• Molecular Basis of Bacterial Pathogenicity

Pathogenicity Islands in Bacterial Pathogenesis

Pathogenicity islands (PAIs) are distinct genetic elements on the chromosomes of a large number of bacterial pathogens. PAIs encode various virulence factors and are normally absent from non-pathogenic strains of the same or closely related species

These elements play a pivotal role in the virulence of bacterial pathogens of humans and are also essential for virulence in pathogens of animals and plants. PAI apparently have been acquired during the speciation of pathogens from their nonpathogenic or environmental ancestors. The acquisition of PAI increases appearance of bacterial pathogens and also may represent a mechanism that contributes to the appearance of new pathogens within a human life span.

Common features of PAI

(i) PAI carries one or more virulence genes; genomic elements with characteristics similar to PAI but lacking virulence genes are referred to as genomic or metabolic islands.

(ii) PAI are present in the genomes of a pathogenic bacterium but absent from the genomes of a nonpathogenic representative of the same species or a closely related species.

(iii) PAI occupy relatively large genomic regions. The majority of PAI are in the range of 10 to 200 kb.

(iv) PAI often differs from the core genome in their base composition and also show a different codon usage. The base composition is expressed as percentage of guanine and cytosine (G+C) bases, and the average G+C content of bacterial DNA can range from 25 to 75%. Most pathogenic bacterial species have G+C contents between 40 and 60%. The reasons for that variation are not known, but the conservation of a genus- or species-specific base composition is a remarkable feature of bacteria

 (v) PAI are frequently located adjacent to tRNA genes. This observation gave rise to the hypothesis that tRNA genes serve as anchor points for insertion of foreign DNA that has been acquired by horizontal gene transfer. The frequent insertion at tRNA loci may be explained by the observation that genes encoding tRNAs are highly conserved between various bacterial species.

(vi) PAIs are frequently associated with mobile genetic elements. They are often flanked by direct repeats (DR). DR are defined as DNA sequences of 16 to 20 bp (up to 130 bp) with a perfect or nearly perfect sequence repetition. DR might have served as recognition sites for the integration of bacteriophages, and their integration resulted in the duplication of the DR. Furthermore, DR act as recognition sequences for enzymes involved in excision of mobile genetic elements, thus contributing to the instability of a PAI flanked by DR.

(vii) PAI often are unstable and delete with distinct frequencies. Virulence functions encoded by certain PAI are lost with a frequency that is higher than the normal rate of mutation. Genetic analyses showed that such mutations are caused not by defects in individual virulence genes within the PAI but, rather, by loss of the large portions of a PAI or even the entire PAI. These mutations can be observed during cultivation of pathogens in vitro, but they are also found in isolates obtained from infected individuals, for example during persistent infections. This indicates that such PAI have an intrinsic genetic instability

 (viii) PAI often represent mosaic-like structures rather than homogeneous segments of horizontally acquired DNA. Some PAI represent an insertion of a single genetic element. Others show a more complex structure, since elements of different origin are present.

(ix)  Integration of PAI into the bacterial chromosome is a site-specific event. Most PAI currently known have inserted at the 3′ end of tRNA loci.

HORIZONTAL TRANSFER OF PAI

The presence of virulence factors in very similar forms in different bacteria may be explained by horizontal gene transfer.

Natural Transformation” Certain bacteria are capable of natural transformation. During certain phases of growth, transport systems are expressed that allow the uptake of free DNA from the environment. Although the majority of this foreign DNA will be degraded, some fragments that harbour “useful” genes are integrated into the genome of the recipient and maintained. It appears possible that this mechanism allows uptake of DNA from distantly related species that will be maintained as the selective pressure selects for the newly acquired features.

PAI and Plasmids: Similar clusters of virulence genes are present in PAI and on virulence plasmids, indicating that episomal and chromosomal locations are possible for the same gene cluster. It was observed that certain clusters of virulence genes are present in PAI of some pathogens but also on virulence plasmids in other bacteria.

Transduction: Bacteriophages have been isolated from virtually all bacterial species; even obligate intracellular pathogens such as Chlamydia spp. contain specific phages. Bacteriophages are able to transfer bacterial virulence genes as passengers in their genomes. The occasional transfer of virulence genes by phages allows the recipient bacteria to colonize new habitats, such as new host organisms or specific anatomic sites. This extension also allows a more efficient spread of the bacteriophages. Thus, the transfer of bacterial virulence genes as passengers in the viral genome can also be an evolutionary benefit for the bacteriophage

Many PAI are too large to be transferred as passengers in bacteriophage genomes.

PAI do not occur only in human pathogens; they have also been found in animal and plant pathogens. Examples are the hrp islands of Pseudomonas syringae and Xanthomonas campestris, and islands in animal pathogenic salmonellae and staphylococci. They are distributed throughout the bacterial world, and horizontal transfer may be facilitated by plasmids and phages or by bacteria, which are competent for the uptake of free DNA by natural transformation.

The PAI of Staphylococcus aureus

Staphylococcus aureusS. aureus is a common commensal found on human skin and respiratory tract mucosal surfaces. However, it is also a pathogen that is associated with a tremendous variety of human diseases ranging from self-limiting skin infections to life-threatening pneumonia or sepsis; nosocomial infections by S. aureus are common. A variety of toxins, such as hemolysins, staphylococcal exotoxins (Set), and superantigens (enterotoxins, exfoliative toxin, toxic shock syndrome toxin), are major virulence factors of S. aureus. These toxins are involved in the pathogenesis of staphylococcal diseases such as food poisoning, scalded skin syndrome, and toxic shock syndrome. Treatment of Staphylococcus infections becomes increasingly difficult, since resistance to a growing number of antibiotics has been observed in clinical isolates.

Genetic analysis of the virulence and resistance genes of various clinical isolates of S. aureus revealed that these genes are clustered in prophages and transoposons, as well as with genomic islands that have characteristic features of PAI. The availability of three genome sequences of methicillin-resistant S. aureus (MRSA) strains allowed a detailed comparison of the structure of PAI in S. aureus. A remarkable difference between PAI in S. aureus and PAI in gram-negative pathogens is the presence of large gene clusters with allelic variations of specific toxins, proteases, and enzymes involved in pathogenesis. The allelic forms may allow adaptation of the pathogen to the various host environments that are colonized during infection. Staphylococcus cassette chromosome mec. A major problem in the treatment of S. aureus infections is the presence of resistance to multiple antibiotics. Methicillin is the first semi  synthetic, β-lactamase-resistant penicillin for therapeutic use. Resistance to methicillin in MRSA strains is conferred by penicillin-binding proteins and is usually accompanied by resistance to variety of other β-lactam antibiotics. It was observed that methicillin resistance is encoded by the chromosomal gene mecA. Analysis of the genome of MRSA strains revealed that antibiotic resistance genes are located within an unstable locus that has certain features of a PAI. The genomic island Staphylococcus cassette chromosome mec (SCC mec) carries the methicillin resistance determinants mecI, mecR, and mecA. The number of resistance genes located in SCC mec can vary among different strains and is dependent on the presence of transposons within SCC mec. In MRSA strains, the insertion of Tn 554 into SCC mec confers resistance to spectinomycin and erythromycin. The availability of genome sequences of several S. aureus strains indicated that the size and gene composition of SCC mec are highly variable and led to the distinction of allelic forms of SCC mec into types I to IV; the size of SCC mec ranges from 20.9 to 66.9 kb depending on the allelic form. Type I and II SCC mec were identified in nosocomial isolates of MRSA. PAI encoding toxic shock syndrome toxins.In addition to SCC mec, a larger number of genomic islands have been identified. It was observed that chromosomal tst genes encoding toxic shock syndrome toxins (TSST) are located on mobile genetic elements that were distributed among S. aureus strains. TSST-1 and enterotoxins of S. aureus function as superantigens. (Superantigens (SAgs) are a class of antigens that cause non-specific activation of T-cells resulting in polyclonal T cell activation and massive cytokine release. SAgs are produced by some pathogenic bacteria as a defense mechanism against the immune system.). Staphylococcal superantigens are associated with food poisoning, toxic shock syndrome, multiple sclerosis, Kawasaki’s disease, and atopic allergy.

Plasmids are extra chromosomal structures in the cell of bacteria which have the ability to self-replicate. They do not combine with genetic material of the host cell but stay independently. They are genetically modified and are used in recombinant DNA technology. They are capable of carrying about 20 genes at a time.

Functions of plasmids

• The main function of plasmids is to carry antibiotic resistant gene and spread them from host to host.
• They carry genes which are involved in metabolic activities and are helpful in digesting pollutants from the environment
• They are able to carry genes which are concerned with increasing bacteria pathogenicity.

Types of plasmids

There are five (5) types of plasmids which are used for different purposes

1. Resistance plasmids
2. Degradative plasmids
3. Fertility plasmids
4. Col plasmids
5. Virulence plasmids

Resistance plasmid

• This type of plasmid  is involved in the bacterial conjugation.
•  They usually carry those genes which code for the resistance of antibiotics.
• They also carry genes which are responsible for the production of pilli
• The main role of conjugation pilli is to transfer the R plasmid from one donor bacterium to the recipient bacterium

Degradative plasmid

• This type of plasmid is capable of degrading or digesting dead organic matter from dead animals or plant.
• They use organic matter to process biosynthesis, make energy and recycle them

Fertility plasmid

• These plasmids carry the tra-genes which are used in the process of conjugation
• It has the ability to form the conjugation bridge (F pillus)
• They are helpful in transferring genetic materials between bacteria
• They are found in F⁺ bacteria with higher frequency of conjugation. Examples include F plasmids of E.coli

Col plasmid

• This type of plasmid produces antibiotics which are involved in killing other strains of bacteria which are in the same environment as them
• The antibiotic is called colicin

Virulence plasmid

• As the name implies, this type of plasmid has the ability to transform a non-pathogenic bacterium into a pathogen one.
• They are responsible for carry genes that encode for virulence in bacteria

Characteristics of plasmids

1. A small circular DNA
2. Contains an origin of replication as such replicates independent of the cell
3. Carries a few genes which are beneficial for the host cells
4. They transfer traits such as drug resistance within cells or resistance to heavy metals
5. Plasmids differ from chromosomal fragments because they have their own origin of replication. Chromosomal fragments have to be incorporated into cell to replicate
6. Plasmids can also provide bacteria with the ability to fix nitrogen.
7. Their size can range from very small mini-plasmids of less than a 1 kilobase pairs (Kbp), to very large mega plasmids of several megabase pairs (Mbp).

CHEMICAL NATURE OF BACTERIAL CELL WALLS

Gram positive cell wall

• In the Gram-positive Bacteria, the cell wall consists of;
•  several Layers of peptidoglycan and; 
• Teichoic acids 
• Lipoteichoic acid which are unique to the Gram-positive cell wall

In the Gram-negative Bacteria, the cell wall is composed of a single layer of peptidoglycan surrounded by a membranous structure called the outer membrane.

The outer membrane of Gram-negative bacteria invariably contains a unique component, lipopolysaccharide (LPS or endotoxin).

In Gram-negative bacteria the outer membrane is usually thought of as part of the cell wall.

Differences in Gram positive and Gram negative cell wall

Gram positive cells have a very thick, multilayered cell wall

They also contain teichoic acids and lipoteichoic acids, Lysozyme

Gram negative cells have a very thin layer of peptidoglycan they also have an outer membrane in addition to the cytoplasmic membrane. The space between these two membranes is called the periplasmic space or periplasm.

Other differences:

Property	Gram-positive	Gram-negative
Thickness of wall	thick (20-80 nm)	thin (10 nm)
Number of layers	1	2-3
Peptidoglycan (murein) content	>50%	10-20%
Teichoic acids in wall	present	absent
Protein/lipoprotein content	0-3%	>50%
Lipopolysaccharide content	0	13
Sensitivity to penicillin	sensitive	resistant
Sensitivity to lysozyme	sensitive	resistant

Cell Wall synthesis in E. coli (Gram negative organism)

In the Gram-negative Bacteria, the cell wall is composed of a single layer of peptidoglycan surrounded by a membranous structure called the outer membrane. The outer membrane of Gram-negative bacteria invariably contains a unique component, lipopolysaccharide (LPS or endotoxin), which is toxic to animals. In Gram-negative bacteria the outer membrane is usually thought of as part of the cell wall. The peptidoglycan layer is substantially thicker in gram-positive bacteria (20 to 80 nanometers) than in gram-negative bacteria (7 to 8 nanometers), It forms around 90% of the dry weight of gram-positive bacteria but only 10% of gram-negative strains. Presence of high levels of peptidoglycan is the primary determinant of the characterization of bacteria as gram-positive.

 The interpeptide bridges are formed by the cross-linking between amino acids in different linear amino sugar chain. This interpeptide bridges occur with the help of the enzyme DD-transpeptidase. The interpeptide bridges results in a 3-dimensional structure that is strong and rigid.

Biosynthesis of Peptidoglycan

• The biosynthesis of peptidoglycan is a complex process that involves three stages.
• The first stage takes place in the cytoplasm and involves the synthesis of the peptidoglycan monomers(UDP-NAG and UDP-NAM) and the tetrapeptide chain.
• The second stage takes place on the cytoplasmic membrane and involves the synthesis of lipid-linked intermediates
•  The third stage takes place outside the cell membrane and involves polymerization reactions.

In stage one Peptidoglycan synthesis can be divided into four sets of reactions:

• Formation of UDP-GlcNAc from fructose-6 phosphate,
• Formation of UDP-MurNAc from UDP-GlcNAc,
• Assembly of the peptide stem   leading to UDP-MurNAc-pentapeptide and
• ‘Side’ or ‘annex’ pathways of synthesis of D-glutamic acid and the dipeptide D-alanyl-D-alanine.

Stage two:

In this stage, a lipid carrier called bactoprenol carries peptidoglycan precursors through the cell membrane. It involves:

• Bactoprenol will attack the UDP-MurNAc penta, creating a PP-MurNac penta, which is now a lipid.
• UDP-GlcNAc is then transported to MurNAc, creating Lipid-PP-MurNAc penta-GlcNAc, a disaccharide, also a precursor to peptidoglycan.
• The precursor is then transported through the membrane to the cell wall.
• How this molecule is transported through the membrane is still not understood. However, once it is there, it is added to the growing glycan chain.

Stage 3: (outside the cell membrane)

• Stage three is known as tranglycosylation.
•  In the reaction, the hydroxyl group of the GlcNAc will attach to the MurNAc in the glycan, which will displace the lipid-PP from the glycan chain.
• The enzyme responsible for this is transglycosylase.