Influenza Virus - Zentrum für Molekulare Biologie
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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 (▲)
