MICR3214 Microbial Interactions2019

9/8/2019
Welcome to MICR3214:
Molecular Microbiology
Paul D. Brown, PhD

Professor of Molecular Biology
(Infectious diseases and basic science)
Course coordinator
Email: paul.brown@uwimona.edu.jm
INTRODUCTION TO MOLECULAR
MICROBIOLOGY – LECTURES
• Microbial interactions: Environmental sensing;
Quorum sensing (4 L)
• Microbe-host interactions (4 L)
• TEST 1 (1 hr; MCQ)
• Microbial pathogenesis (4 L)
• Comparative and environmental genomics (5 L)
• Using whole genome sequencing to track bacterial
and viral pathogens (2 L)
• Stationary phase; Stringent response (2 L)
• (Drug development and reverse vaccinology)
• TEST 2 (1 hr; MCQ)
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MICROBIOLOGY – LABS
• Stress-Induced Mutagenesis (ASM webinar)
– report required
• Tiny conspiracies (ASM webinar) – no report
• Quorum sensing in bacteria (2 wks)
• Lectin agglutination
• Visit to Serology/Immunology section,
Microbiology Department
• Assessing virulence/pathogenesis using a
plant model (2 wks)
• Bioinformatics Exercise (2 wks)
MICROBIOLOGY - STRUCTURE
• Lectures – Thursdays/Fridays at 9 am
• Tutorials – To be decided (Wednesdays at 8 am)
• Labs – Mondays at 1 pm
• Recommended texts:
• Brock’s Biology of Microorganisms. Madigan et al.,
latest edition;
• Jawetz, Melnick and Adelberg’s Medical Microbiology.
Brooks et al. 25th edn. Lange. 2010;
• Medical Microbiology and Immunology. Levinson, 12th
edn. Lange, 2012;
• Bacterial Pathogenesis: A Molecular Approach. Salyers
& Whitt, latest edn.; ASM Press
• Course website: http://ourvle.mona.uwi.edu
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Microbial Interactions
L1. Microbial Interactions
Overall Learning Outcomes:
• Students should be able to discuss

how microbes interact with each
other in their environment.
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Specific Learning Outcomes
Should be able to satisfactorily discuss/describe:

 Environmental sensing and interaction with other
organisms
 Two-component signal transduction
 Koch’s postulates, virulence, virulence factors and

their regulatory networks
 Quorum Sensing in Gram positive and Gram

negative microbes
• The numerous types of microorganisms that exist
in nature rarely occur alone; rather they occur as
populations within complex biological
communities.
• Within these communities, interactions occur

among microbial, plant, animal, and human
populations.
• The categories used to describe these

interactions represent a conceptual
classification system, however, many specific
cases are difficult to classify without ambiguity.
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Neutralism
• This concept implies a lack of interaction
between two microbial populations.
• It occurs when microbial populations are spatially

distant from each other, where one microbial
population does not sense the presence of the
other.
• E.g. a pathogenic microbe invading the root of a

plant vis-à-vis microbes inhabiting leaf surfaces
Commensalism (“at the table together”)
• One population benefits while the other
remains unaffected (not obligatory).
• Microflora of the oral, respiratory, and GI tracts

important for development of immunity.
• E. coli is acquired shortly after birth and this

encounter places some infants at risk for E. coli
meningitis during the first month of life.
• Symptomatic infections with C. albicans can
occur in the first year of life.
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Commensalism “at the table together”
• Host vs Microbe: Microbiome
– 1% of human genome – retroviruses
– 10-100-fold more microbial cells than human (1013)
cells
• Relationship could borders on mutualism

(clownfishes and sea anemones)
– Clownfishes actually may attract other fishes on
which the anemone can feed.
• Epiphytic bacteria living on the surface of

other organisms.
Synergism: “working together”
• Both populations benefit from the

interaction, however, the association is not
obligatory.
• E.g. the relationship between Chlorobium and

Spirillum and Chlorobium and Desulfovibrio,
which is based on the cycling of carbon and
sulfur.
• Chlorobium reduces CO2 and oxidizes H2S;

Desulfovibrio and Spirillum oxidize organic C and
reduce sulfur to H2S.
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Mutualism (Symbiosis)
• This can be considered as an
extension of synergism; however,
this is an obligatory relationship
where both populations benefit.
• Possibly most famous is lichen;

actually hundreds of “species”
• Interaction between the two

microbes allows them to act as if
they were a single organism with a
unique identity
Competition
• This represents a negative relationship
between two populations in which both
populations are adversely affected with
respect to their survival and growth.
• This occurs when two populations utilize the same

resources, whether it is space or limited
nutrients.
• Here no toxins or inhibitory substances are

produced by either organism, but the rapid
growth of one out-competes the other for the
resources.
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Amensalism (Antagonism)
• Microbes that produce substances toxic to
competing populations will naturally have a
competitive advantage.
• The oxidation of sulfur by Thiobacillus

thiooxidans produces sulfuric acid reducing the
pH of aquatic habitats.
• Yeast produces ethanol during fermentation,

which is inhibitory to the growth of other
microbes. Etc…
Parasitism
• The parasite derives its

nutritional requirements
from the population that is
harmed, the host.
• The host-parasite
relationship is
characterized by a
relatively long period of
contact.
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Parasitism
The influenza virus
takes over healthy
cells, spreads through
the body and causes
illness.
Signs and symptoms

include fever, chills,
muscle aches and
malaise.
Parasitism
Infection with Candida

fungus can lead to
problems such as
diaper rash, vaginal
yeast infections and
oral thrush.
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Predation
Predation usually occurs when one organism, the
predator, engulfs and digests the prey.
The two-stage life cycle of

the bacterial predator
Bdellovibrio bacteriovorus
Predation
A Bdellovibrio predator and the killed prey cell
in which it is growing are together termed a
bdelloplast.
As with parasitism, predation is a mechanism of

population control that dampens (or
attenuates) population explosions and
prevents exhaustion of nutritional resources of
the habitat that would lead to severe population
crashes.
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Environmental Sensing
• Bacteria need to sense changes in
environmental conditions and respond
rapidly to ensure their survival by adjusting
their metabolic processes to take advantage
of the current situation.
• Bacteria have a wide range of regulatory

responses to adaptive opportunities, for example,
when nutrients become available transport and
processing machinery are produced to utilize
them.
• Sensory systems mediate gene expression during
host invasion by pathogens.
• Stress conditions such as antibiotics, heavy

metals, starvation, or exposure to UV radiation,
elicit changes in gene expression that lead to
effective coping mechanisms.
• The machinery responsible for these adaptive

responses (stimulus detection, signal processing
and production of output responses) is common
to all cell sensory systems.
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• This recognition and processing of environmental
signals through signal transduction pathways
utilizes reversible protein phosphorylation.
• In simple microbial systems, information
from sensory receptors feeds into a
circuitry of regulatory proteins that
transfer high energy phosphoryl groups
from histidine (His; H) to aspartate
(Asp; D) side chains.
• This phosphotransfer network couples

environmental signals to an array of
response elements that control cell
motility and regulate gene expression.
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• A two component system is called "two

component" for historical and personal
reasons.
• Prior to the use of the phrase "two

component" in this context, most gene
regulation proteins that were known were
single proteins, often homodimers or
homotetramers, which bound to two ligands:
– a metabolic intermediate
– a cis-acting gene regulation element
The first
component
functions as a
sensor; the
second is a
response
regulator.
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• The consequence of ligand binding, an

altered state of the regulatory protein (aka
conformational change), was directed to
the appropriate gene(s) by the protein's
DNA binding activity.
• Eventually, studies of the arabinose

utilization pathway in E. coli showed that
one could also have positive regulation, in
which the protein stimulated gene
expression.
• In the mid-1980s, it had become clear that some

regulatory systems required more than one
protein component.
• The primary examples known at that time were:
 EnvZ/OmpR - osmoregulation in E. coli

 PhoR/PhoB - phosphate scavenging in E. coli
 NtrB/NtrC - nitrogen assimilation in a
variety of bacteria
 DctB/DctD - dicarboxylate transport in
Rhizobium leguminosarum
 VirA/VirG - virulence by Agrobacterium
tumefaciens
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Virulence and its Regulation
• Virulence, or the ability to cause an

infectious disease, is always
multifactorial, since the infectious
disease process is invariably complex.
• Different virulence factors are required by

the pathogenic microorganism at each
stage of the process to cause a disease.
Virulence Factors
• The classical virulence factor is the bacterial

toxin, however, it could be any component of
the microbe that is required for, or that
increases its ability to cause disease.
• A German microbiologist, Robert Koch, realized

that if the new discipline of infectious disease
was ever to become respectable, it had to have a
rigorous scientific basis. (coming out of the era of
miasmic theories of infection)
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Koch’s Postulates:
 Association of the microbe with the lesions of the

disease (diseased tissue) but not in normal tissue
 Isolating the bacterium in pure culture
 Showing that the isolated bacterium causes

disease in humans or animals
 Re-isolating the bacterium from the intentionally

infected animal (or human).
KOCH’S POSTULATES
• Microbe found in diseased tissue but not in

normal tissue
• Microbe can be isolated from diseased tissue

as pure culture
• Microbe can cause disease when inoculated

in animal or man
• Microbe must then be re-isolated in pure

culture from animal or man
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ISSUES WITH KOCH’S POSTULATES

• Microbe found in diseased tissue but not in
normal tissue
• Pathogen can be a colonizer and only sometimes
cause disease (Staph. aureus, Helicobacter pylori)
• Microbe can be isolated from diseased tissue as
pure culture
• Organism may be non-culturable (T. pallidum)PCR
• Disease may require multiple organisms
• Microbe can cause disease when inoculated in
animal or man
• Gonococcus has no animal model
• Microbe must then be re-isolated in pure culture
from animal or man
• Disease requires multiple microbes (periodontal
disease)
• (Therapeutic or preventative measures
eliminates disease)
MOLECULAR KOCH’S POSTULATES:

DEFINING VIRULENCE FACTORS
• Virulence gene is associated with bacteria

that cause disease but is absent or inactive
in strains that fail to cause disease
• Redundant virulence factors
• Ex: PVL+ community-acquired S. aureus (CA-
MRSA)
• Disrupt gene in virulent strainavirulence
• Redundant virulence factors
• Ex: PVL- CA-MRSA
• Introduce cloned gene into avirulent strain
virulence
• Ex: Lab MRSA+PVL
• Gene is expressed during infection
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UP-TO-DATE KOCH’S POSTULATES:

DEFINING NEW PATHOGENS
ASSOCIATED WITH DISEASE
• Metagenomics: bulk sequencing (16S

rRNA or random) to define new species,
new genes, and/or new pathways
• Microarray identification
• Nucleic acid sequence of putative pathogen is
present during disease and at sites of disease
• Nucleic acid sequence of pathogen is absent
or reduced in healthy controls
• Dose response
L2. Virulence Gene Regulatory

Networks
Global Regulation of Virulence in
Bordetella pertussis (Whooping Cough)
The early B.pertussis isolates

• Have distinctive colonial morphology
• Produce a variety of toxins:
– adenylate cyclase, pertussis toxin,
dermonecrotic toxin, and hemolysin
• Possess surface components that are

thought to aid in establishing infection
– pili, filamentous hemagglutinin, capsule,
and several unique outer membrane
proteins.
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Virulence Gene Regulatory Networks

Global Regulation of Virulence in Bordetella pertussis
• Alternates between a virulent +

(bvg+) and an avirulent (bvg-)
state
• Virulent form rapidly becomes biofilm

avirulent if grown at 25oC (close to
the temp of external nares) rather
than 37oC (the internal body temp)
and vice versa
• Concentrations of Mg2+ and

nicotinic acid also modulate the
virulent state Nicotinic acid 0.8 mM 4mM
Global Regulation of Virulence in Bordetella

pertussis
Virulence
genes, fha,
ptx,
siderophore
production
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• A-B toxin: A (active) and B (binding) subunits

• The toxin attaches to the host cell via the B
subunit, allowing the A fragment to enter the cell
and act in the cytoplasm.
• Pertussis toxin is an
ADP-
ribosyltransferase
(Similar to cholera
toxin, diphtheria toxin,
and Pseudomonas
exotoxin A)

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Local damage is caused by a tracheal

cytotoxin, which kills ciliated cells
specifically and leads to their extrusion from
the epithelium as B. pertussis binds to and
multiplies on ciliated cells.
Other Systems involved in Virulence

Regulation
1. Down-regulation of Virulence Factors of
Pseudomonas aeruginosa by Salicylic acid
• Salicylic acid (SA) significantly

diminished P. aeruginosa's
ability to kill Caenorhabditis
elegans worms
• No effect on accumulation of
bacteria inside the worm’s gut,
suggesting that SA negatively
affected factors that influence
the virulence of P. aeruginosa
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Regulation
1. Down-regulation of Virulence Factors of Pseudomonas
aeruginosa by Salicylic acid.
• SA selectively repressed
transcription of exoproteins and
other virulence factors, while it had
no effect on expression of
housekeeping genes.
• In addition to its role as a signal

molecule in plant defense responses,
SA works as an anti-infective
compound by affecting the
physiology of P. aeruginosa and
ultimately attenuating its virulence.

Regulation
2. Virulence control in group A Streptococcus
by a two-component gene regulatory
system
• Group A Streptococcus, the causative agent of

mild infections and life-threatening invasive
diseases.
• A two-component regulatory system,
designated covRS (cov, control of virulence;
csrRS) plays central role in growth and
pathogenesis
• One of 13 two-component signal-transducing
systems (TCS) encoded in GAS genomes
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Regulation
CovRS
• Regulates GAS gene expression in response to
stress in laboratory settings (39°C, a pH of 6,
or a high concentration of salt)
– Under these stress conditions, CovS inactivates CovR
and thus derepresses genes required for growth of the
bacteria
• Acts via repression
• Negatively controls expression of five proven or
putative virulence factors (capsule, cysteine
protease, streptokinase, streptolysin S, and
streptodornase)
• Required for biofilm formation
• Modulates the transcriptome during growth in
human blood
Other Systems involved in GAS

Virulence Regulation
Cole et al., 2011 Nature Rev Microbiol
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Regulation
2. Virulence control in group A Streptococcus by a two-
component gene regulatory system
• Using DNA microarrays and quantitative RT-

PCR, it was shown that CovR influences
transcription of 15% (n = 271) of all
chromosomal genes
• CovR also plays a central role in gene

regulatory networks by influencing
expression of genes encoding transcriptional
regulators, including other two-component
systems.

Regulation
3. Regulation of Virulence by Environmental
Signals
The majority of group A streptococci (GAS,

Streptococcus pyogenes) are aggressive human
pathogens that must recognize and survive
within different environments during infection.
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Regulation
Signals
• The M protein, fimbrial molecule found on the

surface of GAS, thought to play essential role in
establishment and progression of streptococcal
infections.
• Functions by preventing phagocytosis of the

bacteria by polymorphonuclear cells (PMNs) and
allows bacterium to evade host immune response
• Transcription of the emm gene regulated by CO2

Regulation
Signals in GAS
• Increased osmolarity, low temperature, growth

with free exchange of gases, or the restricted
availability of iron resulted in decreased
transcription from the emm promoter
• Transcription of emm and scpA (and several

other virulence surface-associated molecules) in
GAS regulated by the two-component multiple
gene regulator, Mga, in response to CO2 levels
Infect Immun. 1995 Nov; 63(11): 4540–

4542
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Regulation
3. Regulation of Virulence by Environmental Signals
• The GAS CovRS homologs in group B streptococci,

Streptococcus mutans, and Enterococcus faecalis
have also been shown to regulate many virulence
genes and to be important for pathogenesis in
these organisms.

Regulation
Signals
• In Bacillus anthracis, transcription of the

genes encoding the virulence-associated
capsule and the anthrax tripartite toxin is
activated by increased external CO2 levels
and by high temperature.
• The activator AtxA (pXO1) is needed for

toxin and capsule synthesis, while AcpAB
protein (pXO2) is necessary for capsule
expression.
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Regulation
4. The AraC (arabinose) Family
Transcriptional Regulator – E. coli
• Negative auto-regulation
• Complex system for regulating intracellular levels

Regulation
4. The AraC Family Transcriptional Regulator
• Six members of this family (Rv1317c [alkA],

Rv1395, Rv1931c, Rv3082c [virS], Rv3736, and
Rv3833) have been identified in the genome of
Mycobacterium tuberculosis by sequence similarity
to the defining DNA binding domain.
• The first of these, Rv1317c (alkA), has additional

domains conferring a role in the repair of alkylated
DNA, with the regulatory functions residing at the
N terminus.
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Regulation
4. The AraC Family Transcriptional Regulator
• Subsequently, Rv1395 has been shown to

regulate the expression of a cytochrome P450-
encoding gene transcribed divergently from it.
• Another member of this AraC family, Rv3082c

(virS), has been suggested to play a role in M.
tuberculosis pathogenesis on the basis of
sequence similarities and has also been shown to
regulate an operon transcribed divergently from
it.

Regulation
5. DNA Supercoiling
• A number of environmental stresses (such as
osmotic shock and anaerobiosis) have been
shown to induce changes in DNA
supercoiling.
• DNA supercoiling directly affects the

transcription of a specific subset of bacterial
genes, at least some of which (the outer-
membrane porins and type 1 fìmbriae) play a
part in bacterial virulence.
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Other
Systems
involved in
Virulence
Regulation
Examples: virR
gene of
Shigella
flexneri, and
invasin genes
of Salmonella
typhimurium

Regulation
6. DNA Methylation
• Plasmid-encoded fimbriae (Pef)
expressed by S. Typhimurium mediate
adhesion to mouse intestinal epithelium.
• The pef operon shares features with the E.

coli pyelonephritis-associated pilus
(pap) operon
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Regulation
6. DNA Methylation
• These features include conserved DNA GATC
box sites in the upstream regulatory region as
well as homologues of the PapI and PapB
regulatory proteins.
• Unlike Pap fimbriae, which are expressed in a

variety of laboratory media, Pef fimbriae are
expressed only in acidic, rich broth under
standing culture conditions.

Regulation
6. DNA Methylation
• Analysis of the DNA methylation states of pef
GATC sites indicated that, under acidic growth
conditions, which induced Pef production,
most GATC I sites were non-methylated,
whereas GATC II and GATC X were
predominantly methylated.
• The methylation protection at GATC I and

GATC II was dependent on Lrp and was
modulated by PefI.
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Regulation
6. DNA Methylation
• DNA adenine methylase (Ddam) mutants of
Salmonella enterica serovar Typhimurium
contain reduced levels of FinP RNA encoded on
the virulence plasmid.
• Dam methylation appears to regulate finP

transcription, rather than FinP RNA stability or
turnover.
• The finP promoter includes canonical -10 and -

35 modules and depends on the δ70 factor.

Regulation
6. DNA Methylation
• Unexpectedly, a GATC site overlapping with
the -10 module is likewise dispensable for
Dam-mediated regulation.
• These observations indicate that Dam

methylation regulates finP transcription
indirectly and suggest the involvement of a
host factor(s) responsive to the Dam
methylation state of the cell.
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Regulation
• Varying deficiencies in cell growth, antimicrobial resistance and

biofilm formation, alongside low-level increases in gene
expression (recA and papI), and adherence to HEK-293 and HTB-
9 mammalian cells were also detected as a factor of SOS
induction to result in increased mutability. Phenotypic
characteristics of parental strains were restored in dam
complement strains.

Regulation
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Regulation
• Most of the genes involved in attaching-and-effacing lesion

formation are carried within a chromosomal pathogenicity island
called the locus of enterocyte effacement (LEE)

Regulation
• Evaluation of
agrA and sarA
targeted small
RNA silencing of
virulence in
Staphylococcus
aureus in a
Caenorhabditis
elegans model
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Regulation
• Figure 1. HC104A and HC106A inhibit DosRST reporter fluorescence. (a)

Chemical structures of HC104A and HC106A. HC104A (b) and HC106A (c)
inhibited DosR-driven GFP fluorescence signal in a dose-dependent manner, while
having minimal impact of Mtb growth. The EC50 values of fluorescence inhibition
for HC104A and HC106A are 9.8 µM and 2.5 µM, respectively.
L.3: Quorum Sensing
• It was once held that most bacteria function

only as individuals designed to compete with
one another and to multiply rapidly under
appropriate conditions.
• After all, dense cultures of bacteria can be

grown from a single cell.
• This concept has given way to the view that,

like other creatures, bacteria can
communicate with each other and form
communities that represent more than the
sum of the individuals.
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Quorum Sensing
Some, Vibrio fischeri (Beijerinck 1889)/(Aliivibrio

fischeri; 2007) or Vibrio harveyi, can glow in the
dark.
Quorum Sensing
• Others, like Pseudomonas aeruginosa, form
biofilms on the surface of human organs, and
attack virulently those organs multiplying
with tremendous speed, making it practically
impossible for antibiotics to interfere.
What is quorum sensing?
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Quorum Sensing
• Bacteria produce and release chemical signals -
autoinducers - in search of similar cells in their close
surroundings.
– Called "cell-cell communication"
• Other bacteria release the same autoinducers in response.
• One-cell organisms in effect become multi-cellular

organisms and can act together.
• The detection of a minimal threshold stimulatory

concentration of an autoinducer leads to an alteration in
gene expression.
Quorum Sensing
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Canonical bacterial quorum-sensing (QS)

circuits.
Steven T. Rutherford, and Bonnie L. Bassler Cold

Spring Harb Perspect Med 2012;2:a012427
©2012 by Cold Spring Harbor Laboratory Press
Quorum Sensing
• These processes include symbiosis,

virulence, competence, conjugation,
antibiotic production, motility, sporulation,
and biofilm formation.
• In general, Gram-negative bacteria use

acylated homoserine lactones as
autoinducers, and Gram-positive bacteria
use processed oligo-peptides to
communicate.
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Quorum Sensing
Gram Positive System
• Production of some antimicrobial peptides
(AMPs), so-called "bacteriocins", activated by
quorum sensing in several lactic acid bacteria
– Lactacin – against competitive bacteria
– Nicin and pediocin – against spoilage and
pathogenic bacteria
• Important for develop-

ment of genetic
competence (ability to
"pick up" DNA) or
virulence
Quorum Sensing
• Since the animal gut includes a HCD of micro-organisms
consisting mainly of Gram+ve bacteria, we think that a
significant part of these bacteria use quorum sensing
mechanisms involving peptide pheromones.
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Quorum Sensing
Staphylococcus aureus Agr system
• S. aureus can cause skin and soft tissue

infections, as well as pneumonia,
bacteremia, and sepsis
• S. aureus is the leading cause of hospital-
related infections in the US
• Ability to cause disease depends on
expression of an array of adhesins, toxins,
and compounds that affect the immune
system
Quorum Sensing
• QS regulates expression of virulence-

associated genes via the agr locus
• Includes
  a-toxin
  d-haemolysin
  rot (repressor of toxins)
Fibronectin-binding proteins
Protein A, Coagulase
–  secreted toxins (proteases,
lipase, superantigens, urease)
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THOMPSON AND BROWN (2014) PATHOGENS AND GLOBAL HEALTH
90
80
Percentage Frequency (%)

70
60
50
MRSA
40
MSSA
30
20
10
0
TSST LUKE-ED β-HAEMO γ-HAEMO γ-VAR
S. aureus Toxins
Frequency distribution of agr Frequency Distribution of toxins

alleles
Frequency
Distribution of
Staphylococcal
enterotoxin
Quorum Sensing
• agr system inhibits biofilm formation

• Could be due to the fact that establishing
a biofilm community at LCD allows S.
aureus time to grow to HCD and, at that
point, it is optimally poised to secrete
virulence factors
• To facilitate its dispersal, S. aureus
terminates biofilm production and
decreases surface proteins and adhesins
at HCD
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Quorum Sensing
Gram Negative System
The Aliivibrio fischeri LuxI/LuxR System
• LuxI is the protein responsible for

autoinducer production, and LuxR is the
protein necessary for detecting and
responding to autoinducer (acylated-
homoserine lactone).
Quorum Sensing
The Allivibrio fischeri LuxI/LuxR System:
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Quorum Sensing
• Activation of the luxICDABE operon

establishes a positive feedback loop,
increasing the level of autoinducer
production (via luxI) and the amount of
light the bacterium emits (via luxCDABE).
• Conversely, the LuxR-autoinducer

complex inhibits the transcription of luxR,
which provides a compensatory
mechanism for the regulation of light
production.
Quorum Sensing
• In most described cases, acyl-HSL signals
are generated by the activity of a single
enzyme that uses as substrates S-
adenosylmethionine (SAM) and an
intermediate of fatty acid biosynthesis, acyl-
acyl carrier protein.
• Thus Pseudomonas aeruginosa RhlI primarily

catalyzes the synthesis of N-butyryl-HSL
(C4-HSL), and P. aeruginosa LasI directs the
synthesis of N-(3-oxododecanoyl)-HSL
(3OC12-HSL).
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Quorum Sensing
• Quorum sensing was first discovered to
control the luminescence of Allivibrio fischeri,
a bacterium that forms a mutualistic light
organ symbiosis with certain marine animals.
• Acyl-HSL signaling is critical for virulence of

the plant pathogen Erwinia carotovora and
for virulence of P. aeruginosa in mouse
models of lung and burn infections, in
invertebrates, and in plants.
Quorum Sensing
• Both of these systems, LasR-I and RhlR-I,
have linked R and I genes.
• It is estimated that about 4% of the roughly

6,000 P. aeruginosa genes are controlled by
quorum sensing.
• The autoinducers 3OC12-HSL and C4-HSL

bind to a regulatory protein (LasR or RhlR),
activating it to the form that stimulates gene
expression.
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Quorum Sensing
Quorum Sensing
• LasI synthesizes autoinducers and more
activation of LasR, thus the bacterium
responds quickly by producing virulence
factors controlled by LasR.
• These include lasA and lasB, a gene that

encodes an alkaline protease (aprA), the
gene that encodes exotoxin A (toxA), and
two component genes for the export of these
exoproteins (xcpP, xcpR).
44
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Quorum Sensing
Quorum Sensing
• rhlA and rhlB encode enzymes that synthesize rhamnolipid,
which maybe involved in cell damage.
• A LasR mutant strain could

colonize the lung, but it did not
achieve high densities and it
did not cause pneumonia,
bacteremia, or death.
• The mutant did not have a

growth defect under laboratory
conditions, it could invade the
lung and survive but it could
not cause disease.
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ASSESSMENT OF PATHOGENICITY USING

THE ROMAINE LETTUCE LEAF MODEL
Rhl+/Las+ Rhl-/Las+
Rhl+/Las- Rhl-/Las-
ASSESSMENT OF PATHOGENICITY USING

THE ROMAINE LETTUCE LEAF MODEL
 Isolates grouped as rhl-

/las+ and rhl+/las- showed
increased infection potential
due to severe maceration of
tissues as early as day 2
 Isolates grouped as rhl-/las- and rhl-/las+ showed

reduced infection potential since the more severe
effects were not observed until day 4.
 Of significance, isolates that were rhamnolipid
negative/elastase negative could still cause infection
in plants.
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Quorum Sensing
References:
Bacterial Pathogenesis: A Molecular Approach. 2nd Edition by

Abigail A. Salyers and Dixie D. Whitt. ASM Press, Washington , D.C.
Mechanisms of Microbial Disease. 2nd Edition by Moselio

Schaechter, Gerald Medoff and Barry I. Eisenstein. Williams and
Wilkins, Balitmore.
Regulation of finP Transcription by DNA Adenine Methylation

in the Virulence Plasmid of Salmonella enterica.
Eva M. Camacho, Ana Serna, Cristina Madrid, Silvia Marques, Raul
Fernandez, Fernando de la Cruz, Antonio Juarez, and Josep
Casadesus. (2005) J Bact., 187, 5691–5699
Quorum Sensing
References:
Acyl-homoserine lactone quorum sensing in Gram-negative

bacteria: A signaling mechanism involved in associations with
higher organisms.
Matthew R. Parsek and E. Peter Greenberg (2000). PNAS 97, 8789-
8793.
Regulation of Virulence by Environmental Signals in Group A

Streptococci: Influence of Osmolarity, Temperature, Gas
Exchange, and Iron Limitation on emm Transcription.
Kevin S. McIver, Andrew S. Heath, and June R. Scott
Infection and Immunity (1995) 63, 4540–4542
DNA methylation-dependent regulation of Pef expression in

Salmonella typhimurium.
Brad Nicholson and David Low (2000). Molecular Microbiology 35,
728.
47
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Quorum Sensing
References:
Virulence control in group A Streptococcus by a two-

component gene regulatory system: Global expression
profiling and in vivo infection modelling.
Morag R. Graham, Laura M. Smoot, Cristi A. Lux Migliaccio, Kimmo
Virtaneva, Daniel E. Sturdevant, Stephen F. Porcella, Michael J.
Federle, Gerald J. Adams, June R. Scott, and James M. Musser. Proc
Natl Acad Sci U S A. 2002; 99(21): 13855–13860.
The AraC Family Transcriptional Regulator Rv1931c Plays a

Role in the Virulence of Mycobacterium tuberculosis.
Cristiane C. Frota, K. G. Papavinasasundaram, Elaine O. Davis, and
M. Joseph Colston. Infection and Immunity (2004), 72, 5483-5486
48

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MICR3214 Microbial Interactions2019

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9/8/2019

Paul D. Brown, PhD

L1. Microbial Interactions

Overall Learning Outcomes:

• Students should be able to discuss

Specific Learning Outcomes

Should be able to satisfactorily discuss/describe:

 Two-component signal transduction

 Koch’s postulates, virulence, virulence factors and

 Quorum Sensing in Gram positive and Gram

• Within these communities, interactions occur

• The categories used to describe these

• It occurs when microbial populations are spatially

• E.g. a pathogenic microbe invading the root of a

• Microflora of the oral, respiratory, and GI tracts

• E. coli is acquired shortly after birth and this

• Relationship could borders on mutualism

• Epiphytic bacteria living on the surface of

• Both populations benefit from the

• E.g. the relationship between Chlorobium and

• Chlorobium reduces CO2 and oxidizes H2S;

• Possibly most famous is lichen;

• Interaction between the two

• This occurs when two populations utilize the same

• Here no toxins or inhibitory substances are

• The oxidation of sulfur by Thiobacillus

• Yeast produces ethanol during fermentation,

• The parasite derives its

Signs and symptoms

Infection with Candida

The two-stage life cycle of

As with parasitism, predation is a mechanism of

• Bacteria have a wide range of regulatory

• Stress conditions such as antibiotics, heavy

• The machinery responsible for these adaptive

• This phosphotransfer network couples

• A two component system is called "two

• Prior to the use of the phrase "two

• The consequence of ligand binding, an

• Eventually, studies of the arabinose

• In the mid-1980s, it had become clear that some

 EnvZ/OmpR - osmoregulation in E. coli

Virulence and its Regulation

• Virulence, or the ability to cause an

• Different virulence factors are required by

Virulence and its Regulation

• The classical virulence factor is the bacterial

• A German microbiologist, Robert Koch, realized

Virulence and its Regulation

 Association of the microbe with the lesions of the

 Isolating the bacterium in pure culture

 Showing that the isolated bacterium causes

 Re-isolating the bacterium from the intentionally

• Microbe found in diseased tissue but not in

• Microbe can be isolated from diseased tissue

• Microbe can cause disease when inoculated

• Microbe must then be re-isolated in pure