Audrey Smith

The Evolutionary Mechanisms of Bacterial Protein Toxins

OverviewThis presentation will:

Introduce bacterial toxinsIntroduce the field of phylogeneticsIntroduce some basic concepts of bacterial

geneticsDescribe how I constructed a phylogenetic

tree to investigate the evolution of bacterial protein toxins

Discuss the results of that phylogenetic tree

What are bacterial toxins?Bacterial toxins are substances produced

by bacteria that are capable of causing harm to a host.

Many Gram-negative bacteria have lipopolysaccharide toxins associated with their cell walls.

Bacterial protein toxins are usually secreted, and are produced by a wide variety of bacteria.Because they are proteins they are encoded

by genes, and the DNA sequences can be analyzed.

Why are they important?Bacterial protein toxins are responsible for,

or complicate, many human diseases:TetanusMRSAPertussisBotulismToxic Shock SyndromeFood PoisoningScalded Skin SyndromeAnd many more; there are currently over 130

identified bacterial protein toxins

Types of bacterial protein toxinsThey can be grouped according to their

mechanism of action, or pathogenic strategy.

Some examples of categories are:Pore-forming toxinsProtease toxinsProtein synthesis inhibiting toxinsSecond messenger activating toxinsSuperantigen toxins

Pore forming toxinsThese proteins are capable of transforming

from a water-soluble form to a membrane bound form.

Some act as monomers, but most are homo-oligomers, with pentamers, hexamers, and heptamers being common arrangements.

Larger arrangements also occur, especially in the cholesterol-dependant cytolysin family, which may have 50 or more subunits per pore.

A wide variety of microbes, including bacteria produce these toxins.

Pore forming toxinsThese toxins insert into

the membrane of cells and form non-regulated pores.

This can allow ions, water, and small molecules to freely enter and exit, causing death of the cell

To the right is an illustration of pneumolysin, a pore forming toxin produced by Streptococcus pneumoniae

Protease ToxinsThis is a small category, consisting only of

the clostridial neurotoxins tetanus toxin and botulinum toxin, but is interesting because of the diseases they cause: tetanus and botulism, respectively.

Tetanus toxin cleaves synaptobrevin, which prevents the release of inhibitory signals of skeletal muscle contraction, resulting in rigid paralysis.

Botulinum toxin cleaves SNAP-25, which prevents the release of stimulatory signals of skeletal muscle contraction, resulting in flaccid paralysis.

Protease Toxins

An illustration of a man suffering from the rigid paralysis of tetanus

A duckling suffering from the flaccid paralysis of botulism

Protein Synthesis Inhibiting ToxinsProtein synthesis inhibiting toxins block

elongation factors that are required to move RNA transcripts through ribosomes.

When these factors are blocked, new proteins cannot be synthesized, which leads to cell death.

Toxins in this group are responsible for diphtheria and bacilliary dysentery, and contribute to Pseudomonas aeruginosa infection.

Second Messenger activating toxinsThese toxins modify cellular proteins

involved in signaling pathways.The most common targets are G-proteins

and rho.The modified proteins result in either up- or

down-regulation of the normal end product of the pathway, with clinical results varying widely depending on the targeted pathway.

Cholera, Whooping Cough, and Diphtheria are all caused by toxins in this category.

Superantigen toxinsThese toxins non-selectively activate host

T-lymphocytes.This causes a massive over-reaction of the

immune system, causing systemic inflammation and in severe cases, a dangerous drop in blood pressure.

Most known superantigens are produced by bacteria in genus Staphlococcus.

The toxins can cause toxic shock syndrome, rheumatic fever, and food poisoning.

What is phylogenetics?Phylogenetics is the study of evolutionary

relationships between organisms.Sequenced genes are compared to determine

how similar they are.Conserved genes that are found in all of the

organisms to be compared are used.For wide-range studies genes for 16S rRNA or

cytochrome C are often used because they are found in all organisms.

The comparison data is used to construct a graphic representation called a phylogram, or phylogenetic tree.

16S rRNA Phylogram

Bacterial GeneticsBacteria can pass on their genetic material

in two important ways:Vertical gene transfer

This is passing of genetic material from parent cell to daughter cells through simple mitosis. Both daughter cells produced in mitosis are identical clones of the parent cell.

Horizontal gene transferThis is passing of genetic material from one cell to

another unrelated one. Sometimes the cells involved are not the same species. Horizontal gene transfer is sometimes referred to as sexual reproduction in bacteria.

Horizontal Gene Transfer (HGT)There are three primary mechanisms by

which HGT can occur:Transformation

Where genes are transferred as small circular pieces of DNA called plasmids

TransductionWhere genes are transferred by viral bacteriophages

ConjugationWhere genes are transferred via physical contact of

the two cells. Usually the cells are joined by a pilus.

Mobile genetic elementsWhen looking for genes that are likely to be

transferred between bacteria, one should look for genes encoded on plasmids and in bacteriophages, since they are mobile genetic elements that facilitate HGT.

It is known that antibiotic resistance is transferred by HGT, and that genes for some toxins are also transferred this way.

HGT and phylogenetic studiesWhen the objective of a phylogram is to

illustrate the relationship of whole organisms, highly conserved genes are compared.

If less conserved genes were used, the resulting phylogram would show organisms that have undergone HGT as being more related than they really are.

By analyzing such a phylogram for these discrepancies, it is possible to see where HGT has occurred.

Finding out which toxins are undergoing HGTBacterial toxins are a serious public health

concern. It would be advantageous to know if, and which, bacterial toxin genes pass between bacterial populations.

A phylogram to identify points of HGT can be constructed with a multiple sequence alignment tool. For this project I used the ClustalW2 program from the European Bioinformatics Institute to analyze toxin gene sequences that I obtained from the National Center for Biotechnology Information Gene database.

Choosing the toxins to comparePhylogenetic analysis works better when

the genes being compared are somewhat similar. Also, HGT is more likely to occur in genes located on plasmids and bacteriophages.

With this in mind I chose several toxin genes from each functional toxin group already discussed here, and particularly looked for any encoded on plasmids or bacteriophages.

I made phylogenetic trees for each toxin group, then one comparing all of the toxins together.

The results

Interpreting the phylogenetic treeEach ‘branch’ of the tree represents the

evolutionary distance between the toxins it connects.

Therefore, the toxins connected by shorter branches are more related to each other.

It is expected that these toxins would be from the same bacteria, or closely related ones.

If they are from very different bacteria, HGT has likely occurred.

In the tree, we see a group of toxins connected by short branches, all of which are protein synthesis inhibitor toxins

Closely Related Groups

The protein synthesis inhibitor toxins

This tree shows the protein synthesis inhibitor group.The group highlighted in red are all Shiga toxin 1, isolated

from S. dysenteriae, two strains of E. coli and a bacteriophage.

The group in blue are all Shiga toxin 2, isolated from three strains of E. coli and a bacteriophage.

Diphtheria Toxin and Exotoxin 1 are also shown, but do not appear to be particularly related to either Shiga toxin group.

Shiga toxinsShiga toxin 1 and Shiga toxin 2 are two different

proteins that perform the same pathogenic action through the same receptor.

They have one A subunit that stops protein synthesis inside cells, and 5 identical B subunits that help get the A subunit into the cell. These toxins are similar in structure and function to the plant toxin ricin.

These toxins cause bacilliary dysentery; some cases also develop Hemolytic Uremic Syndrome, which can be fatal.

I used the gene for the B subunit for these comparisons because there were more sequenced isolates available than of the A subunit.

Shiga toxin 1The gene for this toxin is found in Shigella

dysenteriae, some strains of Escherichia coli (called Shiga Toxin-producing Escherichia coli or STEC), and in at least one known bacteriophage.

Because nearly identical genes for this toxin are found in unrelated bacteria and in a bacteriophage, it can be concluded that Shiga toxin 1 undergoes HGT at least between S. dysenteriae and E. coli.

Shiga toxin 2The gene for this toxin is not present in S.

dysenteriae. It only appears in strains of STEC, and in at least one bacteriophage.

With just this evidence, it cannot be determined if Shiga toxin 2 undergoes HGT or not.

A closer look at toxin genes from STEC strains may reveal if HGT occurs within E. coli.

ConclusionsBy phylogenetic analysis it has been

confirmed that HGT occurs with Shiga toxin 1 between at least two distinct bacterial species.

HGT may occur with Shiga toxin 2 between different strains of E. coli.

Where to go from hereBecause of the scope of this project, the number

of toxins studied was limited. Repeating the experiment with more toxin genes may reveal incidents of HGT missed here.

The Shiga toxins should be studied in greater detail to determine if there are any other organisms capable of receiving these genes.

Since the plant toxin ricin is very similar in structure and function to the Shiga toxins, it would be of interest to determine the relationship between them, and if some form of genetic transfer occurred in the evolution of either toxin.

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