Phase and antigenic variations in bacteria
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This document discusses phase variation in bacteria. It begins by defining phase variation as a reversible switch in bacterial cells between expressing and not expressing proteins, which allows bacterial populations to adapt to changing environments without random mutations. It then describes 10 common phase variable phenotypes and molecular mechanisms of phase variation, including short sequence repeats, homologous recombination, and site-specific recombination. The document emphasizes that phase variation allows bacterial evasion of host immune responses by varying surface structures like capsules and proteins.
Phase and antigenic variations in bacteria
- 1. Dr. Dinesh Kr Jain, MD., Assistantprofessor, Department of Microbiology, SMS Medical college, Jaipur
- 2. CONTENTS Introduction and definition Phase variable phenotypes and moieties. Biological significance of phase variation Molecular mechanism- 1.Genetic regulation 2.Epigenetic regulation 3.Cell’s regulatory mechanism
- 3. PHASE VARIATION Method of dealing with rapidly varying environments without requiring random mutation. It involves the variation of protein expression . It’s a reversible switch between an “all-or-none”(on/off) expressing phase, resulting in variation in the level of expression of one or more proteins between individual cells of a clonal population. Majority of daughter cells will retain the expression phase of the parent but a minority will have switched expression phase. The switch can be influenced by external factors, the switching frequency can be modulated.
- 4. ANTIGENIC VARIATION Mechanism by which bacteria alters its surface protein in order to avoid host immune response. Bacteria will change the variety of cell surface molecules(proteins and carbohydrates) that can be detected by specific antisera. A single strain may express several antigenic variants of a cellular component. The genetic information for producing a family of antigenic variants is available in the cell, but only one variant is expressed at a given time.
- 5. Phase variation v/s classical mutation In phase variation genetic or epigenetic mechanism. reversible between generations. frequency of this reversion exceeds that of a random mutation. As high as a change in 1 cell/10 generation to 1cell/103 generation.
- 6. Phase variable phenotypes and moieties 1.Colony Morphology and Opacity 2.Capsule 3.Fimbriae and Pili 4.Flagella 5.Other Surface-Exposed Proteins 6.LPS and LOS Modification 7.DNA Restriction-Modification Systems(R/M) 8.DNA Restriction-Modification Systems 9.Regulatory Proteins 10.Metabolism associated genes
- 7. 1.Colony Morphology and Opacity Phenotypic variation in bacteria most commonly observed as colony variation & is associated with the virulence of the strain. Phase variation of a variety of surface-exposed proteins, capsule, cell wall - determines the colony morphology. Dry v/s moist, Ruffled v/s smooth, Opaque v/s translucent. 1.H.influenzae b- 3colony variants( opaque, intermediate, translucent) Due to variation in capsule production and cell envelope protein(encoded by oapA) ;determines the property of colonization in nasopharynx and serum resistance. 2.Streptococcus pneumoniae- opaque more virulent (produce more capsular protein and less teichoic acid) as compared to translucent.
- 8. 1.Colony Morphology and Opacity 3.S.gordonii- colony morphology as well as haemolysin production phase vary. 4.H.pylori-variation in enzyme phospholipase A increases the virulence due to release of ureases. 5.P.aerugenosa-colony variation determines the property of aggregation and motility. Color variation in colonies grown on specific media can be caused by phase variation of proteins that interact with a dye. Staphylococcus epidermidis
- 9. 2.Capsule The capsule can influence interactions with the host cells and host environment, including invasion, adhesion, and serum sensitivity, and is a well-recognized virulence factor. Phase variation of capsule synthesis has been found to occur in both gram-positive and gram-negative bacterial species, including Campylobacter jejuni, Citrobacter freundii, S. pneumoniae, and specific serogroups of Neisseria meningitidis. In Bacteroides fragilis, eight different capsule polysaccharides can be produced per cell. The expression of each is under the control of on/off phase variation. In H. influenzae type b cells, the level of expression of the capsule can be modulated, and an irreversible switch to a nonexpressing phenotype can occur.
- 10. 3.Fimbriae Fimbriae mediated attachment to the host cell is through interaction with receptors. These interactions occur either by structural subunit or by fimbrial adhesin with certain chemical groups of host protein or lipids. Attachment to inorganic solid surfaces by nonspecific intercations- biofilm formation. Phase variation of fimbriae is regulated mostly by mechanisms that affect transcription originating at the major promoter of the operon, resulting in variable (on/off) expression of genes in the fimbrial operon. The S.enterica serotype Typhimurium genome encodes at least 11 fimbrial operons - pef, lpf, fim phase vary. fimbriae in Proteus mirabilis is encoded by pap operon.
- 11. 4.Pili Type IV pili function as adhesins and are involved in interaction with eukaryotic cells, and thus these variations are important for pathogenesis . S. enterica serotype Typhi- phase-variable expression of the PilV affects the pilus-associated property of cellular auto-aggregation . N. gonorrhoeae can produce over a million different, antigenically distinct pilin subunits for its type IV pili, in addition pilus associated protein PilC also phase vary.
- 12. 5.Flagella Flagella mediate bacterial motility; adhesion and virulence are enhanced by flagellar expression and motility. Flagellin is recognized by innate immune system. It’s antigenic property forms a significant part of serological classification scheme. S. enterica serotype Typhimurium -biphasic antigenic variation between H1(FliC protein) and H2(FljB protein). Campylobacter coli-FlhA(flagellin), Helicobacter pylori-fliP(flagellar basalbody), Bordetella pertussis-BvgAS(regulatory system).
- 13. 6.Other Surface-Exposed Proteins Proteins that are integrated in the cell wall in gram-positive organisms or in the outer membrane in gram-negative organisms can have a variety of functions these proteins include. - Transporters, - Porins, - Receptors, - Colonizing factors, - Enzymes. Antigenic or phase variation can occur.
- 14. 6.Other Surface-Exposed Proteins S. pyogenes, expression of the cell wall-associated surface proteins C5a peptidase, M protein, type IIa IgG Fc receptor phase vary, as well as expression of the capsule and pyrogenic exotoxin. N. gonorrhoeae and N. meningitidis-family of outer membrane opacity proteins (opa) that facilitate adhesion. Mycoplasma gallisepticum- phase variation , whereas the homologous proteins in M. synoviae undergo antigenic variation . M. hyorhini , M.hominis, M.penetrans undergoes both phase variation and antigenic variation. E. coli, H. influenzae- phase var. Of colonizing factors, Borrelia spirochetes- multiphasic antigenic variation, Campylobacter fetus-Surface layer proteins(SLP) absence complement activity increased.
- 15. 7.LPS and LOS Modification: Variation in Expression of Surface Epitopes LPS consists - lipid A moiety(endotoxin) - a core of polysaccharide -O antigen-LPS variability among species and serotypes occurs. Modifications impact antigenicity, serum sensitivity and adhesion. Phase-variation between encapsulated (sialyltransferase ) and noncapsulated (glycosyltransferase) H. Influenzae. Helicobacter pylori – phase variation of 3 fucosyl transferase genes(futA,B,C) occurs, due to which variable carbohydrate modifications occur in LPS that resemble structures of the Lewis group of antigens of human blood groups.(host mimicry) Among Neisseria species ChoP expression shows phase-variable and can be expressed either in LPS(commensal) or typeIVpili(pathogenic). Legionella pneumophila, S. enterica serotype Typhimurium -Antigenic or phase variation of LPS.
- 16. 7.LPS and LOS Modification: Variation in Expression of Surface Epitopes In some species, the core lacks the multiple O-linked saccharide units and is often therefore referred to as lipooligosaccharide (LOS) Certain combinations of LOS modification may facilitate colonization or survival in different host environments. Ganglioside mimicry(GM1,GM2) of the LOS by Campylobacter jejuni is an important factor in the development of Guillain-Barre´ and Miller-Fisher syndromes. Expression of the enzymes involved in the modification can phase vary.
- 17. 8.DNA Restriction-Modification Systems Most phase variable genes are predicted to be involved in the biosynthesis of surface structures. Notable exceptions are genes encoding R/M enzymes. Phase-variable expression of R/M enzymes has been found in a variety of bacterial pathogens, including- S. Pneumoniae(modification system), M. Pulmonis(HsdS proteins), H. Pylori(type III R/M system), P. Haemolytica(type III modification system), and H. Influenzae(mod gene). Role of R/M enzyme variation is important during host pathogen interaction, but the actual significance is currently unclear. Phenotypic switching of putative R/M enzymes in H. pylori is induced on contact with human gastric cells. In M. pulmonis a high level of R/M enzyme (HsdS) variation is induced in vivo during infection of the rat trachea.
- 18. 9.Regulatory Proteins DNA binding proteins that function as activators or repressors can be categorized as- 1.Global regulators- virulence-associated regulatory protein, (Mga), BvgS protein in S. pyogenes, two-component BvgAS regulatory system in Bordetella pertussis. 2.Operon specific or Local regulators-E. coli, expression of the local regulator PapB(also affects type 1 fimbrial expression). The expression of multiple regulatory proteins is now known to phase vary and includes representatives of both groups.
- 19. 10.Metabolism associated genes Phase variation of metabolism-associated proteins was identified in the human pathogen Streptococcus pneumoniae. A comparison of protein expression patterns between two colony variants showed that at least three proteins were differentially expressed, pyruvate oxidase (SpxB), a putative elongation factor, and a proteinase maturation protein. SpxB phase variation is related to the hydrogen peroxide that is produced in conversion of the pyruvate to acetyl phosphate. The level is sufficiently high to be lethal to other species and may provide a SpxB isolate with a competitive advantage in a mixed- species environment -bacterial virulence strategy.
- 20. Molecular Mechanism Genetic Regulation a. Short sequence repeat and slipped strand mispairing- SSM b. Homologous recombination c. Site specific recombination Epigenetic Regulation a. PAP phase variation b. Ag43 phase variation Cell’s Regulatory Network a. cross regulation b. environmental regulation
- 21. SHORT SEQUENCES REPEAT AND SLIPPED-STRAND MISPAIRING • SSM is thought to be the most common mechanism of ON/OFF switching of contingency genes. • SSM is a process that produces mispairing of short repeat sequences between the mother and daughter strand during DNA synthesis i.e., DNA replication, repair and recombination. • During DNA synthesis template and nascent strand transiently separates from each other and then reanneal. • In reannealing step, nascent strand or the template strand can be slipped either in forward direction or in backward direction and can result in an increase or decrease in the number of short repeat sequences (1 to 7 nucleotides). • If the location of these repeats is such that either transcription or translation of a gene is affected it can lead to phase-variable expression of a protein.
- 22. SHORT SEQUENCES REPEAT AND SLIPPED-STRAND MISPAIRING Transcriptional regulation: 1. Promoter region 2. Other region 1.Regulation at the level of transcription occurs when the repeats are located in the promoter region between the -10 and -35 sites for RNA polymerase binding . • e.g. H. influenzae has two divergently oriented promoters and fimbriae genes hifA and hifB. The overlapping promoter regions have repeats of the dinucleotide TA in the -10 and -35 sequences. Through SSM the TA repeat region can undergo addition or subtraction which results in the reversible ON phase or OFF phase of transcription of the hifA and hifB.
- 23. SHORT SEQUENCES REPEAT AND SLIPPED-STRAND MISPAIRING Transcriptional regulation: 2. Transcription can also be affected by changes in repeat sequences located outside of the promoter, may be by affecting the binding of a regulatory protein or by affecting posttranscriptional event. • e.g. Phase variation of individual fimbrial genes in B. pertussis is proposed to occur as a result of a change in a poly(C) tract that alters the distance between the binding sites of an activator and RNA polymerase
- 24. SHORT SEQUENCES REPEAT AND SLIPPED-STRAND MISPAIRING Translational regulation: • Translation of a protein can be affected by SSM if the unit repeats are located within its coding sequence . • Open reading frame is disrupted if SSM results in a change in nucleotide number that is not a multiple of three. In this case, a non-functional, usually truncated protein is synthesized. • e.g. phase variation of the expression of the mod gene of H. influenzae, it contains over 30 repeats of the tetranucleotide (5 - AGTC) in its coding sequence . By addition of one tetranucleotide repeat within the coding sequence the reading frame is altered, and, in addition, a premature stop codon is formed. • To summarize, SSM can cause a change in the number of unit repeats consisting of 1 to 7 nt and can affect transcription initiation, a posttranscriptional initiation event, or translation.
- 25. SHORT SEQUENCES REPEAT AND SLIPPED-STRAND MISPAIRING stop codon Truncated protein
- 26. Molecular Mechanism Genetic Regulation a. Short sequence repeat and slipped strand mispairing - SSM b. Homologous recombination c. Site specific recombination Epigenetic Regulation a. PAP phase variation b. Ag43 phase variation Cell’s Regulatory Network a. cross regulation b. environmental regulation
- 27. HOMOLOGOUS RECOMBINATION/GENE CONVERSION • Gene conversion is the process by which one DNA sequence replaces a homologous sequence such that the sequences become identical after conversion event. • It results in a unidirectional exchange of DNA. It involves recombination between one of a repertoire of silent alleles of the gene and the gene located at the expression site. • e.g. 1.Type IV pilin antigenic variation in N. gonorrhoeae. There are several copies of gene coding for this pili but only one is expressed at any given time. This is referred to as pilE gene. The silent versions of this gene is pilS. pilS can use homologous recombination to combine with parts of pilE gene and thus create a different phenotype.
- 29. HOMOLOGOUS RECOMBINATION 2. Antigenic variation of the variable major lipoprotein (Vmp) in Borrelia hermsii and of the VlsE surface proteins in B. burgdorferi 3. antigenic variation of the SLPs in Campylobacter fetus. 4. variation in the level of capsule production in H. influenzae type b. and Streptococcus pneumonia serotypes .
- 30. Molecular Mechanism Genetic Regulation a. Short sequence repeat and slipped strand mispairing- SSM b. Homologous recombination c. Site specific recombination Epigenetic Regulation a. PAP phase variation b. Ag43 phase variation Cell’s Regulatory Network a. cross regulation b. environmental regulation
- 31. SITE SPECIFIC RECOMBINATIONS • Site-specific recombination are non-homologous usually short (no more than 30 bp.) and occur at a single target site within the recombining sequence. • requires specific enzymes that act at cognate DNA sequences known as site-specific recombinase. • Conservative site-specific recombination (CSSR) can lead to inversion, translocation
- 32. SITE SPECIFIC RECOMBINATIONS-Inversion i) Inversion of a DNA element by CSSR : • In DNA inversions ,a segment of DNA is cut, inverted and then rejoined by site specific recombinases. • The inverted DNA segment may contain either a promoter that direct expression of fixed structural genes or structural genes controlled by fixed promoter. • e.g. Type 1 fimbrial phase variation: Type 1 fimbriae, encoded by the fim operon, are the most common fimbrial adhesins in E. coli isolates.
- 34. SITE SPECIFIC RECOMBINATIONS-Inversion Other CSSR-dependent types of phase variation. • antigenic and phase variation of the PilV protein of the type IVB pilus in S. enterica serotype Typhi - Inversion is mediated by the Rci recombinase • flagellar H1/H2 antigenic variation in S. enterica serotype Typhimurium: Hin-recombinase.
- 35. SITE SPECIFIC RECOMBINATIONS-Transposition 2) Insertion and excision of genetic elements from the chromosome – TRANSPOSITION • Transposition mechanism leads to either insertion or excision of transposable element and is restricted to few insertion sequence (IS) elements. • mediated by recombinase enzymes known as transposases. • Classic transposition does not target a specific DNA sequence. In contrast, transposition mediated by the putative transposase MooV lead to phase variation requires short sequence identity between the insertion element and the target sequence.
- 36. SITE SPECIFIC RECOMBINATIONS-Transposition • e.g. eps locus in marine bacterium P. atlantica : P. atlantica contains an eps locus that encodes extracellular polysaccharide. • Phase variation of eps expression affects biofilm formation. • Two recombinases encoded by MooV and Piv mediate the precise excision and insertion of the insertion element IS492 in eps locus respectively. Presence of IS492 in eps locus→ON phase Excision of IS492→→→→→OFF phase
- 37. SITE SPECIFIC RECOMBINATIONS-Transposition • Phase variation of ica expression in Staphylococcus epidermidis: correlates with the insertion and precise excision of an insertion element. Expression of the ica operon results in formation of a polysaccharide adhesin that facilitates cell-cell interactions and biofilm formation. • Phase variation of capsule production in Citrobacter freundii and in Neisseria meningitidis is also regulated by insertion and excision of IS-like elements • Legionella pneumophila, phase variation in expression of LPS is associated with reversible excision and insertion of a 30-kb plasmid into the chromosome.
- 38. Molecular Mechanism Genetic Regulation a. Short sequence repeat and slipped strand mispairing- SSM b. Homologous recombination c. Site specific recombination Epigenetic Regulation a. PAP phase variation b. Ag43 phase variation Cell’s Regulatory Network a. cross regulation b. environmental regulation
- 39. EPIGENETIC REGULATION • Epigenetic regulation of phase variation occurs in the absence of a change in DNA sequence, therefore maintaining the integrity of genome. • It involves differentially methylated sequences in the regulatory regions of the phase-varying gene or operon. • the expressed state is heritable but reversible • The change incurred by methylation alters the binding of transcription factors. The outcome is the regulation of transcription resulting in switches in gene expression. • Methylation-dependent phase variation has been identified in E. coli and S. enterica serotype Typhimurium as Pap phase variation and Ag43 phase variation respectively.
- 40. EPIGENETIC REGULATION Pap (pyelonephritis associated pili) phase variation in uropathogenic E.coli: Expression of the pap operon is dependent on deoxyadenosine methyltransferase (Dam), two sequences (GATCdist and GATCprox) in its regulatory region, leucine-responsive regulatory protein (Lrp), act as both repressor and an activator at pBA • pBA is the main promoter for the pap operon.
- 42. EPIGENETIC REGULATION Ag43 phase variation: Antigen 43 (Ag43) is an outer membrane protein in E. coli encoded by the Agn43 gene • Ag43 causes auto-aggregation and enhances biofilm formation • Phase variation of Ag43 is mediated by two proteins Dam and the oxidative stress regulator OxyR. • The agn regulatory region contains three GATC sequences that are located within a binding site for OxyR • When OxyR is bound to the regulatory region of Agn43 overlaps with the promoter it inhibits transcription OFF phase • Dam dependent methylation of the GATC sequences inhibits OxyR binding allowing transcription of Ag43 ON phase
- 43. Molecular Mechanism Genetic Regulation a. Short sequence repeat and slipped strand mispairing- SSM b. Homologous recombination c. Site specific recombination Epigenetic Regulation a. PAP phase variation b. Ag43 phase variation Cell’s Regulatory Network a. Cross regulation b. Environmental regulation
- 44. CELL’ S REGULATORY NETWORK-Cross Regulation • Cross regulation results in coordinated expression of two cell surface structures. • In P. mirabilis, coordinated expression occurs between phase- varying MR/P fimbriae and non-phase-varying flagella. When the mrp operon is in the “on” phase and fimbriae expressed, transcription of the flagellar operon is repressed. • In E. coli, expression of the phase-varying outer membrane protein Ag43 and that of fimbriae also appears to be coordinated. Fimbrial expression results in a repression of Ag43 on the cell surface.
- 45. CELL’ S REGULATORY NETWORK-Environmental Regulation • Environmental regulation of gene expression allows the bacterium to be optimally suited to its growth environment • Iron starvation, for example, increases the frequency of antigenic and phase variation of N. gonorrhoeae pili • Stimuli such as temperature, pH, carbon source, and amino acid concentration serve as signal and affect the expression of phase-variable fimbriae in E. coli and S. enterica serotype Typhimurium
- 46. References 1. Van der Woude DM. Phase and Antigenic Variation in Bacteria. 2004. American Society for Microbiology 17.3:581–611 2. Ahmad S., Ahmad M. et al. An overview on phase variation, mechanisms and roles in bacterial adaptation. 2017. J Pak Med Assoc . 67.2:285-291 3. Seifert HS., Magdalene SO. Genetic Mechanisms of Bacterial Antigenic Variation. 1988, American Society for Microbiology . 52.3:327-336
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