Introduction

Virulence is the ability of a microorganism to produce disease. Virulence depends on the number of infecting bacteria, their route of entry into the body, the response of the host immune system and any characteristics specific to that bacteria – its virulence factors. Bacterial virulence factors are typically proteins or molecules synthesized by protein enzymes.

Typical Virulence Factors

The general categories for which virulence factors can be broken down into are as follows:

• Adhesion – Virulence factors which facilitate adhesion are known as adhesins, they are what allow bacteria to bind to host cells. For example Bordetella bronchiseptica adhesins include; fimbriae, filamentous haemagglutinin adhesin (FHA) and pertactin. These adhesins bind to specific epithelium receptors and epitopes, which is what derives tissue specificity. Some bacterial cell walls and capsules are also adhesins, these are able to maintain even closer contact.
• Colonisation – Some virulence factors ease colonisation of certain areas of the body, for example Helicobacter species counter the low pH of the stomach by producing urease. This helps to neutralise the stomach acids making the stomach more colonisable for the Helicobacter.
• Invasion – Virulence factors which facilitate bacterial invasion of a host. This is done by disrupting host cell membranes, the result is the facilitation of transport across epithelial layers of tissue and skin. For example, the internalin surface proteins found on Listeria monocytogenes allow them to invade mammalian cells via transmembrane proteins. Thus these virulence factors are facilitating invasion into human epithelial cells.
• Immune Response Inhibitors – These are virulence factors which target the immune system and of the host and inhibit immune responses initiated against the bacteria. Even a low dose of bacterial exotoxin can alter the immune response. For example Escherichia coli heat liable toxin reduces the expression of interleukin 12 (IL-12) a cytokine which is indicative of the immune response.
• Toxins – A large proportion of virulence factors are proteins produced by bacteria which are toxic to the host, these toxins cause damage to hosts cells and tissues.

Toxins

The ability to produce toxins is known as toxogenesis, there are two main forms of toxin (if categorised chemically); lipopolysaccharides (primarily associated with the outer cell membrane structure of Gram-negative bacteria) and proteins. Proteins are secreted from a wide range of bacteria. Cell associated toxins (such as the lipopolysaccharides which are bound to the outer membrane) are known as endotoxins, whilst secreted protein toxins which diffuse extracellularly, are referred to as exotoxins.

Endotoxins

The endotoxin lipopolysaccharide (LPS) consists of a polysaccharide chain with a lipid tail. The toxic component of this molecule is Lipid A, located at the very tip of the lipid tail. When bacteria are damaged or destroyed, this endotoxin is released and causes an inflammatory response.

The innate (cellular-mediated) immune system is able to recognise a broad range of pathogens, including the LPS endotoxin. This ability is mediated by Toll-like receptors (TLR) – a series of receptors able to detect a variety of pathogen epitopes. The TLR responsible for recognising LPS is TLR-4.

Exotoxins

Exotoxins are secreted by living bacteria, with different toxins being secreted by different species. The toxin is the major factor in determining virulence, e.g. strains of E. coli without the exotoxins are low/non-virulent. The toxins can remain toxic even at very low concentrations. Exotoxins are typically named descriptively to show where the toxin acts, for example; neurotoxin, leukotoxin, enterotoxin and haemolysin.

Exotoxins can be categorised into 3 main categories when considering its cellular site of action:

• Type-I – Membrane acting (e.g. Super antigens or Heat stable enterotoxins – typical method of toxicity is to bind to cell surface receptors and trigger intracellular signalling pathways)
• Type-II – Membrane damaging (e.g. Channel forming toxins or Enzymatically active toxins – typical method of toxicity is cell lysis, modulation of signal transduction or enzymatic action)
• Type-III – Intracellular (e.g. bacteria specific toxins such as cholera toxin, pertussis toxin, diphtheria toxin etc. – typical method of toxicity varies, see mechanisms below)

Mechanisms of Exotoxins

Some of the mechanisms of exotoxin actions include:

• Damage to cell membranes – For example, Clostridium perfringens α-toxin has phospholipase C activity which causes degradation of the cell membrane. Staphylococcus aureus α-toxin causes the formation of a pore in the membrane of target cells. This pore alters ion influx/efflux and can lead to swelling/lysis of the cell.
• Inhibition of protein synthesis – Toxins which inhibit protein synthesis target the elongation factors and ribosomal RNA  which are associated with protein synthesis. By targeting these factors, the cell is prevented from synthesising protein and the cell dies. An example of such a toxin is the diptheria toxin.
• Interfere with cell signalling – These toxins target the proteins associated with signal transduction, either blocking or altering the signalling pathways. Such alteration of these pathways disrupts cellular function. For example E. coli cytotoxic necrotising factors modify RHO GTP-binding proteins, their modification disrupts the cell cyctoskeleton and thus the cell membrane.
• Inhibition of neurotransmitters – These toxins target proteins of the synaptic cleft. They prevent the release of neurotransmitters from the presynaptic membrane. For example Clostridium botulinum neurotoxin or the Clostridium tetani tetanus toxin.
• Affecting immune response – One example of altering the immune response is the super antigen TSS-1 released by Staphylococcus aureus which causes Toxic shock syndrome. The toxin interacts with T-cells of the host immune system in an abnormal manner and provokes the release of enormous amounts of inflammatory cytokines, which are harmful to the host.

Secretion of Toxins

The secretion of protein toxins in Gram-positive bacteria, essentially only requires the transportation of the protein through the cell membrane. This process involves:

• An initial secretion assistance protein (SecB) binds to the toxin in the cytoplasm
• SecB facilitates the binding to a secondary secretion assistance protein (SecA) located in the membrane
• Within the membrane is a transmembrane protein complex consisting of 3 secretion assistance proteins, SecY, SecG and SecE
• The toxin binds to the SecYGE complex, a process facilitated by SecA
• A number of small conformational changes occur in the SecYGE complex which requires the phosphorylation of ATP for energy, the ultimate result is the secretion of the toxin via the SecYGE complex

However, in Gram-negative bacteria a much more complex system is required to secrete the toxin. This is due to the structure of Gram-negative bacteria, they have two plasma membranes. A typical method of secreting toxins across both these membranes is to have a large protein made from multiple subunits, which spans both the inner and outer membranes and the periplasm space between them.  This is not the only way for toxins to be secreted from Gram-negative bacteria however, there are 7 types of secretory systems with slightly different properties currently identified:

• Types 1, 2, 3, 4, 6 – Sec independent pathways, where the usual method of secretion consists of some form of protein channel from the inner membrane to the outer membrane.
• Type 5- Also known as ‘autotransporter’ because once secreted from the inner membrane into the periplasm, the protein being transported forms its own beta-barrel in the outer membrane. This structure allows it to pass through the membrane and is often left behind.
• Twin arginine- translocation (Tat) – An alternative pathway to the sec pathway, folding of the transported protein occurs which allows exportation of the protein to the cell envelope.

The genes encoding for type 3 and 4 secretory systems (T3SS/T4SS) are found on pathogenicity islands within plasmids (Small ‘islands’ of genes where the majority of the genes encode for virulence factors). This is due to the ability of T3SS and T4SS to act as virulence factors as their structure consists of a needle like projection, projecting out past the outer membrane of the bacteria. This needle structure is able to puncture membranes of other cells and can act as an adhesin.

For example, Bartonella species use their T4SS as an adhesin, to adhere to host cell, erythrocytes in particular. They also use it to transport proteins (toxins) directly into endothelial cells by puncturing the cell membrane with the needle like structure. Other examples of T4SS usage includes:

• E. coli conjugation
• B. pertussis toxin secretion
• H. pylori pseudopod formation (makes the target cell form a vesicle around the bacterium)
• Agrobacterium plasmid transfer
• Legionella vacuole modification and secretion from within vacuoles

References: Birtles, R (2010), “Evolution of Bacterial Virulence”, BIOV 351, University of Liverpool, Unpublished.