Molecular Biology – Redefining Pathogenesis

Dr. Rajesh Karyakarte MD

Professor & Head,Department of Microbiology,Government Medical college, Akola

Definitions

Molecular Biology is the application of science and technology to living organisms, as well as parts, products and models thereof, to alter living or non-living materials for the production of knowledge, goods, and services (1).

Pathogenicity is the capacity of a microbe to cause damage, in a host.

Pathogenesis is the process of producing this damage.

(1)http://www.oecd.org/document/42/0,3343,en_2649_34537_1933994_1_1_1_1,00.html

Molecular Koch’s Postulates

Falkow (1988) proposed “Molecular Koch’s Postulates,” a set of conceptual tools for dissection of bacterial pathogenesis based on the identification of the genes responsible for causing disease (2).

Many genes required for virulence in bacteria are in discrete DNA segments, e.g., pathogenicity islands. Acquisition of these genes is sufficient for a bacterium to become virulent.

(2) Falkow S. Molecular Koch's postulates applied to microbial pathogenicity. Rev Infect Dis 1988; 10(suppl 2): S274-6.

Molecular basis of host- and species specific tropism

Molecular studies show that the chemistry of the microbial surface is the major distinction between pathogenic and nonpathogenic microorganisms.

Tropism of microorganisms is subdivided into: Tissue and host tropism Species-specific tropism

Tissue and host tropism

Uropathogenic E. coli adhere to human urinary tissue cells by PapG adhesin, which is a component of Pap (pyelonephritis-associated pili).

There are three PapG alleles (Class I, II, and III). Class II PapG is associated with pyelonephritis and Class III with cystitis.

PapG binds Gal-α-1-4-Gal

PapG binds Gal-α-1-4-Gal containing glycolipid present on the surface of renal epithelial cells.

The glycolipid has a digalactoside core linked via a -glucose residue to a ceramide group that anchors the receptor to cell membrane. This receptor is named globotriasylceramide (GbO3).

GbO3 with one additional sugar moiety (GalNAc) becomes GbO4 (Globoside) and

GbO3 with two additional sugar moieties (GalNAc 2) becomes GbO5 (Forssman antigen).(3)

(3) Dodson KW, Pinkner JS, etal. Structural basis of the interaction of the pylonephritic E. coli adhesin to its human kidney receptor. Cell 2001; 105: 733-43.

Species-specific tropism

Neisseria gonorrhoeae, N. meningitidis, E. coli (expressing CFA-I and CFA-II) and group A Streptococci are limited to human infection due to species-specific tropism.

A single strain of E. coli may be able to express several distinct adhesins encoded by respective distinct regions of chromosomes or of plasmids, which helps in adapting to changing environment.

A Couple of Examples of Adhesins and Receptors

Organism Adhesin on the microbe Receptor on host cell Adaptation E. coli Type I fimbriae (FimH) D-mannose residue Allelic variation in fimH gene

helps in differential binding Neisseria gonorrhoeae

Fimbriae with methylated phenylalanine amino terminus residue

Surface glycoprotein CD46 on urogenital cells

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Molecular Biology - Biofilms

Molecular biology has helped in elucidation of the molecular basis of biofilm formation.

Staphylococcus epidermidis forms biofilms and is a major cause of medical-device related infections.

The intercellular adhesion in these biofilms is provided by polysaccharide intercellular adhesin (PIA).

Molecular Biology – Biofilms cont…PIA

PIA is a linear glucosaminoglycan composed of N-acetylglucosamine in -1,6-glycosidic linkages containing deacetylated amino groups and succinate and phosphate substitution.

Molecular Biology – Biofilms cont… Synthesis of PIA

Synthesis of PIA requires expression of icaADBC operon.

These genes are controlled with the help of alternative sigma factor B, icaR, two more regulatory loci, plus phase variation due to IS element insertion.

PIA production can be stopped by inactivational IS element insertion that allows individual S. epidermidis cells to leave the biofilm to colonize new surfaces.(4)

(4) Dobinsky S, Kiel K, Rohde H, Bartscht K, Knobloch JK, Horstkotte MA, Mack D. Glucose-related dissociation between icaADBC transcription and biofilm expression by Staphylococcus epidermidis: Evidence for an additional factor required for polysaccharide intercellular adhesin synthesis. J Bacteriol 2003; 185: 2879-86.

Genetic Loci involved in LPS biosynthesis

In Enterobacteriaceae, the waa locus enzymes required for the sequential assembly of the core oligosaccharide on to the lipid A acceptor. This locus consists of three operons:

The gmhD operon that directs inner core biosynthesis

The central waaQ operon that is responsible for outer core; and

Genetic Loci involved in LPS biosynthesis cont…

The waaA operon that contains the structural genes for a bifunctional KDO transferase, which is required for the addition of KDO to the inner core

With a few exceptions, the enzymes involved in O-polysaccharide assembly are encodes by genes at the rfb locus and are expressed constitutively.

Molecular biology - protein secretion in bacteria

Translocation of protein synthesized in the cell occurs across the inner and outer membranes in Gram-negative bacteria by one of the five main pathways: Type I Mechanism: Utilized for secretion of the

hemolytic toxin HlyA from E. coli. Type II Mechanism: Is the general secretory pathway. Type III Mechanism: Responsible for injection of

proteins into eukaryotic cells Type IV Mechanism: Also responsible for injection of

proteins into eukaryotic cells, known as TFSS (Type four secretion system)

Type V Mechanism: Also called autotransportation

Type III - Subversion of host signal transduction

Pathogenic Yersinia spp. utilize the YopH proteins to cause dephosphorylation of several macrophage proteins to prevent phagocytosis.

Transient tyrosine phosphorylation of macrophage proteins is required for normal phagocytosis.

Type III - Subversion of host signal transduction, Cont…

Salmonellae on the other hand possess two type III secretion systems. These systems are encoded by two distinct gene clusters termed Salmonella pathogenicity island (SPI-1 and SPI-2).

These secretion systems have different roles with SPI-1 involved in mucosal penetration and SPI-2 with systemic spread.

Molecular Biology - Bacterial sensory networks

Pathogenic bacteria encounter countless different environments and therefore need to constantly monitor diverse physical and chemical signals to tailor their responses with the help of regulatory proteins.

Molecular Biology - Bacterial sensory networks, Cont…

The 6.3-MB genome of P. aeruginosa, for example, contains around 5500 genes, some 10% of which encode such regulatory proteins. These regulatory proteins help expression or repression of specific genes depending on environmental cues.

Molecular Biology - Bacterial sensory networks, Cont…

For example, if iron is plentiful in the environment, synthesis of siderophores and their associated export and import pathways can be switched off.

V. cholerae have an inverse correlation between motility and virulence gene expression as it switches between aquatic and human host environment.

Molecular biology - Iron-dependent repression

In E. coli, the 17-kDa Fur repressor protein controls transcription from iron-responsive promoters in an iron-dependant manner, i.e. to be active, Fur requires ferrous ion as a corepressor.

Molecular biology - Iron-dependent repression, Cont…

When iron is plentiful, the Fur-Fe2+ complex interacts with an operator sequence called Fur box and prevents gene expression.

During iron scarcity, ferrous ion is removed from the Fur-Fe2+ complex by the cell. Fur alone cannot act as repressor for expression of iron-repressed structural genes. The entire iron absorption system becomes active.

Two-component signal transduction system (TCSTS)

An enormous number of genes, including many virulence determinants, regulate this system.

This system consists of two regulator proteins, a cytoplasmic-membrane associated sensor and a response regulator in the bacterial cell.

Two-component signal transduction system (TCSTS), Cont…

TCSTC sense a variety of different factors including oxygen, hydrogen, quorum-sensing signal molecules, Mg2+ and phosphate limitation.

Molecular Biology - Quorum sensing

It is crucial in many bacterial infections that the bacterial population attains a particular cell density to overcome host defense and establish an infection.

Pathogens have an ability to communicate between themselves to orchestrate collective attack against the host immune system.

Molecular Biology - Quorum sensing, Cont…

The term ‘quorum sensing’ is commonly used in describing the phenomenon whereby accumulation of a diffusible, low-molecular-weight signal molecule (sometimes called ‘autoinducer’) enables individual bacterial cell to sense when the minimal number, or ‘quorum’ of bacterial cell has been achieved for a concerted response to be initiated.

Molecular Biology - Quorum sensing, Cont…

Bacteria employ a number of different quorum sensing ‘languages’, and several families of signal molecules have been characterized.

At molecular level, quorum sensing requires a synthase plus a signal transduction system for producing and responding to the signal molecule.

Many bacterial species have quorum sensing system consisting of the luxS and a furanone-related molecule AI-2 (for autoinducer-2).

Molecular Biology - Quorum sensing, Cont…

P. aeruginosa has a multisystem quorum sensing. It possesses two AHL (N-acylhomoserine lactone) dependant quorum-sensing systems.

Apart from AHLs, P. aeruginosa has a third quorum-sensing molecule, the pseudomonas quinolone signal (PQS).

Staphylococci produce quorum-sensing molecules termed autoinducing peptides (AIP).

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