Phase and antigenic variations in bacteria

Dr. Dinesh Kr Jain, MD.,
Assistantprofessor,
Department of Microbiology,
SMS Medical college, Jaipur

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

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.

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.

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.

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

 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.

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

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.

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.

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.

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).

 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.

 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.

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.

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.

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.

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.

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.

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

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.

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.

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

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.

MISPAIRING
stop codon
Truncated protein

Molecular Mechanism
a. Short sequence repeat and slipped strand mispairing - SSM
a. cross regulation

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.

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 .

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

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.

fimA
fimA
pA
pA

 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.

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.

• 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

• 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.

Molecular Mechanism
Epigenetic Regulation
a. cross regulation

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.

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.

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

Molecular Mechanism
Cell’s Regulatory Network
a. Cross regulation
b. Environmental regulation

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.

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

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

Phase and antigenic variations in bacteria

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