Dr. Carlos Ríos Velázquez

BIOL 5758

Methods for identifying important genes:

Signature-tagged mutagenesis (STM)

At any time during infection (Attachment, colonization, invasion, multiplication and, persistence), there are factors produced that cause damage to the host (death). Infection processes are coordinately regulated or stimulated by host factors encountered in vivo.

Problems:

A. In vitro systems sometimes do not allow the reconstruction of exact interactions

between bacteria and the host.

B. In vivo analysis of a large number of mutants is very tedious and time consuming.

Definition:

Transposon mutagenesis system, that uses comparative hybridization to isolate mutants unable to survive specified environmental conditions (in vivo).

Purpose:

To study host-bacterium interaction in bacterial pathogenesis. Detection and identification of bacterial virulent genes (critical for survival) in their relevant environment (host) by using a negative selection (loss of function).

What do you need:

1. A collection of transposons (shuttle vector) each one modified by the incorporation of a different DNA sequence tag.

2. DNA sequence tags: short DNA segments that contain a 40 bp variable central region flanked by invariant arms that facilitate the co-amplification and labeling of the central positions by PCR.

3. An animal model system to do the selection.

Method:

1. Mutagenize bacterial strains using the transposons containing the tags.

2. Store individual mutants in arrays (usually in the wells of microtiter dishes).

3. Colony or dot blots are made from those arrays

4. Pools of mutants are subjected to a selective process (input, infection of an animal).

5. Labeled probes are made that represent the tags present in the input preselection (input) by PCR.

6. Recover the “survival” bacteria from the animal.

7. Probes that represent the tags are made from the post selection (output) by PCR.

8. Hybridization of the tags from the input and output to the colony or dot blot are performed.

9. Identify the mutants that are unable to survive the selective process (loss of the signal in the output when compared with the input blot).

10. Recover strains from the original arrays.

11. Sequence the DNA flanking the transposon insertion.

Important details to be considered:

1. Verify that there is no cross-hybridization between tags.

2. Inoculum dose

a. 10⁴ cells (~100 cells/mutants)? ; 10⁵ cells (~1000 cells/mutants) ?

3. Number of pooled mutants to be inoculated in the host (pool complexity)

a. 12, 14, 48, 96, 192?.

4. Route of inoculum administration

a. Intraperitoneal ?

b. Orally ?

5. Duration of the infection

a. Short, long??

6. Multiple animal models?

Genes products identified by this method:

1. ABC transporters

2. secretion systems

3. amino acid biosynthesis

4. lipid metabolism

5. proteases

6. stress response

7. unknown

Advantages:

1. Fast and feasible method for large-scale mutants hunts for virulence genes.

2. “Direct” method to isolate genes required fro survival in the host.

Disadvantages:

1. The method requires the use of animal models. This brings the difficulty in terms of ethical, technical and economical reasons.

2. Complexity of the pools of different mutants

3. Tn mutagenesis random??

4. determination of mutations that cause small or even moderate reduction in survival.

STM used in bacterial and their respective in vivo models:

Pathogen Animal Model

Staphylococcus aureus mouse abscess, bacteremia and wound infection

Pseudomonas aeruginosa rat chronic lung infection

Proteus mirabilis mouse urinary infection

Vibrio cholerae colonisation in mouse model

Streptococcus pneumoniae murine respiratory tract infection

Legionella pneumophila guinea pig pneumonia model

Brucella suis THP1 macrophage intracellular survival assay

Polissi et al., 1998