Bacteria Flashcards
Describe other important classification factors for bacteria
Growth temperature Ability to form heat spores Motility Cell shape Ability to use various carbon and nitrogen sources Special nutritional requirements
How does the gram staining technique work?
Stains the cell wall of gram +ve bacteria
Application of crystal violet➡️ application of iodine➡️ alcohol wash➡️ application of counter stain (safranin)
What are the requirements for the growth of bacteria?
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)
How do medically relevant pathogens acquire carbon?
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
How do medically relevant bacteria acquire nitrogen?
They assimilate nitrate and nitrite reductively by conversion to ammonia
Describe the sources for ‘other’ bacteria requirements eg. Sulphur, phosphorus etc.
Most bacteria can use sulphate
Phosphate is assimilated as free inorganic phosphate
Iron uptake facilitated by production of siderophores that chelate iron
How can different media be used to determine bacterial type?
Blood agar- bacteria that lyse blood
Mannitol salts agar- bacteria that ferment mannitol are yellow
MacConkey agar- bacteria that ferment lactose are pink
Give examples on how you would classify bacteria
Gram -ve or +ve
Genome sequencing
Describe the basic mechanism of transcription in bacteria
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
How can gene expression change?
Programmed mutations
Epigenetics
Changes to sigma factors
Changes in the activity of regulatory proteins
Describe the function programmed mutations
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
How is phase variation achieved by genomic recombination?
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
How is phase variation achieved by strand slippage?
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
How can phase variation be achieved by methylation?
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
What is a regulon?
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
How do bacteria effectively respond to stress?
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
Give an example of a transcriptional regulator that responds to stress inside s bacterium
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
Give some examples of sigma factors
Sigma70- RpoD- housekeeping
Sigma54- RpoN- nitrogen limitation
Sigma38- RpoS- starvation phase
Sigma32- RpoH- heat shock
Describe the production of RpoH
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
What is special about E. coli O157:H7?
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
Describe the production of beta-lactamase
AmpC gene found in n enterobacteriaceae
Requires permease, AmpG, amidase, AmpD, and local regulator AmpR
What causes complementary interactions between host and bacteria cells?
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
Describe skin infections
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
Describe the respiratory tract and infections
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
Describe the oropharynx and disease
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
Describe the gastrointestinal tract and infection
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
Describe the urogenetal tract and infection
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
What can MSCRAMMS bind to?
Collagen Elastin Fibronectin Fibrinogen Laminin Thrombospondin Vitronectin
What are the factors that effect whether a microbe can cause disease?
Host factors- genetics, vaccination status, health, nutrition, co-infections
Pathogen factors- strain (virulence factors) infectious dose
Describe routes of entry across surface barriers
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
Describe some active invasion strategies
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
Describe spread through local tissue
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
Describe the spread of bacteria within tissues
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
Describe abscess formation using the example of Staph aureus
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
What is neutrophil netosis?
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
Outline the strategies utilised by different bacteria to evade death within phagolysosomes
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
Describe the spread of bacteria by the lymphatics
Sone bacteria can grow in lymphatic endothelium Y pestis, M tuberculosis, Mycobacterium bovis
Describe the spread of bacteria via blood
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
How can bacteria evade RES?
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
What is sepsis and it’s implications?
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
What is the importance of iron in bacterial replication and spread?
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
Describe meningitis
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
Why do bacteria secrete proteins?
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
How can proteins be secreted across the cytoplasmic membrane?
Sec- chaperone mediated and srp
Tat- twin arginine translocon
Describe the sec pathway
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
What are the components of the Sec system?
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
Describe the SRP pathway
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)
Describe the Tat pathway
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
Describe the chaperone-usher pathway
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
Describe the type 2 secretion pathway
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
Describe the type 5 secretion system
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
Describe the type 4 pathway
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
Describe the type 1 secretion system
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
Describe the type 3 secretion system
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
What are the similarities between type 2,3,4 secretion systems?
Uses pilus systems
What are the similarities between type 2,3 secretion systems?
Homologous secretin-type proteins
What are the similarities between type 1,3,4?
Do not use Sec (4?)
What are the similarities between type 2,5,4 secretion systems?
Do not use Sec (4?)
What are the similarities between type 1,5 secretion systems?
Possess outer membrane beta barrel proteins that form pores
How do bacteria overcome barriers?
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
How do bacteria avoid complement mediated death
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
How do bacteria avoid phagocytosis?
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
What are superantigens?
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
How do bacteriostatic antibiotics clear infections?
Stop growth of bacteria and the immune system clears up the rest
List some targets for antibiotics
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
Describe the action of polymixin and Cationic peptides
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
Briefly describe the action of some inhibitors of cell wall biosynthesis
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
Describe the action of protein synthesis inhibitors
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
Describe inhibitors of nucleic acid synthesis
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
List some mechanisms of resistance
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
Describe enzymatic resistance to antibiotics
Enzymes that destroy or alter an antibiotic
Beta-lactamases
Aminoglycosides modifying enzymes
Chloramphenicol acetylating enzymes
Describe alterations in target site as a mechanism for antibiotic resistance
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
Describe the mechanism of metabolic bypass in antibiotic resistance
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
Describe prevention of access as a form of antibiotic resistance
Active efflux- efflux pumps- multi drug resistance (MDR/MAR)
Overexpression if efflux pumps often happens together with porin repression
