Infection (Keevil) Flashcards
What did Hippocrates theorise?
That disease might be associated with the physical environment
E.g.
- Touching things
What did Anton Van Leeuwenhoek do?
Developed microscopes
Described the first microorganisms in water and dental plaque in 1683
Named the single cell bacteria and protists as “Animalcules”
What did Edward Jenner do?
Pioneered clinical trial for vaccination to control spread of smallpox using the similar cowpox
Jenners work influenced many others, including Louis Pasteur who developed vaccines against rabies and other infectious diseases
What did Ignas Semmelweis do?
Pioneered hand washing to help prevent the spread of septic infections in mothers following birth
Infection rates in Vienna Obstetrics Clinic decreased from 18% to 2%)
What did John Snow do?
Father of epidemiology
Careful mapping of cholera cases in London during cholera epidemic of 1854
Traced source to a single well on Broad Street, Soho that had been contaminated by sewage
What did Louis Pasteur do?
Disproved spontaneous generation;
fermentation is caused by microbial
growth
Nothing grew in boiled broths when a filter in place or tortuous tube, therefore living organisms that grew in such broths came from outside, as spores on dust, rather than being generated within the broth.
Developed weakened anthrax vaccine, immunised cattle; rabies etc
Often regarded as the father of Germ Theory and Bacteriology, together with
Robert Koch
What did Joseph Lister do?
Pioneered antiseptic surgery
Most had assumed that chemical damage from exposure to bad air was responsible for infections in wounds
Pasteur suggested three methods to eliminate the micro-organisms responsible for gangrene: filtration, exposure to heat, or exposure to chemical solutions
Lister confirmed Pasteur’s conclusions and then used carbolic acid (phenol) to sterilise surgical instruments and clean wounds; reducing post-operative infections
What did Robert Koch do and what are his postulates?
He came up with a way to define a pathogen
Postulates:
1) Isolate the organism from every case of disease
2) Propagate in pure culture in vitro
3) Reproduce disease by exposing suitable host to organism
4) Re-isolate the organism
If these are met, can be defined as pathogen
Turned out to not be accurate, as chronic or minor conditions, multiple causes and pathogens that arent able to be grown in the lab were hard to define with these postulates
What did Hans Christian Gram do?
Pioneered Gram stain
Developed to detect bacteria in stained lung sections in the Berlin hospital morgue but became universal method of differentiating bacteria
Based on the chemical and physical properties of their cell walls. Detects peptidoglycan, a thick layer in Gram-positive bacteria to which crystal violet becomes trapped in the presence of iodine; ethanol dehydrates/stabilises this but washes CV+I-out of Gram-negative bacteria. Counterstain.
- Gram positive gives a purple/blue color,
- Gram negative results in a pink/red color.
Who is Typhoid Mary?
She is the first documented case of ASYMPTOMATIC carrier of typhoid fever
She caused multiple typhoid fever outbreaks over many years when she worked as a cook, she was never ill and completely well but carried typhoid fever asymptomatically
What are some host defences against pathogenic attachment?
Microbes can be rinsed away from epithelial surface by host secretions
Also, ciliary activity in respiratory tract can remove pathogens
We can also produce secretory immunoglobulin (IgA)
What are some microbial strategies for attachment?
They can bind firmly to epithelial surface (and if relevant, interfere with ciliary activity) via surface molecules on the microbe attaching to receptor on epithelial cell (and, if relevant, production of ciliotoxic/ciliostatic molecule)
They can also inactivate IgA via production of IgA proteases
What are some examples of pathogen attachment mechanisms
Enteropathogenic E. coli (EPEC) adhering to a tissue culture cell. EPEC are closely related to enterohaemorrhagic E. coli but don’t express a Shiga-like Toxin
Type 1 fimbriae on E. coli. These filamentous appendages bind to D-mannose residues on the eukarvotic cell surface. They are often termed ‘mannose-sensitive’ fimbriae as the presence of D-mannose in the growth medium prevents binding.
What is a host defence against pathogenic invasion?
The host cell membrane poses barrier to intracellular microbe
What are some microbial strategies of pathogenic invasion?
Traverse host cell membrane via fusion proteins in viral envelope
Endure or trigger uptake by phagocyte and resist killing
via injecting proteins that trigger uptake and/or block intracellular killing
What are some pathogenic invasion examples?
The first image is of a transmission electron microscope image of a tissue culture cell line, infected with human immunodeficiency virus (HIV)
HIV particles are 90-120 nm in diameter. The virus attaches via the CD4 molecule on the surface of a lymphocyte, which acts as a surface receptor for HIV.
After fusion of the virus envelope with the membrane, the nucleocapsid, containing the RNA, is carried into cytoplasm by endocytosis.
The second image is of salmonella associated with membrane ruffles on the surface of an epithelial cell.
After initial adherence, this pathogen injects proteins into the host cell (e.g. SipA to staple actin filaments together, rearrange cell and engulf bacterium) to bring about its uptake - a process termed invasion. Looser actin filaments also surround stabilize the vacuole for intracellular survival.
What are some host defences against intracellular survival of pathogens
Ingestion and killing of microbe by a phagocyte
What are some pathogenic strategies of intracellular survival?
STRATEGIES:
Block phagocyte chemotaxis;
Kill phagocyte before or after phagocytosis; inhibit phagocytosis;
Inhibit lysosome fusion; resist killing and multiply in phagocyte
MECHANISMS:
Release leucocidins, antiphagocytic haemolysins etc.
Use of microbial cell wall or capsule components to inhibit phagocytosis
Intracellular microbe inhibits one or more cell components needed for fusion of phagosome with lysosome
Diversion of toxic compounds away from subcellular compartment containing microbe
Can escape from phagosome into cytosol
What is a salmonella intracellular survival example?
Salmonella is a facultative intracellular pathogen that survives and even replicates within macrophages.
Transposons bearing reporter genes can be used to probe the intracellular like of such pathogens.
In this experiment a transposon expressing Green Fluorescent
Protein as a reporter has inserted into a Salmonella gene that is induced when the bacteria enter macrophages.
Thus, the Salmonella cells within macrophages fluoresce (GREEN) external Salmonella (RED) are also visible.
What is a listeria intracellular survival example?
LEFT PANEL:
- Intracellular Listeria monocytogenes (RED) escape from their phagosomes into the cytosol. They are then able to catalyse the polymerisation of host F-actin at one of their poles using their Act surface protein; actin-based motility by “comet tails”. This ‘rocket’ propulsion mechanism pushes the bacterial pathogen towards the cytoplasmic membrane which eventually ruptures allowing their spread to a neighbouring cell. Mimics neutrophil actin rearrangement pseudopodia movement.
RIGHT PANEL:
- Vaccinia virus (RED) also polymerises actin in order to move within a cell
What is the importance of Fe(III) in intracellular survival?
The host restricts Fe(III) to pathogens
The microbe pathogens scavenges iron in competition with the host.
Microbial mechanisms:
Microbe secretes molecules - siderophores - that bind Fe(III) in the host with extremely high affinity
The complexes are imported to the cytosol where the captured iron is released
Iron is essential to majority of life
What do pathogens do to overcome the host defence of production reactive oxygen and nitrogen species to pathogen intracellular survival
Microbe avoids oxidative burst or removes ROS
and RNS
Microbial mechanisms:
Microbe diverts vesicles bearing NADPH oxidase so that they don’t fuse with phagosome;
They produce enzymes (catalse; superoxide dismutase etc.) to inactivate ROS and RNS
How do pathogens combat production of complement and antimocrobial peptides in extracellular survival?
Microbes alter their cell surfaces; inactivate complement; or bind complement non-productively
Microbial mechanisms:
Sialylation of bacterial cell surface or alterations in LPS structure; production of proteases
C3b receptor on microbe competes with that on phagocyte to hinder production of membrane attack complex
How do pathogens combat production of antimocrobial antibodies in extracellular survival?
Microbes can destroy antibody
They can
- Prevent induction of protective antibody
- Express F receptor
- Prevent antibody (or complement) from binding near cell surface
- Avoid immune recognition
Microbial mechanisms:
- Secretion of IA protease
- Infection of lymphoid cells
- Bind antibody so that it is oriented 180° from normal
- Produce long chain LPS to keep antibodies and complement at ‘arms length’
- Acquire coating of host molecules (e.g. fibronectin).
How do pathogens combat antimocrobial cell-mediated response in extracellular survival?
Invasion of T cells to block their function or to kill them; switch on T cells or B cells non-specifically, or non-productively
Microbial mechanisms:
- Virus envelope component binds CD4 on helper
T cell surface
- Polyclonal activation of B cells
- Polyclonal activation of T cells by release of T cell mitogens
How do pathogens combat antimocrobial immune response in extracellular survival?
Infect glands or epithelial surfaces that are relatively inaccessible to circulating antibody or immune cells
Suppress immune responses
Vary microbial antigens either in a single host or during spread in host community
Microbial mechanisms:
- Trophism for cells in glands or on surfaces
- Invade immune tissues
- Switch on different surface antigens
- Use of mutation and/or genetic recombination
Structure of Cholera Toxin (CT):
- The A subunit is enzymatically active and consists of two parts: A1 and A2
- The B subunit is responsible for binding to the host cell receptor and is composed of five identical B subunits
The B subunit pentamer binds specifically to the GM1 ganglioside receptor on the surface of intestinal epithelial cells
Retrograde endocytosis occurs where B subunits are left behind and the A subunits enter the cell
A1 causes a large increase in adenylate cyclase activity
This causes a very large increase in cAMP activity in the cell
This causes massive loss of water and solutes out of the cell, causing diarrhoea
GM1 receptor mechanism
The GM1 receptor, a seven-loop transmembrane receptor, has an associated molecule called Gsα
Normally, Gsα diffuses along the cell surface and binds to adenylate cyclase, suppressing its activity
However, when cholera toxin binds to this complex, it overcomes the suppression
This results in a large increase in cAMP concentration, converting ATP into cyclic AMP.
What are some E. Coli type mechanisms?
Noninflammatory diarrhoea:
- Enterotoxigenic E. coli (ETEC) - attach to small intestinal mucosa and elaborate one or both of heat labile and heat stable toxins
- Enteropathogenic E. coli (EPEC) - attach firmly to intestinal mucosa causing dissolution of brush border by inducing vesiculation of microvilli: attaching-effacement.
Inflammatory diarrhoea:
- Enteroinvasive E. coli (EIEC) - attach to colonic enterocytes, penetrate by an endocytotic mechanism and replicate therein. Causes necrosis and stripping of large areas of colonic mucosa and a dysentery.
- Enteroaggregative E. coli (EAggEC) - damage and blunt colonic villi by haemorrhagic necrosis, precise pathogenic mechanisms unclear.
- Enterohaemorrhagic E. coli (EHEC) - produce attaching-effacement to terminal ileal and colonic mucosa; release shiga-like toxins 1 or 2 which kill colonic enterocytes and produce haemorrhagic colitis; but diarrhea maybe caused by bacterial invasion of host cells and interference with normal cellular signal transduction, rather than toxin production.
Key features of Enterohaemorrhagic E. Coli
Major food-borne infectious pathogen
Causes diarrhoea, haemorrhagic colitis, haemolytic uraemic syndrome
Destruction of red blood cells (hemolytic anemia), destruction of platelets (responsible for clotting; low counts = thrombocytopenia), and acute renal failure.
Most common cause of acute renal failure in children
Significant health risk due to disease severity, lack of effective treatment and the potential for large-scale outbreaks from contaminated food
950 reported cases in UK in 2005 (36% increase on previous year); USA estimates: ~75,000 p.a.
Typically caused by E. coli 0157:H7 serovar
What are pathogenicity islands?
Genetic studies show EHEC has a >30 kb cluster of pathogenicity genes not present in E. coli
K-12
Such clusters, termed Pathogenicity Islands, are common in disease-causing bacteria.
Pathogenicity islands are acquired by horizontal gene transfer from other bacteria and typically have a C + G content that differs from the rest of the genomic DNA
What is Locus of Enterocyte Effacement (LEE)
Major pathogenicity island in EHEC is termed Locus of Enterocyte Effacement (LEE)
LEE encodes proteins that form a specialized T3SS secretion system that injects EHEC proteins such as Tir (translocated intimin receptor) into the host cell as well as other the injected proteins e.g. EspD facilitates host pore formation.
HIV Structure?
HIV retrovirus (lentivirus family):
Contains RNA as genetic material.
HIV genome contains 2 molecules of single-stranded RNA, each bound by a molecule of reverse transcriptase.
Within genome are also a p10 protease and a p32 integrase.
Genome is surrounded by nucleocapsid consisting of inner layer of protein p24 and outer layer of protein p17 (both part of Gag (group specific antigen) polyprotein complex).
Outer portion of virus consists of lipid envelope derived from host cell membrane
Contains two viral proteins, gp120 and gp41, which collectively are called viral envelope proteins.
HIV genome structure
LTR-long terminal repeats; repetitive sequence of bases
gag-group specific antigen gene, encodes viral
nucleopcapsid proteins: p24, a nucleoid shell protein, MW=24000; several internal proteins, p7, p15, p17 and p55.
pol-polymerase gene; encodes the viral enzyme, protease (p10), reverse transcriptase (p66/55; alpha and beta subunits) and integrase (p32).
env-envelope gene; encodes the viral envelope glyocproteins g120 (extracellular glycoprotein, MW=120 000) and gp41 (transmembrane glycoprotein, MW=41000).
tat: encodes transactivator protein
rev: encodes a regulator of expression of viral protein
vif: associated with viral infectivity
vpu: encodes viral protein U
vpr: encode viral protein R
nef: encodes a ‘so-called’ negative regulator protein
HIV Particle structure
HIV is an enveloped retrovirus and has two single-stranded RNA molecules as its genetic material
The RNA is associated with reverse transcriptase, integrase and polymerase enzymes, which are necessary for viral DNA and RNA synthesis
Surrounding this is the nucleocapsid, which consists of an inner layer of p24 protein and an outer layer of p17 protein
The outer portion of the virus consists of a lipid layer derived from the host cell into which is inserted the viral gp41 envelope protein
Each gp41 protein molecule is associated with a molecule of the gp120 envelope protein.
Life cycle of HIV?
1) Binds to CD4 +ve cells through gp120 to CD4; interactions between virus and chemokine co-receptors.
2) Nucleocapsid enters the cell, unfolds, releasing viral RNA, which is reverse-transcribed to DS DNA.
3) The viral DNA integrates in the host genome, where it lies dormant as a provirus.
4). Following cell activation, viral DNA directs the transcription of viral RNA.
5) Viral proteins are translated from the RNA.
6) Viral proteins and single-stranded viral RNA assemble to form new viral particles.
7) Virus buds from the cell, picking up some of cell membrane, complete viral particles can infect other cells.
HIV infected dendritic cell mechanism
An HIV-infected dendritic cell shoots out thin projections called filopodia (red) with virus particles (white) at their ends. Actin filament rearrangement projects filopodia in a waving arc towards T-cells at um/sec, faciltating 800 dendtritic cell - T-cells interactions/hour.
What are the 4 main ways of CD4 T cell killing?
(a) HIV may directly cause lysis of CD4 T cells.
(b), (c) Infected cells bearing HIV antigens may be killed by antibody and complement (b) or killed by antibody-dependent cell-mediated cytoxicity (c).
(d) HIV-infected cells presenting HIV peptides on their class I MHC molecules may be killed by CD8
T cells
CD4 T cell death overview
