Host-Pathogen Interaction and Human Disease Pylori And... · 2013. 10. 20. · Stanley Falkow, May 2007 Host-Pathogen Interaction and Human Disease, Part 2 The American Society for

Stanley Falkow, May 2007 Host-Pathogen Interaction and Human Disease, Part 2

The American Society for Cell Biology 1

HostHost--Pathogen Interaction and Human DiseasePathogen Interaction and Human Disease

Stanley FalkowStanley FalkowProfessor of MicrobiologyProfessor of Microbiology

Stanford UniversityStanford University



HostHost--Pathogen Interaction and Human DiseasePathogen Interaction and Human DiseasePart 2. Part 2. Helicobacter pyloriHelicobacter pylori and Gastric Cancerand Gastric Cancer

Trying to Understand Human Biology Trying to Understand Human Biology by the Study of Persistent Bacterial by the Study of Persistent Bacterial

InfectionInfection



Twenty Years Ago We Thought of Stomach Ulcers as a Stress Related Disease



We treated ulcers with antacidsWe treated ulcers with antacids



And tried ways to reduce stressAnd tried ways to reduce stress



But not everyone thought that ulcers were stress related…..



But not everyone thought that ulcers were stress related…..

Barry MarshallRob Warren



A bit of time away from a problem can sometimes help you see things more clearly




Sometimes a friend can help you see things better too….



Of course, scientists like to talk about their work



Of course, scientists like to talk about their work

But not everyone wants to But not everyone wants to listen!!listen!!



HOWEVERHOWEVER



Helicobacter pylori is a remarkable microorganism

It is a spiral shaped, flagellated gram negative bacterium



Helicobacter pylori is a remarkable microorganism

transmitted by the fecaltransmitted by the fecal-- oral or oraloral or oral--oral routeoral route



H. pylori has evolved with humans



Infection is usually acquired during Infection is usually acquired during childhood and persists for life.childhood and persists for life.

Prevalence of Helicobacter pylori

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40 50 60 70 80 90

Age in Years

Mexico

Italy

Ethiopia



Helicobacter pylori colonizes the

mucus layer of the human stomach



The Stomach is an acid environment



www.immediart.com

The Gastric Epithelium is Protected by a Mucus Layer

Mucus Layer



www.immediart.com

And H. pylori lives solely within the mucus layer near theEpithelial surface

Mucus Layer

Schreiber, et al PNAS, 2004,101 (14)5024Schreiber, et al PNAS, 2004,101 (14)5024––50295029



Helicobacter pylori

protection against esophageal cancer?

Asymptomatic**colonization (> 75%)

chronic atrophic gastritis (15%)

Ulcer disease (10%)

gastricadenocarcinoma (1%)

MALT lymphoma(



Humans Respond in Various Ways to Infection with H. pylori



Helicobacter pylori




Ulcer disease (10%)


MALT lymphoma(



Humans Respond in Various Ways to Infection with H. pylori

Gastric Cancer



H. pyloriH. pylori and Cancerand Cancer

The risk of getting gastric cancer from H. pylori is as high as getting lung cancer from smoking.




The risk of getting gastric cancer from H. pylori is as high as getting lung cancer from smoking.




H. pylori has been clasified as a Definitive Carcinogen (Type 1) by the WHO (IARC).




Even today, more than 50% of the world is exposed to this carcinogen since childhood.



H. pylori has a Number H. pylori has a Number of Virulence Factorsof Virulence Factors



H. Pylori produces ureaseand assembles a H+ gatedurea channel so that ammonia production neutralizes the bacterialcytosol and the immediatesurrounding environment



Ureaseis essentialfor virulence



Motility is Essential for Virulence



H. pylori swims through the mucus to the epithelial surface



H pylori has Multiple Adhesins



H pylori has Multiple Adhesins

The best characterized H. pylori adhesin is BabA, a protein that binds the Lewis b blood group antigens on the gastric epithelium.



‘‘PlanktonicPlanktonic’ ’ H. pyloriH. pylori

AttachedAttached H. pyloriH. pylori

About 1/3 of H. pylori are attached tothe gastric surface at any given time



About 1/3 of H. pylori are attached to the gastric surface



H . pylori secretes a toxin called VacA



Effect of VacA Toxin on Epithelial Cells



Two Types of Two Types of H. pyloriH. pylori

•One Type Produces the CagA Antigen

•Both CagA+ & CagA-Strains Cause Gastritis

•Cag A+ Associated with Ulcers & Other Severe Disease



The Genetic and Genomic analysis of CagA+ & CagA-strains provided the answer



CagA + strains had extra DNA not found in Cag- strains



The Cag Island Encodes a Secretory System



Molecular signalling alterations induced by Cag secretion system-dependent delivery of CagA or peptidoglycan (PGN)

Peek, R. M. Am J Physiol Gastrointest Liver Physiol289: G8-G12 2005;doi:10.1152/ajpgi.00086.2005



Growth factor effect of H. pylori infection epithelial cells

Infected 6 hours with CagA- strain

Infected 6 hoursInfected 6 hourswith CagAwith CagA++ strainstrain



To determine the possible function of

Cag A we used the host cells transcriptional

activity to tell usthe differences between a

Cag+ and aCag- infection



Reference RNA from uninfected gastric epithelial cells

Host cell Transcriptional Response

RNA from time points of an infection ofgastric epithelial cellswith wildtype and mutant H.pylori

mRNA decreased in infected cells

mRNA unchanged

mRNA increased in infected cellsKarenGuillemin

Karen Guillemin applied the microarray technology to examine the effect of infection on AGS cells by looking at the transcriptional response of the host cells e.g. messenger RNA. In the assay, the reference RNA from uninfected cells is mixed with the RNA from cells infected by wildtype bacteria or by mutant bacteria. Hybridization takes place on the spotted microarray. Can look at the whole human genome spotted on the array. If a gene is downregulated, the spot is green (more reference cDNA); if the gene is upregulated with infection, the spot is red.



There were clear differences in the a number of genes

‘turned on’ by infection with a wildtype Cag + strain as

compared to the exact same strain carrying a mutation in

Cag A



“Among the Cag A –specific induced genes were a striking number of tight junction-associated clones including claudins 1, 3, and 4; occludin;and the junctional adhesion molecule.”



Does Helicobacter pylori

hijack the tight junction of gastric

epithelial cells?

Manual Amievaasked:



Tight Junction



The Tight Junction between epithelial cells is complicatedAnd multi-functional



The Tight Junction between epithelial cells is complicatedand participates in a number of cellular functionsIncluding:

BarrierPolarityCell morphologyCell movementCell divisionCell differentiation



Tight junctions bar the movement ff material from the apical surface (the lumen). Different rows of proteins form a complex ‘meshwork’.

.



Some adhesion structures: do not allow passage of anything from one cell to another.

•Adhesion belts ‘glue cells’ together.



A 3-dimensional view of apolarized epithelium



H. pylori with CagA is found at the tight junction of cells as compared to the same strain without a functional CagAprotein.

Tight Junction

H. pylori

Amieva, et al., Science 300:1430-34, 2003



H. pylori with CagA is found at the tight junction of cells as compared to the same strain without a functional CagAprotein.

Amieva, et al., Science 300:1430-34, 2003

H. pylori

Tight Junction



Green Green –– ZOZO--11

Blue Blue –– EE--cadherincadherin

H. pyloriH. pylori

H. pylori is closely associated with thetight junction protein ZO-1



H. pylori is closely associated with the tight junction protein ZO-1



Phosphorylated CagA protein is found atthe site of attachment of H. pylori to the host cell

H. pylori

PhosphorylatedCag A protein

Host Cell Membrane



CagA - CagA +

Infection with a CagA+ cell opens up the tight junction



What are CagA’s Functional Domains?

aa871-1216aa1-877

Fabio Bagnoli

Bagnoli, F. et al. Helicobacter pylori CagA induces a transition from polarized to invasive phenotypes in MDCK cells. PNAS 2005 102: 16339-16344

Fabio Bagnoli then joined the laboratory to dissect out the functional domainesof CagA.In order to do these experiments, he transfected MDCK epithelial cells with genes encoding a fragment of the CagA protein—the N-terminus versus the C-terminus. The C-terminus contains a series of amino acid repeat sequences called the EPIYA sequence. This is the site which becomes phosphorylated by the host cell tyrosine kinase.



aa1-877

CagA N-terminus is localized to the host cell membranes




CagA C-terminus causes host cell elongation


aa871-1216



The full length CagA molecule induces cell motility and induces the cell to leave the polarized epithelial elongation





CagA breaks down the apical junctions

Polarity is lost




And cells migrate



Gastrulation During Embryogenesis-An Epithelial- Mesenchymal Transition

This Cag A mediated process resembles a phenomenon seen during embryonic development



Nature Reviews Cancer 2, 442 -454 (2002)June 2002 Vol 2 No 6

Such transitions are suspected to be important in the evolution of cancer



Such transitions are suspected to be important in the evolution of cancer



So, how can we relate these changes to carcinogenesis?

What is the role of What is the role of CagA in malignant transformation?CagA in malignant transformation?

www.gastrointestinalatlas.com



The answer we were surprised to find out is that moesin is present from the beginning, actually first as a maternally derived protein inherited by the embryo through the oocyte.The embryos were obtained with the help of Mark Johnson, MD OB/GYN by hyperovulation and flushing from the fallopian tubes. I was able to compare several stages from unfertilized eggs to 16 cell-stage.I show here a vey interesting phenomenom. First as you can see moesin is localized to the cell cortex in the oocyte, and one can visualize the fine microvilli on the surface.Moesin is present throughout the surface of the oocyte. At the two, four and early 8 cell stage, moesin staining continues to be as seen here surrounding the entirety of the cell. But at the stage of compaction, when the first polarized epithelium forms, moesin becomes polarized as well. Staining is lost in the basolateral aspects of the cells as can be seen by this optical cross section.Similar findings have recently been published for ezrin by aFrench group. They did a more careful study than me, since they also determined that the maternally derived ezrin declines in total amount during early embryogenesis, but a particular tyrosine phosphorylated form increases in relative amounts after compaction. They also point out



There is no doubt that CagA is associated with a cancer risk




But it takes decades! But it takes decades!



What’s in it for the Bug?



H. pylori isn’t the only microbe that targets the tight junction



Enteropathogenic E. coli(EPEC) also targets the TightJunction and effects polarity



MDCK cell ZO-1 & H. pylori

Does CagA Have a Colonization function?



Pathogenesis is the reflection of ongoing evolution between a parasite and a particular Host



Pathogenesis is the reflection of ongoing evolution between a parasite and a particular host

Disease is the result of a microbial adaptive strategy, an experiment or a mistake.

What we call virulence factors may have a biological role in a non-pathogenic context.



Reflections



Medical progress has drastically reduced the mortalitydue to infectious diseases



Percentage of deaths due to infectious diseases worldwide.

At least in the technologically advanced countries of the world but……



The leading cause of childhood mortality worldwide isstill infectious diseases



Coincident with the reduction in infectious disease and the carriage rate of H. pylori has been a reduction in gastric cancer

Approximate Incidence of H. pylori (Reviewed Passaro et al. CID 2002)

0.40%

3%

16%

0%

2%

4%

6%

8%

10%

12%

14%

16%

18%

Adults DevelopedCountries

ChildrenDevelopedCountries

ChildrenDevelopingCountries



Coincident with the reduction in infectious disease and the carriage rate of H. pylori has been a reduction in gastric cancer



However Esophageal Disease has increased enormouslyIn parallel



And there is evidence that beinginfected with H. pylori actuallyprotects you against esophageal Disease!



Should we have considered that H. pylori has been essentially part of the normal human?



After Jeff GordonAfter Jeff Gordon



Is H. pylori a Surrogate for Other Indigenous Microbial Species that are being Wiped Out by Human “Progress”?

What Might the What Might the Effects Be on Human Effects Be on Human Health?Health?



Infectious diseases

USA 1950-2000

Measles Mumps

‘Immune disorders’

Multiplesclerosis

Type 1diabetes

Asthma

From: Bach JF, N Engl J Med. 2002; 347:911-20.



The Battle Between Microbes and The Battle Between Microbes and Humans & Other Hosts TooHumans & Other Hosts Too

To regard any form of life as slave or foe will one day be considered poor philosophy, for all living things constitute an integral part of the cosmic order.

Rene Dubos



Do Remember…

The Microbes Always Have the Last Laugh



AcknowledgementsHelicobacter Group

Tompkins/Falkow Lab at StanfordManuel Amieva Sahar El-Etr Stanley Falkow Anne Mueller

Scotty Merrill Maria Goodrich Lucy Thompson

Laser Capture MicrodissectionJan Grimm, Rinat Neuroscience, Palo Alto

Cell Biology at StanfordJames Nelson

Roger Vogelmann

Chiron/Siena, ItalyAntonello Covacci

Stefano CensiniFabio Bagnoli

Sydney, AustraliaAdrian Lee

Jani O’Rourke



Is Disease a Distraction?

• Human Medicine (and granting agencies) demand that we focus on disease and its cure or amelioration.

• But does this focus sometimes distract us from understanding the biology of the pathogen and the evolution of the host parasite relationship?

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Host-Pathogen Interaction and Human Disease Pylori And... · 2013. 10. 20. · Stanley Falkow, May 2007 Host-Pathogen Interaction and Human Disease, Part 2 The American Society for