Influenza Virus - Zentrum für Molekulare Biologie

MICROBIAL AND VIRAL DESEASES
Virulence Factors
The following are types of virulence factors:
Adherence Factors: Many pathogenic bacteria colonize mucosal sites by
using pili (fimbriae) to adhere to cells.
Invasion Factors: Surface components that allow the bacterium to invade
host cells can be encoded on plasmids, but more often
are on the chromosome.
Capsules:
Many bacteria are surrounded by capsules that protect
them from opsonization and phagocytosis.
Endotoxins:
The lipopolysaccharide endotoxins on Gram-negative
bacteria cause fever, changes in blood pressure,
inflammation, lethal shock, and many other toxic
events.
Exotoxins:
Exotoxins include several types of protein toxins and
enzymes produced and/or secreted from pathogenic
bacteria. Major categories include cytotoxins,
neurotoxins, and enterotoxins.
Siderophores:
Siderophores are iron-binding factors that allow some
bacteria to compete with the host for iron, which is
bound to hemoglobin, transferrin, and lactoferrin.
Gene Transfer to Acquire a Selective Advantage
Person to Person Transmission
Airborne pathogens
Diseases Caused by Streptococcous pyogenes
Disease:
Entry
Exit
Meningitis
Spread
Otitis
Sinusitis
Tonsilitis and
Pharyngitis
Adenitits
Pneumonia
Endocarditis
Skin:
Impetigo
Erysipelas
Scarlet Fever
Puerperal Fever
Myositis
Fasciitis
Sequelae (nonsuppurative)
Rheumatic Fever
Glomerulonephritis
Uterus
Reemergence of Severe Streptococcal Disease?
The existence of highly virulent “Flesh eating bacteria”
•
Invasive streptococcal disease have been life-threatening diseases for
many centuries.
•
A marked attenuation in their incidence and severity became apparent
by the late 1800s.
•
The mortality rate described for severe GAS disease at the beginning
of the 1900s century was with 5-10% significantly lower than the 30-60%
seen today.
•
The recent emergence of severe GAS disease despite modern medicine
and antibiotics indicate an enhanced virulence of the organism.
Significance of the Pathogen
•
Most cases of severe GAS disease are caused by M types 1, 3, 12 and 28
•
To date an estimated incidence of 1-5 cases / 100.000 of severe GAS
disease in the US and Europe
•
WHO estimates (1998) that 12 million people in developing countries will
be affected during the coming decade by rheumatic fever/rheumatic
heart disease
400.000 deaths are expected annually
2.000.000 will require repeated hospitalization
1.000.000 will require heart surgery
•
Estimated annual direct health costs in the US only for pharyngitis
exceed $1 billion
Mode of Action of Superantigens
T cell
α
V
β
J
J
Most SAGs share a common MHC class II binding
site facilitating the TCR-SAG MHC class II complex
D
V
SAG
α
Ag
Binding of SAGs to an extracellular domain of a TCR
creates a signal that initiates a massive cytokine
release
β
MHC
Antigen
Presenting
Cell
Direct cytotoxicity
Interference with endotoxin clearance
Interference by TNFα with PMN mobilization
Hypersensitivity
Pathogenic Activity of Superantigen Toxins
SPEs
T cell
Liver Damage
APC
Decreased Endotoxin
Clearance
T cell Activation
and Proliferation
Endotoxin
Cytokine Release
Capillary Leak
and Hypotension
Septic Shock
Streptococcal Toxic Shock Syndrome (STSS)
Defined as any group A streptococcal infection associated
with the early onset of shock and organ failure
Necrotizing Fasciitis
Necrosis of the soft tissues with involvement of the fascia
plus serious systemic diseases including shock, DIC,
failure of organ systems and death
Autoimmunity – Rheumatic Fever
Gross pathology of rheumatic fever involving mitral valve
Meningococcal Septicemia
4 month old female with gangrene of hands and lower extremities
due to meningococcemia.
Bordetella pertussis-Whooping Cough
In the United States, there has been an increase in the number of annual cases of whooping cough.
From an average of less than 2000 pertussis cases per year in the 1970s, the number of cases has
now risen to over 8,000 per year. Inadequately immunized children are at high risk for acquiring pertussis.
World-wide mortality rate: 350,000
Mycobacterium tuberculosis : Tuberculosis: 1.6 Mill.
Deaths/year
Mycobacterium leprae : Leprosis
Tuberculosis is one of the most prevalent and dangerous single
diseases in the world. Its incidence is on the increase in developed
countries, in part because of the emergence of drug-resistant strains.
The pathology of tuberculosis and leprosy is influenced by the
cellular immune response.
M. tuberculosis survives in macrophages;
Aggregation of macrophages: tubercles
Drug:
Airborne viral infections
Viruskrankheiten - Influenza Virus
Influenza: RNA Orthomyxovirus
Schematische Darstellung des Influenza Virus
• Influenza Viren A oder B verursachen Grippe Epidemien, Gruppe C
Viren nur eine milde Erkrankung
• Die Grippeimpfung schützt vor Infektion mit A und B Viren
• Tamiflu inhibiert das Protein Neuraminidase; dadurch wird das
Freisetzen der Grippeviren und damit die Vermehrung verhindert; das
Immunsystem kann daraufhin die Infektion erfolgreich bekämpfen
•Infektion mit 2 versch. Virusstämmen führt durch Neuverteilung der
RNA Segmente zu einem neuen Stamm
Influenza Type A, B, and C
Influenza types A or B viruses cause epidemics of disease almost every
winter.
In the United States, these winter influenza epidemics can cause illness
in 10% to 20% of people and are associated with an average of 20,000
deaths and 114,000 hospitalizations per year.
Getting a flu shot can prevent illness from types A and B influenza.
Influenza type C infections cause a mild respiratory illness and are not
thought to cause epidemics.
Influenza type A viruses are divided into subtypes based on two
proteins two on the surface of the virus. These proteins are called
hemagglutinin (H) and neuraminidase (N). The current subtypes of
influenza A viruses found in people are A(H1N1) and A(H3N2).
Influenza B virus is not divided into subtypes. Influenza A(H1N1),
A(H3N2), and influenza B strains are included in each year’s influenza
vaccine.
Antigenic Shift and Drift
- Drift: Mutations These are small changes in the virus that happen continually over time.
Antigenic drift produces new virus strains that may not be recognized by
the body’s immune system.
This process works as follows: a person infected with a particular flu virus
strain develops antibody against that virus. As newer virus strains appear,
the antibodies against the older strains no longer recognize the "newer"
virus, and reinfection can occur.
This is one of the main reasons why people can get the flu more than one
time. In most years, one or two of the three virus strains in the influenza
vaccine are updated to keep up with the changes in the circulating flu
viruses.
Antigenic Shift and Drift
- Shift: Reasortment of RNA segments Antigenic shift is an abrupt, major change in the influenza A viruses,
resulting in new hemagglutinin and/or new hemagglutinin and
neuraminidase proteins in influenza viruses that infect humans.
Shift results in a new influenza A subtype.
When a shift happens, most people have little or no protection against
the new virus.
While influenza viruses are changing by antigenic drift all the time,
antigenic shift happens only occasionally.
Type A viruses undergo both kinds of changes; influenza type B viruses
change only by the more gradual process of antigenic drift.
Spanish Flu 1918/19
1918-19, “Spanish flu,” [influenza A(H1N1)], caused the highest number of
known flu deaths: more than 500,000 people died in the United States, and
20-50 million people may have died worldwide.
The flu virus that caused it was very deadly. Many died within the first few
days after infection and others died of complications soon thereafter.
The Spanish flu was unique because almost half of the people who died
were young, healthy adults.
Asian Flu 1957/58
“Asian flu,” [influenza A(H2N2)], caused approximately 70,000 deaths in the
United States.
The Asian flu was first identified in late February, 1957 in China and spread to
the United States by June, 1957.
Hong Kong Flu 1968/69
“Hong Kong flu,” [influenza A(H3N2)], caused approximately 34,000 deaths
in the United States.
This pandemic H3N2 virus was first detected in Hong Kong in early 1968
and spread to the United States later that year. A(H3N2) viruses still
circulate today.
Influenza in Animals
Influenza A viruses are found in many different animals, including birds,
pigs, ducks, whales, horses, and seals.
Wild birds are the reservoir for all subtypes of influenza A viruses.
Wild birds usually do not become ill from the influenza virus. However,
birds that are not wild, such as chickens and domesticated turkeys, can
get very sick if they get infected with certain influenza A viruses.
Pigs in the United States have been infected with influenza A (H1N1),
A(H1N2), and A (H3N2) viruses. Infected pigs get symptoms similar to
humans, such as cough, fever, and runny nose.
Are New Outbreaks possible?
The Avian Flu Outbreak
“Avian flu” outbreak, [influenza A(H5N1)].
In 1997, 18 people in Hong Kong were hospitalized because of infection
with a new type of virus that was previously seen only in birds.
Six of those people died, and officials in Hong Kong ordered the slaughter
of all chickens in the area, as chickens were widely infected by the virus.
Studies found that this H5N1 flu spread from poultry to people but not
easily from person to person.
DIRECT CONTACT
TRANSMISSION OF
DISEASES
Staphylococcus aureus: proteolytic enzymes, hemolysin, leucocidin,
toxic shock toxin
Helicobacter pylori
HBV
HEPATITIS VRUSES:
HIV
Loss of CD4 cells during HIV infection
ANIMAL-TRANSMITTED DISEASES
Rabies: Rhabdovirus: Treatment with anti-Rabies serum from
(salvia)
survivor
Hantavirus Pulmonary Syndrome: Fatality 50%: No treatment
(Rodent excreta)
Rickettsial deseases: Rocky Mountain spotted fever
(head louse)
Typhus
Lyme disease: Borrelia burkdorferi
(ticks)
Plaque: Yersinia pestis
West Nil Virus /Enzephalitis
Lyme disease
Yersinia pestis
Bubonic plaque can be treated
Pneumonic plaque is fatal
West-Nil-Virus
Das West-Nil-Virus ist ein seit 1937 bekanntes, behülltes Einzel(+)-Strang-RNA-Virus der
Flaviviridae-Familie, das sowohl in tropischen als auch in gemäßigten Gebieten vorkommt.
Das Virus infiziert hauptsächlich Vögel, kann aber auch auf Menschen, Pferde und andere
Säugetiere übergreifen.
Übertragung: Das Virus wird durch Stechmücken übertragen, in Nordamerika z.B. die gemeine kleine Hausmücke
(Culex pipiens) und ein Vogel und Menschen mögender Hybrid aus Culex Pipiens und Culex molestus.
Die Anfälligkeit für das West-Nil-Virus wird durch eine Mutation des Gens CCR5 (genannt CCR5Δ32) massiv begünstigt.
Die Deletion von 32 Basenpaaren bewirkt auch eine Resistenz gegen das an gewöhnliche CCR5-Rezeptoren andockende HIV.
Die Mutation stellt daher seit Entdeckung 1996 die Grundlage für die Entwicklung neuer CCR5-Inhibitoren im Kampf
gegen AIDS dar.
Symptome: Grippe-ähnliche Symptome, bekannt als West-Nil-Fieber. Das Virus ist in der Lage die
Blut-Hirn-Schranke zu passieren und kann dadurch eine lebensbedrohliche Enzephalitis oder Meningitis auslösen
(0,7% der Fälle).
.
Geschichte: Einer These folgend soll schon Alexander der Große vom West-Nil-Virus dahingerafft worden sein,
da Plutarch berichtete, dass vor Alexanders Ableben Raben tot vom Himmel fielen. Mit dem ersten Auftreten des
West-Nil-Virus in Nordamerika 1999 rückte die Thematik in das mediale Rampenlicht. In den USA begann der
Virusausbruch im Gebiet von New York City. Es gibt eindeutige Hinweise dafür, dass das Virus von einer
infizierten Mücke aus einem israelischen Flugzeug der Linie Tel Aviv - New York eingeschleppt wurde.
Die ersten Anzeichen waren Vögel, die tot von den Bäumen des Central Parks fielen. Bald darauf wurden
ältere Menschen in der Gegend infiziert und erkrankten. Mittlerweile ist das Virus in ganz USA verbreitet.
Statistik: In den USA sind von 1999 bis 2001 149 Infektionen mit 18 Todesfällen dokumentiert.
Im Jahr 2002 stieg diese Zahl auf 4156 Infektionen und 284 Tote; 2003 9.858 Infektionen und 262 Todesfälle.,,,,,,,,,,,,,
1999
2002
2006
Waterborne Microbial Diseases
Vibrio cholera
Legionellosis
Legionella pneumophila
Legionellosis
Was first described in 1976 – occured during a meeting of the
American legion.
Gram-negative organism called Legionella pneumophila
L. pneumophila is an intracellular organism and muliplies within
alveolar macrophages
Clinical picture:
• incubation time 2-10 days
• flu-like symptoms, severe pneumonia
Prognosis:
Without treatment 80%, otherwise 5-10%
Foodborne Microbial Diseases
Enteropathogenic E. coli (EPEC) and
Enterohemorrhagic E. coli (EHEC, E. coli O157:H7)
EHEC or E. coli O157:H7 is an emerging cause of diarrhea in the developed
world.
In the United States, 10 000 to 20 000 cases are reported yearly.
It has been found in ground beef, unpasteurized milk, bottled juices and
sewage contaminated water.
EHEC causes a bloody diarrhea which can lead to kidney failure especially
in young children and the elderly.
Pathogenesis of EHEC is similar to EPEC but EHEC also secretes a
verotoxin thus causing a more serious disease.
Enteropathogenic E. coli (EPEC)
EPEC is a major cause of neonatal diarrhea in developing countries,
killing close to 1 million children each year due to dehydration,
malnutrition and other complications of the disease.
In North America and Europe there have been occasional nursery
school outbreaks of diarrhea attributed to EPEC.
Pathogenesis of EPEC requires intimate attachment of the bacteria to
cells in the host's small intestine.
Bioterrorism: Bacillus anthracis
The anthrax bacillus, Bacillus anthracis, was the first bacterium shown to
be the cause of a disease.
In 1877, Robert Koch grew the organism in pure culture, demonstrated its
ability to form endospores, and produced experimental anthrax by
injecting it into animals.
Bacillus anthracis. Gram stain. The
cells have characteristic squared
ends.
The endospores are ellipsoidal
shaped and located centrally in the
sporangium.
The spores are highly refractile to
light and resistant to staining.
Anthrax and Human Disease
In humans, anthrax is fairly rare; the risk of infection is about 1/100,000. The most
common form of the disease in humans is cutaneous anthrax, which is usually
acquired via injured skin or mucous membranes. In severe cases, where the blood
stream is eventually invaded, the disease is frequently fatal.
Another form of the disease, inhalation anthrax (woolsorters' disease), results most
commonly from inhalation of spore-containing dust where animal hair or hides are
being handled. The disease begins abruptly with high fever and chest pain. It
progresses rapidly to a systemic hemorrhagic pathology and is often fatal if
treatment cannot stop the invasive aspect of the infection.
Gastrointestinal anthrax is analogous to cutaneous anthrax but occurs on the
intestinal mucosa. Intestinal anthrax results from the ingestion of poorly cooked
meat from infected animals. Intestinal anthrax, although extremely rare in developed
countries, has an extremely high mortality rate.
Meningitis due to B. anthracis is a very rare complication that may result from a
primary infection elsewhere.
Bacillus anthracis Infection of the Skin
Pulmonary Form of
Bacillus anthracis
Lethal toxin (LT) is a major virulence factor secreted
by anthrax bacteria. It is composed of two proteins,
PA (protective antigen) and LF (lethal factor). PA
transports the LF inside the cell, where LF, a zincdependent metalloprotease cleaves the mitogen
activated protein kinase kinase (MAPKK) enzymes of
the mitogen activated protein kinase (MAPK)
signaling pathway, thereby impairing their function.
This disruption of the MAPK pathway, which serves
essential functions such as proliferation, survival
and inflammation in all cell types, results in
multisystem dysfunction in the host. The inactivation
of the MAPK pathway in both macrophages and
dendritic cells leads to inhibition of proinflammatory
cytokine secretion, downregulation of costimulatory
molecules such as CD80 and CD86, and ineffective T
cell priming. The net result is an impaired innate and
adaptive immune response. Endothelial cells of the
vascular system undergo apoptosis upon LT
exposure, also likely due to inactivation of the MAPK
pathway. The activity of various hormone receptors
such as glucocorticoids, progesterone and estrogen
is also blocked, due to inhibition of p38 MAPK
phosphorylation, thus affecting the body's response
to stress.
Figure 3. A ribbon repre
The Anthrax Toxin
Hospital
Aquired
Infections
Sepsis
Septic shock is the most common cause of mortality in the intensive
care unit.
It is the 12th leading cause of death overall (1997) and is the most
common cause of shock encountered by internists in the U.S.
Despite aggressive treatment mortality ranges from 16% in patients
with sepsis to 40-60% in patients with septic shock.
There is a continuum of clinical manifestations from SIRS to sepsis to
severe sepsis to septic shock to Multiple Organ Dysfunction Syndrome
(MODS).
Role of Hfq and small RNAs in
P. aeruginosa virulence gene expression
Pseudomonas aeruginosa is an opportunistic pathogen. It causes urinary
tract infections, respiratory system infections, dermatitis, soft tissue
infections, bacteremia, bone and joint infections, gastrointestinal
infections…… PAO1 isolates from hospital settings are multi-resistant
towards commonly used antibiotics. PAO1 is a serious problem in IC
units and patients hospitalized with cancer, cystic fibrosis, and burns.
The case fatality rate in these patients is > 50 percent.
-
QscR
3O-C12-HSL
Hfq
LasR
+
GacA/GacS
+
+
LasI
+
+
+
RsmY RNA
-
+
+
+
+
-
+
lasB
lasA
toxA
apr
...
+
-
LasR
RsmA
RhlR
PQS
(-)
+
C4-HSL
+
+
+
RhlR
+
RhlI
hcn
(-)
-
Neue Antibiotika
Unveränderter Text aus ZCT Heft 6, 2001 (ZCT 2001; 22: 42-44)
Linezolid – erstes Antiinfektivum aus der Klasse der Oxazolidinone
Oxazolidinone sind eine neue Klasse synthetischer Wirkstoffe zur Therapie bakterieller Infektionen. Das erste in der Humanmedizin
verwendbare Oxazolidinon ist Linezolid, das im April 2000 in den USA zugelassen wurde und das nun auch in Europa unter dem
Namen ZYVOXID zur Verfügung steht.
Antibakterielle Aktivität
Linezolid entfaltet seine antibakterielle Wirkung durch Hemmung der bakteriellen Proteinsynthese.1 Es bindet – ähnlich wie
Chloramphenicol – an die 50S-Untereinheit der bakteriellen Ribosomen, verhindert die Bildung eines funktionstüchtigen
Initiationskomplexes und interferiert so offenbar mit einem frühen Schritt der Proteinbiosynthese. Im Gegensatz zu Chloramphenicol
wirken Oxazolidinone offenbar nicht durch Hemmung der Peptidyltransferase
Vaccination: surface antigens-inhibition of functional surface proteins
Bacterial virulence and attenuation
Salmonella typhimurium
Identify surface antigen
Specific antibodies directed
Against a surface antigen
Identify antigen
Clone gene
Overproduce protein
Vaccination
Protection?
Specific inhibitor of growth
Accute treatment possible
DNA vaccines
New concepts of antigen delivery
Phage and Phage-therapy: The Discoverers
Felix d´Herelle (1873-1949)
Frederick Twort (1877 – 1950)
„Genetically modified phage and phage-derived proteins
as antimicrobial agents“
Merril et al., 2003; Nature Rev. Drug Discovery
Westwater et al. (2003)
Hagens et al, (2003/2004)
genetically
engineered phage
New chances and limits
Phage-induced lysis: λ
Rz:
R:
S:
Endopeptidase (?)
17500 Mr soluble Transglycosylase
holin
Avoidance of side effects of phage-induced bacterial
lysis such as septic shock through the use of genetically
modified phage (...is likewise a problem of antibiotics
that compromise the cell envelope)
> Manipulation of the phage lysis operon
- endolysin / + optimized holin
Potential problem: Propagation of genetically modified phage!
Effective phage-killing of the target pathogen without
simultaneous release of exo- and
endotoxins
> Equip phage with a `kil-function´ which does not
compromise the membrane
Restriction endonuclease
BglIIR cleaves non-methylated
DNA ⇒ irrepairable break
BglIIR
DNA
Phage propagation
BglIIM
Phage therapy of Pseudomonas
aeruginosa infections
• Opportunistic pathogen causing a
variety of diseases
• Common cause of hospital
aquired infections
• Notoriously resistant to antibiotics
• Pf3: filamentous phage of P. aeruginosa
I. Genetically-modified phage encoding a lethal
protein, the action of which is silenced at the post
-translational level
Construction of a nonlytic, nonreplicating Pf3 phage encoding
BglIIRand its propagation in P. aeruginosa O1(pUM430)
Rescue of mice after infection with 5xMLD
100
90
80
70
60
50 % Survival
40
30
20
time in hours
0
144
96
48
0
10
neg
n = 20
Pt1
Pf3R
5x108 (60 min)
II. Phage derived-proteins with bactericidal activity
IIa. Phage encoded
proteins
Phage structural
proteins
Use of phage endolysins: Enzymbiotics
(Fischetti; Rockefeller University)
S.aureus phage φ68
Lytic activity of phage structural proteins
identified by Zymography
Separate phage proteins by SDS page
in the presence of S. aureus cells
electroelution
concentration
of
eluted sample
Methylen-blue
Lysostaphin
Mass spectrometry
Genome organization of S.aureus bacteriophage f68
Minor structural protein
Clone gene, purify protein, test for lytic activity,
find „lytic domain“;
Protein 17 displays antimicrobial activity on
clinical S. aureus isolates
0
(□) 5 pg/ml
(■) 50 ng/ml
(○) 50 µg/ml
(▲)