BMS662 Microbial Pathogenicity

CHAPTER 5: REGULATION OF VIRULENCE FACTORS

 Learning outcome(s)
Student must be able to:

1. Understand the regulation of virulence factors.

 Introduction
Microbes have evolved sophisticated regulatory systems,
function to control expression of virulence factors and
optimize their effectiveness.

One of the most widely observed schemes used by bacteria to

control production of virulence factors is the two-component
regulatory system.
5.1 Two-component regulatory systems
A system that allows prokaryotes to regulate cell metabolism in response to
environmental fluctuations, including:
temperature changes
changes in pH and oxygen availability
changes in the availability of nutrients

Involves a mechanism by which cells receive signals from the environment

and ______________________________
transmit them to the specific target
to be -

.
regulated
5.1 Two-component regulatory systems
However, the external signal is not transmitted directly to the regulatory
protein but instead is detected by a sensor that transmits it to the
regulatory machinery, a process called signal transduction.
chemical
→ physical .

Most signal transduction systems contain two parts and are thus called
two-component regulatory systems
(i) the sensor kinase located in cell membrane and
(ii) the response regulator protein located in cytoplasm
5.1 Two-component regulatory systems
Sensor kinases detect a signal from the environment and phosphorylate themselves
a specific histidine residue
(process called autophosphorylation) at ______________________________on
their cytoplasmic surface.

histidine kinases
Thus, sensor kinases are also called ______________________________.

The phosphoryl group is then transferred to another protein inside the cell, the
response regulator.

The response regulator is a DNA-binding protein that regulates transcription, which

can function as an activator and/or repressor
fir to transcribe new mRNA
important
.

opium
The control of gene expression
by a two-component
regulatory system
 ☆ How

A balanced regulatory system must have a feedback loop

regulatory circuit } terminate the initial signal
to complete the ______________________________. .

This feedback loop involves a phosphatase, an enzyme activity that

removes the phosphoryl group from the response regulator protein.

In many systems, this reaction is carried out by the response regulator

itself.

In many other cases, separate proteins are present to stimulate this

reaction.
 Organism and Sensor/ Signals Regulated Virulence Properties
Manifestation Regulator
Bordetella pertussis BvgS/BvgA Mg+2 SO4-2, Pertussis toxin, adenylate cyclase
Whooping cough temperature Toxin/hemolysin, filamentous
hemagglutinin
Salmonella PhoQ/PhoP Mg+2, cationic Macrophage survival, invasion of epithelial
Gastroenteritis, typhoid antimicrobial cells, resistance to innate immune
fever, bacteremia peptides mechanisms

Staphylococcus aureus AgrC/AgrA Peptide Exoprotein production, including activation

Abscesses Pheromone of hemolysins, proteases, and toxins;
repression of coagulase and protein A
Enterococcus faecalis VanS/VanR Vancomycin Enzymes for vancomycin resistance
Urinary, biliary,
cardiovascular infections
Streptococcus pyogenes CsrS/CsrR Mg+2 Repression of hylaruronic acid capsule and
Invasive skin infection; (CovR/CovS) other virulence factors; activation of some
necrotizing fasciitis genes associated with pathogenicity

Explain process right atn ( only choose one

process) .
 5.2 Global regulators
Is a regulatory mechanism that respond to environmental signals by
regulating the expression of many different genes.
opium → promote

§ Operator
.

Also called global control systems.

Stmtneal gone .

Includes sets of operons and regulons scattered in chromosome but all

coordinately controlled in response to a particular stress.

Examples catabolite repression and stringent response.

 5.2 Global regulators: i) Catabolite Repression

Occurs when cells grown in a medium containing more than one substrate.

a better carbon
} energy source
The substrate which is ______________________________would
.

repress
the use of other substrates.

Thus catabolite repression is a system which ensures that the organism

uses the best available carbon and energy source first.
 5.2 Global regulators: i) Catabolite Repression

shown to initiate this response

when E.coli was grown in medium containing glucose and lactose.

T -galactosidase, are
required for using lactose and are induced in its presence.
 5.2 Global regulators: i) Catabolite Repression

However, their synthesis is subject to catabolite repression.

As long as glucose is present, the lac operon is not expressed and lactose is
not used. → Phosphotransferase system .

But when glucose is exhausted, catabolite repression is abolished, and after

a brief lag period, the lac operon is expressed and the cells grow on lactose.
 5.2 Global regulators: i) Catabolite Repression

Thus, catabolite repression leads to two exponential growth phases, a

situation called diauxic growth.

Diauxic growth, diauxie or diphasic growth is any cell growth characterized

by cellular growth in two phases.

Diauxic growth, meaning double growth, is caused by the presence of two

sugars on a culture growth media, one of which is easier for the target
bacterium to metabolize
During the first phase of
exponential growth, the bacteria
utilize glucose as a source of
energy until all the glucose is
exhausted.

Then, after a secondary lag

phase, the lactose is utilized
during a second stage of
exponential growth.
 5.2 Global regulators:
. E E?i i m
.

Mechanism of the catabolite repression

¥
The catabolite repression transcription is controlled by an activator protein
and is actually a form of positive control.

The activator protein is called the cyclic AMP receptor protein (CRP) or CAP
(the catabolite activator protein).

A gene that encodes a catabolite-repressible enzyme is expressed only if

CAP protein binds to DNA in the promoter region.

This then allows RNA polymerase to bind to the promoter.

 5.2 Global regulators:
Mechanism of the catabolite repression
However, CAP would binds to DNA only if it has first bound a small molecule
called cyclic adenosine monophosphate (cyclic AMP or cAMP).

Thus, the key molecule in this metabolic control systems is the cAMP.

cAMP is synthesized from ATP by an enzyme called adenylate cyclase.

 5.2 Global regulators:
Mechanism of the catabolite repression
However, glucose inhibits the synthesis of cAMP.

Glucose also stimulates cAMP transport out of the cell.

Thus, when glucose enters the cell, the cAMP level is lowered, CAP protein
cannot bind DNA, and RNA polymerase fails to bind to the promoters of
operons subject to catabolite repression.
 5.2 Global regulators:
Mechanism of the catabolite repression
Therefore, catabolite repression is actually an indirect result of the
presence of a better energy source (glucose).

The direct cause of catabolite repression is a

low level of CAMP
______________________________.
.
 Why is catabolite repression considered
a mechanism of global control?
When glucose is the preferred energy source, catabolite repression prevents
expression of most other catabolic operons as long as glucose is present.

Thus, many other operons are affected, including those for lactose, maltose, other
sugars including other commonly used carbon and energy sources of E. coli.

In addition, genes for the synthesis of flagella are controlled by catabolite

repression
if bacteria have a good carbon source available, there is
no need to swim around in search of nutrients
______________________________. .
5.2 Global regulators: ii) Stringent response
A mechanisms of which genes are regulated in response to nutrient levels
in the environment (in reaction to amino-acid starvation, fatty acid
limitation, iron limitation - a conserved stress response in bacteria that
controls the adaption to nutrient deprivation).

Found in bacteria and plant chloroplast

e

When a culture is transferred from a rich medium to a poor medium with a

single carbon source
the synthesis of rRNA and tRNA and proteins is stopped.
5.2 Global regulators: ii) Stringent response
However, the biosynthesis of new amino acids is activated
new proteins must be made to synthesize the amino acids no longer
available in the environment; these are made by existing ribosomes.

After a while, rRNA synthesis (and hence, the production of new

ribosomes) begins again but ______________________________.

This course of events is called the ______________________________

another example of global control.
 Mechanism of the Stringent Response ones:
alarm

The stringent response is triggered by a mixture of two regulatory

nucleotides, guanosine tetraphosphate (ppGpp) and guanosine
-

pentaphosphate (pppGpp).
lain .

ggnama
In E. coli, these nucleotides, which are also called alarmones, accumulate
during a shift down from amino acid excess to amino acid starvation.

Alarmones are synthesized by a specific protein, RelA which is associated

with 50S ribosome.
 Mechanism of the Stringent Response
When amino acids is limited, the pool of uncharged tRNAs increase relative to
charged tRNAs.

Eventually, an uncharged tRNA is inserted into the ribosome instead of a charged

tRNA during protein synthesis.

The ribosome would be shut down, and this leads to synthesis of a by RelA using
ATP as a phosphate donor.

By adding two phosphate groups from ATP to GTP or GDP, thus producing pppGpp
or ppGpp
 Mechanism of the Stringent Response
The protein Gpp then converts pppGpp to ppGpp so that ppGpp is the
major overall product.

The alarmones ppGpp and pppGpp have global control effects by inhibiting
rRNA and tRNA synthesis.

initiation of
HOW?: By to RNA
binding polymerase
______________________________ 4 prevent
transcription
-
 Mechanism of the Stringent Response
Alarmones also activate the biosynthetic operons for some amino acids
synthesis.

In contrast, operons that encode biosynthetic proteins whose amino acid

present in sufficient amounts
products are ______________________________remain shut down.
 Mechanism of the Stringent Response
In addition to RelA, another protein, SpoT, helps trigger the stringent response.

The SpoT protein can either make (p)ppGpp or degrade it.

It is generally responsible for degrading (p)ppGpp

However, SpoT also synthesizes (p)ppGpp in response to certain stresses or when

there is a shortage of energy.

Thus the stringent response is the result when the absence of precursors for
protein synthesis and also the lack of energy for biosynthesis.
(p)ppGpp modulates bacterial physiology at all
growth stages.

During adaptation (lag) and exponential (log)

growth phases in the absence of stresses,
basal levels of (p)ppGpp are important to
maintain a balanced metabolism avoiding
uncontrolled consumption of energy stores
and toxic accumulation of metabolic by
products.

Entry into stationary growth phase or

adverse conditions such as nutrient
starvation, heat shock or selected antibiotic
stresses triggers the accumulation of
(blue line increase )
(p)ppGpp✗that rapidly reaches the threshold
necessary to mount the Stringent Response
responsible for remodeling cell physiology
from a growth mode to a survival mode.
 5.3 Quorum sensing
Is widespread among Gram negative bacteria but is also found in Gram
positive bacteria.

of all all communication

Quorum sensing is a process of ______________________________
-

that
allows bacteria to share information about cell density and adjust gene
expression accordingly.

A mechanisms of which prokaryotes regulate genes in response to signals in

the environment.
 5.3 Quorum sensing
Why is quorum sensing in bacteria important?

Quorum-sensing allows individual bacteria within colonies to coordinate

and carry out colony-wide functions such as:
Sporulation
Bioluminescence
Virulence
Conjugation
Competence
Biofilm formation
 5.3 Quorum sensing
The signal here refers to the presence of other cells of the same species in their
surroundings.

Thus quorum sensing is a mechanism by which bacteria assess their population

density to ensure that sufficient cell numbers present before initiating a response.

As the response requires a certain cell density to have an effect

e.g., a pathogenic bacterium that secretes a toxin can have no effect as a single
cell; production of the toxin by that cell alone would be a waste.

But, if sufficient number of cells present, enough expression of the toxin may
successfully initiate disease.
 5.3 Quorum sensing
Quorum-sensing activity results from the production of a specific signal
molecule called autoinducers by the bacteria.

This molecule diffuses freely across the cell envelope in either direction.

Therefore, when many cells nearby making the same autoinducer, the
compound accumulates and reaches high concentrations inside the cell.

Once they reach a threshold level, autoinducers can bind to a regulatory

activate transcription of number of genes
protein and together ______________________________.
a .
 5.3 Quorum sensing
There are several different classes of autoinducers.

The first to be identified were the acyl homoserine lactones (AHLs).

Several different AHLs, with acyl groups of different lengths, are found in
different species of gram-negative bacteria.

In P. aeruginosa up to 10% of genes may be regulated by quorum sensing,

and many encode virulence factors e.g. in biofilm formation.
 5.3 Quorum sensing
auto inducer
.

Many Gram negative bacteria also make AI-2 -

apparently used as a common autoinducer between many species of
bacteria

In Gram positive bacteria, certain short peptides function as autoinducers.

Quorum sensing.

(a) General structure of an acyl

homoserine lactone (AHL)

(b) A cell capable of quorum sensing

expresses acyl homoserine
lactone synthase
 5.4 Other regulators
Heat Shock Proteins
An example of a global response where bacteria response to high
temperature.

Most proteins are relatively stable.

However, at higher temperature, some proteins are less stable and

tend to unfold
______________________________.
.

These proteins are recognized by protease enzymes in the cell and are
degraded.
 5.4 Other regulators
Consequently, cells that are heat stressed induce the synthesis of a set of
proteins, the heat shock proteins, to help counteract the damage and assist
the cell in recovering from stress.

However, heat shock proteins are not only induced by heat, but also by
other stress factors.

These include exposure to high levels of

certain chemicals, such as ethanol and
exposure to high doses of ultraviolet radiation
 5.4 Other regulators
In most prokaryotes, there are three major classes of heat shock protein,
Hsp70, Hsp60, and Hsp10

The Hsp70 protein of E. coli is DnaK, which prevents aggregation of newly

synthesized proteins and stabilizes unfolded proteins.

The Hsp60 and Hsp10 proteins in E. coli include GroEL and GroES,
respectively
these are molecular chaperones that catalyze the correct refolding of
misfolded proteins.
 What does heat shock do to bacteria?

Heat-shock proteins are involved in several processes in bacterial cells,

including assisting the folding of newly synthesized proteins, preventing
aggregation of proteins under stress conditions and recovering proteins that
have been partially or completely unfolded by stresses such as a sudden
temperature increase.
 Heat Shock Response
In most bacteria, such as E. coli, the heat shock response is controlled by
the alternative sigma factors RpoH RpoE.

expression of heat shock proteins

The RpoH sigma factor controls ______________________________. in

cytoplasm
the
.

heat shock proteins

A in the periplasm
expression of different set of
RpoE regulates the ______________________________
and cell envelope.
 Sigma factors:
Involved in regulation of expression of heat shock genes.
Are initiation factors that promote the attachment of RNA polymerase to specific
initiation sites and are then released.

Intracellular concentration of free RpoH protein increases in response to heat

shock, causes
association with RNA polymerase and
initiation of transcription of heat shock genes, including numerous global
transcriptional regulators and genes involved in maintaining membrane
functionality and homeostasis.
degraded
✗
quickly
RpoH is then ______________, leading to a decrease in the rate of synthesis of
heat shock proteins and shut-off of the heat shock response.
 untnk final / test
DA panting
.

Heat Shock Response

At normal temperature, RpoH is degraded by proteases within a minute or
two of its synthesis.

This is stimulated by binding of the DnaK protein to RpoH.

The rate of degradation of RpoH depends on the level of free DnaK protein,
which binds to and inactivate RpoH.

In unstressed cells, the level of free DnaK is relatively high and the level of
intact RpoH is correspondingly low.
 * final .
☆ draw simple
diagram
Heat Shock Response words .

☆
give
a
key

However, at high temperature some proteins are denatured, DnaK

recognizes and binds to the unfolded proteins.

Thus, DnaK removes from RpoH, which slows the degradation rate of RpoH.

Thus, the more denatured proteins there are, the lower the level of free
DnaK and the higher the level of RpoH; the result is heat shock gene
expression.

When the temperature goes back to normal, RpoH is rapidly inactivated by

DnaK, and the synthesis of heat shock proteins is greatly reduced.
 Heat Shock Response
The synthesis of heat shock proteins in stressed cells is important for cells
to survive not only excessive heat but also against chemicals, or physical
agents.

These stresses can generate large amounts of inactive proteins that need to
4 in the process reactivated
be refolded ______________________________or
, degraded to release
free amino acids for the biosynthesis of new proteins.
 this
done
24/5/23