BIMM 120 Final Exam Rvw

Significance of Human microbiome

• Humans are covered on all exposed surfaces by microbes, gut + skin + vaginal are of particular importance

• Studied through metagenomics, sampling (fecal, skin), DNA sequencing

• Not all humans have same microbiome

How has modern life shifted the microbiome?

• More antibiotics, lack of diverse diet, shift in pharmaceutical practices have reduced our microbiome diversity and as a consequence has lead to more obesity, infections

Inner membrane protein translocation

• Protein is translated, N terminus signal recruits SRP

• SRP brings protein to FtsY

• FtsY moves protein to SecYEG, feeds through channel

• SecYEG, powered by ATP, pumps proteins throughout the inner membrane and terminates once complete

Periplasm protein translocation

• N terminal signal sequence appears

• polypeptide folds around SecB, is brought to SecA and associates

• SecA, SecB, protein complex interacts with SecYEG which exports protein into periplasm ATP powered

• LepB cuts the protein away from SecYEG to terminate process

• chaperone assists protein in folding once in periplasm

Outer membrane protein translocation

• N terminal signal sequence appears, Sec Dependent path way seen in the inner membrane occurs

• Protein makes its way to loading membrane protein (e.g. HlyB), is pumped through and into TolC with ATP

• Protein passes through periplasm by traveling through TolC

• Upon arriving at BAM, protein is folded and woven into the outer membrane of the cell

Type I Secretion System

• cytoplasm to outside of cell channel

• TolC outer membrane channel = nonpolar and uncharged

• ABC is specific to the types of proteins or molecules being exported

• ABC transports through periplasm and out of cell. Similar to outer membrane translocation without BAM step

Type III Secretion System

• “injectosome”, syringe like structure that is used to transport genetic material cell to cell

• only delivers genetic payload when in contact with another cell

Locus of Enterocyte Effacement

“Pathogenicity island” seen in some E. Coli, present multiple times within Salmonella

EPEC, EHEC

E. Coli which bind to epithelial gut cells to cause infection, “effacement” occurs to affected cells with membranes raising and engulfing E Coli cells upon contact.

Transformation

• exogenous or extracellular DNA is picked up and utilized for DNA repair or energy by host cell

• Competence factors are secreted (in gram positive cells) to signal to other cells via quorum that Transformation should occur

  • transformasome, CF peptides, sigma factor, and sensor kinase involved

• gram positive need CF Quorum sensing, gram negative always competent

Conjugation

• transfer of genetic material from one cell to another through sex pilus

  • oriV, oriT, sex pilus, relaxosome, and IS involved

• When plasmid is integrated into the host chromosome, recombination can lead to integration. Tra genes inserted into element

Transduction (generalized)

• phage transfer of DNA, carry DNA from a previous host bacteria into another

• host genes are packaged by chance, when capsids are packing they don’t have a discriminating process for DNA it picks up

Transduction (specialized)

• specific location of integration (e.g. phage lambda). Phage integrates genes at this site and may be active or dormant.

• Same chance for Host DNA to be picked up as a consequence of random capsid packing

Phase variation

• allows 2 different versions of a gene to be present within genome, varying what invaders like phage may bind to or disable. This assists in resisting invader attacks (e.g. H1 vs H2 flagellin)

Transcriptional regulation (activator)

• activator protein has inducer binding, binds to operator and enables transcription

Transcriptional regulation (activation by depression)

• repressor binds to operator sequence with corepressor, as corepressor diminishes transcription is unblocked and reactivated

Transcriptional regulation (activation by induction)

• repressor binds to prevent transcription, eventually inducer binds repressor and the repressor falls off.

structure and function of an operon (transcription & translation)

• promoter : region for transcription, RNAP bind and begins to transcribe from this point

• ribosome binding sequence : region for RNAP to bind and begin transcription, factors may block or enhance binding to this region

• start codon : region for translation, AUG start codon for fMet, starts the chain of polypeptide that leads to formation of protein

• coding region : translation/transcription related, mRNA is coded for coding region which is translated into protein

• stop codon : typically UAG, UGA, etc, signals protein to terminate translation

• terminator : region for transcription, RNAP stops transcription here

Lac operon inducing

• allolactose is catabolized by beta galactosidase, then binds the lac repressors (LacI), leading to transcription

cAMP + CRP effect

• cAMP and CRP bind and are recruited to the transcription site, cAMP + CRP contact RNAP and induce transcription

mRNA stability control

RNA instability due to RNases, sRNAs can inhibit degradation

Translational control

• translational repressor proteins bind mRNA

post translational control

• cleavage, phosphorylation, methylation

• activation, inhibition, destruction

How does phage deliver DNA into cells?

phages bind an extracellular receptor, then are transported inside the cell

Phage Structure

• nucleic acid (RNA or DNA) genome

• head, tail, fibers

Lytic Phage life cycle

• Phage makes early proteins to regulate the processes for viral replications, later genes are structural and make capsids/viral structural elements

• doesn’t integrate into host chromosome, viral enzymes lyse to release progeny

Lysogenic phage life cycle

• plasmid integrates into the genome, lays dormant as a prophage

• when excised, enters lytic phase through replication of viral DNA and construction of capsids

Mutation (bacterial defense)

• disallows temperate phages from integrating, changing the recognition site

CRISPR (bacterial defense)

• protein (Cas) and genomic repository (CRISPR) system that identifies and cuts invader DNA. tracr+crRNA form a double zipper from the CRISPR repository data and look for DNA adjacent to a PAM sequence to cut and disable invader DNA

DNA restriction + modification

• restriction enzymes are used to recognize palindromic sequences and cut invading DNA. modification (methylation) is used for the host to self-identify what DNA is host and what is invader

How are new phages found for phage therapy?

• “phage hunts” are conducted. A test of phages on bacterial confluent lawns is conducted to see how effectively certain phages can kill the target pathogen.

How is phage therapy different from antibiotic therapy?

• phage therapy uses bacteriophages to kill specific bacteria, antibiotics are indiscriminate in their killing of bacteria and can throw off microbial balance in the host

• bacteriophages can work synergistically with antibiotics and replicate after initial introduction.

How is evolutionary resistance against phage dealt with?

• phage cocktails, engineering of phage genome to avoid or circumvent resistance

How do vaccines work, and do they always prevent infection?

• vaccines aim to build immunity against a disease without having to expose the host to the full disease and the complications that may arise from it.

• introduction of a modified phage that does not give the host the disase generates a biphasic response, resulting in partial or full immunity over time

• infection is not always avoided

Describe the phases of antibody response after vaccination.

• 1st, moderate, slow, builds enough antibodies to resist disease if introduced

• 2nd, fast, grater antibodies, generally seen as the phase where immunity is built

What was insufflation, variolation?

• insufflation: dried scabs of smallpox were ground and blown into the sinuses of receivers of the treatment to confer immunity

• variolation : smallpox pus scratched into the skin of patients to confer immunity, 1-2% death rate

Edward Jenner’s Cowpox Vaccine

• different since it used a related disease (cowpox) instead of the actual disease (smallpox) for vaccination, resulting in a much lower fatality rate

Who was the last person to have been infected with smallpox?

Ali Maow Maalin

Live Attenuated Vaccine

- replicates in host but weakened

Whole Inactivated Viral Vaccine

• virus is dead, may not give as strong of an immune response and may require a booster

Virus-like particle

• contains no genetic information, also dead, may struggle to make immune response

Protein Subunit Vaccine

• safest option, not self replicating, weak immune response

Live viral vector

• live virus, harmless variety

  • can be replicating or non replicating

  • genetically engineered to have gene from a pathogenic virus, makes a strong immune response

mRNA

• mRNA in lipid bubble, non replicating, quick to design and manufacture