Chapter 14: Virulence Gene Regulation

• Bacterial pathogens use strategies to control their disease-causing potential while waiting for opportunities to activate virulence mechanisms.

Virulence Gene Regulation

• Pathogens adapt to different environmental conditions inside and outside the host by sensing signals and inducing or repressing specific genes.
• Environmental signals include temperature, pH, iron abundance, carbon/nitrogen sources, signaling compounds, osmolarity, oxygen, carbon dioxide, and light.
• Genes regulated in response to the host environment are virulence genes, often regulated positively or negatively, concomitantly or sequentially.

Mechanisms of Regulation

• In bacterial operons, genes are transcribed together as a single mRNA from a single promoter and are typically regulated and expressed together.
• In regulons, genes at different locations on the chromosome have promoters controlled by the same regulatory signaling protein (regulator).
• Coordinate regulation involves regulating multiple genes in response to a condition or signal.
• Global regulators control a broad range of physiological processes through a large number of genes.
• The PrfA protein positively regulates transcription of virulence genes in Listeria monocytogenes.
• The PrfA regulon includes three multigene operons and several single-gene operons, such as plcA-prfA, mpl-actA-plcB, hly, inlA-inlB, inlC, and hpt.

Activators and Repressors

• Transcription factors bind to DNA sequences to control gene expression.
• Transcriptional activators stimulate gene expression by recruiting RNA polymerase, sometimes requiring a coactivator, ligand, or modification.
• Transcriptional repressors bind to DNA sequences (operators) and block RNA polymerase, sometimes requiring a corepressor, ligand, or modification.
• The lac operon of Escherichia coli regulates genes for lactose transport and metabolism.
• The lac operon consists of lacZ, lacY, and lacA under a single promoter and LacI repressor control.
• Catabolite repression, controlled by cyclic adenosine monophosphate (cAMP), represses genes for secondary carbon sources when glucose is present.
• cAMP activates the cAMP activator protein (CAP), recruiting RNA polymerase.
• LacI repressor binds to the operator in the absence of lactose, preventing transcription; allolactose releases LacI, enabling transcription.
• Iron regulates virulence genes through iron-binding transcriptional repressors like the ferric uptake repressor (Fur) of E. coli.
• Diphtheria toxin gene (tox) expression in Corynebacterium diphtheriae is regulated by the DtxR repressor.
• The Fe2+-DtxR complex represses tox gene transcription when iron levels are high; low iron levels allow transcription.
• Staphylococcus aureus senses its metabolic state through the catabolite control protein E (CcpE), a LysR-type transcriptional regulator.
• CcpE regulates virulence factors like staphyloxanthin, staphyloferrin, capsular polysaccharide, superantigens, fibronectin-binding proteins, and exoproteases.
• CcpE activates citB transcription, encoding aconitase, involved in citrate to isocitrate conversion in the TCA cycle.
• Deletion of citB diverts acetyl-CoA flux to the mevalonate pathway, increasing pigment biosynthesis and staphyloferrin production.

Two-Component Regulatory Systems

• Two-component regulatory systems (TCSs) control many bacterial virulence and metabolic genes.
• A TCS consists of a sensory histidine kinase and a response regulator.
• Histidine kinases sense signals and autophosphorylate histidine residues.
• Response regulators recognize phosphoryl-histidine and transfer the phosphoryl group to an aspartate residue.
• Phosphorylation of the response regulator modulates cellular processes, usually through transcriptional activation or repression.
• Many histidine kinases have phosphatase activity to remove phosphoryl groups, fine-tuning responses.
• Some TCSs involve multiple phosphorylation steps in a phosphorelay.
• The BvgAS system in Bordetella pertussis is a phosphorelay, acting as a global regulator of virulence genes.
• BvgAS regulation has three phases: Bvg+ (active BvgS, phosphorylated BvgA, virulence gene expression), Bvg– (inactive BvgS, unphosphorylated BvgA), and Bvgi (intermediate levels of modulating signals).

Sigma Factors

• Sigma (σ) factors are subunits of RNA polymerase, directing it to specific DNA promoter sequences.
• Sigma factors regulate the expression of a large number of bacterial genes.
• Examples include σ70 (housekeeping genes) and σ38 (stress responses) in Gram-negative bacteria.
• Bacteria have varying numbers of sigma factors; each RNA polymerase complex contains only one at a time.
• Anti-sigma factors regulate sigma factors posttranslationally by sequestering them.
• Anti-anti-sigma factors negatively regulate anti-sigma factors.
• In Pseudomonas syringae, the type III secretion system (T3SS) is controlled by HrpL, which is regulated by σ54.
• The PrfA regulon in L. monocytogenes is controlled by σB under stress conditions and then positively regulates itself using σ70 RNA polymerase.
• Mycobacterium tuberculosis has a complex regulatory cascade with multiple sigma factors, each with positive feedback loops and anti-sigma factors.

Transcriptional Terminators and Antiterminators

• Bacteria control transcription termination using Rho-dependent and Rho-independent mechanisms.
• Rho-dependent terminators involve Rho (ρ) factor binding to mRNA and disrupting the RNA polymerase complex.
• Rho-independent terminators involve hairpin loops in the mRNA, causing RNA polymerase to stall.
• Antiterminators bypass terminators, enabling transcription of additional genes.
• Some antiterminators prevent terminator hairpin formation by stabilizing alternative secondary structures.
• Riboswitches sense metabolites and control biosynthesis of the ligand they sense.
• Ribosomes trailing RNA polymerase can act as antiterminators, with translation influencing mRNA secondary structure.
• Attenuation regulates amino acid biosynthesis in Gram-negative bacteria.
• T-boxes in Gram-positive bacteria bind to uncharged tRNAs and prevent terminator hairpin formation.

Regulation of Translation Initiation

• RNA secondary structures regulate translation by sequestering ribosome-binding sites (RBSs).
• Secondary structures can be altered through small molecule ligand riboswitches or binding of regulatory proteins.
• Thermostable riboswitches sense temperature changes, forming stable secondary structures at low temperatures.
• LcrF expression in Yersinia pestis is regulated by a thermosensor, with more expression at 37°C than at lower temperatures.
• Translation of PrfA in Listeria is increased at higher temperatures due to an RNA thermosensor.

Regulatory Small RNAs

• Small RNAs (sRNAs) regulate cellular processes, ranging from 50 to 500 nucleotides in length.
• sRNAs can bind directly to mRNA transcripts (base-pairing sRNAs) or to DNA/RNA-binding proteins (protein activity-modifying sRNAs).
• Base-pairing sRNAs bind to mRNA transcripts to inhibit translation or enhance translation by preventing inhibitory secondary structures.
• Hfq protein in Gram-negative bacteria serves as a chaperone for sRNAs.
• DsrA from E. coli negatively regulates translation of H-NS and positively regulates translation of RpoS.
• Antisense sRNAs are transcribed from the antisense strand and regulate the gene they arise from; an example is RNAβ in Vibrio anguillarum.
• Protein activity-modifying sRNAs modify protein activity by binding to target proteins.
• CsrA family of translational repressors bind to 5′ upstream regions of regulated mRNAs, preventing ribosome access and regulated by sRNAs CsrB and CsrC.
• RsmA in Pseudomonas aeruginosa controls switching between acute and chronic infection by negatively regulating chronic infection genes, negatively regulated by RsmY and RsmZ.
• Gac/Rsm pathway enables large sets of genes to be coordinately activated in response to multiple environmental signals through the two-component system GacSA and two associated sensor histidine kinases, RetS and LadS.

Bacterial Chromatin

• Bacterial chromosomes are organized into bacterial chromatin or the nucleoid.
• Nucleoid-associated proteins (NAPs) bind chromosomal DNA, shaping chromatin structure and regulating gene expression.
• H-NS protein of E. coli binds to AT-rich regions of DNA, interfering with gene transcription.
• Enteropathogenic E. coli (EPEC) carries the locus of enterocyte effacement (LEE) pathogenicity island, silenced by H-NS at ambient temperatures.
• Ler, a paralog of H-NS, relieves H-NS silencing of the remaining LEE operons at higher temperatures.

Responding to Environmental Signals

• Bacteria respond to their external environment through various regulatory mechanisms.
• Phase variation involves heritable and reversible phenotypic switching in bacterial populations.
• Antigenic variation, a form of phase variation, leads to variability in bacterial components presented to the host immune system which presents a challenge for vaccines.
• Bistable switches, regulatory mechanisms, control the transition between two phenotypic states.
• In Bacillus subtilis, competence is controlled by ComK, which drives its own expression.
• Positive feedback loops and mutually repressing repressors can create bistable switches: CI and Cro proteins in bacteriophage λ.
• Bistable switches control sporulation, biofilm formation, motility, and virulence gene expression.

Hypermutability, Intragenomic Recombination, and Positive Selection

• Bacterial genomes undergo genomic rearrangements (genomic plasticity) that generate variability.
• Helicobacter pylori exhibits high diversity due to nonrandom, repetitive sequences and intragenomic recombination.
• Gene duplication and amplification (GDA) events, intragenomic recombination, result in multiple copies of a gene.
• GDA events enable adaptation to environmental stresses, particularly antibiotics, through increased production of antibiotic-modifying enzymes, drug efflux pumps, or molecular targets of the antibiotics.

Coordinate Virulence Regulation

• Bacterial pathogens coordinate virulence through complex regulatory networks.
• Vibrio cholerae's virulence gene regulation revolves around ToxT activation and is a transcriptional activator.
• ToxT controls ctxAB, tcp, acf, tag virulence genes, and regulatory sRNAs TarA and TarB.
• ToxT expression depends on pH, oxygen levels, nitric oxide, osmolarity, temperature, catabolite levels, mucus, and bile salts, sensed by regulators.

Quorum Sensing

• Bacteria coordinate activities through autoinducers, resulting in cell-cell communication.
• At high cell densities, autoinducer concentrations increase, activating receptors.
• Quorum sensing involves direct or indirect activation of a cognate receptor protein by the autoinducer, which in turn directly or indirectly upregulates or downregulates a specific set of genes.
• It affects many pathogens, coupling virulence factors to bacterial population density.
• Enables coordinately regulated expression: bioluminescence, sporulation, DNA conjugation, toxins, adhesins, flagella, capsules, siderophores, antibiotic resistance, biofilm formation, and secretion systems.
• Gram-negative bacteria use N-acyl homoserine lactones (AHLs), while Gram-positive bacteria use small posttranslationally modified peptides called autoinducing peptides (AIPs).
• Autoinducer-2 (AI-2) is used for interspecies cell-cell communication, indicating the presence of competing for colonization and growth.

The Lux System of Vibrio fischeri

• Quorum sensing first described in Vibrio fischeri.
• V. fischeri bioluminescence results from transcriptional activation of the bacterial Lux operon, luxCDABEG
• V. fischeri bioluminescence results from transcriptional activation of the bacterial Lux operon, luxCDABEG
• AinR and LuxPQ are sensor kinases responding to C8-HSL and AI-2, respectively, linked to LuxU, LuxO, Qrr1, and LitR regulators.

The Agr System of S. aureus

• Staphylococcus aureus's Agr system is a quorum-sensing system elucidated in Gram-positive bacteria.
• agr locus controls virulence factors, comprising agrAC (two-component signaling), agrDB (ligand biosynthesis and export), and RNAIII (regulatory sRNA).
• Mature AIPs interfere with signaling through variations in AgrB and AgrC.

Chemotaxis

• Bacteria actively seek favorable environments through chemotaxis.
• Bacteria bias movement toward chemoattractants and away from chemorepellents.
• E. coli chemotaxis system is well-understood.
• Phosphorylated CheY response regulator protein binds flagellar motor, switching flagellar rotation to clockwise.
• CheA, a histidine kinase, associates with chemoreceptors via CheW. CheZ is a phosphatase.
• CheR methylates glutamate in chemoreceptors, increasing the CheA kinase.