Bact Path 2

What are cytokines?

Small signaling proteins released by host cells, especially immune cells, that communicate between cells and coordinate immune responses.}

When are pro-inflammatory cytokines beneficial?

Early during infection, because they recruit and activate immune cells, increase blood flow and vascular permeability, and help the body eliminate pathogens.}

When are anti-inflammatory cytokines beneficial?

When inflammation needs to be controlled, because they reduce immune activity and prevent excessive inflammation from damaging host tissues.}

What is a modulin?

A bacterial molecule or component that stimulates or modifies the host immune response, including the release of cytokines.}

Where is lipid A found?

Lipid A is the lipid portion of lipopolysaccharide (LPS), which is located in the outer membrane of Gram-negative bacteria.}

What are the three parts of LPS?

O antigen, core polysaccharide, and lipid A. Lipid A is responsible for most of LPS's toxic effects.}

How and why does lipid A in LPS cause septic shock?

1. Lipid A acts as an endotoxin and is recognized by host immune receptors, especially TLR4. 2. Recognition activates immune cells, triggering excessive release of pro-inflammatory cytokines such as TNFα and IL-1. 3. These cytokines cause widespread vasodilation (blood vessels widen) and increased vascular permeability (fluid leaks out of vessels). 4. Widespread vessel dilation and fluid loss cause blood pressure to drop. 5. Low blood pressure reduces blood flow to organs, potentially causing organ failure and septic shock.}

Why is excessive cytokine release dangerous during septic shock?

Cytokines normally help fight infection, but widespread excessive release causes inflammation throughout the body. This produces dangerous vasodilation, fluid leakage, low blood pressure, and inadequate organ perfusion.}

What are three ways bacteria evade cytokines?

1. Inhibit cytokine production so the host releases fewer signals. 2. Block cytokine activity or signaling so host cells cannot respond. 3. Degrade cytokines so they cannot continue signaling.}

How does Yersinia block TNFα expression?

1. Yersinia injects the effector protein YopJ into host cells through its Type III secretion system. 2. YopJ blocks host signaling pathways, including NF-κB and MAPK activation. 3. It prevents IκB phosphorylation and degradation, so NF-κB remains inhibited in the cytoplasm. 4. NF-κB cannot activate TNFα transcription in the nucleus, reducing TNFα production.}

Why does blocking NF-κB reduce TNFα production?

NF-κB is a transcription factor needed to activate TNFα gene expression. If it remains inhibited in the cytoplasm, it cannot enter the nucleus and promote TNFα transcription.}

Would bacteria benefit from inducing pro- or anti-inflammatory cytokines? Why?

Anti-inflammatory cytokines, because they suppress or limit the host immune response. This reduces immune-cell activity and makes it easier for bacteria to survive and multiply.}

What is gene rearrangement?

A change in the physical arrangement or orientation of DNA that can switch gene expression ON or OFF. It allows bacteria to change surface characteristics and evade host defenses.}

What is phase variation?

A reversible switch between different gene-expression states, commonly ON and OFF, allowing bacterial populations to vary their traits.}

How does phase variation turn fimA expression OFF?

1. In E. coli, the fimS DNA segment contains the promoter for the fimbrial operon, including fimA. 2. When the promoter faces the operon, RNA polymerase can transcribe the genes, so expression is ON. 3. Recombinases invert the fimS segment. 4. The promoter now faces away from the operon, preventing transcription, so expression is OFF.}

Which proteins control fimS inversion?

FimB and FimE are recombinases. FimB can promote inversion in either direction, while FimE mainly switches expression from ON to OFF.}

Why is fimA phase variation useful to E. coli?

It allows bacteria to switch fimbriae on when attachment is useful and off when avoiding immune recognition or other host defenses is beneficial.}

What is slipped-strand mispairing?

A mutation mechanism in which repeated DNA sequences misalign during DNA replication, causing repeat units to be inserted or deleted.}

How does slipped-strand mispairing cause phase variation?

1. Repeated DNA sequences misalign during replication. 2. DNA polymerase adds or loses repeat units. 3. The altered repeat number changes the DNA sequence or spacing. 4. This can cause a frameshift or alter promoter function, switching gene expression ON or OFF.}

Why is slipped-strand mispairing useful to bacteria?

It creates reversible or rapidly changing surface structures and other traits, helping bacteria adapt to different environments and evade host immune recognition.}

What is an operon?

A group of adjacent genes controlled by one promoter and transcribed together into a single polycistronic mRNA.}

Why do bacteria organize genes into operons?

Genes involved in the same process can be switched on or off together, allowing coordinated and efficient expression.}

What is a regulon?

A group of genes or operons located in different parts of the genome but controlled by the same regulatory protein or environmental signal.}

What is the difference between an operon and a regulon?

An operon contains neighboring genes transcribed together from one promoter. A regulon contains separate genes or operons controlled by the same regulator.}

What is Fur?

Ferric uptake regulator, a protein that senses intracellular iron levels and controls the expression of iron-acquisition genes.}

How does Fur regulate iron-acquisition genes?

1. When intracellular iron is high, Fe²⁺ binds Fur. 2. Iron binding activates Fur's ability to bind regulatory DNA. 3. Fur binds near iron-acquisition genes and blocks transcription. 4. Siderophore production and iron-uptake proteins decrease. 5. When iron is low, Fur cannot effectively bind DNA, so repression is removed and iron-acquisition genes are expressed.}

Why does Fur repress iron-acquisition genes when iron is abundant?

The bacterium already has enough iron. Turning off additional uptake prevents unnecessary energy expenditure and toxic iron accumulation.}

How is diphtheria toxin expression regulated by iron?

1. The classic regulator is DtxR, an iron-dependent repressor in the Fur family. 2. When iron is high, iron binds DtxR and activates it. 3. Active DtxR binds the tox operator and blocks transcription. 4. Diphtheria toxin production decreases. 5. When iron is low, DtxR cannot bind the operator effectively, allowing toxin transcription.}

Why is diphtheria toxin expressed when iron is low?

Low iron prevents active DtxR from repressing the tox gene. Toxin production is therefore permitted under iron-limited conditions.}

What is the distinction between Fur and DtxR?

Fur is a major regulator of iron uptake in many bacteria. DtxR is the specific iron-dependent repressor classically responsible for diphtheria toxin regulation; it belongs to the Fur family.}

What are ToxR and ToxS?

Membrane-associated regulatory proteins in Vibrio cholerae that help detect environmental conditions and activate virulence-gene expression.}

How does ToxR/S regulate cholera toxin (CtxAB)?

1. ToxR/S activates expression of tcpPH. 2. TcpP and TcpH work together with ToxR to activate the toxT promoter. 3. The toxT gene is transcribed, producing ToxT. 4. ToxT acts as a transcriptional activator and binds regulatory DNA. 5. ToxT activates ctxAB expression, leading to cholera toxin production.}

What is the order of the cholera toxin regulatory pathway?

ToxR/S → TcpP/TcpH → ToxT → ctxAB → cholera toxin production.}

What is a two-component signal transduction system?

A bacterial signaling system made of a sensor histidine kinase and a response regulator. It detects environmental signals and changes gene expression in response.}

How does a two-component system work?

1. A sensor histidine kinase detects an environmental signal. 2. The sensor autophosphorylates using ATP, adding phosphate to a histidine residue. 3. The phosphate transfers to an aspartate residue on the response regulator. 4. Phosphorylation changes the response regulator's activity. 5. The regulator often binds DNA to activate or repress target genes.}

Why is phosphorylation important in a two-component system?

Phosphorylation changes the response regulator's activity, allowing the bacterium to alter gene expression in response to an environmental signal.}

What is a sigma factor?

A protein that binds the core RNA polymerase and directs it to specific promoters so transcription can begin.}

How do alternative sigma factors regulate bacterial genes?

Different sigma factors recognize different promoter sequences. When conditions change, bacteria use different sigma factors to direct RNA polymerase toward genes needed for that condition, such as stress-response genes.}

What is an RNA thermosensor?

A temperature-sensitive mRNA structure that controls translation by changing whether the ribosome-binding site is exposed or hidden.}

How does an RNA thermosensor work?

1. At low temperature, the mRNA folds into a hairpin that hides the Shine-Dalgarno sequence or start codon. 2. The ribosome cannot access the mRNA, so translation is blocked. 3. At higher temperature, the hairpin melts or opens. 4. The Shine-Dalgarno sequence becomes exposed, allowing ribosome binding and translation.}

Why do RNA thermosensors help bacteria regulate genes?

They allow bacteria to respond directly to temperature changes by controlling translation without needing to first make a regulatory protein.}

Why must the Shine-Dalgarno sequence be single-stranded and accessible?

The Shine-Dalgarno sequence must pair with the complementary anti-Shine-Dalgarno sequence on the 16S rRNA of the small ribosomal subunit. This positions the ribosome correctly at the start codon. If the sequence is hidden in a hairpin, the ribosome cannot bind efficiently.}

What are sRNAs?

Small regulatory RNAs that bind complementary sequences on mRNA and control translation or mRNA stability.}

How do sRNAs repress translation?

1. An sRNA base-pairs with the mRNA near or over the Shine-Dalgarno sequence. 2. This blocks the ribosome-binding site. 3. The ribosome cannot bind efficiently, so translation decreases.}

How do sRNAs activate translation?

1. An sRNA binds a region of mRNA that forms an inhibitory hairpin. 2. Base-pairing disrupts the hairpin. 3. The Shine-Dalgarno sequence becomes exposed. 4. The ribosome can bind, increasing translation.}

How can sRNAs change mRNA stability?

An sRNA can protect mRNA from RNase degradation, increasing its stability, or promote RNase access and mRNA degradation, decreasing its stability.}

Why can sRNAs regulate protein production without changing transcription?

They act after mRNA has been produced by changing whether ribosomes can translate it or whether the mRNA survives long enough to be translated.}

How does Staphylococcus aureus use multiple regulation mechanisms?

S. aureus combines quorum sensing, a two-component system, transcriptional regulation, and sRNA regulation. As AIP accumulates, AgrC/AgrA signaling activates gene transcription, including RNAIII production. RNAIII then changes translation and stability of target mRNAs to alter virulence-factor production.}

How is the Agr signal produced in S. aureus?

1. AgrD is produced as an AIP precursor. 2. AgrB processes AgrD into the mature autoinducing peptide (AIP). 3. AgrB also helps export AIP outside the cell.}

How does S. aureus detect AIP?

1. AIP accumulates outside cells as population density increases. 2. AIP binds AgrC, a membrane sensor histidine kinase. 3. AgrC autophosphorylates. 4. AgrC transfers phosphate to AgrA.}

What does phosphorylated AgrA do?

Phosphorylated AgrA activates the Agr P2 and P3 promoters. P2 increases expression of agr genes, while P3 produces RNAIII.}

What is RNAIII?

A regulatory small RNA produced by the Agr system that controls translation and stability of target mRNAs involved in virulence.}

How does RNAIII affect virulence-factor production in S. aureus?

RNAIII base-pairs with target mRNAs, changing their translation or stability. It generally reduces some surface-associated proteins and promotes production of secreted toxins and other virulence factors.}

How does RNAIII affect Rot?

RNAIII represses translation of Rot, a protein that normally represses toxin production. Less Rot allows toxin expression to increase.}

Why is the Agr system an example of multiple levels of regulation?

It combines AIP-based quorum sensing, AgrC/AgrA two-component signaling, transcriptional activation by AgrA, and post-transcriptional regulation by RNAIII.}

What is Gram-negative quorum sensing?

A common bacterial communication system using acyl-homoserine lactone (AHL) signals to detect population density and coordinate gene expression.}

How does Gram-negative AHL quorum sensing work?

1. LuxI-type enzymes produce AHL signals. 2. AHL diffuses across bacterial membranes and accumulates as cell density increases. 3. AHL binds a LuxR-type receptor inside the cell. 4. The AHL-LuxR complex binds regulatory DNA. 5. Target genes are activated or repressed.}

Why does AHL concentration increase as bacterial population density increases?

More bacterial cells produce more AHL. As the population grows, the signal accumulates until it reaches a concentration sufficient to activate the response.}

What is Gram-positive quorum sensing?

A bacterial communication system that commonly uses secreted peptide autoinducing signals (AIPs) to detect population density.}

How does Gram-positive peptide quorum sensing work?

1. Bacteria produce peptide signal precursors. 2. The precursors are processed into mature peptides and exported. 3. Peptides accumulate outside cells as population density increases. 4. A peptide binds a membrane sensor histidine kinase. 5. The kinase autophosphorylates and transfers phosphate to a response regulator. 6. The response regulator changes gene expression.}

What is the main difference between Gram-negative and Gram-positive quorum sensing?

Gram-negative bacteria commonly use AHL signals that bind cytoplasmic LuxR-type receptors. Gram-positive bacteria commonly use secreted peptide signals that bind membrane histidine kinases.}

Why is iron important to bacteria?_Iron is needed for essential enzymes, electron transfer, redox reactions, and processes such as the electron transport chain. It helps proteins transfer electrons during cellular metabolism.}

Why is iron difficult for bacteria to obtain inside a host?_The host uses nutritional immunity to keep free iron extremely low. Iron is bound to host proteins, making it unavailable for bacterial growth.}

What is nutritional immunity?_The host's defense strategy of restricting essential nutrients, especially iron, to limit microbial growth.}

Which host proteins restrict iron availability?_Transferrin binds iron in the blood; lactoferrin binds iron in secretions and tissues. Both reduce the amount of free iron available to bacteria.}

What are four ways bacteria acquire iron from eukaryotic hosts?_1. Proteases degrade host iron-binding proteins. 2. Siderophores bind iron. 3. Iron reduction converts Fe³⁺ into Fe²⁺. 4. Specific receptors take up iron directly from host proteins.}

How do bacterial proteases help acquire iron?_Proteases break down host iron-binding proteins, such as transferrin and lactoferrin, helping release their bound iron so bacteria can access it.}

How do siderophores help bacteria acquire iron?_Bacteria secrete siderophores that bind iron with high affinity. The iron-bound siderophore can then be recognized by bacterial receptors and transported into the cell.}

How does iron reduction help bacteria acquire iron?_Bacteria reduce Fe³⁺ to Fe²⁺. Fe²⁺ is generally more soluble and accessible, making it easier for bacteria to acquire.}

How can bacteria directly acquire iron from host proteins?_They produce specific receptors that bind iron-containing host proteins, such as transferrin or lactoferrin, allowing the bacteria to obtain the bound iron.}

What is a siderophore?_A secreted molecule with a very high affinity for iron, especially Fe³⁺, that captures iron from the environment.}

Why do siderophores have a high affinity for iron?_Iron is tightly controlled by the host and often unavailable. High-affinity siderophores allow bacteria to compete for and capture even very limited iron.}

What is a ferri-siderophore?_A siderophore bound to iron; the iron-bound complex is recognized by bacterial uptake receptors.}

How does Pseudomonas aeruginosa acquire ferri-pyoverdine?_1. The bacterium secretes pyoverdine. 2. Pyoverdine binds Fe³⁺ outside the cell. 3. Ferri-pyoverdine binds the outer membrane receptor FpvA. 4. TonB-dependent transport moves the complex across the outer membrane.}

What is FpvA?_A TonB-dependent outer membrane receptor in Pseudomonas aeruginosa that recognizes and binds ferri-pyoverdine.}

How does ferri-pyoverdine binding change FpvA?_Ferri-pyoverdine binds FpvA and causes a conformational change in the receptor. This helps initiate TonB-dependent transport and activates signaling through FpvA's signaling domain.}

What is TonB's role in siderophore transport?_TonB transfers energy from the inner membrane ExbB/ExbD complex to the outer membrane receptor FpvA, allowing energy-dependent transport across the outer membrane.}

What are ExbB and ExbD?_Inner membrane proteins that work with TonB to harness the proton motive force (PMF) and provide energy for outer membrane transport.}

How does the proton motive force power siderophore transport?_1. ExbB/ExbD use the energy stored in the proton motive force. 2. This energizes TonB. 3. TonB interacts with FpvA. 4. TonB transfers energy to FpvA, causing receptor changes that allow ferri-siderophore transport.}

What is the order of ferri-pyoverdine transport across the outer membrane?_1. Pyoverdine binds Fe³⁺. 2. Ferri-pyoverdine binds FpvA. 3. FpvA changes conformation. 4. ExbB/ExbD use the PMF to energize TonB. 5. TonB interacts with FpvA and transfers energy. 6. FpvA undergoes changes that facilitate transport across the outer membrane.}

What does the FpvA signaling domain do?_It detects ferri-pyoverdine binding through a conformational change and initiates signaling that increases expression of pyoverdine-related genes.}

When does the FpvA signaling domain move or change conformation?_When ferri-pyoverdine binds FpvA. The resulting receptor conformational change transmits a signal to the regulatory pathway.}

How does FpvA signaling increase pyoverdine-related gene expression?_1. Ferri-pyoverdine binds FpvA. 2. FpvA changes conformation and initiates signaling. 3. The signal relieves FpvR inhibition of PvdS. 4. PvdS associates with RNA polymerase. 5. PvdS activates transcription of pyoverdine-related genes.}

What is FpvR?_An anti-sigma factor that normally inhibits PvdS. FpvA signaling relieves this inhibition, allowing PvdS to activate gene expression.}

What is PvdS?_An alternative sigma factor that associates with RNA polymerase and activates transcription of pyoverdine-related genes.}

How are siderophore and outer membrane receptor levels regulated by iron?_Low iron increases their expression because the bacterium needs to acquire more iron. High iron activates Fur, which represses iron-acquisition genes and decreases their expression.}

What is Fur's role during high iron availability?_1. Fe²⁺ binds Fur. 2. Iron activates Fur's DNA-binding ability. 3. Fur binds regulatory regions of iron-acquisition genes. 4. Transcription is repressed, reducing siderophore and receptor production.}

What happens to Fur when iron is low?_Fur lacks its iron cofactor and cannot effectively bind regulatory DNA. Repression is removed, so iron-acquisition genes are expressed and siderophore production increases.}

Why don't bacteria continuously express iron-acquisition systems?_When iron is abundant, additional uptake is unnecessary and could cause toxic iron accumulation. Fur represses uptake genes to conserve energy and prevent iron toxicity.}

Why is excess iron toxic to bacteria?_Free iron promotes reactive oxygen species (ROS) formation. These highly reactive molecules damage DNA, proteins, and membrane lipids.}

How does iron produce reactive oxygen species?_Fe²⁺ reacts with hydrogen peroxide through the Fenton reaction, producing Fe³⁺, hydroxide, and a highly damaging hydroxyl radical (•OH).}

What is the Fenton reaction?_Fe²⁺ + H₂O₂ → Fe³⁺ + OH⁻ + •OH. The hydroxyl radical can damage DNA, proteins, and membrane lipids.}

Why are hydroxyl radicals dangerous?_They are highly reactive and can damage cellular molecules, disrupting DNA integrity, protein function, and membrane structure.}

Are siderophores found in Gram-positive, Gram-negative, or both?_Both Gram-positive and Gram-negative bacteria can produce siderophores.}

How do Gram-negative bacteria transport siderophores?_They can use TonB-dependent outer membrane receptors to move ferri-siderophores across the outer membrane, followed by transport across the inner membrane.}

How do Gram-positive bacteria transport siderophores?_They lack an outer membrane, so they use cell-surface binding proteins and membrane transport systems to bring iron-bound siderophores into the cell.}

Why don't Gram-positive bacteria use TonB-dependent outer membrane receptors?_They do not have an outer membrane. Their siderophore uptake systems instead use surface-associated and membrane transport proteins.}