Infectious Disease Shouguang Jin, Ph'D'3928323 sjinmgm'ufl'eduR1293

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Infectious DiseaseShouguang Jin,
Ph.D. 392-8323sjin_at_mgm.ufl.edu R1-293
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Introduction to bacteria

• Haploid organisms small circular genomes, (E.
  coli 4.5 X 106 bp.)
• Rapid growth (E. coli 20 minutes per generation
  therefore, 1 cell to 1,000,000 in less than 7
  hr.)
• Grow to high population density liquid 109-
  1010 at saturation, bacterial paste 1012
  cells/cc.

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Bacterial classification

• Wall structure
• Gram
• Staphylococcus, Streptococcus, Clostridium,
  Bacillus
• Gram -
• Enteric, respiratory and others
• Acid-fast
• Mycobacterium
• Wall-less
• Mycoplasma
• Unusual
• Obligate intracellular
• Rickettsia, Chlamydia, Treponema

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Bacterial growth

• Lag phase
• Exponential phase
• Stationary phase
• Death phase

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Bacterial Metabolism

• Carbon, hydrogen, oxygen, nitrogen, phosphate
• Aerobic respiration
• Anaerobic respiration
• Anaerobic fermentation
• Microaerobic
• Aerotolerant
• Anaerobic
• Facultative
• Prototroph and auxotroph

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Bacterial metabolism
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Production of Energy
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(No Transcript)
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Mutations A. Genotype vs. Phenotype
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B. Mutation frequency (10-5 to 10-6 for
auxotrophic mutation) Random (Lederberg
technique)
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C. Genetic code (redundant and is a triplet
code)
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D. Types of mutations (chemical
sense) Transitions - Purine-pyrimidine bp to
purine-pyrimidine bp (TA to CG) Transversions
- Purine-pyrimidine bp to pyrimidine-purine
bp (TA to AT)
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E. Types of mutations a. Silent b. Missense c.
Nonsense d. Frame shift (small insertions
or deletions) e. Insertions and deletions (large)
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F. Types of mutations (phenotypic sense) a.
Recessive b. Dominant c. Auxotrophic
mutation d. Lethal mutation e. Conditional
mutation (Tm sensitive)
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G. Types of suppressors a. Reversion (back
mutation) b. Nonsense suppressors - Mutation in
anticodon of tRNA genes. c. Intragenic
suppressors - 1 frame shift to restore
reading frame of close by -1 frame shift. d.
Intergenic suppressors - Usually occur in
genes which encode subunits of multi-subunit
enzymes.
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Genetic selections A. Positive - select for -
drug resistance - prototrophic marker e.g.
TrpE in trpE background B. Negative -
select against - phage receptors in
bacteria - sacB gene and sucrose
sensitivity
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DNA insertion elements, plasmids and
episomes A. Mobile genetic elements a.
Insertion sequences (I.S. elements).
Small discrete segments of DNA ranging in
size from 750bp to 1600bp.
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Insertion sequences
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Mechanism oftransposition Nonreplicative
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I.S. elements can act in pairs to mobilize
intervening DNA. Nature has used many such pairs
of I.S. elements to mobilize important
determinants such as antibiotic resistance genes,
genes for lactose utilization, and genes for
bacterial enterotoxins. In E. coli the ST
enterotoxin gene is encoded by a transposon and
is sometimes found on plasmids and sometimes on
temperate phages.
b. Transposons
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Transposons anatomy
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Transposons genesis
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Transposons types
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Transposons replicative transposition for
Tn3-like transposons (non-replicative for Tn5
and Tn10 type)
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c. Conjugal plasmids
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Conjugal plasmids
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Genetic exchange

• Transfer of DNA from one bacterium to another is
  of obvious medical importance. For it is this
  transfer that is responsible for the
  dissemination of drug resistance determinants
  among and between bacterial species.
• Three mechanisms
• Transformation
• Conjugation
• Transduction

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A. Transformation

• a. Discovered by Griffith in 1928 during the
  course of his studies of virulence in
  Streptococcus pneumoniae

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Transformation

• b. Avery, MacLeod, and McCarthy (1944)
  fractionation studies led to conclusion that
  transformation principle is DNA.

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Transformation

• 1) Streptococcus
• 2) Staphylococcus
• 3) Bacillus
• 4) Acinetobacter
• 5) Hemophilus
• 6) Neisseria

• c. Physiological transformation occurs in nature
  in a wide variety of genera which include

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Transformation

• d. Competence. The ability to take up DNA varies
  regularly during the cell cycle. In Streptococcus
  competence is highest shortly after cell
  division.
• e. Entry integration. Cell components required
  for uptake.
• f. Heteroduplex formation with homologous
  recipient DNA.

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B. Conjugation

• a. Direct transfer of DNA from one strain to
  another mediated by fertility factor (F). Best
  studied in E. coli, Approximately a third of
  freshly isolated E. coli have plasmids.
  Conjugative plasmids have been found in
  approximately 30 genera of bacteria, mostly
  gram-negative. Antibiotic-resistance plasmids
  RP4 R68.45 can propagate and promote
  conjugation in virtually any gram-negative
  bacterium. Some gram-positive conjugate such as
  Streptococci, Staphylococcus, Streptomyces,
  Clostridium, and Bacillus.

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Conjugation

• b. Fertility factor is a large (95kb) DNA
  plasmid.
• c. Conjugative plasmids can exist
  extrachromosomally or integrated into the host
  chromosome. Intergration occurs at a frequency of
  10-5 per generation.
• d. Integration occurs via homologous
  recombination between IS-elements on the plasmid
  and IS-elements in the chromosome.

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Conjugation

• d. Conjugative plasmids when integrated into the
  chromosome can mobilize the host genome. F can
  mobilize any marker at a frequency of 10-5 to
  10-6 per donor. Strains with F plasmids
  integrated into the chromosome are known as Hfr
  strains.
• e. DNA transfer is in the direction of F to F-.
• f. Interspecies transfer. F can be transferred
  from E. coli to Salmonella, Shigella, and
  Proteus.

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Conjugation

• Consider an experiment

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Conjugation

• g. Can be used to map genes via interruptive
  matting experiments.

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Conjugation

• h. Excision can occur via homologous
  recombination between the same IS-elements on the
  plasmid and IS-elements in the chromosome that
  were involved during integration to regenerate an
  episomal F plasmid, or F can excise via
  homologous recombination between different
  IS-elements than were used during integration
  resulting in an F episome containing bacterial
  genomic DNA.

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Conjugation

• i. R factors - Drug-resistance plasmids first
  isolated in late 1950's in Shigella during an
  outbreak of dysentery. The first plasmid isolated
  carried resistant determinants to four different
  antibiotics chloramphenicol, tetracycline,
  streptomycin, and sulfonamides. Latter the same
  plasmid was found in E. coli.
• In patients given oral tetracycline, the
  predominant fecal E. coli isolates carry
  tetracycline-resistance R plasmids within one
  week.

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Conjugation

• j. Conjugal plasmids are involved in the
  mobilization of DNA from one organism to another
  and are an important mechanism by which genes
  conferring antibiotic resistance are propagated
  from one bacterium to another.
• k. Homologous recombination between IS-elements
  which serve as portable regions of homology allow
  for the generation of many different R plamids.

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C. Transduction

• a. Bacteriophage mediate transfer of DNA from one
  organism to another. Occurs within and between
  species, for example phage P1 infects Salmonella,
  E. coli, and Shigella. Transduction has been
  shown to occur in genera as varied as
  Pseudomonas, Staphylococcus, Bacillus, and
  Proteus.

Phage T4
Phage lambda
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Transduction

• b. Overview of bacterial phage life cycle

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Transduction

• c. Generalized Transduction - Transducing
  particle does not contain phage DNA. Stable
  transductants result when incoming bacterial DNA
  is recombined into the recipients genome.

In generalized transduction, bacterial DNA is
packaged into phage particle by mistake.
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Transduction

• d. Overview of generalized transduction
• In generalized transduction bacterial DNA is
  packaged into phage particle by mistake.
  Frequency 1/1000 normal page particles.
• Homologous recombination is required for stable
  maintenance in the recipient cell.

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Transduction

• e. Specialized Transduction - Transducing
  particle contains both phage and bacterial DNA
  covalently joined.

Phage lambda is the classic example of a
specialized transducing phage however, there
are medically important examples of specialized
transducing phage. (Gene encoding diphtheria
toxin is phage encoded. Only strains of
Corynebacterium diphtheria that are lysogenic
for beta- phages produce toxin.)
Phage lambda
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Transduction

• f. Specialized Transduction Genesis of
  Specialized transducing particle, containing both
  phage and bacterial DNA covalently joined,
  results from an aberrant excision event of the
  phage genome from the bacterial genome.

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Genetic exchange - Summary

• Transfer of DNA from one bacterium to another is
  of obvious medical importance. For it is this
  transfer that is responsible for the
  dissemination of drug resistance determinants
  among and between bacterial species.
• Three mechanisms
• Transformation transfer of free DNA
• Conjugation cell to cell contact
• Transduction phage mediated
• Generalized
• Specialized

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Genetic recombination

• Homologous recombination
• Neisseria gonorrhoeae pili phase variation
• Site-specific recombination
• Salmonella flagella phase variation
• Illegitimate recombination

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Gene Regulation and Operon Structure
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Operon structure

• A. Monocistronic
• Promoter, ribosome binding site, start codon,
  structural gene, stop codon, terminator.
• B. Polycistronic
• Promoter, ribosome binding site, start codon,
  structural gene, stop codon, ribosome binding
  site, start codon, structural gene, stop codon,
  terminator. (See Trp operon, below)
• a. If polycistronic gene products tend to be
  involved in the same pathway.
• b. Means of achieving coordinate regulation (only
  one transcriptional unit).

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Promoters

• A. Frequence of transcription initiation can vary
  over 4-orders of magnitude. Some genes are
  transcribed once per second, while other genes
  are transcribed less than once per generation.

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Promoters

• B. Compilations of promoter sequences in E. coli
  have revealed a consensus sequence.
• TTGACA----17bpTATAAT-----5 to 9bp RNA start
• -35 sequence -10 sequence
• (Bases in large type most important for function)
• Promoters that match the consensus are generic.

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• C. Mutations that affect promoter strength
  generally affect the consensus base pairs which
  make up the -10 and -35 sequences.

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• D. Kinetics of Initiation cartoon view.

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Regulation

• A. Global - Regulons (C, O, N, PO4,
• temperature, DNA damage, growth rate)
• Regulons are groups of operons that are
  coordinately regulated.
• B. Specific - Operons (catabolites or
• metabolites)
• C. Temporal - Phage gene expression
• during infectious cycle

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• A. Positive (Role of CAP in lac gene expression)

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• B. Negative (Role of repressor at lac)
• - tend to block either the binding or
  isomerization step of
• transcription initiation.

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C. Two-component regulatory system

• Sensor
• Response regulator

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D. Quorum sensing

• Discovery
• Model
• Importance in pathogenesis
• LuxR/I of Vibrio fisheri
• LasR/I of Pseudomonas aeruginosa
• TraR/I of Agrobacterium tumefaciens

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Bioluminescence in Vibrio fischeri
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LuxS/AI-2 quorum-sensing system
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Quorum sensing in EHEC
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Control of gene expression

• A. Promoters that are positively regulated tend
  not to look like generic promoters.
• Many regulated promoters do not have sequences
  that match the -35 consensus sequence.
• Many regulated promoters have binding sites for
  regulatory proteins in place of the 35 sequence
  element (TTGAC). For example the CAP binding site
  in the lac promoter.

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Control by sigma factor

• RNA polymerase holoenzyme, ?2???. Is required
  for transcription initiation
• RNA polymerase core enzyme, ?2??. Core enzyme
  can elongate but not initiate

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• Different ? subunits of RNA polymerase
• Different ? subunits used for different
  promoters. ? subunits plays role in promoter
  selection. Generic promoters use ?70. Heat shock
  promoters use a different ? subunit, Genes
  involved in nitrogen regulation and assimilation
  use a different s subunit. Different ? subunits
  are used to achieve global regulation.
• Genes which are transcribed with RNA polymerase
  with different ? subunits tend to have -10
  sequences that are different than the generic -10
  sequence.

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Control at DNA level

• Gene amplification
• Recombination
• Transposon
• Gene rearrangement
• DNA inversion
• S.t. flagella H1-H2 E.c. pili FimA-FimB
• DNA rearrangement
• N.g. pilE and pilS recombination
• P.a. chromosomal DNA rearrangement
• DNA mutation
• Gene replacement
• Transformation (S.p. N.g. H.I.)

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Control at DNA level

• DNA supercoiling
• DNA gyrase (nalidixic acid, novobiocin)
• B.p. PTOX synthesis (activated by novobiocin,
  repressed by gyrase)
• DNA bending
• Histone-like protein mediated
• DNA methylation
• On/off control of pap promoter activity in E.coli

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RNA structure mediated control

• Alternative secondary structure
• Trp operon
• His operon
• Transcriptional termination
• Rho-dependent
• Rho-independent
• Anti-termination protein
• Anti-sense RNA
• Cleavage of mRNA

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Environmental stimuliand sensing mechanisms
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Nutritional signals

• Iron starvation
• Fur mediated de-repression
• Phosphate starvation
• PhoU sense free Pi, signal through PhoR/B to
  alkaline phosphatase
• Nitrogen starvation
• PIII sense Gln?-ketoglu ratio, signal through
  NtrB/C
• Amino acid starvation
• stringent response, RelA/SpoT sense uncharged
  tRNA, ppGpp
• Aerobic to anaerobic switch
• ArcB/A system, signal??
• Catabolite repression
• PTS system sense external glucose and regulates
  Cya which makes cAMP
• lac, ara, mal operons etc.
• Chemotaxis
• MCP, CheA, CheY etc.

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Non-specific

• Temperature
• Temperature sensitive regulator (?32 and TlpA of
  Salmonella)
• Detection of mis-folded intracellular proteins
  (chaperons in heat shock proteins)
• Osmolarity
• EnvZ/OmpR mediated induction of ompC and micC or
  repression of ompF (OmpC makes smaller porin)
• pH
• Proton gradient?
• Trace metal ions?

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Stress signals

• Toxic agents
• DNA damaging (SOS response)
• Any anti-microbial agents (signal)
• Anti microbial peptides
• Low nutrition
• Low amino acids (stringent response)
• Extreme conditions
• Extreme pH, temperature, osmolarity, etc.

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Adaptation

• Regulatory adaptation
• Transcription
• Positive regulator
• Negative regulator
• Anti-terminator (Control of transcription
  termination)
• Translation (mRNA structure, processing, half
  life etc.)
• Positive regulation
• Negative regulation
• Posttranslational regulation
• Regulation of enzyme activity
• Allosteric interactions
• Feedback regulation
• Covalent modification of enzyme reaction

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Adaptation

• Genetic adaptation
• Mutation
• Deletion
• Duplication
• Inversion
• Translocation
• Base changes
• Genetic exchange
• Transformation
• Transduction
• Conjugation
• Recombination (homologous, site specific,
  illegitimate)

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Adaptation

• Morphological changes
• Cell shapes
• Unicellular vs. multicellular or mycelium
• Spore formation
• Vacule formation
• Marine bacteria to float (get light for
  photosynthesis)
• Surface appendages
• Flagellar, pili, LPS etc.

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Chemotaxis net work