Posts Tagged ‘ virulence factor ’

Bacterial Pathogens of the Respiratory Tract

Mannheimia haemolytica

M. haemolytica is responsible for causing contagious bovine pleuropneumonia, a bacterial disease which causes pneumonia and inflammation of the lung membranes. It is a Gram-negative coccobacillus (elongated, rod spheres) which shows mild haemolysis when plated on blood agar plates. This species comprises of 12 capsular serotypes (of which some are more responsible for disease than others). Serotype A2 is associated with sheep pneumonia.

Typically, diseases associated with M. haemolytica present themselves as fever, along with nasal discharge, coughing, inappetance and weight loss. Death associated with this bacteria is typically due to acute fibrous pleuropneumonia. Pathological observations would reveal an obstruction of the bronchioles with a fibrous exudate and an accumulation of neutrophils and fibrin in the alveoli. There will also be thrombosis and distension of the lymphatic vessels.

Mannheimia haemolytica as an Opportunistic Pathogen

M. haemolytica resides in the nasopharynx and tonsils of healthy cattle. A balance between the host and bacteria, under good environmental conditions maintains homeostasis between the host and bacteria (i.e. no disease). Should environmental conditions alter however, the balance may be tipped in favour of the bacteria, removing the status of homeostasis and resulting in disease. Key inciting events for disease include; weaning, adverse weather conditions, dehorning, feed changes and transportation i.e. causing stress. Stress provokes the bacteria in the nasal/tonsillar mucosa allowing them to be inhaled in to the lungs.

In healthy cattle clearance of the bacteria from the lungs is efficient enough to prevent disease. This suggests that stress induced alterations in immune functions can lead to host susceptibility and thus development of disease.

Close contact with other animals increases exposure to microorganisms which upsets the natural balance of commensal bacteria within the host. Changes in the natural microflora can cause M. haemolytica to revert to a pathogenic state. Slight changes in the environment are responsible for creating a favourable situation for M. haemolytica to colonies the lung and thus cause disease.

Mannheimia haemolytica Virulence Factors

Virulence factors of M. haemolytica include:

  • Adhesins – Required for initial colonization and adherence to host cells
  • Capsular polysaccharide – Helps prevent opsonisation and thus has anti-phagocytic properties
  • Sialoglycoprotease – An enzyme which cleaves IgG and thus reduces opsonisation of the bacteria
  • Neuraminidase – An enzyme which reduces the viscosity of respiratory mucus, reducing the chance of the bacteria being excreted via mucus
  • Iron-binding proteins – Removes iron from the host, for the benefit of the bacterium
  • Leukotoxin – A primary toxin involved in the pathogenesis of M. haemolytica

Mannheimia haemolytica Leukotoxin

Leukotoxin (LKT) is an actively secreted exotoxin which targets leukocytes, primarily neutrophils. The toxin is encoded for by four genes (lkt A-D) which can be found in the RTX toxin operon.

  • lktA encodes for the active toxin
  • lktB and lktD encode for the secretion of the toxin
  • lktC encodes for proteins responsible in the transportation and activation of the toxin

LKT help bacteria survive by allowing them to evade phagocytes. It is a type 2 exotoxin (membrane-damaging) which binds to leukocytes via the cell surface receptor CD18 (an integrin). It forms pores in the cell membrane of leukocytes which leads to an influx of K+/Ca2+ ions. This promotes swelling of the leukocyte and ultimately lysis of the cell.

Neutrophils affected by the toxin also become overly active, they overproduce certain mediators, reactive oxygen species and proteases, all of which promote further cellular and tissue damage within the host. Lesions develop which are filled with fibrous exudate and thrombosis of lymphatic vessels occurs. The alveolar epithelium also becomes damaged which is believed to be associated with neutrophil infiltration.

Controlling Mannheimia haemolytica Infections

Methods which could be implemented to reduce cases of M. haemolytica infections include:

  • Livestock management – Reduce stress of cattle by effectively managing when and how calves are weaned, sold and transported
  • Antibiotic use – Injectable antibiotic regimes are extensively used, especially in the intensive farming seen in large feedlots. However evidence shows that M. haemolytica are developing resistance to many of the common antibiotics used, such as; penicillin, ampicillin, tetracycline and sulphonamide
  • Vaccines – Both killed and live attenuated vaccines are used as well as cell-free supernatants such as the leukotoxin or capsular polysaccharide
  • Genetically Modified plants – A suggestion has been made for the use of GM crops to feed cattle in the future. The GM plants would produce M. haemolytica antigens which would essentially act as edible vaccines

These control methods could help to restore balance to the situation (previously unbalanced by negative environmental factors). Thus homeostasis between host and bacteria would be restored and disease prevented.

Bordetella bronchiseptica

Bordetella bronchiseptica is an evolutionary progenitor of B. pertussis and is one of the organisms responsible for causing kennel cough in dogs. Kennel cough is the term used to describe a disease which causes coughing in a dog due to inflammation of the trachea and lower airways. Although kennel cough is primarily caused by infection of the airways with B. bronchiseptica it can also be caused by viruses such as canine parainfluenza. Clinical signs of kennel cough include; an intense cough, mucus, nasal discharge and breathing difficulties.

Kennel cough gets its name from the frequency of infections which arise in dogs temporarily kenneled for example whilst the owner is on holiday. A number of dogs can carry the infection and the close contact in the kennels promotes transfer of the infection. Infection is caused either airborne transmission or via direct contact and has an incubation period of 3-10 days. Infected dogs can carry and shed the infection for up to four months after recovering from the disease. Because it require a only a few B. bronchiseptica bacteria to establish and infection in the airways (i.e. low infectious dose), B. bronchiseptica are considered highly infectious.

Bordetella Bronchiseptica Virulence Factors

Adhesins

  • Fimbriae – Associated with the initial adherence of the bacteria to epithelia. This form of adhesin allows the bacteria to latch on to host cells and begin to proliferate and form infectious colonies.
  • Filamentous Haemagglutinin Adhesin (FHA) – A large, filamentous protein which serves as a dominant attachment factor for adherence to host colliery epithelia cells of the respiratory tract. It is associated with biofilm formation and possesses at least four binding domains which can bind to different cell receptors on the epithelial cell surface.
  • Pertactin – An ‘autotransporter protein’ capable of getting to the cell surface without the need of accessory proteins. Pertactin also acts as an adhesin.

Adenylate Cyclase (cyaA)

  • Structurally similar to M. haemolytica leukotoxin
  • Different function, cyaA enters host cells and catalyses large amounts of cyclic AMP
  • This disrupts cell signaling pathways, impairing cellular function and inducing apoptosis whilst allowing the bacterium to avoid phagocytosis
  • It also inhibits the expression of interleukin 12 (IL12) an inflammatory cytokine

Dermonecrotic Toxin

  • A type 3 exotoxin which acts intracellularly
  • Precise mechanism is unknown however the overall effects on the cell include; actin reorganisation, increased DNA synthesis and binulcleation
  • Its overall contribution to the pathogenesis of B. bronchiseptica is currently unknown

Not all these virulence factors are active at the same time however, only some are active depending on the temperature. At 37˚C all are active except the flagellum, at at 27˚C none are active except the flagellum. What this shows is that there is a global regulation of gene expression depending on the requirements of the bacteria’s survival.

For example, when B. bronchiseptica is required to colonise the respiratory tract (indicated by a 37˚C temperature i.e. body temperature) it is not going to need to use its flagella for movement so the genes encoding for flagella motion are not expressed. In contrast when B. bronchiseptica is outside of its target destination e.g. the nasal passageway (indicated by a lower temperature) the flagella genes are expressed again as movement will be required to reach the target destination (the airways). Genes encoding for virulence factors will not be expressed as there is no need when the bacterium is not at its target site of pathogenesis.

Treatment of Kennel Cough

Kennel cough is susceptible to many antibiotics (it is however resistant to erythromycin). There are bivalent canine vaccines available which vaccinate against B. bronchiseptica and canine parainfluenza virus. Many of B. bronchiseptica outer proteins are highly immunogenic which makes good vaccines (e.g. fimbriae, FHA, pertactin)

In Summary

M. haemolytica and B. bronchiseptica are two important veterinary pathogens as they both inhabit the same ‘niche’. This is interesting because they also share similar virulence factors. Both diseases result directly from the disruption of the respiratory epithelium and from immunopathology and both diseases are often polymicrobial, involving mutliple agents.

Diagnosis relies on isolation and cultivation of bacteria on agar plates after which the correct antibiotic treatment can be given. Vaccinations are available for both diseases.

These diseases show how the management of animals can influence disease epidemiology.

Evolution of Bacterial Virulence

Point Mutations

Much like humans, bacteria are subject to evolution. Evolution occurs through beneficial mutations in the DNA. Because bacteria have much shorter generation times than humans, the process of evolution occurs much more rapidly. An example of how bacteria evolve through mutation is point mutation.

Point mutation is the random mutation of DNA nucleotides which occurs due to incorrect replication of DNA. The types of mutation that can occur are substitution of a nucleotide, deletion of a nucleotide or the addition of a nucleotide. For example, in a sequence of DNA a fragment of which codes for something (such as a protein) consists of multiple codons (sections of 3 nucleotides) if one of these nucleotides is altered in a point mutation, the codon (which codes for a specific amino acid) will be altered. Some alterations of codons will mean they encode for a different amino acid:

Initial DNA:                  … … TTA … …

After point mutation: … … TAA … …

In this case the selected codon in the initial DNA encodes for the amino acid leucine, however after the mutation, it no longer encodes for an amino acid at all as TAA is a stop codon (this closes the reading frame, meaning the rest of the gene is not read).

Point mutations may sometimes have no effect however, as multiple codons can encode for the same amino acid, for example:

Initial DNA:                  … … GCT … …

After point mutation: … … GCA … …

Both GCT and GCA codons encode for the amino acid alanine, so there are no alterations to the final protein which is encoded for by this gene.

Horizontal Gene Transfer

Bacteria are also able to ‘evolve’ by means other than mutation. Horizontal gene transfer is the sharing of genetic information between individuals in the population. Typical gene transfer occurs vertically i.e. parent to offspring, however with horizontal gene transfer genetic information can be spread to completely unrelated individuals.

There are three primary forms of horizontal gene transfer, these are:

  • Transformation – DNA is taken up from the environment
  • Conjugation – DNA is transferred directly between individual bacteria
  • Transduction – DNA transfer is mediated by bacteriophages (a virus which infects bacteria)

Transformation

Many bacteria are naturally transformable and have various means of DNA uptake from their environment. Bacteria able to do this have genes which encode for protein machinery which transports the DNA across the cell membrane from the environment. It is then possible for this DNA to be incorporated into the bacterial chromosome.

This ability has important implications in bacterial pathogenicity for example; Neisseria gonorrhea are able to uptake DNA from the environment which enables them to undergo possible conformational changes that make them more pathogenic. Another example would be Streptococcus pneumoniae and its possibility to acquire penicillin resistance.

Conjugation

Conjugation is the direct transfer of DNA between bacteria, this is of major advantage as advantageous genes (such as those which encode for antibiotic resistance) can be easily spread throughout the population.

DNA is transferred in the form of a plasmid (a circular DNA structure) in a unidirectional manner, that is only the donor requires the plasmid transfer-associated genes (i.e. those which encode for plasmid transfer mediating pili) for horizontal transfer via conjugation to occur.

Plasmids

Plasmids are typically circular in structure and consist of double stranded DNA. They replicate independently of the bacterial chromosome and are located in the cytoplasm, they are usually much smaller than the bacterial chromosome ranging from 1-100kb long. The genes found within the plasmid can be expressed by the bacterium and so proteins encoded for by the plasmid can be formed in the bacterium.

The most common plasmids are those which encode for antibiotic resistance. They are highly prevalent because of the great advantage they give to bacterial survivability. The are multiple genes found on plasmids which promote antibiotic resistance all with different mechanisms of producing resistance.

For example β-lactam antibiotics (such as penicillin derivatives) work by inhibiting cell wall synthesis, however β-lactamase is a bacterial enzyme which breaks the ring structure of these antibiotics are removes their antibacterial properties. These enzyme is encoded for by genes found within plasmids, which can easily be spread around the population thus increasing the resistance of the population to β-lactam antibiotics.

Other forms of plasmid-conferred attributes include:

  • Bacteriocins – Proteinaceous toxins which inhibit the growth of competitor species a specific example of this is colicin, a bacteriocin produced by Escherichia coli which exerts a cytotoxic effect within competitor species’ cytoplasm.
  • Toxin production – Toxins which affect other species such as the heat-labile enterotoxin produced by E. coli which causes diarrhoea or the Clostridium tetani tetanus toxin, the neurotoxin responsible for causing tetanus.
  • Protein delivery systems – Such as the Yersinia pestis secretion system, which allows them to inject protein materials into other organisms (such as immune cells), these injected proteins are known as YOPs (Yersinia outer proteins).
  • Adhesins – Typically pili (protein surface projections) which allow adherence to bind to cells, which is important in pathogenicity. Such adhesins can be found in E. coli and Shigella.
  • Metabolic activities – For example, the ability of Salmonella species to ferment lactose

Transduction

Transduction is the transfer of DNA which is mediated by bacteriophages (viruses which infect bacteria, combining their DNA with the bacterium host). Many bacteriophages have both lytic and lysogenic cycles of reproduction (typically viruses are one or the other). The lytic cycle is where the virus enters the cell, replicates using the cell’s own machinery and then lyses the cell releases large numbers of virus. The lysogenic cycle is where the virus incorporates its genetic material into the cell where it lies dormant until a later date, the viral genes are replicated when the cell replicates allowing them to reactivate and continue into a lytic cycle (formation of phages).

Bacteriophage-encoded virulence factors include:

  • Streptococcus pyogenes toxin (which causes scarlet fever)
  • Corynebacterium diphtheriae toxin
  • Clostridium botulinum toxin

E. coli O157:H7 contains around 1.4Mb of extra DNA when compared to E. coli K12 (a much less pathogenic strain which is used in laboratories). It is suggested that around 50% of this extra genetic material has been derived from bacteriophages.

Vibrio cholera Toxin

Another bacteriophage encoded virulence factor is the Vibrio cholera toxin, however for this toxin to be produced by the bacterium, genetic material from two bacteriophages must be acquired.

The initial bacteriophage (VPI phage) infects a non-toxic Vibrio cholera bacterium, its transduced genetic material encodes for pili proteins which act as an attachment site for the secondary bacteriophage (CTX phage). When CTX phage binds, the genetic material it incorporates encodes for the production of the cholera toxin and thus the bacterium becomes toxogenic.

Recombination

Transformed and transduced (i.e. non-plasmid DNA) is inserted into the recipient bacterial chromosome by recombination. Recombination is also responsible for the rearrangement of DNA fragments around the genome (genomic plasticity) which enables the bacteria to survive in rapidly changing environments due to the alterations in the genes which are expressed. Recombination can be either homologous (RecA dependent – a DNA repair and maintenance protein) or non-homologous.

Homologous Recombination

DNA inserted into the bacterium can be incorporated into the bacterial chromosome if there are regions of homology expressed on both the foreign transmitted DNA and the bacterial chromosome DNA. The differences between the foreign and bacterial DNA will cause RecA to attempt to ‘repair’ the chromosome. In doing so, the foreign DNA may be incorporated into the bacterial chromosome, (there is also the chance that it will be disregarded).

Non-homologous Recombination

Non-homologous recombination allows genetic material to enter the bacterial chromosome without RecA. Small fragments of DNA known as insertion sequences (ISs) are what enter the chromosome. ISs typically consist of DNA fragments from bacteriophages or elsewhere, however, all that an IS codes for is its motility and insertion. ISs integrate into the bacterial chromosome through self-encoded transposition enzymes (as opposed to RecA) and when within the chromosome they facilitate replication of themselves. The short ISs then become numerous within the bacterial chromosome.

The presence of multiple ISs within the bacterial chromosome is what allows genetic plasticity (The movement or deletion of short fragments of DNA within the chromosome). Genetic plasticity leads to alterations in genes or the removal of genes altogether, this has the potential to increase pathogenicity. For example, Bordetella bronchiseptica is a gram negative bacterium responsible for causing infectious bronchitis, which rarely infects humans. B. pertussis on the hand is the major causative agent of pertussis (whooping cough) and is an obligate human pathogen. It is believed that genetic plasticity caused a reshuffling of the B. bronchiseptica chromosome which lead to the formation of the obligate human pathogen B. pertussis.

Transposons

Transposons are transmitted by similar means to ISs, however they are more structurally complex and much larger, due to the greater amount of genetic information they carry. This extra genetic material encodes for attributes such as antibiotic resistance, toxins and enzymes.

Some transposons (known as conjugative transposons) excise themselves from the bacterial chromosome to form circular intermediates of DNA, much like plasmids. These can also be transmitted around a population, again like plasmids, to spread resistance to antibiotics etc.

Pathogenicity Islands

Pathogenetic islands are also laterally-transmitted. They are large integrated DNA elements which carry multiple virulence-associated genes such as those which encode for adherence factors, secretion systems, toxins and invasion factors. A bacterium may have multiple pathogenicity islands and due to the incorporation of multiple virulence factors on one island, the transmission of one island often causes a non-toxogenic bacterium to become pathogenic.

They may be incorporated into the main bacterial chromosome or any plasmids which may be present in the cytoplasm, the DNA found within these islands may contain remnants from bacteriophages and IS elements. The GC-content (guanine-cytosine content) is often different from the rest of the genome.