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Pathogenicity Islands (PAIs) are a subset of genomic islands and are characterized by a distinct set of virulent genes that were acquired through horizontal gene transfer (HGT). PAIs are unique to pathogenic bacteria and can be found in both Gram positive and Gram negative species. PAIs can carry 1 or more genes and occupy large regions of the genome, ranging from 10 to 200 kb. PAIs are responsible for encoding the virulence factors, and their secretion and regulatory systems.

Pore-forming toxins

Pore-forming toxins (PFTs) are the most commonly used virulence factor among pathogenic bacteria, making up 25-30% of virulent proteins, and are encoded by the bacterial PAI. A pore are characterized as a water-filled lesion through the target membrane, which allows the bacterium to communicate with the host cell, either by interfering with cell signaling or by triggering apoptosis. One way bacteria create pores is by secreting water soluble PFTs into environment which bind specifically and polymerize in at the host cell surface. The proteins then form a protein channel through the host membrane. Bacteria can also form pores by fusing its membrane with the host’s membrane, creating a lipid based opening. After the pore has been formed, the bacteria can release effector proteins into the cell to release nutrients from the host, modulate cellular process, or to trigger cell death.

Type III secretion system

Secretion systems are a major component of pathogenesis, and are encoded by the locus of enterocyte effacement (LEE) PAI. The Type III secretion system uses a protein complex that forms a syringe like structure that extends outward from the bacterium and breaks through the host cell’s membrane to directly inject virulent proteins into the host’s cytoplasm or membrane, depending on the function of the effector protein.

The structure and function of the Type III apparatus is conserved among all pathogens; however, the released effectors are specific to the target cell. The base of the Type III apparatus spans the entirety of the bacterial membrane and protrudes into the bacterial cytoplasm, and the tip extends out into the environment, forming a narrow channel inside the apparatus for proteins to pass through. A protein sits on the tip of the apparatus and blocks effector proteins from being secreted until contact with a host has been made. After successful contact, the tip is released, and bacterial enzymes unfold the effector proteins before sending them through the apparatus, as the channel is too narrow for a folded protein to pass through. The secretion process takes only a few minuets at a rate between 7 to 60 proteins a second.

Adherence

The 1^st step of infection for pathogenic E. coli is to attach to epithelial cells of the gastrointestinal tract by forming structures known as, attaching and effacing lesions (AE lesions). AE lesions are characterized by intimate bacterial attachment to host cells, the destruction of intestinal microvilli and the formation of cytoskeleton structures made from actin called pedestals.

All the genes needed for AE lesions are encoded by the LEE PAI. The LEE PAI encodes the Type III secretion apparatus which is used to inject the protein, Tir, directly on the surface of the host membrane. LEE also encodes the protein, Intimin, which is secreted from the bacterium onto the bacteria’s outer membrane. Tir has a hairpin-loop structure and binds intimin to form an intimate connection between the bacterium and the host cell.

Eukaryotic cells utilize the Arp2/3 signaling pathway to control actin polymerization that makes up the eukaryotic cytoskeleton. However, after successful adhesion, pathogenic E. coli can exploit the eukaryotic Arp2/3 pathway. Effector proteins are released into the adhesion site and activate the Arp2/3 pathway to build a cytoskeleton structure called a made of actin filaments, known as pedestal, directly below the bacterial attachment site. The pedestal partially covers the bacterium, providing some protection.

There are 2 types of E coli that use AE lesions, typical-Enteropathogenic E. coli (tEPEC) and atypical-EPEC (aEPEC), which are defined by the use of the EPEC adherence factor (EAF) plasmid. tEPEC strains encodes virulence factors from genes found on LEE and from EAF while aEPEC strains encode virulence factors from LEE and from the core genome. There are 3 types of adhesion, localized adherence, diffuse adherence and aggregation adherence, and the EAF is necessary for encoding the genes needed for localized adherence.

Localized Adherence (LA) is when bacteria bind in tight clusters to localized areas of a host’s cell surface, forming a micro-colony. EAF encodes the bundle-forming pili (BFP) that interconnects bacteria in the cluster to promote stability of the micro-colony, however, EAF is not essential for successful adhesion. LA is prevalent in younger children and results in diarrhea, however, LA is rarely seen in older children and adults as humans gradually develop a resistance to LA.

Diffuse Adherence (DA) is mediated by the Afa adhesion protein. Bacteria expressing diffuse adherence are commonly associated with urinary tract infections, pregnancy issues and diarrhea. The bacterial adhesion is similar to LA, but lacks BFP to create tight colonies, this results in DA being less virulent than LA.

Aggregation Adherence (AA) is mediated by aggregative adherence fimbriae (AAF) and is characterized by bacteria clustering together in a stacked brick-like formation on intestinal mucosa cells. After adhesion, they enhance mucus secretion to create a thick bacterium-mucus biofilm. This leads to cytotoxic effects such as, shortening of villi and a mild inflammatory response in sub mucosa. AA is associated with diarrhea in the small intestine of both children and adults and can sometimes occur in the urinary tract, resulting in UTIs.

Siderophores

Iron is not only essential for all microbial survival but is also an essential prerequisite for pathogenesis. Iron in microbes acts as an important global regulator for synthesis of DNA, ATP and heme. Iron also acts as a cofactor for various enzymes and can regulate the electron transport system and nitrite reduction pathways.

Iron in nature is found as iron III which can be converted into an insoluble ferric oxyhydroxide, which is difficult for bacteria to absorb. So bacteria have developed strategies to uptake iron III from the environment. Siderophores are a low weight, water soluble molecule that have a high affinity for iron III, and are common produced by PAIs of pathogenic bacteria. In iron limiting conditions, bacteria release siderophores into the environment. The siderophores bind to free iron, and then outer membrane proteins that were also encoded by the PAI, bind and uptake the iron-siderophore complex.

Because the levels of free iron in host bodies is too low for bacterial survival, siderophores are released in mammalian hosts to compete with host iron carriers. S. flexineri can release the siderophore, aerobactin, which can remove iron from host iron carriers, and deliver the iron to bacterium.

Shigella bacteria possesses the Crb phenotype which enhances shigella’s ability to invade cells. Crb encodes outer-membrane proteins that bind to the host iron carrier, heme. Shigella can utilize the iron within the heme for its own metabolism but also the heme coated bacteria can be use for virulence by traveling to host cells; the heme on the bacteria’s membrane surface binds to heme receptors on the host outer-membrane and then Shigella can trigger endocytosis of the host cell in a Trojan horse-like effect.

Invasion

Invasion Genes allow bacteria to enter eukaryotic cells and mediate host activities. A common method of invasion is to modify the host’s cytoskeleton. The bacteria first injects effector proteins via Type III secretion to modify the host’s signaling pathways of the actin cytoskeleton and of the lipid metabolism pathway, resulting in the formation of a ruffled looking outer-membrane organelle, known as a macropinosome. The ruffles of the macropinosome fold back on themselves, encapsulating and protecting the bacteria. Bacteria then use specific defense strategies to maintain its survival, by releasing proteins to fight the antimicrobial particles released from the host, breaking through the membrane to reside with in the host’s cytoplasm, or by residing in a non-microbicial organelle.

PAI formation

PAIs develop randomly through a relatively rapid natural selection process. The DNA that makes up PAIs are acquired through horizontal gene transfer, which is accomplished in 3 ways, natural transformation, conjugation and transduction. PAIs have a mosaic structure as opposed to a homogeneous sequence; the genes that make up the PAI were acquired at different times from different donor cells, the resulting PAI will have an inconsistent guanine-cysteine concentration compared to the core genome that can easily be identified when sequencing bacterial genomes.

During specific times of growth, some bacteria express genes that allow the cell to uptake free DNA from the environment. This process is called natural transformation. Most DNA collected will be degraded by the cell as if it has degraded over time, has no homologous sequences with the genome or serves no beneficial purpose to cell. However, DNA with homologous sequences containing beneficial genes can be integrated in the bacterial genome.

Conjugation is the process that most often occurs between bacteria of the same species. A donor cell extends a sex pilus to the recipient cell, and transfers a copy of a plasmid. The plasmid will replicate independently of the core genome, however, under certain conditions, the plasmid can integrate in the genome.

Transduction is the transfer of DNA from one bacteria to another via bacteriophages, and is the biggest contributor to PAI formation. During the phage lytic cycle, the phage may accidently package bacterial DNA. Because of the phage’s small size, it can only package DNA equivalent in size to its own genome. The packaged bacterial DNA replaces the viral genes within the phage, preventing it from killing the next bacterium it infects. When phage finds another bacterium, it injects the bacterial DNA into the bacterium. If the foreign DNA is homologous to the new bacterium’s genome, the DNA can be inserted via homologous recombination.

PAIs are typically found adjacent to tRNA genes on the core genome. tRNA genes are highly conserved among all bacterial species, and therefore, serve as an ideal insertion point for foreign DNA that happen to contain a homologous tRNA gene. Integrase genes located on the foreign mediate site-specific recombination by targeting the genomic 3’ end of tRNA loci, and using site-specific recombination with the tRNA gene on the foreign DNA to insert the DNA segment into the genome. Bacterial cells that continue to collect foreign DNA with homologous tRNA sequences will continuously add nonspecific genes to the same tRNA site, so long as the genome has room to expand. Over time, some of the new genes may be successfully be expressed. The genes that benefit the bacterium will be positively selected for while inert or harmful genes will be removed. Removing genes regardless of whether or not they harm the bacteria is advantageous because the cell must spend extra time and energy replicating its genome. If the new genes contain genes encoding virulence factors, the cell will eventually evolve regulatory pathways or die trying, either by utilizing innate regulatory systems, or by acquiring regulators horizontally. The PAI continues to develop into an increasingly complex system of genes for effectors proteins, secretion systems and regulatory systems.