Bacterial Pathogenesis: Virulence Factors

• Recombinant DNA technology has enabled the discovery of many virulence-associated genes in pathogenic bacteria.
• Molecular genetic technologies have accelerated the discovery of virulence genes and previously unsuspected virulence traits.
• Next-generation sequencing, automation, and bioinformatics are transforming bacterial pathogenesis research.
• Comparative genomic analysis of virulent and avirulent bacterial strains reveals many new virulence factor candidates.
• Characterizing new candidates, annotating gene functions, and determining their roles in pathogenesis are current research challenges.
• Combining multi-omics with classical biochemical and genetic techniques is effective for identifying virulence factors and understanding their functions.
• Genetic and biochemical analyses alone are insufficient for understanding how information plays out in the living cell.

Biochemical Approaches

• Bacterial protein toxins were among the first virulence factors identified.
• Purification methods for toxins include filtration, centrifugation, selective precipitation, and chromatography.
• Purified toxins can be reintroduced into animals or host cells to reproduce disease symptoms.
• Diphtheria toxin (Corynebacterium diphtheriae) and cholera toxin (Vibrio cholerae) are historical examples.
• Separation methods separate proteins based on intrinsic charge through ion exchange chromatography
• Size exclusion or gel filtration chromatography separates proteins based on molecular size
• Affinity chromatography separates proteins using specific affinity tags or ligands.
• Metal-chelation chromatography separates proteins based on the binding affinity of a recombinant protein.
• Friedrich Loeffler discovered diphtheria toxin in 1884.
• Émile Roux and Alexandre Yersin showed cell-free bacterial culture filtrates could kill animal cells.
• Emil von Behring developed diphtheria antitoxin from horse serum and received the Nobel Prize in 1901.
• Anna Wessels Williams and William H. Park improved diphtheria antitoxin production.
• Vaccination with inactivated diphtheria toxin (toxoid) provides protection.
• John Snow connected cholera to contaminated water in 1854.
• Robert Koch identified V. cholerae as the cause of cholera in 1883.
• Cholera toxin affects intestinal cell signaling without damaging the cells.
• Intradermal or intramuscular injections with the cholera toxin leads to a humoral (IgG antibody) immune response
• Cholera toxin produces diarrhea specifically in intestinal cells.
• Deleting the cholera toxin gene reduces virulence in V. cholerae.
• A rabbit ileal loop assay was used to demonstrate that cholera toxin is responsible for cholera diarrhea.
• Other secreted proteins, like proteases, nucleases, glycosidases and lipases, can be isolated via biochemical methods.
• Mass spectrometry can determine a protein toxin's amino acid sequence.
• The toxin gene can be cloned and expressed in a laboratory strain like E. coli.
• Affinity tags can be added to cloned proteins to facilitate purification.
• Other virulence factors include cell surface glycolipids or carbohydrates and secreted molecules.
• Nuclear magnetic resonance (NMR), mass spectrometry, and X-ray crystallography can determine the structure of carbohydrates, lipids, and other secondary metabolites.
• Pseudomonas aeruginosa produces phenazine-derived compounds with antibiotic properties and host toxicity.
• Organic solvents and crystallization were used to first isolate these compounds.
• High-pressure liquid chromatography (HPLC) is a current biochemical separation method.
• Mycobacterium ulcerans causes Buruli ulcers, which are associated with little pain or inflammation.
• Mycolactones, lipid-like polyketide-derived macrolides, are the toxins responsible for the disease.
• NMR and mass spectrometry determined the structure of mycolactones.
• The biosynthetic genes were identified based on their similarity to other polyketide biosynthetic genes.
• A 174-kb plasmid in M. ulcerans contains gene clusters for mycolactone production

Molecular Genetic Approaches

• Cloning genes from a pathogen into an avirulent strain can identify virulence traits.
• Cloning the toxin gene from a pathogenic strain into a nontoxigenic E. coli strain can demonstrate virulence.
• Screening E. coli clones containing Salmonella DNA for adherence and invasion can identify Salmonella adhesins and invasins.
• Introducing the hlyA gene into a laboratory strain of Bacillus subtilis can facilitate its escape from the phagolysosomal degradation pathway.
• Cloning is limited to small portions of the bacterial genome and requires expression in E. coli.
• Reporter genes can identify virulence genes based on differential regulation under selective conditions.
• Reporter fusions (operon fusions) link the regulatory circuit of a virulence gene to a reporter gene.
• Reporter genes include antibiotic resistance, colored pigment production, luminescence, and fluorescence.
• A transcriptional reporter gene system fuses a virulence gene promoter/operator to a promoterless reporter gene.
• Transcription initiation and regulation from the vir P/O promoter is reflected in the expression of the reporter gene.
• A translational reporter gene system fuses a virulence gene promoter/operator and the vir gene RBS and AUG start codon to the reading frame of the reporter gene.
• Transcription and translation control of the vir gene is represented in the expression of the reporter gene.
• Translational fusions with phoA can identify secreted or surface-expressed virulence factors.
• PhoA is active when secreted onto the surface in the periplasm or medium
• β-galactosidase activity is readily detected using chromogenic substrates like indolyl-galactoside (X-gal).
• Lactose-MacConkey agar contains bile salts, an important signal in vivo for gastrointestinal pathogens.
• Careful attention must be given to the medium and growth conditions used during experiments to determine whether putative virulence genes are expressed in vitro as well as in vivo.
• Translational fusions can provide information about subcellular location and secretion properties of expressed virulence genes
• Translational fusion reporters can study post-transcriptional regulation of virulence genes.

Mutagenesis Screening

• Mutagenesis links specific DNA sequence elements to measurable phenotypes.
• Mutagenesis involves observing a phenotype, mutagenizing the organism, isolating mutants lacking the phenotype, and identifying the DNA changes.
• The phenotype of interest is the ability to produce toxic compounds or colonize and/or cause disease.
• Individual mutants are tested for virulence or screened for toxic compounds.
• P. aeruginosa produces toxic phenazine-based compounds that screens were used to identify biosynthetic pathways for.
• Mutagens include chemical mutagens (EMS, ethidium bromide), UV irradiation, and transposons.
• Chemical mutagenesis and UV irradiation are less biased.
• Identifying altered genes in mutants was labor-intensive but is now easier with whole-genome sequencing.
• Spontaneous mutations can be rapidly identified by whole-genome sequencing.
• Transposon insertion mutagenesis is another widely used strategy for identifying virulence genes.
• Insertion mutants are screened for loss of virulence
• Transposons carry a selectable marker (antibiotic resistance gene) or a reporter gene.
• Transposon insertion disrupts a gene in a library of mutants.
• Current approaches make it easier to identify disrupted genes than older genetic mapping methods.
• The approach is important for bacterial species lacking sophisticated genetic mapping tools.
• Transposons can carry transcriptional terminators and have polar effects
• Aberrant increased expression from genes downstream of transposon insertion sites can cause phenotypes.
• Genetic complementation tests may be performed, where a wild-type copy of the transposon-disrupted gene is reintroduced at a second location.
• Insertions can only be obtained in genes not essential for growth in vitro.
• Separate bacterial genes with redundant functions can lead to no observable phenotype when one copy is knocked out
• Tn-Seq can identify functionally redundant genes
• Transposons carrying a promoterless lacZ are often used to locate genes that respond to specific conditions or signals.
• Colonies with transposon insertions are screened for regulated expression of lacZ
• Regulatory genes that control the expression of putative virulence genes can also be found using transposons.
• After tagging a gene with a lacZ fusion or transposon insertion, it can be identified by sequencing.
• Illumina and PacBio sequencing technologies enable low-cost, high-coverage whole-genome sequencing.
• Nanopore sequencing technology is a low-cost, portable sequencing platform.
• Nanopore sequencers can be used to identify microbes and generate draft scaffold genome sequences

Genome-wide Sequencing Approaches for Identifying Virulence Genes

• Tn-Seq is a powerful approach for discovering in vivo-expressed virulence genes.
• Tn-Seq studies the requirement of specific genes for bacterial survival in certain environmental niches.
• Tn-Seq combines transposon mutagenesis with in vivo selection.
• Mutants are pooled, grown in vitro, and used to inoculate an animal.
• Infected organs or blood are harvested, and the bacteria growing in it are recovered.
• DNA is extracted and sequenced to determine the relative ratio of mutants in the input and output pools.
• Sequencing reads are initiated from the edge of the inserted transposons to the adjacent bacterial chromosome.
• Tn-Seq can determine nonessential genes that were disrupted
• Genes that cannot tolerate disruption when the bacteria are grown in vivo are required for host colonization or survival.
• These putative virulence genes are involved in processes ranging from in vivo nutrient acquisition to gene regulation to adherence of toxin production.
• Mutant bacteria can be constructed with deletions in putative virulence factors for comparative studies of virulence properties.
• The technique is fast, applicable to a wide range of bacteria, and requires only a small number of host animals.
• Tn-Seq is a negative selection method for identifying putative virulence genes.
• Genes that suppress pathogenesis cannot be identified via Tn-Seq.
• Standard Tn-Seq can only be used to identify virulence genes with nonredundant functions.
• Secreted factors can be complemented in trans, masking the essentiality of certain virulence factors.
• Determining the fitness cost of knocking out a bacterial gene in an animal model is complicated.
• Bacterial generation time in vivo needs to be calculated, and bottlenecks caused by random stochastic processes need to be considered.
• RNA-sequencing (RNA-Seq) is a preferred method for genome-wide identification of bacterial genes expressed during infection.
• Differentially regulated genes are candidates for virulence factors and possible targets for drug or vaccine development.
• RNA-Seq technology can analyze and profile the relative quantity of mRNA in a biological sample at any given time or condition.
• Dual RNA-Seq technology involves optimized sample preparation and quantitation of relative transcript amounts from pathogens and host cells together simultaneously.
• Fluorescently labelled bacteria are allowed to infect host cells
• Host cells are then sorted by fluorescence-activated cell sorting (FACS) to enrich for a population of bacterially infected host cells.
• Total RNA is extracted from the bacterially infected host cells, and the highly abundant host and bacterial rRNAs are removed.
• The remaining RNA is converted into cDNA and sequenced
• Dual RNA-Seq allows researchers to follow the dynamic interaction networks occurring within and between pathogens and hosts during the course of an infection.

Comparative Genomic Sequence Analysis for Identifying Virulence Genes

• By comparing the genomes of pathogenic and nonpathogenic strains of the same bacterial species, researchers can identify genes exclusively found in pathogenic strains but not in their nonpathogenic relatives.
• Strain-specific genes represent strong candidates for being virulence factors.
• Similar comparative genomic approaches can also be applied toward identifying genes associated with other biological properties, such as adhesion, metabolite production, and antibiotic resistance.
• Microbial genome sequence information and the ability to analyze the expression activity of every gene in a cell are powerful tools that are accelerating vaccine development
• All potential antigens can be identified sing software that predict secreted or expressed cell surface proteins.
• These putative vaccine targets are then expressed as recombinant proteins in E. coli.
• Purified proteins are used to immunize mice where results are screened and tested for their ability to provide protective immunity.
• Newly available methods allow for much faster sequencing of whole bacterial genomes

Proteomics Approaches for Identifying Virulence Factors

• Proteomics technologies include analytical instrumentation with high-throughput separation and enhanced sensitivity capabilities.
• There has been an explosion in recent years of vast amounts of structural and functional data and information about bacterial proteins and their analytical properties.
• Tools are enabling the identification of proteins and changes in protein properties and profiles during infection.

Protein Microarrays (Proteoarrays)

• Microarray technology has been extended to proteins.
• After the genome sequence of a pathogen is determined, every protein reading frame is cloned into an expression vector.
• Corresponding proteins are expressed using an E. coli-based cell-free in vitro transcription-translation system.
• Expressed proteins are printed onto nitrocellulose membrane microarrays to generate protein microarrays (proteoarrays).
• The phage display approach can be used to express the proteins from the pathogen.
• Proteoarrays are used to determine the antibody-binding profiles of serum from vaccinated humans or animals.
• Antibody-binding profiles can identify cross-reactive antigens that might serve as potential vaccine candidates.
• The antibodies against a particular antigen can be covalently immobilized on a microarray glass surface to generate an antibody proteoarray.
• Biotinylated protein antigens from patients, vaccines, and infections are screened.
• Biotinylated antigens can be screened for the antigen(s) of interest from vaccinated, convalescent, or naïeve patients.
• Whole-proteome microarrays of Chlamydia trachomatis were generated from genomic DNA using cell-free protein expression and PCR amplification directly on a chip.
• Bacterial protein antigens were used for the immunoprofiling of patients to identify antigens binding to disease-related serum antibodies.

In Vivo-Induced Antigen Technology (IVIAT)

• IVIAT is an antibody-based genomic method that can be used to identify genes induced during human infections.
• IVIAT avoids the use of animal infection models.
• Pooled serum from patients with a protective immune response is absorbed with cells or extracts from bacteria grown in vitro.
• The remaining serum contains antibodies reactive against in vivo-induced (IVI) antigens.
• An expression library of E. coli clones that express genes from the pathogen is probed with the serum.
• Clones expressing proteins that are cross-reactive with the remaining antibodies in the serum are identified as putative IVI antigens.
• IVIAT identifies virulence factors involved in human infection and for identifying virulence factors of human-specific pathogens for which there is no suitable animal model.
• Antigenic fingerprinting occurs when the serum is not pre-absorbed.
• Antigenic fingerprinting identifies antigenic surface proteins that are expressed both in vitro and in vivo, including essential surface proteins involved in cell division and signaling.
• IVIAT technologies have also been combined with protein microarrays for determining the complete antigen-specific humoral immune response profile from vaccinated or infected humans and animals.

The Importance of Understanding Bacterial Physiology

• S. typhimurium experiments screened transposon-generated mutants for the ability to survive and grow inside macrophages.
• At least 200 genes were involved in the ability of S. typhimurium to survive in macrophages, and these genes were scattered all over the bacterial chromosome.
• Genome sequence comparisons found genes important for virulence in one strain also located in a nonvirulent strain of the same species.
• Genes identified in approaches have proven to be housekeeping genes or stress response genes.
• The entire physiology of the bacterium, not just a few genes, is important.
• Most gene products act in concert with other gene products in interaction networks.
• Clues gained from Tn-Seq or RNA-Seq should be followed up with other biochemical, analytical, or microscopy studies to determine the functional role of the gene in the context of its interaction network.
• About one-third of all genes are not recognizable.
• Most putative virulence genes have no significant matches in the known databases.
• It will take a new initiative to advance our knowledge of bacterial physiology to the point that the sequence information becomes truly decipherable
• There also needs to be a better understanding elucidated regarding the interactions between different pathways
• The expression of different virulence genes and the relationships of the proteins they encode are highly interactive.
• Bacterial physiology will be the field most likely to get at the essence of what it means to be a living organism.
• The genome sequence of Mycoplasma genitalium, which contains about 300 genes, stands as the simplest genome of a free-living organism.
• Scientists hope that by understanding the function and interactions of this small number of gene products, they might finally have an insight on what defines “life,” at least at the single-cell level.
• High-throughput methods coupled with robotic handling are making the functions of the unknown genes decipherable.
• Genome-wide methods using CRISPR-Cas9 interference show great promise in determining the functions, interactions, and redundancies of genes in a broad range of pathogens.