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Bio films and Quorum sensing
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DR.T.V.RAO MD 1
Dr.T.V.Rao MD
SOCIO-MICROBIOLOGYBIOFILMS AND QUORUM SENSING IN
INFECTIOUS DISEASES
DR.T.V.RAO MD 2
• 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.
SOCIOMICROBIOLOGY
DR.T.V.RAO MD 3
SOCIOMICROBIOLOGY
• The term "sociomicrobiology" was introduced by Matt Parsek & Peter Greenberg in 2005 (Trends in Microbiology, 13:27-33) 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).
DR.T.V.RAO MD 4
INTRODUCTIONQuorum 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 .
DR.T.V.RAO MD 5
• 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
SOCIOMICROBIOLOGY ADVANCES THE UNDERSTANDING MICROBES
DR.T.V.RAO MD 6
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
DR.T.V.RAO MD 7
• 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?”
MICROBES RUN IN OUR BODY AS NORMAL FLORA
DR.T.V.RAO MD 8
• 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
MICROBES DO HAVE SOCIAL LIFE AND WELL ADOPTED WHY DO WE WORK
ON IT?
DR.T.V.RAO MD 9
• In the past decade, significant debate has surrounded the relative contributions of genetic determinants versus environmental conditions to certain types of human behavior
GENETICS X ENVIRONMENT
DR.T.V.RAO MD 10
• 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.
WHAT IS A BIOFILM?
DR.T.V.RAO MD 11
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.
DR.T.V.RAO MD 12
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
DR.T.V.RAO MD 13
• 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.
WHERE ARE BIOFILMS FOUND?
DR.T.V.RAO MD 14
BIOFILMS ARE CONCERN IN EVERY ASPECT OF LIFE
DR.T.V.RAO MD 15
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?
DR.T.V.RAO MD 16
• 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.
BIOFILMS ARE ADVANTAGEOUS TO MICROORGANISMS
DR.T.V.RAO MD 17
• 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.
BIOFILMS IN MEDICINE
DR.T.V.RAO MD 18
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.
DR.T.V.RAO MD 19
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).
DR.T.V.RAO MD 20
• 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.
QUORUM SENSING
Henke and Bassler, 2004
DR.T.V.RAO MD 21
Environmental Pseudomonas
PA colonization-ASYMPTOMATIC
Model of Chronic Pseudomonas aeruginosa Infection in Cystic Fibrosis
Bacterial Adaptation
• Alginate/mucoidy• Auxotrophy
Increased bacteria - SYMPTOMATIC
Lung
Disease• surface modifications• Increased PQS
(biofilm, virulence,
antibiotic resistance)
Innate Immune Selective Pressure
• Structuring of multicellular communities
• Stress survival
• Production of
• Antibiotics
• Pigments
• Host tissue degrading enzymes
BEHAVIORS CONTROLLED BY QUORUM SENSING
DR.T.V.RAO MD 23
• Cell-cell communication can occur within and between bacterial species, and between bacteria and their eukaryotic hosts.
QUORUM SENSING CAN OCCUR …..
DR.T.V.RAO MD 24
• Bacteria use Quorum sensing to mastermind
behaviors including
• Mating
• Releasing toxins
• Causing disease (virulence )
QUORUM SENSING HELPS IN …
DR.T.V.RAO MD 25
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
DR.T.V.RAO MD 26
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.
DR.T.V.RAO MD 27
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
DR.T.V.RAO MD 28
THE CHAIN OF COMMAND IN BACTERIAL COMMUNICATION
LuxRMaster Regulator
Protein
Qrr1
Qrr2
Qrr3
Qrr4
Sm
all
RN
A m
ole
cule
s (s
RN
As
)
Qrr = Quorum Regulatory RNA
Signals from sRNAs turn LuxR on and off
Signals from LuxRcontrol quorum-sensing
AI-2AI-2 attaches to LuxR to
initiate cellular communication
LuxRMaster Regulator
Protein
Qrr1
Qrr2
Qrr3
Qrr4
Sm
all
RN
A m
ole
cule
s (s
RN
As
)
Qrr = Quorum Regulatory RNA
Signals from sRNAs turn LuxR on and off
Signals from LuxRcontrol quorum-sensing
AI-2AI-2 attaches to LuxR to
initiate cellular communication
DR.T.V.RAO MD 29
QUORUM SENSING IN P. AERUGINOSA
P. Aeruginosa
•Major cause of hospital infection in the US.•Major cause of deaths in intubated CF patients, and IV fed patients.
Quorum Sensing: The ability of a bacterial colony to sense its size and regulate its activity in response.
Examples: P. aeruginosa
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 bythe immune system.
DR.T.V.RAO MD 30
QUORUM SENSING IN P. AERUGINOSA
Planktonic
Loosely Bound EPS secreting
DR.T.V.RAO MD 31
“WALL SENSING” IN P. AERUGINOSA
Wall Sensing: The ability of bacteria to differentiate in response toContact with a wall (the substratum).
Planktonic Loosely Bound EPS secreting
DR.T.V.RAO MD 32
• 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.
INTRASPECIES VS. INTERSPECIES COMMUNICATIONS
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 regulatorspecific binding sites for AHL and DNA (sensor/transducer)
Genetic basis regulatory circuit involving both regulatory genes accumulation of AHL - activating gene transcription
DR.T.V.RAO MD 34
LABORATORY MADE MOLECULES HAVE SOLUTIONS TO COUNTER 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
Autoinducer 2 may hold the key to disrupting quorum-sensing.
The three general classes of quorum-sensing systems
Modified oligopeptides
Processing and secreation
SHKA R
ATPADP
Class Autoinducer Strain
P. aeruginisaV. fisheriE. carotovoraA. tumefaciensY. enterocolitica
E. coli O157:H7V. harveyiV. choleraeV. vulnificusS. Typhimurium
B. subtilisS. aureusS. pneumoniaeS. epidermidisL. lactis
QS1
G(+)
QS2
AI1
PAI
AI2
DR.T.V.RAO MD 36
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.
37
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.
38
WHAT IS THE NEED FOR QUORUM SENSING INHIBITORS ?
39
STRATEGIES FOR QUORUM SENSING INHIBITION
3 strategies can be applied
Targeting AHL signal
dissemination
Targeting the signal
receptor
Targeting signal
generation
Signal precursor
Signal
Signal receptor
Signal precursor Signal precursor
Signal Signal
Signal receptor Signal receptor
X
X
X
DR.T.V.RAO MD 40
• 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
FURTHER RESEARCH IS NEEDED IN SOCIOMICROBIOLOGY ON ….
DR.T.V.RAO MD 41
VISIT ME FOR MORE TOPICS OF INTEREST IN INFECTIOUS DISEASES
DR.T.V.RAO MD 42
• Created by Dr.T.V.Rao MD for ‘ e’ learning resources for Medical Microbiologists in the
Developing world • Email.