Molecular Biology of Infectious Disease

dr. Tri Wibawa, PhDDept. of Microbiology

Gadjah Mada Univ. Sch. of Med.

Application of Molecular Biology in Infectious Diseases

• Molecular epidemiology

• Pathogenesis

• Diagnosis

• Treatment

• Prognosis

• Vaccine development

Molecular Epidemiology

Molecular epidemiology application:

Accessing the burden of recent transmission False positive culture. Risk factor for recent transmission (ethnic, ages, etc) Reinfection case (endogenous or exogenous?) Phenotyphic difference between strain: virulence, growth, transmisibility Relative transmisibility between drug sensitive and resistance strain.

Molecular epidemiological markers for M. tuberculosis

Molecular epidemiological markers used: IS6110 restriction fragment length polymorphism (RFLP). Secondary markers:

☺ Polymorphic guanin-cytosine-rich repetitive sequence (PGRS) RFLP

☺ Spolygotyping Mycobacterial interspersed repetitive units (MIRUs)

IS6110 restriction fragment length polymorphism (RFLP): Described initially in 1989 1,555 bp IS3 family Two ORF encoding transposition function Number of copy range 0-25 (mean: 8-18) Most widely applied. Gold standard for other methods High Stability over time Problems arise when only small amount band appear Restriction enzyme: pvuII Probe: PCR product of Mtb IS6110

IS6110-based DNA fingerprinting

Polymorphic guanine-cytosine-rich repetitive sequence (PGRS) RFLP

Discriminatory power close to IS6110 Comprise many imperfect repeats Encode some unknown function protein

Spoligotyping

PCR-based technique. No need to culture the pathogen Can be carried out directly to the clinical specimen Repetitive 36 bp sequence were separated by non repetitive sequence/spacer. Absent-present the spacers in southern blotting is the individual determinant.

Mycobacterial interspersed repetitive unit (MIRU)

VNTR (variable number tundem repeat) The number of repeats at different loci varies between strains. PCR result : different length of amplified product. Technical difficulty : sizing the PCR product.

Pathogenesis

Model of G domain of laminin-M.leprae interaction

Laminin - 2

Genome of S. enterica serotype typhi (CT18)

Salmonella Virulence Genes

Clustered in certain area of chromosomes: “Salmonella pathogenicity islands” (SPI) SPI-1 and SPI-2 encode: Type III Secretion Systems (TTSS)

SPI-1:

Encoded TTSS translocates effector proteins into the cytosol of host cells Invasion of nonphagocytic host cells Enteropathogenesis

SPI-2

Intracellular survival in murine macrophages

Model for the structure of S. typhimuriumSPI-2 TTSS

Translational arrest in rotavirus infected-eukaryotes cells

eIF : eukaryotic initiation factorPABP: polyadenosine binding proteinGTP : Guanosine 5’-triphosphate

Rotavirus

Rotavirus mRNA posses of 5’ cap structure, but lack of polyadenosine tail in its 3’ end. 3’ end mRNA contained a tetranucleotide motif. Viral NSP3 protein bind specifically to the tetrancleotide motif. NSP3 bind to the binding site of PABP in the eIF4G. NSP3 has higher affinity to the eIF4G than PABP. Concequencies:

Increase the translation of viral mRNA Disrupt the host cell translation process.

Diagnosis

Molecular Diagnostic Techniques of Tuberculosis

Molecular Diagnostic Techniques are indicated: Detection of organisms that cannot be grown in vitro or for which current culture techniques are too insensitive. Require complex media and prolong incubation time.

Molecular Diagnostic Techniques consideration: high sensitivity high specificity speed simplicity clinical relevance.

Basic principle of any Molecular Diagnostic Techniques is the detection of specific nucleic acid (DNA) sequence of the pathogens.

Example: Polymerase chain reaction (PCR) Southern blot hybridization Nested PCR Multiplex PCR Reverse transcriptase PCR (RT PCR) Trancription-mediated amplification (TMA) Ligase chain reaction (LCR) Nucleic acid sequence-based amplification (NASBA)

Technical aspects: (for MTB)

Target: Insertion sequences/repetitive element: IS986; IS6110Antigens : 32 kDa; 38-kDa; 65-kDa.Genes: dnaJ; groEl; mtb-4

Sample preparation:Boiling; freezing-boiling; shaking with glass bead; sonication; chloroform; proteinase K; resin treatment; more complicated techniques (kit).

PCRs techniques

Internal controls

Studies using in house PCR techniques with IS6110 as a targets from sputum specimen

ResumeNone of the studies observed a statistically significant difference between culture and PCR Specificity : 85-100% Sensitivity : 74-97% Discrepancy of sensitivity in smear neg – culture pos Human resources dependent.

Treatment

Rifampisin (RIF)

Action: Inhibition of DNA-dependent RNA polymerase. RNA polymerase: 4 subunit: α ; ß ; ß’ ; σ Genes : rpoA; rpoB; rpoC; rpoD Target: bind to the ß subunit resulting in transcription inhibition Mutation in the rpoB gene responsible to rifampisin resistance.

Identified mutations in rpoB genes of MTB

Isoniazid (INH)

Action and target: Not clearly known Candidate; INH or INH metabolite block the synthesis of mycolic acids. Genes : katG. Encoding catalase-peroxidase enzym INH resistance MTB had decreased catalase activity. Mutation in the katB gene responsible to isoniazid resistance.

Isoniazid (INH)

Genes : inhA. Encoding protein for fatty acid biosynthesis. inhA has correlation with resistance to INH and ETH Polymorphisms were found in the upstream of orfI

Ethambutol (EMB)

Action and target: Not clearly known Candidate;

Inhibition of RNA metabolism Inhibition of phospholipid synthesis Inhibition of transfer of mycolic acid Inhibition of spermidine synthesis Inhibition of first step of glucose conversion.

Genes : No genes were identified.

Pyrazinamide (PZA)

Action and target: Not clearly known Candidate; Pyrazinamidase convert PZA to pyarzinoic acid. PZA-resistance MTB lack of the pyrazinamidase activity. Genes : No gene were identified

Prognosis

T7809C polymorphism of the LAMA2 gene study

χ2 = 8.07; p < 0.025

T/T C/CT/CGTA GCA

Val Ala

Tuberculoid group

Lepromatous group

T/C genotype is strongly associated with the TT (OR = 6.73). (χ2 8.73; p < 0.005).

SIRS/SEPSIS

Vaccine Development

Subunit vaccine (recombinant)

Advantages No risk of pathogenicity Defined composition Various delivery system available Simplified large-scale production Possibility of further engineering

Disadvantages Multiple doses typically required Adjuvants needed

Recombinant Subunit Vaccine

Reduction side effect of other components/epitopes Often poorly immunogenic Short in vivo half lives Often elicit only strain-specific production

Recombinant Subunit Vaccine

Protein immunogens production in heterologous hosts (bacterial, yeast, insects, mammalian system vector) Live delivery systems Construct recombinant live viral and bacterial carrying foreign immunogens - Vaccinia virus of rabies, - Attenuated recombinant vacinia virus vector for plasmodium

- Adenovirus- Bacteria gram negative (Salmonella sp)- Bacteria gram positives

Nucleic acid vaccines

Live delivery system

S typhi TY21a as a vector for vaccine antigens

Antigen

Bacterium A S typhi TY21a S typhi TY21aExpressing antigen

of bacterium A

plasmid

ReassortmentQuadrivalent Vaccine

Common strain in human: P[8]G1 P[8]G3 P[8]G4; P[4]G2

P= VP4G= VP7