BACTERIAL PATHOGENESIS I

• responsible for recycling of compounds essential for growth
• they are great at adapting to environments and can quickly recover from population loss
• they can colonise a wide range of organisms 
• genome plasticity

Bacteria are consistently associated with most body surface. Ones which are constantly associated with a host are called normal flora. Human are the perfect host because of the intestinal tracts and respiratory tract. 

Requirement for colonisation 

• C, O, N, H, P, S, K, Na, Ca, Mg, Cl and Fe
• trace element (cabalt, copper, manganese)
• organic growth factor (AA, vitamins, fatty acids)
• physiochemical (pH, temperature, humidity, CO2)
• Host biological factors - bacterial removeral mechanism (coughing, saliva, urine)
• anti microbial factor

successive colonisation - primary colonisation, secondary colonisation etc. this is determined by environmental conditions such as nutrients, physiochemical and host biological factors. 

1, Beneficial - both benefits

2, Neutral - no apparent benefits or harm to either members

3, Parasitic - one member lives at the expense of the other member

Mutualism of normal intestinal microflora 

 It helps in the development of immune tolerance to allergens and self-Ag. It also helps with food digestion and synthesis of vitamin K, B12, biotin and riboflavin.

It stimulates maturation and fluid secretion.

Provide defence against bacterial pathogens:

• filling available colonisation niche
• competing with nutrients
• produce anti-microbial chemical substances

 Parasitism 

• infectious dose
• virulence trait
• newly emerged bacteria              BAD ABCETRAIL --> either neutralisation or diseas
• inoculating material
• antibiotic resistance 

A healthy individual can defend against pathogen at different stage, early onset or late. The host mechanism is divided into 2: non-specific (early) and specific (late). 

None specific defences

This is common to all healthy animals. It provides general protection against colonisation.

E.g. Physical or biochemical barrier 

1, skin and other barrier                                     E.g. Phagocytosis and bacterial killing

2, washing action of fluids                                  E.g. complement cascade in serum

3, acidic environment                                                 - opsonisation

4, antimicrobials                                                        - inflammation

5, low nutrients level (Fe level)                                     - cytolysis 

6, Normal flora

Specific defences

Activated by host exposure to a pathogen. It is only activated upon appropriate exposal to the pathogen. This leads to memory cells productions. 

This system involves immune system and specific Ag recognition to induce Ab and cell-mediated immunity (T-cells, macrophages). 

• weak immune responses (AIDS)
• extreme age and physiological imbalance
• predisposition disease such as cancer
• under going immuno-supressive chemotherapy for organ transplant
• genetic defects of complement system
• compromised skin or mucosa - especially in case of severe wounds 

2 types of bacterial pathogens

Opportunistic pathogens

• causes disease in a compromised host - not occur in healthy individual
• part of normal flora or free-living environmental bacetria
• do not cause disease unless they have an opportunity brought by weakness in the immune system

Obligate / strict pathogen

• those that cannot survive outside the host
• survival inside the host often result in disease 

Starts with single-cell protozoan which fed on bacteria and archaea - similar to human phagocytic cells. The bacteria develop resistance to killing and may be fore-runner to today's bacterial pathogens. 

Why are they pathogenic 

• produce surface located or secrete virulence factors
• bacterial product or strategy that contribute to pathogenicity

All helps the bacteria to invade host cells, gain access to nutrients, evade immune system, spread to other site or tissue damage. 

Route of transmission 

• biological vector (animal bites)
• aerosol
• food/water borne
• body fluid (blood, saliva, breast milk)
• nosocomial infection

Route of entry

• break of skin
• respiratory/ urine / gastronintestinal tract
• eyes
• surgical intervention (implants)

Adherence factors: many colonises mucosal sites using hair like structure called pili or ligands to adhere

Invasion factors: surface or secreted components that allow bacterium to invade host cells

Evasion strategies: Some have capsules which protect bacteria from host defence system. 

Endotoxins: 1, naturally occurring components of all bacteria 

                  2, causes fever, change in temperature, inflammation, lethal shock

Exotoxins: 1, secretion of toxins or enzymes such as cytoxins and neurotoxins 

                2, attack cell membrane or signalling process in the cell  that causes damage to                     host cells 

Nutrients acquistion: able to compete nutrients with host (normal flora)

New-New --> newly discovered disease caused by newly discovered bacetria

New-Old --> new disease caused by bacterium known for a long time

Old-New --> Old disease which is thought to have been eliminated but has now reappear

Old-Old --> old disease with established cause that is now recognised by the media 

Terminology 

• infection - colonisation of body by bacterium
• disease - infections which causes a symptoms
• colonisation - bacterium occupying and multiplying in particular area of the body
• symptomatic carrier - infected person with no symptoms
• symptoms - effects of the bacterial infection to the infected person
• virulence (pathogenicity) - the ability for the bacterium to cause disease
• virulence factor - products or strategy that contributes to virulence
• opportunist - bacterium which causes infection in those with weak immune system but not in a healthy individual 

It's a cause and effect relationship

How to prevent the bacterium from causing infections

• First: bacterium should be found in diseased tissue and not healthy ones
          1, secondary symptoms may allow bacterium to gain entry  
          2, bacterium may be associated with diseased tissue but not always
          3, asymptoms carrier 
• Second: bacteria can be isolated from a pure culture
          1, bacteria have diverse metabolisms - difficult to cultivate
          2, some cannot be culture in  vitro 
• Third: the pure culture of bacteria should cause the same symptoms if innoculated into human or animals
          1, absence of good animal models
          2, some research, therefore, involves study of bacteria that are closely related                     natural pathogens of animals

• Forth: bacteria must be re-isolated in pure culture from diseased lesions of intentionally infected human or animal 

        1, disease lesion could results from a secondary infections - increased in lesion             formation 
        2. satisfy that the bacteria causes the disease in the infected host 

• (Fifth): development of antibacterial that eliminates the symptoms 

          1, antibiotics that kill the bacterium in lab may not have the same affect in animal                     because of antibiotic distribution in the host and the local destabilising conditions 

Animal models 

Human are the best possible model, however, there are ethical issue implications - problematic to cure life-threatening disease. The suitable models must develop the same symptoms, have similar bacterial distribution and acquire the disease by the same route of infection. E.g. rabbits, guinea pigs, primates.

ADVANTAGES 

• cost and ease of care and maintenance
• clonal
• controlled environment
• transgenics

DISADVANTAGES 

• anatomically different
• different microbiota
• behavioural differences
• human disease can take different course 
• a virulence determinant may be missed - targeted at human

Fruit fly, nematodes, protozoan, plants, zebrafish

• cheap
• can be culture to a high number
• non-expert carers
• no ethical considerations
• conserved innate immunity
• controlled environment
• ease of mutagenesis
• established mutant libraries

Tissue culture models 

Advantages 

• one cell type
• growth in defined media under reproducible conditions
• cheap 

Disadvantages 

• tumour derives with no growth control
• undefined genetic mutations - accumulates in culture
• loses the traits in original derivatives
• absence of bathing solutions found in intact animal - altered gene expressions
• lack polarity and typical cell shape

• ID50 measures infectivity. It measures the number of bacteria needed to infect 50% of the animals exposed to bacterium
• LD50 measure lethality. It measures the number of bacteria needed to kill 50% of the animals exposed to bacterium

Limitation 

• crude
• may fail to pick up the virulence factor
• competition assay can improve sensitivity
• different bacteria cannot be compared
• V.cholerae requires 1000x more bacetria than S. dysentariae to infect human (ID50) but V.cholerae is more virulent (LD50)
• S. is more able to survive passage through the stomach 

To prove that a particular gene contribute to virulence:

Molecular Koch's postulates:

1, the gene or its product should be found in pathogenic bacteria causing the disease

2, disrupting the gene should reduce virulence or isolated and introduced to avirulent bacterium to increase its virulence

3, gene should be expressed during infection of host 

Virulence factor hunting

Vitro techniques 

1, transoson mutagenesis

2, gene cloning

3, transcriptional gene fusion

4, deciphering genome sequences --> Microarray technology or bioinformatic approaches

Transposon mutagenesis

• DNA segments carrying random markers in its genome - create a pool of bacetria, each colony carrying selectable transpson mutations inserted into a gene disrupting expression
• this 'Mutant library' is used to screen loss of virulence properties
• the transposon also serve as a marker to locate the gene of interest - generate polar mutation
• gene essential for growth are not removed 

Gene cloning 

• DNA from virulent bacteria may convert ordinary E.coli into pathogen
• however, this is restricted by the amount of DNA that can be cloned into a plasmid expression vector
• the virulent phenotype must be conferred by closely linked genes
• the gene must be expressed in E.coli - work best with DNA derived from closely related bacteria 

Transcriptional gene fusion

• this method exploit the fact that virulence expression is tightly regulated
• the functional genes are often organised into regulon regulated by the same regulatorsor environmental stimuli
• novel genes regulated in the same way may function in virulence 
• Identification - by generating a transcriptional hybrid that contains the potentail virulence gene promoter fused to promoterless structural gene encoding some easily assayable enzyme reporter
• to search for genes randomly - use a transposon-based transcriptional reporter system 

Microarray technology 

• chip containing individual segments of ssDNA representing all genes of the genome
• RNA is isolated from bacteria grown under particular conditions mimicing the host environment 
• the mRNA binds to DNA of the gene from which it is transcribed
• the label on the the chip is detected to determine which genes were expressed under the chose growth conditions

Bioinformatics approach 

•  selected sequences conserved in a number of bacterial pathogens 
• restrict to search ORF of hypothetical or unknown function
• assay for virulence in animal model 

1, IVET (In vivo expression technology)

• detect genes the gene that enable the bacteria to survival in mouse based on the inability of purA (purine) to auxotrophs it infect mice
• this generate a bacterial library in a purine auxotroph in which gene promoter are transcriptionally fused to a promoterless purA gene that is also linked to a promoterless lacZ gene
• the library is used to infect the mouse
• promoter turned onin the animla will be able to express purA, complementing the purine auxotrophy and enabling bacetrial survival 
• surving bacteria recovered and screen for Lac- phenotype in vitro

2, STM (Signature-tagged mutagenesis)

• uses a mixture of transposons each having its own tag
• this generates a library of mutants with each colony containing unique tag saved on a reference plate

• the mixture of transposon-containing isolates is used to innooculate an animal
• bacteria are recovered and the tag is amplified suing PCR and used to probe the original collection of transposon mutants
• original mutants that don't hybridise with the mixture of probe tags represents a mutant that was lost during infections - less virulent 

3, BLI (Bioluminescence imaging)

• real time monitoring of spatial and temporal progression of the infection in the same animal
• pathogens or mice engineered to express genetically encoded luciferase enzymes
• luminescence detected microscopically
• molecular events can be studied in vivo directly - yielding important insight into mechanisms of pathogenesis 

The rise in death by disease after the reduction is caused by the bacterial resistance to drugs, new virulence traits, aging population and modern medicine. 

To search for a new antibacterial drugs..

• isolate anti-bacterial that target virulence determinant
• ones that is least likely to evolve a resistance
• specific for pathogenic bacteria
• leaves normal flora unaffected 

We live in an environment that is filled with diverse populations of dangerous bacteria. The goals are two to understand the natural immune mechanisms of the host and to identify and characterise bacteria virulence mechanisms. In the future, this will allow us to design an effective treatment based on the interruption of the virulence mechanisms and to improve host resistance to bacteria  infections.