Modulation of Lung Epithelial function by Pseudomonas

aeruginosa

JOURNAL :
Lau GW, Hassett DJ, Britigan BE. Modulation of Lung Epithelial
function by Pseudomonas aeruginosa. Trend in Microbiology,
2005;13(8):389-99.
 Objectives
Introduction
Pseudomonas aeruginosa of normal and Cystic
Fibrosis Airways
PA product and their effects on the epithelium
Secreted Virulence Determinants
Benefits and limitations of experimental systems to
study PA-epithelial cell interaction
Genetic approaches to dissect the modulation of
lung epithelia by PA
Therapy and Prevention
 Introduction
Normal breathing gives access to most
microorganisms and harmful agents.
Ciliary motion removes most of the
microorganisms. The bacterium that
reaches the alveolar space are
deposited on the pulmonary epithelium.

Innate and acquired defense mechanisms

are initiated to counter the infection.
Pseudomonas aeruginosa of normal and Cystic
Fibrosis Airways
Effects of PA on the epithelial cell function, including
direct cyto-toxicity, activation of cellular signaling
and pro-inflammatory response have been examined
in vivo and vitro. DNA microarray has revealed the
epithelial gene expression varies with different
derived products.

Abnormalities in the cystic fibrosis transmembrane

regulator results in cystic fibrosis which enhances
sodium, chloride and water absorption,
hypersecretion of mucous results, which deposits on
the epithelial cilia, either the cilia beats harder or
does not beat.
PA growing on the cystic fibrosis airways is
known as biofilms.

The biofilm formation requires:

 flagella,
 type IV pili,
other gene products.
PA biofilms mainly results in the high resistance
to antibiotics and phagocytic killing.
Two models of biofilms are known:
steps leads bacterial attachment to the
surface,
second model is characterized by bacterial
rafts.

At the chronic stage the biofilm model 2

predominates. This involves
overproduction of alginate,
loss of flagella,
mutation in antigenic B-band O-antigen genes
reduces expression of the extracellular
virulence determinants.
Microscopic examination of the airways
suggests that the PA rarely colonizes in the
airways at the chronic stage. Instead of
colonizing the bacteria embeds itself in thick
mucus. Within the mucus there are low levels
of oxygen therefore creating toxic gradients.
PA product and their effects on the
epithelium
Membrane Bound Virulence Determinants

LPS (lipopolysaccharide)
lipid A is a component of LPS and is a regulator of
epithelial function through interaction with toll like
receptor 4 signaling complex (TRL4, TRL2, CFTR)
lipid A is a hexa-acylated substances that induce
inflammatory action
Flagella and Pili
required for motility and lung infection of P. aeruginosa
enable attachment to cells (mucin, glycolipid,
asialoGM1) and causes release of interleukin(IL-8) through
Src-Ras-ERK1/2NF-kB pathway.
Flagellin is a globular protein component of flagella that
activates matrilysin transcription
Alginate
is a linear co-polymeric exopolysaccharide
comprised of alpha-L-guluronate & O-acetylated beta-
D-mannuronate residue and responsible for mucoidy
in P.aeruginosa
its primary role is to moisten local immune response
towards P.aeruginosa
Secreted Virulence Determinants
Pathogenic interaction between PA and hosts are often
guided by exoproducts secreted by the bacteria that interact
with specific host targets.
What these exoproducts?
Quorum sensing molecules
 Communication between PA cells by small, diffusible acylated homoserine
lactones: Pseudomonas autoinducers (PAIs)
 PAI have two types
 PAI-1: stimulate production of IL-8 always deactivated host
 PAI-2: does not modulate host immune function not inactivated
 Pseudomonas quinolone signal (PQS)
 Intertwine with PAI-1 and PAI-2 and associated with production of
numerous virulence factors.
Type 3 secretion system (T3SS)
allows P. aeruginosa to translocate or to deliver protein
toxins (effectors) directly into the cytoplasm of targeted host
cells.
 Secretion is triggered by cell contact.
Only seen in early stage of infection
Pseudomonas produce four T3SS proteins.
ExoU
 a phospholipase that inhibit survival pathway and activated
proapoptotic pathway
ExoS and ExoT
 The N-terminal of ExoS & ExoT encode for GTPase activating protein
activity
 The C-terminal domains – ADP-ribosyltaranferase activity
ExoY
 Increase lung epithelial intracellular cAMP
 Cell rounding and detachment.
 Inhibit phagocytosis
 Disrupts pulmonary endothelial barrier function
Phenazines
secondary metabolites which function as microbial
competitiveness and virulence
Phenazines: pyocyanin-1-hydroxyphenazine and
phenazine-1-carboxylic acid
Pseudomonas aeruginosa secretes phenazine into the
CF airways copiuos amounts. These secretions
penetrates biological membranes and cause damage to
lung epithelia, alters epithelial cytokine production, and
modulate cellular signaling pathway.
Exoproteases
Secretes elastases (LasA and LasB), alkaline protease,
and protease IV.
Elastases activates the mitogen-activated protein
phase (MAPK) pathway which increases IL-8 expression
and augument respiratory epithelium permiability
Phopholipases
Pseudomonas aeruginosa elaborates several
phospholipase: hemolytic PlcH and non-hemolytic PlcN,
PlcB and PldA.
Anti-PLC antibody can be detected in CF patients, and
the isolation of PA strains is capable of secreting PLCs
which is usually associated with poor clinical status
Exotoxin-A (ETA)
Most PA isolates secretes ETA as a potent inhibitor of
protein synthesis via ADP-ribosylation of elongation
factor 2.
ETA can be expressed by the availability of iron which
is chelated by two siderophores pyochelin and
pyoverdin.
ETA can bind to 2-macroglobulin receptor
ETA can kill airway epithelia non-apoptotically via
mitochondrial dysfunction , superoxide production and
DNA degradation
Rhamnolipid
Rhamnolipid contains: rhanose and glycolipid biosulfactant

Nitrate reductase (NirS)

Lysed bacteria can release NirS which directly stimulates
the expression of IL-8 and monocyte chemoreactant protein-
1 (MCP-1) but not tumor necrosis factor-α (TNF-α) or IL-1β
However, NirS can stimulate alveolar macrophage
production of TNF-α or IL-1β
Benefits and limitations of experimental systems to study
PA-epithelial cell interaction
The agar or alginate bead model should not be constructed
as one that represents CF airway disease because it is simply
a chronic lung infection.
The acute pneumonia model probably serve well as a
reasonable representation of infection in non-CF airways
and perhaps early stages of CF airway colonization by
environmental isolates
The model host systems represent a convinient method to
screen for conserved PA virulence determinants that are
required to infect evolutionarily diverged hosts, but any
findings require confirmation in mammalian system
Genetic approaches to dissect the modulation of
lung epithelia by PA

Genomic sequence – PA01 and PA14 , available and

most widely used.
Genomic approach : Microarrays and Proteomics
 Therapy and Prevention
PA highly resistant to antibiotic, due to anti-PA
coumpounds that block QS.
Furanones – marine macro alga Delisea pulchra :
Inhibit QS in PA
Suprress virulence factors expression and biofilm formation
Accelerat bacterial clearance
Render PA within preformed biofilm susceptible to
Polymorhonuclear leukocyte killing.
Combination of PMNs,QS inhibitors, and antibiotics could
eliminate PA before chronic infections.
The PAI-1 could be remove by human acyl-homoserine
lactone acylase.
Antioxidants such as gluthathione aerosol, thioredoxin,
and anti-oxidant supplement.
Vaccination : Mucoid exopolysaccharide commony -
found in sera of older CF patients. Only for reducing the
opsonic antibodies.
LPS O-antigen have been encouraging but not clinical
efficacious.
Polysaccharide –protein conjugates and passive
immunization with immune sera offer additional
immunotherapeutic reagents .
 Any
Questions ?