Mycobacterium Tuberculosis Flashcards
What is tuberculosis, how long has it been around?
An ancient disease of mankind
Causative agent is mycobacterium tuberculosis
It has afflicted humans for about 70,000 years
Comment on the prevalence and mortality
Causes 8.6 million cases
Causes 1.4 million deaths
Why have we not been able to control tuberculosis
Even through significan changes in lifestyle and upgrades of modern medicine they havent been enough to compate the agility and toughness of tuberculosis
Paradigm for human-host pathogen adaptation:
- ‘Great white plague’
-‘the captain of all thos men deaths’
Comment on trends in incidence rates of TB
Countries with socio-economic problems tend to be more affected
High levels in Southern africa and india
Numbers decreased with covid as people werent coughing on each other and there was a decline in testing but numbers are now high
High numbers post covid as we are still detecting some of those cases we missed during lockdown
What are the three important steps in ending TB?
Integrated, patient-centred care and prevention
Bod policies and supportive systems
Intensified research and innovation
What was the plan to eradicate TB, how did we fair against targets?
To reduce TB incidence rate by 20% between 2015-2020 (we only got a 9% reduction)
To reduce TB deaths by 35% between 2015-2020 (we only got a 14% reduction)
To reduce % of people with TB facing catastrophic costs to 0% (49% still face catastrophic costs)
NB: high economic countries were going to support low economic countries etc
How much research is done on TB in Ireland
Ireland spends about 10million on research on TB bovis done mostly in UCD while only spending about 1 million on human TB
What is the strategy for addressing tuberculosis over the next few years?
The 2030 Global Strategy: they set out the three steps to addressing tuberculosis:
- integrated, patient centred care and prevention
- bold policies and supportive systems
- intensified research and innovation
What is the TB strategy for 2030s target, how do we meet this?
80% reduction in tuberculosis by 2030 met through:
- multidiscipinary TB workforce meetins
- MDTs
- presenting case studies etc
A lot has already been done in Ireland so its interesting to see if we will meet these targets
How does mycobacterium establish infection?
Spred is solely from person to person via inhalation of contaminated droplets
Mycobacterium evades upper respiratory airway defensins such as mucocillary action
Mycobacterium then reaches deep airways and alveoli from there it gets into macrophages
What is normally the role of alveolar macrophages?
They recognise foreign proteins on cell surfaces
They phagocytose foreign material
They fuse phagosomes and lysosomes -> this creates acidic conditions where bacteria cannot survive
Their hydrolytic enzymes break down foreign molecules
What happens to the macrophages in TB
Macrophages recognise foreign TB proteins on cell surfaces
Foreign material is phagocytosed
TB then inhibits fusion of the phagosome and lysosome -> lysosome action inhibited by TB
TB then proliferates inside macrophages
TB creates local infection in the lung and eventually crosses over the mucous barrier
Its important that TB invades what particular type of macrophage?
TB can only proliferate in naive macrophages, as these cannot work well
What is primary tuberculosis
Flu-like symptoms
asymptomatic of tb like symptoms
Patients often think they have a normal head cold
After 3 weeks a ghon focus is formed
What is a ghon focus
A ghon focus is formed approximately 3 weeks after infection
It is a product of the activation of cell mediated immunity
Its the production of a granuloma in the lung
also known as caseous necrosis
Tb is able to proliferate within this and cause local infection
How is a ghon focus formed and how does this affect pathogenicity?
Ghon focus begins when a barrier is formed around the infected macrophage
All cells inside the ghon begin to die -> this is what causes the caseous necrosis
TB love this invironment and will proliferate and cause infection
The focus will get bigger and bigger until it reaches a lymph node -> contain more naive cells for infection
TB infects these cells and thus forms a ghon complex
What is a ghon complex
This is where TB has spread to the hilar lymph node to infect immune cells
It is the extension of the ghon focus
What does the ghon complex progress to?
Ranke complex or disease eradication or latentinfection
What is the ranke complex?
This is where fibrosis and calcification of the tissue encapsulating the granuloma form
Scar tissue is layed down
-> this can have two results -> the cells inside will either die but in most cases will survive and result in a latent infection
How can the Ranke complex result in disease eradication?
If the cells trapped inside the fibrotic tissue die then the tb will be eradicated
How does the ranke complex progress to latent infection
If viable cells are trapped in the complex and cannot be killed they will remain inside waiting for an opportunity to reactivate
These patients wont be infections, and will be auramine O negative but between 2-5 years down the line they will have a reactivation
When will latent tb reactivate?
2-5 years down the line
What % of latent TB becomes active?
Between 5-20% become active within 5 years
What are some risk factors for disease progression into active TB
Immunosuppression
Malnutrition - particularly vitamin D deficiency
Diabetes
Alcohol abuse
Give an example of a TB outbreak in Ireland
Massive outbreak in Baggot street
Between 20-30 people infected
All traced back to the one guy coughing in a pub
How does active infection occur?
Active infection occurs when the immune system is compromised and:
Activation of Ghon Focus
Exogenous re-infection -> secondary infection if unlucky patient
Spread to one/both upper lobes
Oxygenated area
What happens upon secondary exposure to tb
Memory T cells release cytokines
Formation of areas of caseous necrosis
Formation of cavities
Allow dissemination of bacteria
Memory T cells are already formed so theyll recognise TB much quicker -> these release cytokines -> lots of caseous necrosis form quickly which causes cavities in the lung often seen on autopsy -> these cavities allow bacteria to then spread to the blood and cause millerary TB
What is milliary TB?
Occurs in secondary infection
Whereby TB spreads from the lung to the blood
Give an example of where miliary TB was missed?
A baby was born with TB -> CSF positive for TB on biofire
However mother and rest of family were negative
Staff and everyone involved were screened and all were negative
The mother had milary TB (latent TB) i.e. it had spread to reproductive organs and baby was then born with TB
How does latent TB become symptomatic disease?
Bronchopneumonia
Miliary disease
What five features make mycobacterium tuberculosis unique?
Dependent on human host for survival and evolution
Lacks classical virulence factors
Used by mucosal pathogens for colonisation - selective colonisation advantage
Uses macrophages to trasverse mucosal barrier and to persist in these
Employs cell wall lipids as a novel mechanism
shat virulence factors does tb lack?
Virulent capsule to avoid phagocytosis
Pilli or other adhesions to adhere to host tissue
Flagella for motility
Enzymes and toxins
Biofilms to persist
TB lacks a lot of significant virulence factors, why is this important
Virulence factors tend to be taregts for vaccines
-> without these we cant produce vaccines against it etc
What three things must mycobacterium do to evade the innate immune system?
Optimum host niche
Avoid microbicidal macrophages
Recruit growth permissive macrophages
What four things must Mtu do to be successful?
Evade the host innate immune response
Multiplication within the host cell
Exit the host cell
Transmit to a new host
What does tb need to do to multiply within the host cell?
TB needs to develop strategies to avoid or modulate the immune response in their favour
How does tb exit the host cell?
Apoptosis
Necrosis
Then transmission to a new host
What is the mtb cell wall composed of?
Outermost layer/capsule
Cell wall-tripartite complex
Conventional plasma membrane
What four things do we know about the mycobacterial cell wall?
Acid fast bacilli
Low permeability - contributes to resistance of the organism
High lipid content -> has a whole lipid layer of mycolic acids
Slow growers -> takes 8-12 weeks to grow in conventional culture
Why are antibiotics generally not affective on the TB cell wall?
Low permeability
But also cause its really slow growing and takes ags to form -> so antibiotics that affect cell wall synthesis their affects arent as evident as this occurs over a longer period etc
what is the plasma membrane of tb made of/
Phospholipid bilayer
Glycolipids that extend into the periplasmic space
NB: nothing special, same in all other bacteria
What is the mycobacerial cell wall composed of?
Cell wall
Tripartite complex:
- cross linked polymer of peptdoglycan
- highly branched arabinogalactan polysaccharides
- long chain mycolic acids
What are long chain mycolic acids?
Free lipids and glycolipids such as PGM and PDIM which make the cell wall waxy
NB: they are the most important structure of TB
What is the Mytb capsule made of?
Proteins and polysaccharides
Pathogenic mycobacterium - primarily glucan and proteins - small amount of lipids
Non-pathogens primarily proteins
What are the two phases of tb growth
Acive phase and stasis phase
What is the active phase of tb growth?
Abundant during active growth in early infection
The initial phase of infection where everything is in abundance
Lots of proteins are secrete here etc hence its a good point where we can target treatment but patients usually only think they have a cold at this point
What is the stasis phase of tb growth?
Metabolism of the core wall layers is reduced -> seen in chronic infection
Metabolism slows down
We dont have many targets of treatment here but this is when we usually diagnose patients
What are our treatment targets for tb
Glucans and secrete protein that have a role in host interactions and virulence - treatment targets
Talk about the mtb cell wall
There are chemically distinct cell envelopes that exist in different cells at different infection stages
Particular cell envelope of a mycobacterial cell at any given moment partly determines how that cell will respond to the immune system and antibiotics
How do they do this, we still dont know but its probably going to be a possible treatment option in the future
What two things are needed to make an optimum host environment for tb
An environment in which the pathogen can rapidly replicate
Small aerosol droplets
What is needed to make an optimum host environment for tb?
Alveolar space deep within the lung
Circument host commensal aliance
- host defenses and the immune system as well as
- commensal microbiata which confer colonisation resistance
Talk about tb in small aerosol droplets
1-3 bacteria in each droplet
These can reach the lungs if the circumvent upper respiratory tract commensal microbiota
Talk about the research of tb on zebra fish
Much of our knowledge comes from zebra fish -> these are transparent fish in which we can fluorescently tag white cells and track through the fish
Can be done on alveolar cells deep in the lungs
Why does TB keep its aerosols small?
The smaller the aerosol the further down in the lung it can get before being blocked etc
Its much harder to compete in the upper resp tract as there is a lot of non specific immune responnses up here and TB can often get killed in the cross fire
TB avoid this by keeping aerosols small so they can get down to the alveoli
We found this out by experimenting on zebra fishes and following them down into the lung etc
What do microbicidal macrophages do normally?
They default to respond to comensal pathogens
TLRs activated by PAMPS determine this activity
How does tb interact with macrophages
There are abberant respons by Mt to avoid microbicidal macrophages
TB inhibits its on detection as well as the activation of cytokine responses
Instead tb recruits naive marophages to sites of infection
Talk about TB PAMPS
TB still has normal PAMPs which stimulate normal immune responses but it also has PDIM
PDIM is a surface lipid which sits ontop of PAMPs present on TB to cloak them
This prevents TLRs from recognising PAMPs
Macrophages cant recognise the TB then
This is one of the main virulence factors of TB
Only clinical isolates have this - knowckout strains will not e virulent etc
What specific molecule is responsible for PAMP cloaking in TB?
PDIM
What does PDIM stand for?
Pthiocerol dimycoceroserate
What is the role of PDIM
It inhibits toll-like receptor 2 wih sulfoglycolipids
It masks surface antigens
How does MTB recruit naive macrophages?
It produces PGL which induces macrophage chemoine CCL2 which recruits macrophages for infection
PGL is not required for virulence unlike PDIM but its expression increases infectivity by enhancing the recruitment of permissive macrophage thus is called an indirec virulence factor
What does PGL stand for?
Phenolic glycopolipid PGL
What does PGL induce expression of?
CCL2
Why is PGL considered an indirect virulence factor?
It is not expressed by all clinical isolates i.e. it is not absolutely required for virulence
What is the tripartite response?
Avoiding microbicidal macrophages
Recruiting permissive macrophages
Under what scenario might TB fail to get down into the lung?
In a commensal rich niche
-> this means there is an endless suplly of PAMPS stiulating macrophages
-> this means MTU gets killed as collateral damage
So even if host is optimal/niche etc it might not always result in infection
What are the three steps in the tripartite nnate immune evasion strategy?
- avoiding microbicidal macrophages
- recruiting permissive macrophages
- optimum host niche
Now once recruited tb has the opportunity to survive within macrophages and transvers host epithelial barriers
What is significant about the ability of tb to replicate?
Its ability to replicate defines the pathogenesis of the mycobacterium
what is the normal pathogenic activity response in the lung
pathogens are phagocytosed by macrophages and enter the endocytic pathway
How do intracellular pathogens resist immune responses
They thwart/modulate/exploit or avoid the endocytic pathway of phagocytosis
They resist lysosomal fusion to reside in non-acidified endosome-like compartments
They survive acidification to be able o reside in acidified lysosome-like compartments (tolerant organisms)
Break out of the phagosome altogether to reside in the cytosol
NB: intracellular organisms have to be able to do at least one of these three things but TB can do all three
Talk about the life of tb inside the macrophage
TB can resist lysosomal fusion to reside in non-acidified endosome-like compartments
TB can survive acidification to be able to reside in acidified lysosome-like compartments
Break out of the phagosome altogether to reside in the cytosol
TB can break out of the phagosome altogether to reside in the cytosol
How can TB resist lysosomal fusioin to reside in non-acidified endosome-like compartments
Serine-threonine protein kinase PknG- inhibits lysosomal fusion
Trehalose dimycolate is also thought to have a role in blocking phagosome-lysosome fusion and promoting granuloma formation
What is responsible for inhibiting lysosomal fusion in TB?
Serine-threonine protein kinase PknG
What is responsible for blocking phagosome-lysosome fusion and promoting granuloma formation in tb?
Trehalose dimycolate
How can TB break out of the phagosome to reside in the cytosol?
TB has an ESX-1 Bacterial Secretory System
This initiates the rupture of phagosomes and directly protects against reactive nitrogen species
What is a TB granuloma
It is a hallmark of TB infection
It has an essential host protective strucutre
Its essentially a fortress containing a complex mixture of diverse host cells that wall off bacteria
The more complicated the granuloma the more TB in the granuloma
What has research on granulomas shown?
Research has shown that even though host immune responses form the granuloma it is actually TB controlling the granuloma
TB mediated granuloma not our immune response as we previously thought
TB exploits granulomas that host immune system produces
TB can multiply within and then spread from them
What three things can TB do in a granuloma?
Avoid or modulate the immune response in their favour
Exploit granulomas
Expansion and dissemination in innate granuloma
In what two ways can TB undergo expansion from a granuloma
Spread to new macrophages from dying macrophages
Macrophage cell death and re-phagocytosis allows for expansion of bacterial niche
How can TB spread to new macrophages from dying macrophages
- bacterial nnumbers increase in macrophages
- programmed cell death - apoptosis -> viable bacteria remain encased within the dying cell
- recruitment of new uninfected macrophages
- dying cell components are engulfed by new macrophages
Explain how TB can spread using granulomas
From here TB can now be picked up by more macrophages and can spread disease
TB can trigger apoptosis of cells in order to spread etc
TB has a whole new sense of life etc and begins again and again etc etc
What does the ESX-1 bacterial secretory system do?
Mediates macrophage cell death and rephagocytosis
How is macrophage cell death and rephagocytosis by ESX-1 secretory system
Done through secretory factor ESAT 6
- this induces apoptosis of infected cells
- this recruits macrophages by inducing metalloproteinase 9 (MMP9)
What does induction of MMP9 by ESAT6 do?
Increased MMP9 associated with increased severity and mortality e.g. in meningitis
Decreased MMP9 associated with attenuated infection
What does ESAT 6 do other than inucing MMP9 and inducing apoptosis?
It can delay migration of T cells to the granuloma through immunosuppressive regulation of T cell populations
What changes occur in macrophages as granuloma matures and how does TB combat this
Macrophages become more microbicidal as granuloma matures
However TB rapidly adapts to this hotile environment and transcribes new genes to aid intraccellular survival e.g. efflux pumps -> TB make the best of a bad situation
What are the two systems TB has in place to recruit macrophages?
PGL-CCL2 initially -> to recruit naive cells at start of infection
But this transitions to ESAT-MMP9 later on -> for the recruitment of macrophages to allow TB to spread
Why is escape of the granuloma so important in TB
The evolutionary success of tb is dependen on is ability to exit the granuloma (host) and enter a new host and spread
How does TB spread to a new host?
Necrotic areas rupture into the bronchial tree
TB is then aerosilised in cough droplets
What does escaping the granuloma depend on?
Depends on macrophage death either through aptosis or necrosis
Apoptosis: tb remains encased in dead cell
Necrosis: tb released into extracellular millieu-caseum - the ideal bacterial growth medium
What promotes necrotic caseation in mature granulomas?
trehalose dimycolate
What does trehalose dimycolate promote?
Necrotic caseation in mature granulomas
How does necrosis enable spread?
Necrosis essentially blows everyhing up
But some bacteria survive
This bacteria can then spread into the bronchial tree and spread when a person coughs etc
What can induce necrosis in tb
Over or under production of tumour necrosis factor
Talk about the steps in tb spread
TB increases multiplication - serpentine cord like growth occurs -> this is not readily engulfed by macrophages
Granuloma shifts to favour necrosis - this is not fully understood
Disseminates infection through the egress of infected macrophages to new sites
Either over or under regulation of tumor necrosis factor
What does serpentine cord-like growth in tb mean
This means tb is trying to spread
This form cant be readily engulfed by new macrophages -> instead looking to spread
Talk about the role of tumour necrosis factor
It is either over or under regulated
Over production -> results in increased ROS which has a dual effect - this kills macrophages and mycobacterium - some mycobacterium escape extracellularly - replicate exponentially
TNF deficiency -> mycobacterium grow exponentially within the macrophage, macrophage dies and releases them to extracellular
How is the granuloma modulated?
Modulate the host response to infection to build and modify the formation of the granuloma:
1. intracellula growth
2. extrtacellular growth that also favours transmission
What % of infections are cleared?
Estimated to be 90%
Dependent on:
- initial immobilisation of innate immune mechanisms
- adaptive immunity
Talk about the paradigm of host-adaptive microorganisms
Co-evolved with the human immune system
Deletion and insertion of genes to adapt to evolution of the host immune response
Trump cards such as host immune evasion, mmodulation and exploitation
