EHEC , EPEC spread of infection

Seite 1

EHEC virulence factor includes the ability to adhere to plant materials, acid tolerence and the attachment and effacement to the intestinal endothelium, and the production of endotoxins and shiga toxin .This hyper- adherence is caused by tdcR and OmpA which are expressed on the outer membrane of the protein.

Survival of E.coli 0157:H7 during the passage through the stomach or encounter with acidic conditions in the environment is due to 4 distinct acid tolerance systems.There are 4 acid resistance gene systems.Mechanism for AR1 is unknown but AR2-4 each depend on amino acid decarboxylation and consumption of protons which results in pH homeostasis. The expression of AR is usually induced by the acidic environment,anaerobiosis and entry into a stationary phase.One or more of these systems is on when the EHEC is exposed to acid

A cardinal virulence factor of EHEC is the Locus of Entrocyte Effacement LEE which is found on the pathogenicity island that encodes all the gene products needed for attaching and effacing the colonic epithelium.Following the attachment of EHEC to the epithelial cells there is proteins secreted which are encoded by the LEE into the host cell.these proteins include EspA , B and D. The translocation of these proteins into the host cell is nessecary for the changing the host physiology.

EspA may be a part of translocation machinery because it forms filamentous appendages between the bacterium and the host cell.

EspB is translocated and alters the host cell signal transduction pathways

These LEE proteins are secreted by type III secretion system which is also encoded by LEE genes.

The next stage of intimate attachment and effacement involves the EHEC cell attachment to the host cell.

This is accomplished by EHEC which translocates its receptor protein ,translocated intimin receptor (TIR) to the host cell where it can be expressed on the host cell membrane

EHEC expresses an outer membrane protein called intimin at is encoded by the eae gene.

EHEC encodes both its receptor and its adhesin.

Following the tight association between EHEC and host cells actin pedestals are formed in the host cell which allow the push of host membranes to lengths up to 10 micrometer away from the host cell surface. The role of these pedestals in pathogenesis is not fully understood

Like all the gram-negative bacteria EHEC outer membrane has a outer facing leaflet of LPS, this LPS consists of Lipid A , Core polysaccharide and O-antigen which consists of 40-80 repeating subunits of 4 sugars that in the case of EHEC is serotype O157 which contains Nacetyl d perosamine , I-fucose , d -glucose and N acetyl -d-galactose.

Core polysaccharide is found in all ecoli serotypes.

Lipid A is a toxic component of LPS, it is also known as a endotoxin which is a heat stable toxin.Lipid A consists of a phosphorylated disaccharide of glucosamine linked by a beta 1,6 linkage and modified by fatty acids in addition to ketodeoxyoctanoate of the core polysaccharide.

Endotoxin is released by cell lysis rather than being secreted , endotoxin is less potent and less specific than exotoxins. Endsotoxins cause fever hemorrhagic shock and diarrhea

The second cardinal virulence factor of EHEC is shiga toxin which tends to cause diarrhea and hemolytic uremic syndrome which is a giveaway of EHEC infection.

EHEC produces Stx-2 which is an A-B toxin comprised of a single A subunit noncovalently associated with pentamer of B subunits.

B subunits bind to globotrioacyl ceramide on the host cell cytosolic membranes and facillitate A subunit uptake by endocytosis.

Stx is an N-glycosidase that targets the 28S rRNA, which it depurinates at a specific adenine residue, causing protein synthesis to cease and infected cells to die from apoptosis.

Stx receptors are found on intestinal endothelial cells and also on glomerular endothelial cells. Death of colonic cells and lesions in small blood vessels causes bloody diarrhea.

In the kidney, Stx damages glomerular endothelial cells by binding to globotrioacyl ceramide. Consequent to glomerular endothelial cell damage is a loss of glomerular barrier function that allows the passage of red blood cells and proteins into the urinary space.

Thus, Stx causes glomerular endothelial cell dysfunction, inflammation, and microvascular thrombosis. The resulting compromise in blood supply is thought to contribute to loss of kidney function.

Seite 2

Escherichia coli O157:H7 is an emerging pathogen that causes hemorrhagic colitis. E. coli is a Gram negative rod, a facultative anaerobe motile by means of peritrichous flagella, and found in the GI tract of mammals. Most E. coli are harmless and actually are an important members of a healthy human intestinal microbial community.

However, E. coli is a very diverse group of bacteria consisting of many ecotypes (the harmless variety) and pathotypes. The pathogenic strains of E. coli can cause diarrheal diseases, urinary tract infections, and even meningitis.

The E. coli pathotypes that cause diarrhea are transmitted via contaminated food or water, or through contact with infected animals or people. E. coli O157:H7 was first reported by the CDC in 1982. [1] E. coli O157:H7 is one of several pathotypes classified as enterohemorrhagic E. coli (EHEC). EHEC cause disease by producing a toxin called Shiga toxin, Shiga toxin causes bloody diarrhea and in approximately 5-10% of cases hemolytic uremic syndrome (HUS), which can lead to complete kidney failure.

Patients with bloody diarrhea usually recover within ten days and those who develop HUS usually recover within a few weeks, but in cases of kidney failure the patient may die.

While people of any age are susceptible, the young and elderly are more often affected. Treatment of EHEC typically involves rehydration without administration of antibiotics and hospitalization in severe cases, especially HUS

Seite 3

EPEC

Intro:

EPEC is the major cause of fatal diarrhoea in infants around the world in developing countries.

The pathovar belongs to the family of attaching and effacing (A/E) lesions which attach to the epithelial cells of the intestine

There is also other members form this family which include EHEC and RDEC, the murine pathogen Citrobacter Rodentium and recently identified escherichia albertii which is assoicated with diarrhoe in humans.

The bacteria efface the microvilli and divert the host cell actin to form pedestals beneath the site of attachment.

This phenotype is afforded by the 35kb PAI known as the LEE.

Pathogenesis :

LEE is highly regulated and encodes the type 3 secretion system that translocates bacterial effector proteins into the host cell cytoplasm.

There is 7 effectors encoded by LEE.

Initial attachment of EPEC to the enterocyte of the small bowel is thought to involve bundle forming pili that are encoded on the EPEC adherence factor (EAF) plasmid.

Bundle forming pili are rope like fimbraie which interact with other EPEC bacteria to form microcolonies for localised adherence and with N-acetyl lactosamine containing receptor host cells.

Recently demonstrated that e.coli pilus which is found in many isolates of ecoli may act in concert with bundle forming pili to stabilize interactions between EPEC and host cells.

Intimate attachment is mediated through the interactions of the bacterial outer membrane protein intimin and translocated intimin receptor (Tir)

EPEC uses the T3SS to rapidly translocate the TIR into the cytoplasm of the host cell by the process which possibly is initiated by CA+ sensing.

TIR is then displayed on the surface of the host cell this then acts as a receptor for intimin.

Phosphorylation of TIR recruits Nck to the site of attachment which then activates the (N-WASP) and actin related protein 2/3 (ARP 2/3), this mediates the actin rearrangements and the pedestals formation

Phosphorylation of TIR dispensable in A/E lesion formation ex vivo.

EPEC has a large repertorie of effectors that are translocated into the host cell by T3SS and cause a subversion of host cell processes

Example of this is the cause for the cytoskeletal rearrangement and immune modulation as well as causing diarrhoea.

Many of these translocated effectors have many functions, example is the mitochondrial associated protein (MAP) this belongs to the family of proteins which share WXXXE motif and is thought to mimic the active form of CDC42 a small G protein

MAP is shown to act as a guanine nucleotide exchange factor for CDC42 in which it regulates the actin dynamics which results in the formation of filopodia which surrounds the bacterial microcolonies

MAP also targets the mitochondria where it disrupts mitochondrial structure and function.

ESpf is another effector which is targeted to the mitochondria and triggers the mitochondrial death pathway .

ESpf is also implicated in the inhibition of phagocytosis.

disruption of tight junctions and the mimicing the aspects of the host cell signalling pathway that is involved in membrane trafficking.

ESpB s has a dual role as a T3SS translocation protein and as an effector that prevents phagocytosis.

NLE proteins have roles in the EPEC virulence.

NLeA for example reduces the protein trafficking and disrupts tight junctions.

EspI inhibits opsonophagocytosis by the red blood cells cycle inhibiting factor (Cif) is a cyclomodulin that prevents the progression of the cell cycle and later induces the apoptosis.

There are several other Nle proteins which have been identified but there characterisation still remains a cursory.

EHEC

Intro:

A key reservoir for EHEC is cattle , EHEC is a highly infectious A/E pathogen which colonises the distal ileum and large bowel of humans.

This is the causative agent responsible for gastroenteritis in developed countries.

Transmission in humans occurs through contaminated food as well as water sources.

In north america and japan and parts of europe the serotype of EHEC which affects many people is the O157:H7 serotype

The adults and kids affected with EHEC suffer from haemorrhagic colitis and further complications can lead to a potentially fatal haemolytic uraemic syndrome.

All the EHEC 0157:H7 harbour the 92kb virulence plasmid called the pO157 which is 100 ORFs and which encodes several virulence factors.

The main virulence factor of EHEC is the shiga toxin stx which is the defining characteristic of the shiga producing ecoli (STEC) group to which EHEC O157:H7 belongs to

TWO subgroups of stx , STX1 AND STX2 which are found in various combinations in EHEC isolates with Stx2 being more prevalent in haemorrhagic colitis and HUS than Stx1]

Stx is an AB toxin which consists of a pentamer of the B subunit that is non covaently bound to enzymatically active aA subunit

Pathogensis:

EHEC lacks the secretory mechanism for Stx so the release of Stx occurs through the lambdoid phage mediated lysis in response to DNA damage and the SOS response , this is why antibiotic therapy would be discouraged as the toxin would be released.

STx receptors are the globotriaosylceramides which are found on the paneth cells in the human intestinal mucosa, and also on the surface of kidney epithelial cells.

The cattle lack these receptors on the intestinal tract which is why the colonisation of the EHEC in cattle is asymptomic

Stx B subunit interacts with Gb and induces membrane invagination to facilitate the internalization of the toxin.

The internalized Stx is trafficked through the early endosomes into the golgi where the A subunit is activated by the cleavage event.

This leads to necrosis and cell death

The Stx binding to the paneth cells has not been explored fully

Stx can however be found in Gb3 negative human intestinal cells , this can be possible after they have been taken up by the macropinocytosis inside these cells.

Inside these cell stx does not prevent protein synthesis or induce apoptosis but instead dampens chemokine expression and therefore suppress the inflammatory response.

The initial attachment of EHEC to the coloncytes is not that well defined,

EHEC possesses 16 potential fimbria like operons

Recent studies have shown that a type 5 pilus called the haemorrhagic coli pilus thats involved in adherence and biofilm formation ; the flagella and ecoli common pilus might also be involved in the attachment to the host cells

Like EPEC attachment of EHEC to host cells occurs through the interactions between intimin and TIR

Attachment can also be enhanced by intimin with nucleolin which is a surface localized intimin receptor the expression of which is increased by the Stx2.

As the stx is released on bacterial lysis the increase in nucleolin expression may be important for the attachment of progeny EHEC

The EHEC genome contains the same LEE as the EPEC genome but the EHEC injects twice as many effectors into the host cell as EPEC most of which are redundant

The redundancy provides EHEC with an evolutionary advantage that allows it to compete with other bacteria ,

The mechanism of the pedestal formation of EHEC is slightly different to the one from EPEC ,

Tir is not tyrosine phosphorylated by the host cell and the pedestal formation is Nck independent even though the pedestals are highly similar.

The Subversion of the host cell actin cytoskeleton is mediated by EspF homologue called the Tir cytoskeleton coupling protein (TccP)

This is linked to Tir by host protein insulin receptor tyrosine kinase substrate IRTKS.

Tccp interacts with N-WASP which activates ARP2\3 complex mediated actin assemble.

EHEC can sense the hormones adrenaline and noradrenaline from the host cells as well as quorum sensing molecule auto inducer 3 from gastrrointestinal cells that regulate the motility and T3SS expression.

The sensing of these molecules is required for virulence of EHEC in animal models and presents new interactions that should be taken into account when considering pathogen host interactions.

Seite 4

Ecoli consists of eight pathovars each of which cause infections

These eight pathovars are :

Enteroaggrevative ecoli (EAEC)

Enteropathogenic ecoli(EPEC)

Enterotoxigenic ecoli(ETEC)

Enterohemorrhagic ecoli(EHEC)

Enteroinvasive Ecoli(EIEC)

Diffusely adherent ecoli(DAEC)

Urinary pathogenic ecoli(UPEC)

Neonatal meningitis ecoli( UMEC)

The e.coli pathovars share many virulence strategies.

Adhesion of the bacterium to the host is required except for EIEC ecoli , this adhesion is achieved by the long appendages called fimbraie or pili.

Following attachment the ecoli subverts host cell processes often using the secreted proteins.

Hijacking the hosts cell signalling pathways will allow the ecoli to evade the host immune response and carry out an effective colonisation of the host and lead ultimately to the disease.

Each of the pathovars have there own method in attaching to the cell and exploiting the host cell and its machinery.

Seite 5

ETEC

The most common cause of travellers diarrhoea and can be fatal to children under the age of 5.

ETEC engagement to the epithelial cells in the small bowel is mediated through colonisation factors.These can be fibrial, non fibrial and fibrillar or helical.

There is a large number of CFS the most studied the CFA/I ,CFA/II and CFA/IV

Recent study showed that the flagella are bound to the tip with a secreted adhesin Etpa which can be used as a epithelial adhesion factor

Both the CFs and the flagella anchor the ETEC for initial attachment to host cells but a more intimate attachment is facilitated by the outer membrane proteins Tia and TibA

ETEC mediated diarrhoea has been attributed to the secretion of heat stable enterotoxins the heat labile enterotoxin or a combination of these.

STs are small toxins which can be further classed as STa or STb on the basis of structure and function and are synthesized s 72 amino acid precursors that are processed into active forms 18-19 amino acids for STa and 48 amino acids for STb

The STa which is linked with human disease binds to guanylyl cyclase receptors on the brush border of the intestine and stimulates their activity.

This leads to increased intracellular levels of cyclic GMP and activates the cystic fibrosis transmembrane conductance regulator which results in impaired absorption of Na and H2O efflux into the lumen.

LT is similar to cholera toxin and is also an AB toxin

Secreted from the pole of a bacteria cell and associated with lipopolysaccharide on the surface where it may act as an adhesin and facilitates the attachment of host cells.

The b subunit of LT interacts with monosialoganglioside GM1 on the host cells, this toxin is internalized at lipid rafts, where it is trafficked to the cytosol through the endoplasmic reticulum.

The A subunit ADP-ribosylates the stimulatory guaninenucleotide-binding (G) protein α-subunit (Gsα), which activates adenylyl cyclase and increases the levels of intracellular cyclic AMP

This activates cAMP-dependent protein kinase A (PKA), which in turn activates CFTR38. Intriguingly, activation of PKA and other host cell signalling pathways by LT has also been shown to inhibit expression of antimicrobial peptides

Other virulence factors have been shown to be secreted by ETEC (reviewed in REF. 63).

For example, EatA is a serine protease autotransporter of the Enterobacteriaceae (SPATE) that cleaves cathepsin G and may accelerate fluid build-up.

Other secreted virulence factors include CylA, a pore-forming cytotoxin, and E. coli ST1 (EAST1), which may have similar functions to STa.

Seite 6

EIEC

It is generally accepted that EIEC and Shigella should form a single pathovar, because they have the same mechanisms of pathogenicity.

However, the genus name Shigella is still used owing to its association with the disease shigellosis and is retained in this section.

Shigella are highly infectious bacteria that cause bacillary dysentery and bloody diarrhoea1 .

This pathovar differs from the other E. coli pathovars, because it includes obligate intracellular bacteria that have neither flagella nor adherence factors

Virulence is largely due to a 220 kb plasmid that encodes a T3SS on the Mxi–Spa locus that is required for invasion, cell survival and apoptosis of macrophages

Infection commences in the colon, where the bacteria pass through microfold cells (M cells) by transcytosis to reach the underlying submucosa .

The disruption of tight junctions and the damage that is caused by inflammation also give Shigella access to the submucosa.

Shigella uptake into resident macrophages, escape from the phagosome, caspase-1-dependent inflammasome activation and ultimate release from macrophages have been extensively reviewed.

Shigella are released from dead macrophages into the submucosa, from where they invade the basolateral side of colonocytes with the aid of effectors that are secreted by the T3SS.

Key effectors, such as IpaC, activate SRC kinases at the site of bacterial contact to ultimately recruit the ARP2/3 complex

and cause actin polymerization and ruffle formation for bacterial entry73.

RAC1, which can promote membrane ruffle formation, may be activated by IpgB1 mimicry of RhoG in the ELMO–DOCK180 (engulfment and cell motility–dedicator of cytokinesis protein 1) pathway

or directly, by IpgB1 guanine-nucleotide exchange factor activity.

Other effectors, such as IpgD, IpaA and VirA, are involved in the destabilization of actin and microtubules to promote invasion into a phagosome, and escape from phagosomes is dependent on the effectors IpaB, IpaC, IpaD and IpaH

Once free in the epithelial cell cytoplasm, Shigella promote their survival by using effectors to further subvert host cell processes .

To prevent intestinal epithelial cell turnover, IpaB mediates cell cycle arrest by targeting MAD2L2, which is an inhibitor of anaphase75, and OspE has been shown to interact with integrin-linked kinase (ILK) to prevent epithelial cell detachment

Apoptosis is also prevented through IpgD77, which can stimulate phosphoinositide 3-kinase and activate Akt proteins, which regulate cell survival.

These three mechanisms prevent cell death and sloughing, providing a replicative niche for Shigella to maintain an infection

To persist inside colonocytes, Shigella must also evade innate immune responses, for which they use at least four effectors.

One of these effectors, OspG, was shown to bind ubiquitylated E2 proteins, which prevents degradation of inhibitor of nuclear factor-κB (NF-κB) subunit-α (IκBα) and thus inhibits NF-κB activation78.

Additionally, OspF is targeted to the nucleus, where it irreversibly dephosphorylates mitogen-activated protein kinases that are required for the transcription of genes that are regulated by NF-κB79. IpaH9.8 is also targeted to the nucleus, where it interacts with a splicing factor that is involved in the expression of inflammatory cytokines80.

The fourth effector, OspB, acts with OspF to reduce interleukin-8

(IL-8) levels by recruiting host factors that remodel chromatin81.

Collectively, these four effectors are involved in dampening inflammatory responses and therefore allowing persistence of the bacteria

As Shigella do not have flagella, movement in the host cytosol and cell-to-cell dissemination require manipulation of the host machinery .

The outer-membrane protein VirG (also known as IcsA) localizes to a single pole and recruits and activates N-wASP and the ARP2/3 complex for actin polymerization82. The growth of the actin filaments pushes the bacteria through the cell.

The ability of VirA to stimulate the destabilization of microtubules plays an important part in the efficient intracellular spread of Shigella by providing a channel that enables them to move throughout the cell83.

As a host defence, autophagy targets cytosolic Shigella through autophagy protein 5 (ATG5) recognition of VirG. Interestingly, the secreted effector IcsB can bind VirG and sequester it, thereby avoiding another aspect of host defence84

Seite 7

EAEC

Although it is considered to be an emerging pathogen, EAEC is the second most common cause of travellers’ diarrhoea after ETEC in both developed and developing countries. EAEC is also becoming commonly recognized as a cause of endemic and epidemic diarrhoea worldwide.

Diarrhoea caused by EAEC is often watery, but it can be accompanied by mucus or blood. EAEC colonization can occur in the mucosa of both the small and large bowels, which can lead to mild inflammation in the colon38.

Much like the details of its transmission and epidemiology, the understanding of EAEC and its pathogenesis is limited, in part owing to the paucity of suitable animal models and the heterogeneity of virulence factors.

The characteristic phenotype of EAEC is aggregative adhesion, which involves the formation of a stacked-brick pattern of HEp-2 cells and is mediated by the genes that are found on a family of virulence plasmids called pAA plasmids.

These 100 kb plasmids encode the necessary genes for the biogenesis of the aggregative adherence fimbriae (AAFs), which are related to the Dr family of adhesins and mediate the adherence of EAEC to the intestinal mucosa .

AAF- and flagellin-mediated adherence induces an IL-8 response, which leads to the transmigration of neutrophils85,86. Four variants of AAFs (AAF/I, AAF/II, AAF/III and Hda) have been identified.

The receptors for AAFs are unknown, but recent data show that AAF/II can bind fibronectin89

Fimbrial extension of the positively charged AAFs away from the negatively charged lipopolysaccharide is thought to occur through a secreted protein called dispersin.

Dispersin associates with lipopolysaccharide through electrostatic interactions and is speculated to mask the negative charge of the lipopolysaccharide, thus allowing the AAF to extend away from the bacterial surface instead of collapsing onto it.

This promotes dispersion of EAEC across the intestinal mucosa, by counteracting excessive AAF-mediated aggregation between other EAEC cells.

Biofilms formed by EAEC are distinct from biofilms formed by non-pathogenic E. coli, in that they can form independently of common factors such as curli, flagella and antigen 43 (Ag43)91.

EAEC biofilms on the surface of enterocytes are encased in a thick mucus layer.

EAEC is thought to be able to penetrate this mucus layer through the mucolytic activity of the SPATE Pic92.

A few genes, both plasmid-borne and chromosomal, encoding proteins that are involved in the formation of biofilms have been identified, including genes that encode a type VI secretion system93; the details, however, remain cursory.

EAEC causes mucosal damage by secreting cytotoxins, although not all toxins are found in all isolates.

The plasmid-encoded toxin (Pet) is a SPATE that targets α-fodrin (also called SPTAN1), disrupting the actin cytoskeleton, and induces exfoliation1 .

The toxin is internalized by a clathrin-based endocytosis mechanism and is subsequently trafficked through the endoplasmic reticulum to the cytosol94,95.

Two other toxins, Shigella enterotoxin 1 (ShET1) and EAST1, can be found in other pathogenic E. coli isolates, and their role in pathogenesis is not completely understood.

Seite 8

DAEC

DAEC is a heterogenous group that generates a diffuse adherence pattern on HeLa and HEp-2 cells.

This pattern is mediated by proteins encoded by a family of related operons, which include both fimbrial (for example, Dr and F1845) and afimbrial (Afa) adhesins, collectively designated Afa–Dr adhesins .

DAEC isolates that express any of the Afa–Dr adhesins (which are referred to as Afa–Dr DAEC) colonize the small bowel and have been implicated in diarrhoea in children between the ages of 18 months and 5 years, as well as in recurring urinary tract infections (UTIs) in adults

All Afa–Dr adhesins interact with brush borderassociated complement decay-accelerating factor (DAF), which is found on the surface of intestinal and urinary epithelial cells .

Binding to DAF results in the aggregation of DAF molecules underneath the adherent bacteria.

It also triggers a Ca2+-dependent signalling cascade, which results in the elongation and damage of brush border microvilli through the disorganization of key components of the cytoskeleton96.

Furthermore, along with flagella the interaction between Afa–Dr adhesins and DAF induces IL-8 secretion from entero cytes, which promotes transmigration of polymorpho nuclear neutrophils (PMNs) across the mucosal epithelial layer.

This stimulates the upregulation of DAF on the apical surface of epithelial cells, providing DAEC with more receptors for tighter adherence97.

DAEC interactions with PMNs, mediated by Afa–Dr, lead to an accelerated rate of PMN apoptosis and a decreased rate of PMN-mediated phagocytosis.

A subclass of Afa–Dr fimbriae interact with members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family of receptors that are found on the surfaces of membranes, in particular in lipid rafts96.

Interactions with CEACAMs enhance the activation of CDC42, leading to CEACAM aggregation underneath the adherent bacteria and the effacement of the brush border microvilli99.

These lesions disrupt several brush border enzymes that are involved in intestinal secretion and absorption, which may contribute to diarrhoea.

It was recently shown that interactions between Dr and CEACAMs cause CEACAM dimers to dissociate, so that Dr can interact with the monomeric form of the receptor100.

This may serve as an interesting method to manipulate host pathways through the response that is mediated by the disruption of CEACAM dimers.

Afa–Dr adhesin interactions with CEACAMs and with DAF may be involved in microtubule-dependent uptake of DAEC cells, following which the bacteria can survive in vacuoles

Unlike other pathogenic E. coli, the pathogenesis of DAEC seems to be predominately mediated through Afa–Dr adhesin interactions with host cells.

Secreted autotransporter toxin (Sat) has been implicated in lesions of tight junctions that are found with Afa–Dr DAEC infection and in increased permeability .

No secretion systems or other virulence factors have been identified in typical Afa–Dr DAEC isolates.

Seite 9

UPEC

Uropathogenic Escherichia coli.

UPEC infections account for roughly 80% of all UTIs, causing cystitis in the bladder and acute pyelonephritis in the kidneys.

UPEC has the challenge of moving from the intestinal tract to establish an infection in the urinary tract, where it uses peptides and amino acids as the primary carbon source for fitness. T

he ability to ascend the urinary tract from the urethra to the bladder and kidneys reflects exceptional mechanisms for organ tropism, evading innate immunity and avoiding clearance by micturition.

Several highly regulated virulence factors contribute to this complex pathogenesis, including multiple pili, secreted toxins (for example Sat and vacuolating autotransporter toxin (Vat)), multiple iron acquisition systems and a polysaccharide capsule

Entry of UPEC into the urinary tract is followed by adhesion to the uroepithelium. This attachment is mediated by fimbrial adhesin H (FimH), which is found at the tip of the phase-variable type 1 pili.

FimH binds to the glycosylated uroplakin Ia that coats terminally differentiated superficial facet cells in the bladder.

Interactions between FimH and uroplakin IIIa were recently found to lead to phosphorylation events that are required to stimulate unknown signalling pathways for invasion and apoptosis.

UPEC invasion is also mediated by FimH binding to α3 and β1 integrins that are clustered with actin at the sites of invasion, as well as by microtubule destabilization.

These interactions trigger local actin rearrangement by stimulating kinases and Rhofamily GTPases, which results in the envelopment and internalization of the attached bacteria.

Once internalized, UPEC can rapidly replicate and form biofilm-like complexes termed intracellular bacterial communities (IBCs) or pods, which serve as transient, protective environments.

UPEC can leave the IBCs through a fluxing mechanism; motile UPEC leaves the epithelial cells and enters the lumen of the bladder.

Filamentous UPEC has also been observed fluxing out of an infected cell, looping and invading surrounding superficial cells in response to innate immune responses

During infection, the resulting influx of PMNs causes tissue damage, and UPEC attachment and invasion results in apoptosis and exfoliation of bladder cells.

In addition, sublytic concentrations of the poreforming haemolysin A (HlyA) toxin can inhibit AKT activation and lead to host cell apoptosis and exfoliation.

This breach of the superficial facet cells temporarily exposes the underlying transitional cells to invasion and dissemination of UPEC.

Invading bacteria are trafficked in endocytic vesicles enmeshed with actin fibres, where replication is restricted.

Disruption of host actin permits rapid replication, which can lead to IBC formation in the cytosol or fluxing out of the cell.

This quiescent state may act as a reservoir that is protected from host immunity and may therefore permit long-term persistence in the bladder.

Interestingly, UPEC infection was recently shown to manipulate the differentiation of the urothelium, and when urothelial turnover was chemically induced these quiescent reservoirs were able to reactivate and cause an acute infection of the bladder.

The regulation of urothelial turnover may have important implications in patient predisposition to UTIs and bladder cancer.

UTIs that are left untreated can disseminate to the kidney in an ascending progression of disease.

Ascension to the kidney is mediated by reciprocal regulation of type 1 pili and motility.

Bacteria that express type 1 pili are less flagellated than those that do not, suggesting that when type 1 pili are ‘switched off ’, UPEC can become more motile116.

Futhermore, motility was shown to permit the ascension from the bladder to the kidney117.

UPEC isolates that are associated with pyelonephritis often express the P fimbriae that adhere to Galα(1–4)Galβ moieties of the globoseries glycolipids that are found on the surface of kidney epithelial cells1 .

Similarly to the inverse relationship between type 1 pili and motility, expression of P fimbriae is associated with fewer flagella and repressed motility 118.

Crosstalk between P fimbriae, type 1 pili and other adhesion clusters prevents co-expression of multiple surface organelles119.

The correlation between P fimbriae and virulence, however, remains inconclusive.

Seite 10

NMEC

NMEC, a common inhabitant of the gastrointestinal tract, is the most frequent cause of Gram-negative-associated meningitis in newborns.

Fatality rates can approach 40%1 , and survivors are usually burdened with severe neurological sequelae.

The pathogenesis of NMEC is complex, as the bacteria must enter the bloodstream through the intestine and ultimately cross the blood– brain barrier into the central nervous system , which leads to meningeal inflammation and pleocytosis of the cerebrospinal fluid.

Initial colonization, after the bacteria have been acquired perinatally from the mother, is followed by transcytosis through enterocytes into the bloodstream.

The progression of disease is dependent on high bacteraemia

so survival in the blood is crucial. Protection from the host immune responses is provided by an antiphagocytic capsule, made up of a homopolymer of polysialic acid, and serum resistance, resulting from manipulation of the classical complement pathway by the bacterial outer-membrane protein A (OmpA)120. NMEC has also been shown to interact with immune cells: invasion of macrophages and monocytes prevents apoptosis121 and chemokine release122, providing a niche for replication before dissemination back into the blood.

Maturation of dendritic cells is also inhibited by NMEC123.

Recently, a lambdoid phage that encodes O acetyltransferase was discovered, which acetylates the O antigen to provide phase variation and diversity to the capsule124 and may therefore hide the bacteria from host defences

The blood–brain barrier is a tight barrier formed by brain microvascular endothelial cells.

Attachment of NMEC is mediated by FimH of the type 1 pili binding to CD48 and by OmpA binding to its receptor, ECGP96 , on the surface of brain microvascular endothelial cells.

Invasion occurs through the actions of Ibe proteins, FimH, OmpA and cytotoxic nectrotizing factor 1 (CNF1)127.

The receptors for the Ibe proteins are unknown, but the 67 kDa laminin receptor (67LR; also known as RPSA) was shown to be the receptor for CNF1.

CNF1 is a toxin that deaminates Rho-family GTPases that are involved in myosin rearrangement129.

It is possible that FimH- and OmpA-mediated attachment to brain microvascular endothelial cells may be required before translocation of CNF1 into the host cell can occur. OmpA interaction with its receptor and FimH-mediated increase of intracellular Ca2+ stimulates actin rearrangements125,130 that, along with the CNF1-stimulated myosin rearrangements, are involved in the invasion of NMEC.

The K1 capsule — which is found in approximately 80% of NMEC isolates — also has a role in invasion by preventing lysosomal fusion and thus allowing delivery of live bacteria across the blood–brain barrier 131.

Collectively, these mechanisms allow NMEC to penetrate the blood–brain barrier and gain access to the central nervous system, where they cause oedema, inflammation and neural damage.

Seite 11

AIEC

Pathogensis

Despite the genetic diversity of AIEC, several strains do share common virulence factors, albeit not unique to AIEC.

One of the defining features of AIEC is their ability to adhere to intestinal epithelial cells, which is likely facilitated by several bacterial surface structures.

For example, surface expression of chitin-binding domains by AIEC was found to mediate their adherence to the chitinase-like receptors on the intestinal epithelium

Additionally, long polar fimbriae mediate AIEC attachment to Peyer's patches, allowing AIEC to localize to the terminal ileum from where they are often isolated

And finally, AIEC type I pili can bind carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) via FimH, the terminal subunit of these surface appendages

Unlike commensal E. coli strains, FimH in AIEC has accumulated a small number of non-synonymous mutations that may facilitate binding to CEACAM6

Notably, the abundance of CEACAM6 in the ileum of CD patientsmakes FimH a plausible target for pathoadaptation during AIEC evolution, and is the focus of most of the anti-adhesive strategies in therapeutic development in this space.

Other host surface receptors that were found to be upregulated in the ileum of CD patients include the endoplasmic reticulum stress response glycoprotein Gp96.

This host protein may be clinically relevant because it has been shown to interact with AIEC OmpA, a member of a family of outer membrane porins in Gram-negative bacteria

Given that both CEACAM6 and Gp96 are more abundant during inflammation, the expression of inflammation itself may promote the redistribution of AIEC to sites proximal to the epithelial surface, a process seen by the host as a danger signal that initiates even greater inflammatory responses

E. coli is a diverse bacterial species whose members range from seemingly innocuous commensal strains to quite dangerous human pathogens. Pathovar designations are used to classify E. coli into groups with unique molecular mechanisms that govern their pathogenic behavior.

The genetic determinants that help define E. coli pathovars (including serotype, toxins, and virulence factors) represent the basic tenets for their identification and facilitate the tracking of their evolutionary history

While much is known about the evolution of many E. colipathotypes, the origin of the AIEC group is less clear.

One key challenge in defining the AIEC pathovar is that the genetic factors conferring the adherent-invasive phenotype are not fully defined.

Consequently, the identification of AIEC is done based on a series of in vitro phenotype assays that are laborious, time-consuming, and somewhat non-standardized.

Also, virulence determinants that define other E. coli pathovars at the genetic level (i.e. Shiga toxin, type III secretion systems) are not found in AIEC.

While this fact can be used as exclusion criteria when attempting to classify isolates of E. coli from patients, a molecular genetic signature that distinguishes the AIEC pathovar remains elusive.

In a recent study, comparative whole-genome analysis of 14 AIEC strains identified a potential subgroup within the B2 phylotype that appeared more similar due to three genetic insertions that differentiated them from commensal E. coli

A separate study by a different group did not identify a readily distinguishable genomic signature among 11 different B2 phylotype AIEC strains although these strains were from a different geographic locale

As shown in different genomic studies, AIEC appear to be most closely related to extraintestinal E. coli strains such as UPEC, APEC, and ExPEC, which are also among the B2 clade

Furthermore, many virulence factors were found to be shared between AIEC and UPEC, including genes that are required for iron acquisition and transport.

Together, these findings suggest that AIEC do not arise by parallel evolution and clonal expansion as described for the notorious O157:H7 enterohemorrhagic E. coli

At this point, available evidence suggests that AIEC can evolve from diverse founder populations and use genetically distinct mechanisms to attain the ‘AIEC phenotype’.

This is reminiscent of the genetic variability that exists among uropathogenic E. coli(UPEC), which also lacks a common genetic signature

UPEC are members of the so-called ‘extraintestinal pathogenic E. coli’ to which AIEC are also closely related

The selective drivers of AIEC's evolutionary trajectory remain obscure, yet they likely originate within the host.

Given that individual host environments can drive adaptive bacterial evolution an important question is whether unique host environments (i.e. different CD patients; biogeography within a single individual; or combinatorial risk factors, for example) might select for AIEC emergence and outgrowth.

Considering that research is now mobilizing towards AIEC as a new therapeutic frontier in CD

efforts to understand the selective pressures driving AIEC evolution should be redoubled.

he diversity of virulence factors displayed by multiple AIEC strains, some of them shared with ExPEC, suggests that members of this pathovar have evolved different strategies to colonize their hosts.

This is also in keeping with the genetic variability that exists within the AIEC.

It remains to be determined whether the host environment might favor AIEC strains that harbor a specific set of virulence determinants.

We believe that an important next step in our understanding of AIEC is to broaden the scope of genomic studies to both bacteria and host.

This could reveal potential associations between bacterial genotypes and host genetic backgrounds from which the strains arose.

Figure 1.

A schematic diagram that summarizes some of the inflammation-mediated changes in the gut and their influence on AIEC colonization.

Gut inflammation can be triggered by multiple factors like host genetics, antibiotic administration, diet, and acute gastroenteritis.

The resulting proinflammatory environment mediates pronounced changes in the human gut, which includes hypoferremia, the generation of alternative electron acceptors, increased secretion of antimicrobial peptides, and overexpression of CEACAM6 surface receptors by intestinal epithelia.

During their evolution, AIEC strains have gained traits that confer on them a competitive advantage in the inflamed gut.

This includes the ability to utilize S- and N- oxides as alternative electron acceptors, an abundance of iron acquisition genes, resistance to antimicrobial peptides and the ability to bind to CEACAM6 through a modified FimH protein.

The type 1 pilus is a vital virulence factor that allows bacteria to adhere to host cells [15] [16]. In AIEC, the sigmaE pathway promotes type 1 pilus expression [17].

Therefore, interactions between intestinal epithelial cells and AIEC are enhanced, leading to adhesion of AIEC and increased biofilm formation [18].

Furthermore, the orientation of type 1 pilus of AIEC differs from the E. coli K12 strain and is regulated by various mechanisms.

The expression of fim genes is induced by Fis histone-like proteins. When Fis proteins bind to Fis-binding sites, the transcription of fim genes such as fimE is initiated.

The FimE recombinase is responsible for regulating the on- and off-phases of the type 1 pilus. It has been found that fimE expression is significantly downregulated in the AIEC strain.

This results in an increase of on-phase oriented type 1 pili on the outer membrane [19].

In addition, DsbA is a periplasmic oxidoreductase that catalyzes disulfide bond formation between cysteine residues.

In turn, DsbA promotes on-phase type 1 pilus orientation through posttranslational modification [15]. The increased expression and favorable orientation of type 1 pilus enhances the adhesion of AIEC to epithelial cells.

Differing physiological mechanisms of AIEC not only enhance its ability to adhere to the host, but also to invade the intestinal epithelial cells.

The AIEC strain has enhanced outer membrane vesicle (OMV) formation thus leading to the delivery of more virulence factors to the cell membrane via exocytosis [20].

Outer membrane proteins OmpA and OmpC are present on the OMV membranes [21]. Upon exocytosis, these proteins are transferred to the outer membrane of AIEC cells.

OmpA is responsible for binding to a protein, Gp96, located on the intestinal epithelial cells [22].

Meanwhile, OmpC is responsible for activating the aforementioned sigmaE pathway, resulting in increased expression of outer membrane proteins and type 1 pili [23].

With the increased secretion of virulence factors by OMV formation and movement, the tight binding between OmpA and Gp96 promotes the ability of AIEC to invade the cells and cause disease [22].

Following invasion of the intestinal epithelial cells, the AIEC has mechanisms that allow it to evade intracellular macrophage killing. DsbA, a periplasmic oxidoreductase, is activated in nutrient-deprived environments such as the phagolysosomes of the macrophage.

Bringer et al. suggests that the catalysis of disulfide bond formation by DsbA activity prevents the accumulation of unfolded proteins in the cytoplasm, which would otherwise lead to cell death [15].

In addition, AIEC promotes the secretion of TNF-α by macrophages, which suppresses the function of host immune cells.

Furthermore, increased amounts of TNF-α promote the intracellular replication of AIEC.

As a result, AIEC not only evades intracellular cell death, but also creates an immunosuppressive environment that can promote its intracellular replication [24].