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Bacterial infections
This document discusses bacterial infections and modern molecular biology techniques used to study and combat pathogenic bacteria. It describes how bacteria cause infections through various virulence factors, including adhesins that help bacteria attach to host cells and invasins that facilitate bacterial entry. New diagnostic techniques like PCR and checkerboard hybridization that identify bacteria by their rRNA sequences are faster than traditional culturing methods. The document also examines how bacterial pathogens acquire iron, an essential nutrient, through siderophores and discusses the mobile genetic elements that allow bacteria to gain virulence properties.
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Bacterial infections
- 1. Bacterial Infections Amith Reddy EasternNew Mexico University
- 2. Infections in mankind Infectionsin all manner of living organisms are caused by all sorts of microorganisms ◦ ◦ ◦ ◦ Bacteria Viruses Single-celled eukaryotes Etc.
- 5. Using modern molecularbiology to combat infection Molecular mechanisms for invading pathogens best understood for pathogenic bacteria ◦ Especially those related to E. coli Bacterial methods are the easiest to understand ◦ Viruses interact with host cell genome ◦ Single-celled eukaryotic infections are the most difficult to understand
- 6. Molecular approaches to diagnosis Identificationof pathogenic bacteria is often difficult ◦ Bacteria may grow slowly, or not at all outside host cells Instead of culturing the bacteria, new techniques in nucleic acid technology are being used.
- 7. ssu rRNA Small subunitribosomal RNA ◦ Each species is different ◦ Bacteria have 16S rRNA ◦ Eukaryotes have 18S rRNA ◦ Diagnosing pathogenic bacteria by ribosomal RNA sequences is faster than culturing techniques
- 8. Ribotyping Detailed restriction analysisof rRNA genes DNA from a strain is digested with several different restriction enzymes Fragments separated by gel elctrophoresis Fragments then submitted to Southern Blot test A probe that recognizes part of the 16S rRNA sequence is used. Uses large amounts of DNA
- 9. PCR Uses small amountsof DNA Primers that recognize the conserved region of 16S rRNA The fragment is compared to a database of known organisms Works well with bacteria that cannot be cultured well.
- 10. Checkerboard Hybirdization Allows multiplebacteria to be detected and identified in one sample Probes are applied in horizontal lines across a hybridization membrane ◦ The probes correspond to different bacterial species 16S genes are amplified by PCR ◦ Fragments are labeled with a fluorescent dye, and added vertically to the membrane ◦ After hybridization, the membrane is washed to remove unbound DNA and the hybridized samples appear as bright dots
- 11. FIGURE 21.1 Checkerboard Hybridization Probescorresponding to 16S rRNA for each candidate bacterium are attached to a membrane filter in long horizontal stripes (one candidate per stripe). To quickly identify a group of unknown pathogens, mixed DNA is extracted from a sample and amplified by PCR using primers for 16S rRNA. The PCR fragments are tagged with a fluorescent dye and applied in vertical stripes. Each sample is thus exposed to each probe. Wherever a 16S PCR fragment matches a 16S probe, the two bind, forming a strong fluorescent signal where the two stripes intersect. Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved. 11
- 12. Virulence segments Virulence factorsare properties that allow microorganisms cause infections. ◦ Virulence factors can be broken down into three groups Those required for invasion of the host Those required for life inside the host Those for aggression against the host
- 13. Mobile virulence segments Insome cases, the DNA that encodes for virulence factors are borne by virulence plasmids Some are carried by lysogenic bacteriophages that are inserted into the bacterial chromosomes of some strains Pathenogenicity islands ◦ DNA segments are grouped together and flanked by repeats May move as a unit by transposition
- 14. FIGURE 21.2 Pathogenicity Islandsof Escherichia coli Different strains of E. coli vary greatly in their abilities to cause disease. Pathogenic E. coli have unique regions of DNA that are not found in nonpathogenic strains, called pathogenicity islands (PAI). The regions are designated I–IV, where I encodes alpha-hemolysin; II encodes alpha-hemolysin and fimbriae; III encodes fimbriae; and IV encodes the yersiniabactin iron-chelating system. Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved. 14
- 15. Implications of mobility Closelyrelated bacterial strains are very different in their ability to cause disease. Virulence factors can be transferred to harmless bacteria, creating novel pathogens If the harmless strain is a very close relative, we get a new variant of the old disease If it isn’t, we run the possibility of having a genuinely new pathogen that does not act like the old disease. ◦ Yersinia pestis
- 17. Attachment and entry Attachmentis the first step in many infections There are two type of adhesions : fimbrial and nonfimbrial ◦ Pili are thin filaments from the membrane that incorporate adhesions at the tip ◦ Nonfimbrial adshesions are found on the bacterial cell surface.
- 18. FIGURE 21.3 Bacterial Adhesins (A)The surface of some bacterial cells is covered with pili (fimbriae), composed of helically arranged pilin protein. At the tip of the pili are adhesins, which recognize the surface glycoproteins of the host cell. (B) Nonfimbrial adhesins are found on the surface of the bacterial cell. Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved. 18
- 19. FIGURE 21.4 Assembly ofBacterial Pilus The pilus has two segments, the tip and the shaft, which are assembled on the outside of the bacterium. The protein subunits of the pilus are synthesized in the cytoplasm and exported across both membranes. The proteins are folded in the periplasmic space. The pilus is assembled from the tip to the base by starting with the adhesin protein and other tip proteins and then adding further layers of pilin protein beneath. Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved. 19
- 20. The second step: Invasins Not all bacteria have the ability to enter the host cell ◦ Some only attach to the outside ◦ Some cells (such as phagocytic cells) absorb the bacterium but then fail to destroy the bacterium. ◦ Some bacteria utilize invasins, which induce the host cell into eating them.
- 21.
- 22. Turning the tableson bacteria With the spread of antibacterial resistance, scientists are considering alternative approaches One of these alternatives is to design antiadhesin drugs that will bind to the adhesin and block attachment. ◦ Through binding studies and X-ray crystallography, it has been revealed that pathogenic E. coli adhesins (FimH) bind to mannose residues on mammalian glcoproteins ◦ May be blocked by different alkyl- and aryl-mannose derivatives
- 23. Decoys Another approach wouldbe to use genetically engineered gut bacteria. ◦ Such as nonpathogenic E. coli. These bacteria would express target oligosaccharides for adhesins on their cell surfaces, acting as decoys. Avoid the need of expensive sugar derivatives One decoy could carry multiple adhesin targets.
- 24. Inducing non harmfulcompetition The third possibility may be to equip nonpathogenic strains with genes for adhesins and/or invasins from pathogenic species These engineered strains would then compete for receptor sites By taking away sites from pathogenic bacteria, the effect of these pathogenic bacteria may be lessened. These engineered cells could also be used for delivering protein pharmacueticals or segments of DNA for gene therapy All alternatives are currently in experimental stages.
- 25. Iron acquisition Almost allbacteria need iron ◦ Iron serves as a cofactor for many enzymes Especially for respiration Free iron in the body is kept low due to specialized proteins that tightly bind to it ◦ Surplus iron is bound by transferrin and lactoferrin, two iron transport molecules ◦ Ferritin, an iron storage protein
- 26. Siderophores Siderophores are ironchelators that are excreted by bacteria, bind iron, and return to the bacteria cell by specialized transport systems The best known siderophore is Enterochelin (enterobactin). ◦ It is made by E. coli and other enteric bacteria ◦ The FEP transport system transporrts the enterochelin and FE complex back across the membrane ◦ Enterochelin bind iron so tightly, it must be destroyed by Fes protein ◦ Enterochelin is not strong enough to unbind Fe from transferrin
- 27. FIGURE 21.5 Acquisition andUptake of Iron by Enterochelin FepA protein is the outer membrane receptor for enterochelin. Energy for crossing the outer membrane requires the TonB system, which uses the proton motive force. The FepB protein gets enterochelin from FepA and passes it to the inner membrane permease, consisting of FepG and FepD. The FepC protein uses ATP to supply energy to FepGD for transport across the inner membrane. Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved. 27
- 28. Pathogenic bacteria Pathogenic bacteriaoften possess more potent siderophores that can retrieve iron from transferrin. Two examples are mycobactin and yersinabactin Yersiniabactin is widespread in the enteric family, and part of the pathogenicity island in Yersinia Other bacteria utilize hemolysin, which lyses the red blood cells and frees the hemoglobin (where the iron resides)
- 29. UNN 21.1 Biotechnology byClark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved. 29
- 30. Bacterial toxins Bacteria willmount aggressive attacks against eukaryotic cells by utilizing toxins. Toxins ◦ In the broadest sense, anything that damages eukaryotic cells. ◦ Can be accidental or deliberate
- 31. Endotoxins Endotoxins are actuallythe lipid components of lipopolysaccharides ◦ LPS forms part of the outer membrane of gram negative bacteria. ◦ If bacteria are killed, they released LPS ◦ Immune cells attach to LPS by CD14 receptor, ◦ Triggers the release of cytokines ◦ Simultaneous death of massive amounts of bacteria may result in sepsis.
- 32. Exotoxins Most pathogenic bacteriahave toxins that deliberately harm the host. Secreted by living cells Mostly exotoxins are proteins.
- 33. Type I Exotoxin Donot enter the cell Bind to a receptor on the cell surface Stable ( heat stable toxin a) is made by some strains of e.coli. ◦ Causes overproduction of cyclic GMP
- 34. Type II Exotoxin Acton the cell membrane of the target cell Some degrade the membrane lipids themselves or create holes in the membrane Hemolysin A disrupts the membrane of many types of animal cells.
- 35. Type III Exotoxin Entera target cell Consist of two factors ◦ Toxic protein ◦ Delivery protein ◦ Several interesting examples
- 36. ADP-Ribosylating toxins Large familyof toxins that hydrolyzes the cofactor NAD and ADP-ribose The fragments are transferred to an acceptor molecule (usually one that binds GTP) The target becomes locked in a binding formation, leacing it unable to continue in its normal processes. Both cholera and diphtheria toxins use ADPribosylation, but on different targets ◦ Cholera toxins inactivate the G-proteins that control adenylate cyclase ◦ Diphtheria toxins attack elongation factor EF-2, a translation factor used for protein synthesis
- 37. FIGURE 21.6 ADP-Ribosylating Toxins Nicotinamideadenine dinucleotide (NAD) consists of ADP-ribose linked to nicotinamide. These are split by some bacterial toxins and the ADP-ribose is attached to a GTP-binding protein, thus preventing it from splitting GTP. Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved. 37
- 38. Bacteriophages Certain other bacteriophagescan use enzymes that utilize NAD and ADPribosylate proteins of their hosts Usually, it is several bacterial proteins that are modified so that the target of the protein is uncertain ◦ Blocking key enzymes can cripple host metabolism ◦ Modification of host polymerases Bacteriophage T4, which modifies host E.coli polymerases, which then loses its ability to transcribe E.coli genes but not T4 genes.
- 39. Cholera Vibrio cholerae doesnot enter host tissues ◦ Attaches to the exterior wall of cells lining small intestine The bacterium severely damages the host tissue by excreting cholera toxin The toxin attacks the epithelial cells, causing them to lose sodium ions and water into the intestinal tract Cholera causes loss of body fluids by massive diarrhea and then death by dehydration
- 40. Virulence proteins ofVibrio cholerae Virulence proteins not only include the cholera toxin, but also pilis and cell-surface adhesins The genes for the toxin are carried by a bacteriophage (CTXphi) that lysogenizes cholera bacterium Synthesis of the virulance factors is partially regulated by the ToxR protein in the wall of the inner membrane of the bacteium. ◦ This protein ‘senses’ the correct environment and activates the genes ◦ The internal domain of the protein binds to the promoters of the virulence genes
- 41. FIGURE 21.7 Regulation ofV. cholerae Virulence Genes ToxR of V. cholerae sits in the cytoplasmic membrane, where it senses that the cell is in a human host and directly activates the genes for cholera toxin and for attachment. Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved. 41
- 42. Cholera toxin Cholera toxinconsists of two protein subunits ◦ Encoded by ctxAB genes The original A protein is split into two pieces by a protease and linked by a disulfide bond The B protein forms a ring like sturctue of five subunits which surrounds the A subunit The B protein attaches to the galactose end of a ganglioside glycolipid. After attachement, part of the A protein splits from the protein complex and enters the cell
- 43. FIGURE 21.8 Structure andEntry of Cholera Toxin (A) Cholera toxin consists of an A protein plus five copies of B protein. The A protein is split into two halves (A1 and A2), held together by a disulfide bond. The B protein forms a ring with a central channel for the A1-S-S-A2 protein. (B) Cholera toxin binds to the host cell when the five B-subunits recognize ganglioside GM1. The disulfide bond in A1-S-S-A2 breaks, allowing A1 to enter the cell. Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved. 43
- 44. Cholera toxin After enterinthe cell, the toxin splits NAD into nicotinamide and ADP-ribose ◦ The ADP-ribose is used for ADP-ribosylate target molecules The toxin can actually ADP-ribosylate many acceptors ◦ Free arginine and its derivatives ◦ Many other proteins ◦ Itself, increasing productivity by 50% The true target is a G-protein, which regulates adenylate cyclase
- 45. G-proteins and choleratoxin Normally, a G-protein will be activatated, bind to a GTP. And then bind to adenyl cyclase GTP hydrolysis releases the G-protein and deactivates it. ADP ribosylation of an arginine residue prevents the hydrolysis of the GTP and results in the G-protein being locked in a bound state Causes hyperactivation of adenylate cyclase and overproduction of cyclic AMP Loss of sodium and water GTP analogs that cannot by hydrolyzed show similar effects.
- 46. Heat-labile enterotoxins Cholera toxinsand other heat labile toxins are all variants of the same toxin Some enterotoxins in E.coli are encoded on the Ent-plasmid which may be transferred All of these toxins have similar amino acid sequences and cause the same symptoms (in varying degrees of severity)
- 47. FIGURE 21.9 Mechanism ofAction of Cholera Toxin In their inactive state G proteins bind GDP. When an external signal activates the G protein, the GDP is exchanged for GTP. The G protein then activates adenylate cyclase. Normally, the GTP is hydrolyzed and the G protein returns to its inactive state. Cholera toxin cleaves NAD and attaches the ADP-ribose group to an arginine in the G protein. This prevents the G protein from splitting GTP. Consequently adenylate cyclase does not get turned off and continues to produce cyclic AMP. Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved. 47
- 48. Anthrax toxin Anthrax iscaused by the gram positive bacterium Bacillus anthracis In 1877 Rober Koch grew this organism and demonstrated its ability to grow spores There are two important virulence factors are exotoxins and the capsule, both on different plasmids The capsule protects against immune cells Thispathogen is very similar to other Bacillus species
- 50. Edema factor andLethal factor Anthrax makes two toxins ◦ The edema factor, the first toxin, is an adenylate cylase Not toxic in of itself, but intensifies lethal factor ◦ The lethal factor is a protease Disrupts the domains responsible for proteinprotein signaling Lyses macrophages Excessive release of interluekines results in shock leading to respiratory failure and/or cardiac failure
- 51. Antitoxin therapy Most therapiesrely on antibodies against toxins But now, more gene related approaches are beginning to emerge The dominant-negative mutation is one new approach ◦ Dominant-negative mutations in the binding subunit of the toxins ◦ These mutations typically result in inactive proteins ◦ Occasionally, it will not only inactivate the proteins themselves, but will also interfere with functioning proteins
- 52. Mechanism of dominant-negative mutations Involvesthe binding of a defective subunti to functional subunits resulting in an inactive complex Most of these mutations will affect proteins with multiple subunits Multisubunit B Proteins of A and B protein complexes of cholera and anthrax toxins are a good example ◦ This type of mutation has been deliberately isolated in the protective antigen of the anthrax toxin ◦ Mixture of mutant and active subunits resulted in the binding of A factors which allow the lethal factors to be built, but not transported into the target cell ◦ Treatment with these modified proteins can protect humans and mice from lethal doses of anthrax toxin
- 53. FIGURE 21.10 Activation ofProtective Antigen The pag gene of the pOX1 plasmid encodes the protective antigen (PA) of B. anthracis. PA is synthesized as an inactive precursor that is cleaved and assembled into a ring structure. Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved. 53
- 54. Polyvalent Inhibitor Phage displayis used to isolate nonnatural peptides These peptides bind weakly to single proteins If several of the these peptides are attached together on a flexible backbone (polyvalent inhibitor) Binding to many target proteins occurs, causing an increase in affinity For this to work, the target must be a multisubunit protein
- 55. FIGURE 21.11 Dominant-Negative ToxinMutations The PA63 protein (protective antigen) binds the lethal factor (LF) and edema factor (EF) and transports them into the target cell cytoplasm via an endocytotic vesicle. The dominant-negative inhibitory (DNI) mutant of the PA63 protein (purple) assembles together with normal PA63 monomers (pink) to give an inactive complex that cannot release the LF and EF toxins from the vesicle into the cytoplasm. Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved. 55
- 56. Summary Bacterial infections forthe most part, may be treated by antibiotics Plasmids, bacterial viruses and transposons move genes between species Analyzing toxins may allow us to combat infections