacter does not always cause cancer. Cancer probably

only occurs in long term infection where acid secretion is suppressed or

removed by atrophic gastritis.

When stomach acid is normal or high, as it is in duodenal ulcer patients and

most persons with Helicobacter, cancer risk is actually low. This observation

has also been made in seroepidemiologic studies in California (Parsonnet

NEJM) and Hawaii (Nomura NEJM).

Oct-04 © BJM

9/97

0102030405060708090

100

0 10 20 30 40 50 60

age

% in

fect

edDevelopingWestern

Epidemiology of H. pylori - Western vs. Developing

Whole of life

The upper graph shows that even in a Western country, older persons have a high prevalence of H. pylori because they acquired the infection in childhood. The lower graph illustrates a developing country where the infection rate is

20% per annum and the loss of the infection rate is 3% per annum.

Vietnam is in transition so we expect to see the pattern change over the next

generation.

Things which will decrease the new Hp cases are smaller families and

improved public health measures, particularly drinking water quality.

Oct-04 © BJM

Population Demographics: VietnamAge structure: – 0-14 years: 29.4%– 15-64 years: 65%– 65 years and over: 5.6%

Median age: – total: 24.9 years Life expectancy at birth: – male: 67.86 years – female: 73.02 years (2004 est.)

Demographics

As you know even more than I do, Vietnam has a young population so most

people do not yet experience a cancer risk from Hp. As shown here, in 2004,

only 5.6% of the population is above the age of 65 years. The average age is

25 years.

Oct-04 © BJM

Age related incidence and mortality from gastric cancer for Japanese males

Ferlay J, Bray F, Pisani P, Markin D. 2001. GLOBOCAN 2000: Cancer Incidence, Mortality and Prevalence Worldwide. V. 1.0. Lyon: IARCPress.

As lifespan increasesin Vietnam, cancer risk

is more important

Gastric cancer and Age – Data from Japan More than 2800 new cases of stomach cancer occur in persons under the age

of 44 year but most (10,000 or so) are in persons above the age of 60.

As the population ages, and lifespan increases, gastric cancer will become

more and more important. That is why Hp needs attention now.

helicobacter pylori commensal or pathogen? · pdf fileslide 5 gastric mucosa, silver stain,...

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