Bacteria

Question 1

Describe other important classification factors for bacteria

Answer 1

Growth temperature
Ability to form heat spores
Motility
Cell shape
Ability to use various carbon and nitrogen sources
Special nutritional requirements

Question 2

How does the gram staining technique work?

Answer 2

Stains the cell wall of gram +ve bacteria

Application of crystal violet➡️ application of iodine➡️ alcohol wash➡️ application of counter stain (safranin)

Question 3

What are the requirements for the growth of bacteria?

Answer 3

Growth for bacteria is increase in cell number therefore they need the elements of their composition
Require energy to synthesis of the anhydride bonds that links the macromolecules
Via fermentation of sugars of respiration (Chemical reductant of an oxidant)

Question 4

How do medically relevant pathogens acquire carbon?

Answer 4

Using an inorganic substrate as a reductant and CO2 as a carbon source (hydrogen/thio-sulphate)
Use organic carbon in an assimilable form eg glucose

Question 5

How do medically relevant bacteria acquire nitrogen?

Answer 5

They assimilate nitrate and nitrite reductively by conversion to ammonia

Question 6

Describe the sources for ‘other’ bacteria requirements eg. Sulphur, phosphorus etc.

Answer 6

Most bacteria can use sulphate
Phosphate is assimilated as free inorganic phosphate
Iron uptake facilitated by production of siderophores that chelate iron

Question 7

How can different media be used to determine bacterial type?

Answer 7

Blood agar- bacteria that lyse blood
Mannitol salts agar- bacteria that ferment mannitol are yellow
MacConkey agar- bacteria that ferment lactose are pink

Question 8

Give examples on how you would classify bacteria

Answer 8

Gram -ve or +ve

Genome sequencing

Question 9

Describe the basic mechanism of transcription in bacteria

Answer 9

RNA polymerase bind to gene promoter by the sigma factor which then dissociates and rebinds once transcription is completed and transcribed genes into RNA
Transcription regulators can promote it repress expression of a gene- often dimers with a helix-turn-helix motif that allows the protein to sit in the grooves of the DNA

Question 10

How can gene expression change?

Answer 10

Programmed mutations
Epigenetics
Changes to sigma factors
Changes in the activity of regulatory proteins

Question 11

Describe the function programmed mutations

Answer 11

Phase variation genes can be turned on/off randomly at high frequency- usually code for immunogenic proteins so there is variation in large populations
Can be achieved by genomic recombination, strand slippage, methylation
Phase variation is a passive activity it is not a response to anything

Question 12

How is phase variation achieved by genomic recombination?

Answer 12

Phase variation can be achieved via genomic rearrangement- eg. E. coli change the expression of fimA by inverting the promoter using integrases FimE and B that recognise a pattern of repeats (requires Lrp, IHF, H-NS)
Eg. Salmonella can express two different types of flagella, the expression of one also transcribes the repressor of the other, the the promoter of that gene is inverted by Hin that recognises his sequences then transcription of both genes is stopped to the other flagella is transcribed

Question 13

How is phase variation achieved by strand slippage?

Answer 13

Many phase variable genes are associated with short sequence repeats (SSR) the copying of which by DNA or RNA polymerase is prone to errors due to strand slippage
SSRs can be added or removed depending on is the mismatch occurs on the lagging or leading strand the number of these repeats can lead to altered expression or a different of the gene
The same effect can be achieved by slippage in one base pair

Question 14

How can phase variation be achieved by methylation?

Answer 14

In E. coli after DNA has been replicated it is methylated by dam methylase- recognises GATC sites, it does this to identify the parent strand for proofreading DNA synthesis
Methylation can also affect expression of genes eg. E. coli can express pap (a pilus that helps it binds to bladder epithelia) expression depends on if Lrp binds before or after methylation of certain GATC sites, before activates transcription, after represses transcription

Question 15

What is a regulon?

Answer 15

All the genes in a regulon are controlled by the same regulatory mechanism- they are turned on/off together
Eg. Multiple antibiotic resistance (mar) regulon in E. coli - over 60 genes controlled by MarA, when activated it results in many responses including decreased poring expression and increased efflux pump expression

Question 16

How do bacteria effectively respond to stress?

Answer 16

Common way: two-component systems
One sensor protein, a protein kinase, with a partner which, once phosphorylated, effects a change once the sensor has been activated, alters transcription of genes

Question 17

Give an example of a transcriptional regulator that responds to stress inside s bacterium

Answer 17

Mer operon
MerR binds to DNA, recruits RNAP and initiates transcription of the target gene when Hg is present
MerD can bind the promoter and repress transcription in the absence of Hg

Question 18

Give some examples of sigma factors

Answer 18

Sigma70- RpoD- housekeeping
Sigma54- RpoN- nitrogen limitation
Sigma38- RpoS- starvation phase
Sigma32- RpoH- heat shock

Question 19

Describe the production of RpoH

Answer 19

At room temperature the transcript for RpoD has a secondary structure that prevents ribosome binding and is degraded by DnaK DnaJ-GrpE
Under heat shock conditions the transcript does not form that secondary structure and it is transcribed do it can be used to start transcription of heat shock proteins

Question 20

What is special about E. coli O157:H7?

Answer 20

Carries two deadly shiga toxins associated with bacteriophages- the transcription of which is triggered under stress- turns the phage genes on and accidentally triggers their transcription
Antibiotics can cause patient death

Question 21

Describe the production of beta-lactamase

Answer 21

AmpC gene found in n enterobacteriaceae

Requires permease, AmpG, amidase, AmpD, and local regulator AmpR

Question 22

What causes complementary interactions between host and bacteria cells?

Answer 22

Adhesins

• Flagella, agents of motility
• Fimbriae/pili- can be rigid or flexible- may give a certain amount of elasticity
• Gram -ve outer membrane proteins (Omps) promote more intimate attachment, may promote invasion
• Gram +ve cell wall proteins- MSCRAMMS- Microbial Surface Components Recognising Adhesive Matrix Molecules

Question 23

Describe skin infections

Answer 23

Skin is a natural barrier to infection, commensald prevent colonisation, fatty acids keep pH low, 5.5
Antibacterial products produces by sebum
Hair follicles and nipples can be infected and through bites- flea regurgitation of plague bacteria, mosquito saliva

Question 24

Describe the respiratory tract and infections

Answer 24

Large particles filtered by nasal hairs, small particles reach lungs and only very small particles can reach alveoli
Mucous blanket traps bacteria and macrophages patrol the alveoli
Eg, Mycoplasma pneumoniae has cell surface projections to attach to neuraminic acid receptors on cell surface

Question 25

Describe the oropharynx and disease

Answer 25

Saliva- constant wash and antibacterial substances Bacteria need strong attachment Some attach to mucosal surface, others colonise the gingival crevice eg. Actinobacillus actinomycetemcomitans Most attach to the enamel and then attach to each other eg. Streptococcus mutans

Question 26

Describe the gastrointestinal tract and infection

Answer 26

Few bacteria in stomach Many in the small intestine Very large amount in large intestine Helicobacter pylori in stomach produces urease, has a sheathed flagellum and outer membrane adhesins

Question 27

Describe the urogenetal tract and infection

Answer 27

Women more prone than men Cystitis in the bladder Pyelonephritis in kidney Flushing of urinary tract prevents bacteria establishing Must have strong adhesive factors- E. coli- type1 Fimbriae and P-Fimbriae

Question 28

What can MSCRAMMS bind to?

Answer 28

``` Collagen Elastin Fibronectin Fibrinogen Laminin Thrombospondin Vitronectin ```

Question 29

What are the factors that effect whether a microbe can cause disease?

Answer 29

Host factors- genetics, vaccination status, health, nutrition, co-infections Pathogen factors- strain (virulence factors) infectious dose

Question 30

Describe routes of entry across surface barriers

Answer 30

Arthropod vectors eg. Lyme disease- ticks Plague- fleas Typhus- louse Wounds/punctures/trauma eg. Staph aureus can invade and spread to surrounding tissues or enter the bloodstream➡️ infective endocarditis, abscesses, vertebral osteomyelitis, septic arthritis and meningitis Active invasion- via adhesins and complementary receptor-ligand interactions- determine tropism and pathogen location

Question 31

Describe some active invasion strategies

Answer 31

Invasins- bacterial proteins that are either injected via Type 3 secretion systems into cells- trigger mechanisms (salmonella and shigella) or anchored in the bacterial surface that ligate host surface receptors and initiate endocytosis- zipper mechanisms (listeria and yersinia) Toxins- exotoxins induce cell death of epithelial/endothelial cells eg. Staph aureus- delta toxin for epithelial cells Shigella app- shigella enterotoxin E. coli- shiga-like enterotoxin Trojan horse- infected alveolar macrophages carry bacteria (M tuberculosis) across the alveolar epithelium into the lymph nodes and/or local blood stream

Question 32

Describe spread through local tissue

Answer 32

Spreading factors- tissue degrading enzymes Cellulitis- spread within surface tissues Necrotising faciitis- spread within deeper subcutaneous tissues Eg. Staph aureus (MRSA) Step pyogenes (group A Strep) Most people also have an underlying condition eg. Immunosuppression, diabetes, poor lifestyle Other spreading factors- hylauronase, collagenase, neuraminidase- degrades neuraminic (sialic) acid, the intercellular cement of epithelial cells, DNAases break down of neutrophil released chromatin NETS

Question 33

Describe the spread of bacteria within tissues

Answer 33

They have to evade the effects of the immune system (acute inflammatory response)- the complement system, coagulation and neutrophils They can overcome the fibrin clot traps formed by the clotting cascade by using host plasminogen, using streptokinase to make plasmin and break down fibrin

Question 34

Describe abscess formation using the example of Staph aureus

Answer 34

Staph aureus forms a fibrin wall around a colony This pseudocapsule allows bacteria to persist in tissues and protects them from cell mediated phagocytosis and attack by antibodies/drugs Enzymes released by neutrophils cause the liquefaction of the tissue surrounding the pseudocapsule forming pus The abscess will grow until it reaches a surface and then ruptures- very serious if it ruptures into the peritoneal cavity or s blood vessel Staph aureus can secrete staphylokinase to break down the pseudocapsule when it meets a surface

Question 35

What is neutrophil netosis?

Answer 35

A form of activated neutrophil cell death Nuclear membrane permeabilises, nuclei swell and chromatin dissolves➡️ granules mix with strands of DNA➡️ extruded DNA coated with histones and granule proteins traps and kills microbes

Question 36

Outline the strategies utilised by different bacteria to evade death within phagolysosomes

Answer 36

Escape out of the vesicle into the cytoplasm eg. Listeria Formation of a specialised endosome eg. Legionella Inhibit the fusion of lysosomes eg. Mycobacterium tuberculosis

Question 37

Describe the spread of bacteria by the lymphatics

Answer 37

Sone bacteria can grow in lymphatic endothelium Y pestis, M tuberculosis, Mycobacterium bovis

Question 38

Describe the spread of bacteria via blood

Answer 38

The most effective means Plasma- pneumococcus, resistant to antimicrobial factors in plasma Red blood cells- bartonella bacilliformis White blood cells- M tuberculosis, listeria, M leprae The hey hVe to evade the reticuloendothelial system (RES is comprised of phagocytic cells located in different organs In the body

Question 39

How can bacteria evade RES?

Answer 39

High bacterial loads Intracellular pathogens- survive and replicate inside macrophages Extracellular pathogens can evade by the expression of virulence factors eg. Capsule, complement inhibitors, IgA/G proteases, antigen masking and variation ➡️ reduces opsonisation and toxins which kill phagocytes

Question 40

What is sepsis and it's implications?

Answer 40

Systemic inflammatory response to infection LPS of gram -ve eg E. coli, Pseudomonas aeruginosa Lipotechoic acid of gram +ve eg. Staph aureus, Strep pyogenes Sepsis shock is the result of a decreased blood pressure due to leaky capillaries because of pro-inflammatory cytokine production also clots form in small vessels which can cause single or multiple organ failure

Question 41

What is the importance of iron in bacterial replication and spread?

Answer 41

Bacteria need iron for multiplication Bacteria that express siderophores can compete effectively for Fe3+ bounty to lactoferrin and transferrin Staph aureus can lyse RBCs via an exotoxin so the haemoglobin will be broken down into haem and utilised as an Fe source

Question 42

Describe meningitis

Answer 42

Infection of the meninges Can be caused by Strep pneumoniae, Neisseria meningitidis, Listeria monocytogenes, Mycobacterium tuberculosis They can cross the BBB transcellularly, paracellularly or by the Trojan horse mechanism Specific bacteria ligand-host interactions help the crossing of the BBB N meningitidis- Opc➡️ fibronectin-alpha3beta1 integrin -pili➡️ CD46 E. coli- FimH➡️ CD48 -OmpA➡️ gp96 -IbeA➡️ 45kDa

Question 43

Why do bacteria secrete proteins?

Answer 43

Mediators of motility-flagella Mediators of adhesion- pili Scavengers of nutrients- TbpA (transferrin binding protein) Avoid death by host defences- Opa Avoid death my environmental factors- iceP

Question 44

How can proteins be secreted across the cytoplasmic membrane?

Answer 44

Sec- chaperone mediated and srp | Tat- twin arginine translocon

Question 45

Describe the sec pathway

Answer 45

Proteins have a signal sequence at the N terminus- charged N-domain, hydrophobic H-domain, C-domain that is a signal peptidase recognition site SecB binds to protein to prevent it folding Localises it to SecA Signal sequence binds to SecYEG in the cytoplasmic membrane SecA binds ATP to undergo conformation changes which transport the protein through the pore LepB cleaves off the signal

Question 46

What are the components of the Sec system?

Answer 46

``` SecYEG- cytoplasmic membrane components SecA- cytoplasmic associated ATP binding protein, provides energy for translocation LepB- serine protease SecB- cytoplasmic chaperone SecD, SecF, YidC- auxiliary components ATP ```

Question 47

Describe the SRP pathway

Answer 47

Signal sequence has a larger charged domain and it may or may not have a cleavage site Additional components include Ffh- SRP 4.5S RNA FtsY- SRP receptor GTP SRP binds to the signal sequence on the n terminal of the polypeptide and the ribosome and stops translation This targets them complex to the FftY component in the membrane and the ribosome continues translation through the pore in SecYEG when SRP dissociates (GTP hydrolysis)

Question 48

Describe the Tat pathway

Answer 48

``` Twin arginine pathway Signal sequence contains a motif with conserved twin arginine residues in the N domain followed by a H domain and C domain It moves folded proteins Gram -ve protein secretion Type 1- sec-independent Type2- sec-dependent Type3- sec-independent Type4- dependent/independent Type5- sec-dependent Chaperone/usher pathway- sec-dependent ```

Question 49

Describe the chaperone-usher pathway

Answer 49

Used for the assembly of pili Chaperone protein in the periplasm carries the pilus sub-units to the usher proteins following secretion by the Sec pathway to prevent self-assembly in the periplasm Interactions between the chaperone and usher proteins release the proteins and transports them through the outer membrane for assembly

Question 50

Describe the type 2 secretion pathway

Answer 50

Eg. Pullulanase- PulA in Klebsiella oxytoca Two step process Sec secretion across inner membrane A protein specific translocase for transport across the outer membrane Requires 12-16 accessory proteins

Question 51

Describe the type 5 secretion system

Answer 51

Auto-transporter proteins Eg. Neisserial IgA1 protease Most common secretion system found in gram -ve bacteria The signal sequence is needed to cross the inner membrane then it is cleaved off leaving a periplasmic intermediate The beta domain of the intermediate forms a subunit of a beta-barrel in the outer membrane releasing the domain extracellularly

Question 52

Describe the type 4 pathway

Answer 52

Eg. Agrobacterium Transfer DNA Bordetella pertussis toxin Tra plasmid transfer system Can be one or two step Multiprotein complex can span both membranes Similar system to type 2 secretion- sec➡️ protein specific translocase

Question 53

Describe the type 1 secretion system

Answer 53

``` Eg. E. coli haemolysin Components; HlyA- secreted haemolysin HlyB- inner membrane ATP binding protein HlyD- membrane fusion protein TolC- pore forming outer membrane protein Sec secretes components not substrate Components form a tunnel which collapses to seal the membrane after the protein has been secreted ```

Question 54

Describe the type 3 secretion system

Answer 54

Eg. Yop in Yersinia Approximately 20 components assemble a needle like structure which spans both membranes and translocates effector molecules into host cells Secretion is one step Sec dependent secretion of components

Question 55

What are the similarities between type 2,3,4 secretion systems?

Answer 55

Uses pilus systems

Question 56

What are the similarities between type 2,3 secretion systems?

Answer 56

Homologous secretin-type proteins

Question 57

What are the similarities between type 1,3,4?

Answer 57

Do not use Sec (4?)

Question 58

What are the similarities between type 2,5,4 secretion systems?

Answer 58

Do not use Sec (4?)

Question 59

What are the similarities between type 1,5 secretion systems?

Answer 59

Possess outer membrane beta barrel proteins that form pores

Question 60

How do bacteria overcome barriers?

Answer 60

LPS O chain may help protect against stomach acid and pulsation The protein OmpA helps protect against bike Induce inflammation to enhance host killing of commensals

Question 61

How do bacteria avoid complement mediated death

Answer 61

Conferred by LPS O chain and Vi capsule Some streptococci secreted a capsule that resembles self to hide S. pyogenes have immunoglobulin degrading enzymes like IdeS and SpeB Bing surface Fc receptors on bacteria like protein A makes antibody functionless

Question 62

How do bacteria avoid phagocytosis?

Answer 62

Kill phagocyte by toxins Prevent opsonisation Possess a capsule that prevents contact Inhibit lysosomes fusion with phagosome Escape into the cytoplasm Organism resists killing by producing antioxidants

Question 63

What are superantigens?

Answer 63

Some Strep, Staph, and mycobacterium species secrete extremely potent T cell mitogens Bind to the outside of MHC2 on APC and polyclonal I activate T cells by binding to the Vbeta region if the TCR By activating lots of CD4 T cells they upset the cytokine balance leading to a non-productive immune response and can kill T cells

Question 64

How do bacteriostatic antibiotics clear infections?

Answer 64

Stop growth of bacteria and the immune system clears up the rest

Question 65

List some targets for antibiotics

Answer 65

Cell wall inhibitors- beta-lactams, glycopeptides, bacitracin Protein synthesis inhibitors- aminoglycosides, chloramphenicol, macrolides Nucleic acid/cytoplasmic cell envelope synthesis inhibitors- sulphonamide, quinolones/polymixin, peptides Inhibitors of enzymes in metabolic pathways

Question 66

Describe the action of polymixin and Cationic peptides

Answer 66

Interfere with the integrity of the cell envelope Peptide neutralises a patch in the outer membrane and enters through the crack Peptide binds to the membrane and flip-flops across Translocation into the cell Or Peptide binds at Cationic binding sites in LPS and disrupts the membrane Peptides aggregate into membrane spanning micelle-like structures

Question 67

Briefly describe the action of some inhibitors of cell wall biosynthesis

Answer 67

Cycloserines- inhibit reactions involved in the incorporation of alanine into the cell wall precursor Bactracin- prevents dephosphorylation of the phospholipid carrier which prevents it's recycling necessary for synthesis to continue Glycopeptides- bind to terminal alanine residues which prevent the incorporation of the subunit into the growing peptidoglycan Beta-lactams- bind to and inhibit enzymes which catalyse the link between subunits to form the growing peptidoglycan

Question 68

Describe the action of protein synthesis inhibitors

Answer 68

Aminoglycosides bind irreversibly to the 30S subunit or bacterial ribosomes. They may also prevent peptide chain elongation Tetracyclines- bind reversibly to the 30S subunit and alters the shape of the ribosome so that anticodons on the tRNAs cannot line up with the mRNA codons Macrolides- bind reversibly to the 50S subunit and inhibit peptidyltransferase which forms the peptide binds between adjacent amino acids, therefore elongation of the protein is inhibited Oxazolidinones- bid to the 50S ribosomal subunit and interfere with binding to the initiation complex

Question 69

Describe inhibitors of nucleic acid synthesis

Answer 69

Sulphonamides- analogues if PABA and competes with it for the active site and decreases the concentration of folate and therefore inhibits the synthesis of purines and pyrimidines Trimethoprim- interacts with dihydrofolate reductase, decreasing precursor synthesis Quinolones- attack DNA topoisomerases, destabilising gyrase-DNA complex and leads to dsDNA breaks and cell death

Question 70

List some mechanisms of resistance

Answer 70

Cell wall- prevention of entry, enhanced efflux, target alteration Target proteins- affinity change, new genes acquired, less important to the cell Enzymes- intracellular and extracellular which inactivate antibiotics Metabolic bypass- alternative routes to generate essential components

Question 71

Describe enzymatic resistance to antibiotics

Answer 71

Enzymes that destroy or alter an antibiotic Beta-lactamases Aminoglycosides modifying enzymes Chloramphenicol acetylating enzymes

Question 72

Describe alterations in target site as a mechanism for antibiotic resistance

Answer 72

Mutations at the target site of the antibiotic reduces the interaction of the antibiotic with its target Beta-lactate- penicillin binding proteins Vancomycin- D-ala ➡️ D-lac Erythromycin- altered ribosomal 50S subunit Quinolones- altered isomerases Rifampicin- altered DNA-dependent RNA polymerase

Question 73

Describe the mechanism of metabolic bypass in antibiotic resistance

Answer 73

Acquisition of new genes that give bacterium an alternative route along a metabolic pathway allowing them to bypass the target of the antibiotic Eg. dhfr gens from transmissible elements confer trimethoprim resistance MRSA resistant to methicillin due to a new PBP allele

Question 74

Describe prevention of access as a form of antibiotic resistance

Answer 74

Active efflux- efflux pumps- multi drug resistance (MDR/MAR) | Overexpression if efflux pumps often happens together with porin repression