BIOFILMS AND QUORUM SENSING , SOCIAL MICROBIOLOGY

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Socio-microbiologybiofilms and quorum sensing in
infectious diseases

• Dr.T.V.Rao MD

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Sociomicrobiology

• Sociomicrobiology" is part of the broader
  discipline "Microbiology", the study of organisms
  (bacteria, yeast, molds, viruses and protists)
  that cannot be observed with the naked eye, but
  having critical mechanisms for propagation.

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Sociomicrobiology

• The term "sociomicrobiology" was introduced by
  Matt Parsek Peter Greenberg in 2005 (Trends in
  Microbiology, 1327-33) and refers to the group
  behavior of micro-organisms, Two topics that
  form the core of sociomicrobiological research
  are microbial biofilm formation and cell-cell
  communication (quorum sensing).

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Introduction

• Quorum sensing is cell to cell signaling
  mechanism that enables the bacteria to
  collectively control gene expression.
• This type of bacterial communication is achieved
  only at higher cell densities.
• Bacteria release various types of molecules
  called as auto inducers in the extracellular
  medium, these molecules are mediators of quorum
  sensing.
• When concentration of these signaling molecules
  exceed a particular threshold value, these
  molecules are internalized in the cell and
  activate particular set of genes in all
  bacterial population, such as genes responsible
  for virulence, competence, stationary phase etc .

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Sociomicrobiology advances the understanding
microbes

• The study of group behavior in microbes
• Debate over environmental vs. genetic
  determinates
• Biofilms and quorum sensing
• Model for dissecting social behavior at a
  genetic level

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Bacteria are dynamic creatures

• Bacteria are dynamic creatures that are able to
  regulate their metabolism and lifestyle in
  response to a variety of environmental cues.
  These cues include changes in their chemical,
  physical, and biological surroundings. In recent
  decades, microbiologists have come to appreciate
  that bacteria are even able to recognize changes
  in their own population density. Cell
  density-dependent regulation has been termed
  "quorum sensing."

• iosynthetic and regulatory prodigiosin mutants
  of Serratia

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Microbes run in our body as normal flora

• Microbes run much of our body. The human micro
  biome in our gut, mouth, skin, and elsewhere,
  harbors 3,000 kinds of bacteria with 3 million
  distinct genes. (Our own cells struggle by on
  only 18,000 genes or so.)This biotech century
  will be microbe enhanced and maybe microbe
  inspired.Confronting a difficult problem we
  might fruitfully ask, What would a microbe do?

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Microbes do have social life and well adopted

Why do we work on it?

• Models to understand biology of sociality
• To develop new medicines to treat devastating
  bacterial infections
• ?Understanding bacteria
• Sociomicrobiology
• The new science of
• Tools for synthetic biology

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Genetics x environment

• In the past decade, significant debate has
  surrounded the relative contributions of genetic
  determinants versus environmental conditions to
  certain types of human behavior

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What is a Biofilm?

• A structured community of bacterial cells
  enclosed in a self-produced polymeric matrix.
• -Biofilms are a protective mode of growth that
  allows survival in hostile environments.
• -Bacteria in biofilms are inherently resistant to
  killing.

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Biofilms are .

• Biofilms are multicellular aggregates of bacteria
  and yeast that congregate on surfaces.
• Biofilm may form on any surface exposed to
  biofilm-forming bacteria and some amount of
  water.
• Biofilms are formed to protect the bacteria from
  host defenses, antibiotics, and from harsh
  environmental conditions.

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Biofilms are communities of Microorganisms

• Biofilms are communities of microorganisms that
  develop on surfaces in most natural and
  artificial environments. Biofilm maturation
  requires cell contact with a surface and
  cell-cell adhesion counteracting the shear forces
  of the environment. Biofilms are characterized by
  a surface covered by a high number of cells (a
  film) encased in a self-produced extra cellular
  matrix, are highly heterogeneous environment,
  both at structural, physiological and specific
  levels and biofilm bacteria express still
  under-explored specific biological properties
  such as a characteristic increased tolerance to
  biocides

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Where are Biofilms Found?

• Biofilms are found almost everywhere in nature,
  including rivers, lakes, soil, water pipes, and
  even inside the human body
• Bacterial biofilms are often a cause of
  infections associated with medical implants such
  as catheters and IV lines and other medical
  devices.

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Biofilms are concern in every aspect of life
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The Dynamics of Growing Biofilm

• Quorum Sensing
• What is it?
• How does it work?
• Heterogeneous structures
• How do these cells use polymer gel for
  locomotion?
• What are the mechanisms of pattern (structure)
  formation?
• Why is polymer gel so effective as a protective
  environment?

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Biofilms are advantageous to microorganisms

• Biofilms are important survival mechanisms for
  bacterial cells. According to in vitro studies,
  they can avoid attack by host defenses. it is
  difficult for phagocytic cells to engulf bacteria
  in biofilms. Also, biofilms are much more
  resistant than planktonic cells to antimicrobial
  agents. The bacteria within the biofilm remain
  healthy, and the biofilm can regrow. Repeated use
  of antimicrobial agents on biofilms can cause
  bacteria within the biofilm to develop an
  increased resistance to biocides.

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Biofilms in medicine

• In medicine, biofilms spreading along implanted
  tubes or wires can lead to pernicious infections
  in patients. Biofilms on floors and counters can
  make sanitation difficult in food preparation
  areas.
• Dental plaque is a yellowish biofilm that build
  up on the teeth. If not removed regularly, it can
  lead to dental caries.

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Why Research on Biofilms?

• Due to the morphology of biofilms, bacteria
  capable of forming them are highly resistant to
  antibiotics, making treatment very difficult.
• In the US alone, one million nosocomial (hospital
  acquired) infections each year are caused by
  bacterial biofilms, leading to longer
  hospitalization, surgery, and even death.

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Biofilms and Infections

• Biofilms are responsible for Otitis Media, the
  most common acute ear infection.
• Biofilms play a role in Bacterial Endocarditis
  (infection of the inner surface of the heart and
  its valves).
• Biofilms form frequently in patients with Cystic
  Fibrosis (a chronic disorder resulting in
  increased susceptibility to serious lung
  infections).
• Biofilms also play a role in Legionnaire's
  disease (an acute respiratory infection resulting
  from the aspiration of clumps of Legionnella
  biofilms detached from air and water
  heating/cooling and distribution systems).

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Quorum Sensing

• A process that enables bacteria to communicate
  using secreted signaling molecules called auto
  inducers
• This process enables a population of bacteria to
  regulate gene expression collectively and
  therefore, control behavior on a community-wide
  scale.

Henke and Bassler, 2004
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Model of Chronic Pseudomonas aeruginosa
Infection in Cystic Fibrosis
Environmental Pseudomonas
Lung Disease
Bacterial Adaptation

• Alginate/mucoidy
• Auxotrophy

• surface modifications
• Increased PQS
• (biofilm, virulence,
• antibiotic resistance)

Innate Immune Selective Pressure
Increased bacteria - SYMPTOMATIC
PA colonization-ASYMPTOMATIC
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Behaviors controlled by quorum sensing

• Structuring of multicellular communities
• Stress survival
• Production of
• Antibiotics
• Pigments
• Host tissue degrading enzymes

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Quorum Sensing can occur ..

• Cell-cell communication can occur within and
  between bacterial species, and between bacteria
  and their eukaryotic hosts.

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Quorum Sensing helps in

• Bacteria use Quorum sensing to mastermind
  behaviors including
• Mating
• Releasing toxins
• Causing disease (virulence )

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How pathogenic Bacteria Use Quorum Sensing

• These changes culminate in an infection that can
  ambush and overwhelm our immune system defenses.

The bacteria appear relatively innocuous as they
quietly grow in number.

• When their population reaches a certain level,
  instant changes occur in their
• Behavior
• Appearance
• Metabolism

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Quorum Sensing Systems Gram negative bacteria

• Lux I/R systems
• Auto inducers acylated homoserine lactone
• Lux I-type enzymes synthesize acylated homoserine
  lactone (AHL) auto inducers by ligating a
  specific acyl moiety to the homocysteine moiety
  of S-adenosylmethionine (SAM)
• LuxR-type proteins bind their cognate
  autoinducers and control transcription of target
  genes.

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Quorum Sensing Systems Gram positive bacteria

• Two-component systems involved
• Autoinducers modified oligopeptides
• The signals are synthesized as precursor
  peptides, which are subsequently processed and
  secreted
• Sensor histidine kinases detect the extracellular
  peptide autoinducers, autophosphorylate and
  transmit sensory information via phosphorylation
  of a response regulator
• Response regulator changes gene expression

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The Chain of Command in Bacterial Communication
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Quorum Sensing in P. Aeruginosa
Quorum Sensing The ability of a bacterial
colony to sense its size and regulate its
activity in response.
Examples P. aeruginosa
P. Aeruginosa

• Major cause of hospital infection in the US.
• Major cause of deaths in intubated CF patients,
  and IV fed patients.

P. Aeruginosa in planktonic (non-colonized) form
are non-toxic, but as a biofilm, they are highly
toxic and well protected by the polymer gel in
which they reside. However, they do not become
toxic or begin to form polymer gel until the
colony is of sufficient size to overwhelm the
immune system. Before this, they cannot be
detected by the immune system.
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Quorum sensing in P. Aeruginosa
Planktonic
Loosely Bound
EPS secreting
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Wall Sensing in P. Aeruginosa
Wall Sensing The ability of bacteria to
differentiate in response to Contact with a wall
(the substratum).
EPS secreting
Planktonic
Loosely Bound
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Intraspecies vs. interspecies communications

• AHL type autoinducers are for intraspecies
  communications
• AI-2 and its synthase, LuxS, are widespread,
  existing in many bacterial phyla. AI-2 is
  suggested to serve as an interspecies bacterial
  communication signal.

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How quorum sensing works? Signaling compounds,
auto inducers AI synthases (luxI gene
products) cell density indicators -
non-essential aa, acyl homoserine lactones
lactone ring part - binding to a receptor site
acyl chain tail determining the species
specificity - oligopeptides -
diketopiperazines - quinolone - furanones
Recognition systems LuxR transcriptional
regulator specific binding sites for AHL and DNA
(sensor/transducer) Genetic basis regulatory
circuit involving both regulatory genes
accumulation of AHL - activating gene
transcription
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Laboratory made molecules have solutions to
counter quorum sensing
Autoinducer 2 may hold the key to disrupting
quorum-sensing.

• AI-2 contains the element boron

AI-2 and similar boron-containing molecules made
in the laboratory could serve as decoys to
subvert virulence and other quorum-sensing
behaviors
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The three general classes of quorum-sensing
systems
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Communication Between Prokaryotes and Eukaryotes

• Chemical communication extends to the eukaryotic
  hosts with which bacteria engage in pathogenic
  and symbiotic relationships.
• P. aeruginosa AHLs enter eukaryotic cells and
  stimulate production of chemokine interleukin 8
  (IL-8), which in turn induces the NF-kB
  transcription factor.
• These responses cause recruitment of neutrophils
  to the lung, in which they contribute to
  pulmonary inflammation and tissue deterioration.

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Need for Inhibition of quorum sensing

• Inhibition of quorum sensing has been proved to
  be very potent method
• for bacterial virulence inhibition.
• Several QS inhibitors molecules has been
  discovered.
• QS inhibitors have been synthesized and have
  been isolated from several
• natural extracts such as garlic extract.
• QS inhibitors have shown to be potent virulence
  inhibitor both in in-vitro
• and in-vivo, using infection animal models.

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What is the need for Quorum sensing inhibitors ?
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Strategies for quorum sensing inhibition
3 strategies can be applied
Targeting AHL signal dissemination
Targeting the signal receptor
Targeting signal generation
Signal precursor
Signal precursor
Signal precursor
X
Signal
Signal
Signal
X
X
Signal receptor
Signal receptor
Signal receptor
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Further research is needed in sociomicrobiology
on .

• Further studies are needed on quorum sensing
• regulated gene expression
• Onset of QS
• Affects on Onset
• Architecture and physical flow
• Functional consequences on biofilm
• community
• Role in mixed species systems
• Effects of signal consumption

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infectious diseases
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• Created by Dr.T.V.Rao MD for e learning
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  Developing world
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