The life and death of Helicobacter pylori

in stomach

Davoud azadi

Introduction

• Colonisation of the normal stomach has been achieved only

by Helicobacter spp., such as H.pylori, H. felis or H.

mustelae

• Gram negative, motile, microaerophilic, spiral shaped

organisms that dwell on the gastric surface and within the

gastric mucus

• They are found more frequently in the antrum than in the

fundus and often within antral glands

• grow between pH 6.0 and 8.5 and survive between pH 4.0

and 8.5 in the absence of urea

The gastric environment

• The pH of the gastric lumen:

median pH is 1.4 .The pH can fall to below 1.0 with acid

secretion at night

The pH at the gastric surface(neutral) is thought to be

significantly higher than in the lumen(pH:2)

The pH of the lumen of the fundic glands, is much lower, The

pH on the antral surface likely to be higher

HCO3 secretion is able to neutralise 10% of maximal acid

secretion.( gastric surface pH:6)

Microbial acid adaptation• pH of periplasmic space, that is important for their survival or

growth.

• Neutrophiles bacteria like HP that can only survive but not grow in

acid are acid resistant

• Neutrophiles bacteria grow best at neutral pH but are able to survive

and grow in acidity and to increase their periplasmic pH

1. All are designed to maintain a tolerable electrochemical

gradient of H+ across the inner membrane of these bacteria.

This can be achieved by alterations in transmembrane potential

lead to reducing the effective gradient

2. Acid tolerance can be achieved by the generation of neutralising

buffers either inside the cell or in the periplasmic space.

3. Many bacteria respond to acidity by changes in gene expression

of specialised proteins.

Molecular mechanisms of acid adaptation of Helicobacteria

STRUCTURAL ASPECTS OF MEMBRANE PROTEINS

• The outer membrane of Gram negative containing a variety of proteins

• some membrane spanning, porins are able to mediate the flux of various small molecules across the outer membrane.

• some associated with only one face(LPS)

STRUCTURAL ASPECTS OF MEMBRANE PROTEINS

• A means of acid protection would be to alter the isoelectric point

of these proteins as a means of reducing the transport of protons

across the bilayer.

• Helicobacteria porins and some inner membrane proteins have an

isoelectric point significantly more alkaline than that of bacteria

rarely exposed to acid thereby retarding flux of protons into the

periplasmic space

• In H pylori the c subunit of the F1F0ATP synthase. loss of four

carboxylic amino acids in the sequence of the compared with that

of E coli or B subtilis.

• increase in isoelectric point suggests that both the outer and inner

membrane of the organism can be exposed to high acidity

• increase in numbers of positively charged amino acids or

decrease in the number of carboxylic acids in the membrane

proteins

• Helicobacter pylori use of this adaptive mechanism is acid

resistance when is exposed directly to luminal acidity.

• This mechanism provide transient protection. Thus other

mechanisms must be present to allow both long term survival

and growth in the gastric environment.

ATP SYNTHESIS AND MEDIUM pH• The survival of aerobic bacteria depends on their ability to

synthesise ATP, by hydronium flux inward across the F1F0 ATPase driven by an inwardly oriented electrochemical gradient of H3O+

• The gradient related to pH gradient and the transmembrane potential difference (PMF)

In E coli, in a medium of pH 7.0, the cytoplasmic pH is about 7.5 to 7.8 and the transmembrane potential is −160 mV, to give an PMV: −200 mV.

H pylori in the absence of urea at pH 7.0 gave a value of −131 mV and an internal pH of 8.4(lower proton permeability of the membranes of this organism) PMV: −220 mV.

• H.pylori is able to maintain a membrane potential between

pH 4.0 and 8.5. If the pH was outside these limits there was

a relatively rapid and irreversible loss of the membrane

potential

• The addition of urea restored the membrane potential

within about pH 3.5.

• Addition of urea at neutral pH in the absence of buffer

resulted in medium alkalinisation to a pH > 8.5

• Urease activity is a two edged sword for H pylori, enabling

survival in acid, preventing survival in the absence of acid

REGULATION OF PERIPLASMIC pH IN ACID

• Above mechanism that used by H pylori would not

ensure its survival in the stomach

• this organism is synthesis of a neutral pH with urease at

high concentrations.

• 15% of the protein synthesis is devoted to the production

of ure A or ure B.and active urease enzyme need Ni2−

Urease activity

• H pylori has a prokaryotic carbonic anhydrase in its genomic

sequence, hence the eventual effect of urease activity at a pH less

than the pKa of NH3 (9.5) and greater than 4.8 therefore is able to

alkalinise the environment of the urease

• urease activity rapidly showed was organism either in solution or

bound to the cell surface.

• surface urease activity reduces the acidity of the microenvironment

of the organism below the mucus layer of the gastric mucosa

• H. pylori have two urease :internal urease and surface urease

• This microenvironment located external to the organism or in the

periplasmic space

• Internal urease was designated as the responsible urease

compartment, it was neutralisation of the external

microenvironment

Urease activity

• External urease activity low at pH > 6.5 At pH < 6.5, there is a

notable increase in urease activity(about 10-fold) reaching maximum

at pH 5.5 and remaining steady until pH 3.0

• internal urease is activated at a pH < 6.5 and cytoplasmic pH

remains steady

• 95% of the urease is found inside and not on the surface or outside

majority of the protein is found in the cytoplasm thus inaccessible

for immune system elements

• internal urease is responsible for acid protection

• The inner membrane potential of H pylori at fixed medium pH

between 3.0 and 6.0 rose to −105 mV with the addition of 5mM

urea,

• internal urease activity was responsible to periplasmic pH was

elevated to pH 6.2

• Addition of urea also enabled protein synthesis at a fixed medium

pH of 3.0 to 6.0 where normally no protein synthesis is found.

RESPONSE TO ELEVATED pH

• external urease is toxic at neutral pH

• ureas elevates medium pH to 8.5 or greater

resultant irreversible loss of membrane potential

and death

• This latter effect may explain the absence of H

pylori infection in pernicious anaemia

• H.pylori show no Internal urease activity at a pH >

6.5 prevents alkalinisation in the absence of acid.

• In summary, internal urease is active at a gastric

pH of < 6.5 and enables survival and growth at pH

between perhaps 2.0 and 6.0 depending on the

gastric urea concentration

• urea concentrations for effective internal enzyme

activity is about 1 mM

• External urease does not function as a

gastroprotective mechanism but reducing the

immune response in the gut

Detoxification

• H. pylori microaerophilic and able to protect themselves from the

toxic products of oxygen metabolism

• H. pylori possess a superoxide dismutase and a catalase

• H. pylori has two genes encoding peroxidases (JHP991/HP0390 and

JHP1471/HP1563) located adjacent to the superoxide dismutase

gene

• Theability to isolate catalase-negative mutants of H. pylori suggests

that at least one of the peroxidases can function as a catalase

The ecological niche of H pylori

• pH 6-8 Optimum for H.pylori growth(growth or

protein synthesis)

• for effective colonisation, the periplasmic pH

must be kept within those pH 6-8

• H pylori would inhabit only those regions of

the gastric mucosal surface that remain largely

within this pH range

• population of Fundus of stomach is sparse but

population of is high Antral(absence of acid

secretion and the presence of HCO3)

• The organism is motile but pH not affect on chemotaxis of

Helicobacteria

• If the gastric pH is changed, intragastric population would tend

to redistribute on the gastric surface

• the ability of the bacteria to colonise the gastric surface where

the pH is between 3.0 and 6.0

• Hence with proton pump inhibition the antrum would tend to

lose organisms but the fundus would tend to acquire organisms

where the surface pH would exceed 3.0

Chemotaxis

• H. pylori motile bacteri and have chemotaxi like

other motile bacteria

• Three homologues of the chemotaxis pathway in

E. coli (CheW, CheA, and CheY)

• four methyl-accepting chemotaxis proteins

(MCPs), which mediate specificity for

ligands,sens environment conditions

Strategies for eradication

2013 regimens • Triple therapy:

• proton pump inhibitor (PPI) (lansoprazole 30 mg twice daily, omeprazole 20 mg twice daily, pantoprazole 40 mg twice daily, rabeprazole 20 mg twice daily, or esomeprazole 40 mg once daily), amoxicillin (1 g twice daily), and clarithromycin (500 mg twice daily) for 7 to 14 days. We suggest treatment for 10 days to two weeks.

• Quadruple therapy

• bismuth (525 mg four times daily) and two antibiotics (eg, metronidazole 250 mg four times daily and tetracycline 500 mg four times daily) given for 10 to 14 days. Quadruple therapy is appropriate as initial therapy in areas in which the prevalence of resistance to clarithromycin or metronidazole is ≥15 percent, or in patients with recent or repeated exposure to clarithromycin or metronidazole [18]. If tetracycline is not available, doxycycline (100 mg twice daily) may be substituted

1. PETER DOIG,1* BOUDEWIJN L. DE JONGE,1 RICHARD A. ALM.et al. Helicobacter pylori Physiology Predicted from Genomic Comparison of Two Strains. MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Sept. 2006, p. 675–707

2. David j. KellyThe Physiology and Metabolism of the Human Gastric PathogenHelicobacter pylori. Advances in Microbial Physiology. Volume 40 .1998, Pages 137–189

3. D Scott, D Weeks, K Melchers, et al. The life and death of helicobacter pylori . Gut 1998;43(suppl 1):S56–S60

4. Marco Romano, and Antonio Cuomo. Eradication of Helicobacter pylori: A Clinical Update. MedGenMed. 2004; 6(1): 19.

5. Sheila E Crowe. Treatment regimens for Helicobacter pylori. Gut2013 . : 19,