ROLE OF PERSISTER CELLS IN CHRONIC INFECTION,CLINICAL RELEVANCE AND

PERSPECTIVES ON ANTI PERSISTER THERAPIES

Persisters: cells that entered a dormant,

multidrug-tolerant state

Antimicrobial tolerance: a property of dormant cells that

survive killing by bactericidal antibiotics in the absence of drug resistance mechanisms

Antimicrobial resistance: an

ability to prevent the interaction of an

antibiotic with a target by a variety

of resistance mechanisms

Bactericidal antibiotics kill cells by forcing the active

target to produce corrupted products.

Streptomycin interrupts translation, producing toxic

misfolded peptides .

Resistance versus tolerance

PERSISTER ARE NON GROWING OR SLOW GROWING CELLS WHOSE FORMATION IS FAVOURED UNDER GROWTH LIMITING CONDITION.

PERSISTER CELLS HAVE BEEN OBSERVED IN E.COLI,C.ALBICANS,M.TUBERCULOSIS,P.AERUGINOSA,S.AUREUS

Antibiotic treatment of microbial populations

typically results in a biphasic killing pattern.

When the antibiotic concentration exceeds a

certain threshold, only so-called ‘persister’ cells

survive

.

Persister proteins act by blocking the target, so no corrupted product can be

produced.

Bactericidal antibiotics also lead to the production of reactive oxygen species,

contributing to cell death .

ANTIBIOTIC PRESSURE DROPS,

THE CELLS WILL GIVE RISE TO A POPULATION THAT IS AS SUSCEPTIBLE AS THE ORIGINAL ONE

THAT POSSESSES A SMALL PROPORTION OF PERSISTER CELLS.

THIS DISCRIMINATES PERSISTER CELLS FROM RESISTANT MUTANTS, WHICH EXHIBIT STABLE, INHERITABLE DRUG INSENSITIVITY.

A microbial population

(confined by a light-grey ellipse)

consists of mainly

antibiotic-sensitive cells (dark-grey). (top panel) ,

the population may contain resistant cells (black), due to

permanent change at the genetic level.

Antibiotic treatment

(+Ab), only resistant cells

remain. On regrowth (−Ab),

the entire population is composed of

resistant individuals.

(bottom panel)

The population may contain

persister cells (black),

resulting from a reversible phenotypic switch to a

tolerant state. After antibiotic treatment, only persister cells

remain.

Upon regrowth,

the population will exhibit the same sensitivity

as the original

population.

Antibiotic resistance versus persistence

No actual mechanism of persister formation.

Accidental formation of various misfolded proteins causes stasis, producing persisters.

Persister formation

Few persisters are present in an early exponential culture and then rise sharply at around mid-exponential state.

Maintaining populationinearlyexponentialstate by serialreinoculation resulted in complete disappearance of persisters

A more advanced method isolating native persister was hypothesis that dormant cells with diminished protein synthesis

PERSISTERS FORM A SMALL AND TEMPORARY POPULATION, MAKING ISOLATION CHALLENGING.

SIMPLEST APPROACH IS TO LYSE A POPULATION OF

GROWING CELLS WITH A Β-LACTAM ANTIBIOTIC AND

COLLECT SURVIVING PERSISTERS

Persister Isolation andTranscriptome Analysis

AN ESCHERICHIA COLI STRAIN EXPRESSING A DEGRADABLE GFP UNDER THE CONTROL OF A RIBOSOMAL PROMOTER WAS CULTURED TO EXPONENTIAL STATE, AND THE CELLS WERE SORTED BASED ON THE GREEN SIGNAL.

THE LARGER GREEN POPULATION IS MADE OF REGULAR CELLS SMALL SUBPOPULATION IS MADE OF DIM CELLS THAT HAVE DIMINISHED TRANSLATION.

CLINICAL RELAVANCE OF PERSISTER CELLS

Cystic fibrosis is a common recessive genetic disease which affects the entire body, causing progressive disability and often early death.

CF is caused by a mutation in the gene for the protein cystic fibrosis transmembrane conductance regulator.

Difficulty breathing is the most serious symptom and results from frequent lung infections that are not cure by antibiotics.

CYSTIC FIBROSIS PNEUMONIA

CYSTIC FIBROSIS PNEUMONIA

BACTERIAL PERSISTER

CELLS

RECALCITRANT NATURE OF CHRONIC

INFECTIONS.

Candidiasis or thrush is a fungal infection of any of the Candida species (all yeasts), of which Candida albicans is the most common.

Candidiasis infections that range from superficial, such as oral thrush and vaginitis, to systemic and potentially life-threatening diseases. confined to severely immunocompromised person

Candidiasis or moniliasis or oidiomycosis

C. albicans are a serious threat to human health today.

Incidence of

candidiasis in hospita

l

Candida biofilms

formed on medical

indwelling devices such as catheters, heart valve

replacements

Tuberculosis, MTB or TB (short for tubercle bacillus) is a common and in many cases lethal infectious disease caused by various strains of mycobacteria, usually Mycobacterium tuberculosis.

It is spread through the air when people who have an active MTB infection cough, sneeze, or otherwise transmit their saliva through the air.

TUBERCULOSIS

M. TUBERCULOSIS IS ALSO CAPABLE OF FORMING COMPLEX BIOFILMS CONTAINING DRUG-TOLERANT CELLS THAT SHARE IMPORTANT FEATURES WITH PERSISTER CELLS OF OTHER SPECIES .

FIRST, A SMALL FRACTION OF BIOFILM CELLS ARE INSENSITIVE EVEN TO ANTIBIOTICS THAT TARGET NON-REPLICATING BACTERIA, AT DRUG CONCENTRATIONS OF UP TO 100 TIMES THE MIC.

THE PRESENCE OF M. TUBERCULOSIS BIOFILM PERSISTERS, REFLECTED BY A PLATEAU IN THE KILLING CURVE, GIVING RISE TO THE BIPHASIC KILLING PATTERN THAT IS TYPICAL OF PERSISTENCE TO ANTIBIOTICS.

A model of a relapsing biofilm infections

REGULAR CELLS AND PERSISTER CELLS FORM IN THE BIOFILM AND ARE SHED OFF INTO SURROUNDING TISSUE AND THE BLOODSTREAM.

ANTIBIOTICS KILLS REGULAR CELLS, AND THE IMMUNE SYSTEM ELIMINATES ESCAPING PERSISTER CELLS.

THE MATRIX PROTECTS PERSISTER CELLS FROM THE IMMUNE SYSTEM, AND WHEN THE CONCENTRATION OF THE ANTIBIOTIC DROPS, THEY REPOPULATE THE BIOFILM, CAUSING A RELAPSE.

BIOFILMS ARE CREATED WHEN BACTERIA OR FUNGI ATTACH TO A SURFACE AND FORM A PROTECTIVE POLYSACCHARIDE COATING

BIOFILM MAY FORM ON A PROSTHETIC DEVICE OR MAY FORM IN INFECTED BONE OR ON THE SURFACE OF A HEALING SURGICAL AREA.

THE IMMUNE SYSTEM RESPONSE THAT THESE BACTERIA IS INEFFECTIVE SINCE IT CANNOT PENETRATE THE SURFACE, IT MAY ACTUALLY BE HARMFUL TO SURROUNDING HEALTHY AREAS.

ANTIBIOTICS MAY TREAT THE INFECTION THAT SPREADS BEYOND THE BIOFILM BUT OFTEN CANNOT GET AT THE ROUTE CAUSE.

CAUSE OF POOR RESPONSE TO ANTIBIOTICS.

ANTIMICROBIAL

AGENTS FAIL TO

PENETRATE BEYOND

THE SURFACE

LAYERS OF THE

BIOFILM.

OUTER LAYERS OF

BIOFILM CELLS

ABSORB DAMAGE.

ANTIMICROBIAL AGENTS

ACTION MAY BE IMPAIRED IN AREAS OF

WASTE ACCUMULATI

ON OR ALTERED

ENVIRONMENT (PH, PCO2, PO2, ETC).

ANTIMICROBIAL AGENTS

MAY BE TRAPPED

AND DESTROYED BY ENZYMES

IN THE BIOFILM MATRIX.

ANTIMICROBIAL AGENTS MAY

NOT BE ACTIVE AGAINST

NONGROWING MICROORGANISMS (PERSISTER

CELLS)

EXPRESSION OF BIOFILM-SPECIFIC

RESISTANCE GENES (E.G.,

EFFLUX PUMPS).

STRESS RESPONSE TO HOSTILE

ENVIRONMENTAL CONDITIONS (E.G.,

LEADING TO AN OVEREXPRESSION OF ANTIMICROBIAL

AGENT-DESTROYING

ENZYMES)

BIOFILMS HAVE BEEN LINKED

DENTAL DISEASE

ENDOCARDITIS

CYSTITISURINARY TRACT

INFECTIONS

DEEP-SEATED INFECTIONS

INDWELLING DEVICE

A TEAM LED BY JAMES COLLINS AT THE UNIVERSITY OF BOSTON FOUND KILL BACTERIAL PERSISTERS BY ADDING SUGARS TO AMINOGLYCOSIDE ANTIBIOTICS.

'MOST ANTIBIOTICS WORK AGAINST GROWING BACTERIAL CELLS', SAYS COLLINS.

WE COULD TRIGGER THE BACTERIAL PERSISTERS WITH SUGARS OR METABOLITES TO GET THEM TO GROW, AND THEN KILL THEM WITH THE ANTIBIOTICS.'

A SUGAR-ANTIBIOTIC COMBO COULD TARGET BACTERIA RESPONSIBLE FOR PERSISTENT INFECTIONS

ADDING SUGAR TO ANTIBIOTICS CAN BOOST THEIR EFFECTIVENESS AND PREVENT RECURRENT AND CHRONIC INFECTIONS

THE TEAM INVESTIGATED THE EFFECT OF A NUMBER OF SUGARS COMBINED WITH GENTAMICIN, AN AMINOGLYCOSIDE, ON S.AUREUS OR ESCHERICHIA COLI  BY LABELLING THE ANTIBIOTIC WITH A RED FLUORESCENT DYE.

THEY ANALYSED THE BACTERIA USING FLUORESCENCE-ACTIVATED CELL SORTING.

THE TEAM FOUND THAT THE SUGARS LED THE BACTERIA TO TAKE UP THE ANTIBIOTIC, KILLING A HIGH PROPORTION OF THEM.

COLLINS EXPLAINS THAT WHEN THE BACTERIA WERE TREATED WITH GENTAMICIN AND SUGARS, SUCH AS MANNITOL, FRUCTOSE OR GLUCOSE, THE BACTERIAL PERSISTERS DID NOT START GROWING AGAIN, BUT STILL DIED.

THE RESEARCHERS THEORISE THAT, INSTEAD OF PROMOTING GROWTH, THE SUGARS ALTER THE ELECTROCHEMICAL GRADIENT IN THE BACTERIA, ALLOWING THE ANTIBIOTIC IN.

THE SUGAR-ANTIBIOTIC COMBINATION WAS SUCCESSFULLY TESTED ON BACTERIAL BIOFILMS - POPULATIONS OF BACTERIA, ENCAPSULATED IN A POLYSACCHARIDE MATRIX, THAT ARE EXTREMELY DIFFICULT TO KILL.

TOXIN–ANTITOXIN (TA) LOCI ARE PRIMARILY KNOWN AS PLASMID-

BORNE OPERONS THAT PROMOTE PLASMID

INHERITANCE BY THE EXPRESSION OF A STABLE

TOXIN, WHICH IS COUNTERACTED BY AN UNSTABLE ANTITOXIN.

EVIDENCE SUPPORTING THE

INVOLVEMENT OF TA MODULES IN

PERSISTENCE COMES FROM THE

EXPRESSION PROFILE OF PERSISTER CELLS

PERSPECTIVES ON ANTI-PERSISTER THERAPIES

TA MODULES WERE FOUND TO BE HIGHLY OVEREXPRESSED IN PERSISTER CELLS COMPARED TO THE REST OF THE POPULATION.

TWO MOST PROMINENT TA LOCI INVOLVED IN PERSISTENCE, HIPBA AND TISAB.

Mutant allele hipA7 increases the persister fraction from 10−6 to 10−2 without altering the MIC.

hip locus revealed that it consists of two genes, hipA and hipB .

HipA is a member of the phosphatidylinositol 3/4-kinase superfamily and can phosphorylate the translation factor EF-Tu .

hipBA

The hipB gene product is a small Cro-like protein with a helix–turn–helix DNA-binding domain.

It functions as a repressor for the hipBA operon by binding to four operator sites on the promoter region of hipBA and inhibits HipA activity through covalently binding to the toxin.

Upon exceeding a threshold level, unbound HipA triggers growth arrest and thus entry into the persistent state .

crystal structure of HipA and the binding mechanism with HipB make this a very interesting target for the rational design of an anti-persistence therapy.

A model of TisB-dependent persister formation in Escherichia coli.

Fluoroquinolone antibiotic causes DNA damage by

converting the DNA gyraseand topoisomerase

to endonucleases.

Activates the RecA protein, which in turns

activates the LexA repressor, causing it to

cleave.

SOS response is induced, and repair enzymes that contain lex boxes in their

promoter regions are transcribed.

The Lex repressor also controls the expression of the TisB toxin, a small cationic membrane-acting agent.

Decrease in the proton motive force (pmf) and ATP shuts down target functions, including DNA topoisomerase and gyrase, and a dormant persister is formed.

The SOS response leads to overexpression of the TisB toxin and persister formation.

TisB toxicity is inhibited by an antisense RNA antitoxin, IstR-1. TisA contains the binding site for this antisense RNA molecule .

TisB

Persister level increases equally in a istR deletion mutant, which is correlated with an overexpression of the toxin TisB.

A tisAB mutant displays a significantly decreased number of persister cells after treatment with ciprofloxacin

Quorum sensing (QS) molecules are well-known extracellular factors that mediate cell–cell-communication and affect many bacterial processes.

LasR and LasI mutant strains of P. aeruginosa, which are disabled in their QS response

P. aeruginosa QS molecules pyocyanin and homoserine lactone.

Extracellular factors and surface modifications

QUORUM SENSING

BECAUSE OF ITS CRUCIAL ROLE IN REGULATING CLINICALLY RELEVANT PROCESSES SUCH AS BIOFILM FORMATION AND VIRULENCE, INHIBITION OF QS AS A NON-LETHAL MEANS OF FIGHTING INFECTIOUS DISEASE.

P. aeruginosa persistence genes revealed the involvement of surface-associated components in persistence.

Mutation of edpA resulted in a decrease in persisters of 10–1600-fold .

ECTOPIC EXPRESSION OF THE CHAPERONE DNAJ FROM E. COLI OR PMRC, A S. ENTERICA ENZYME,BOTH TOXIC WHEN OVERPRODUCED IN E. COLI, INHIBITED CELL GROWTH AND RESULTED IN MULTIDRUG TOLERANCE

GLOBAL REGULATORS

THE MAJORITY OF HITS WERE IN GLOBAL REGULATORS,DKSA, DNAKJ, HUPAB, AND IHFAB

Redundant pathways of persister formation. Candidate persister genes and their targets, when known, are

indicated. Abbreviations: FMN, flavin mononucleotide; pmf, proton motive force; TA, toxin/antitoxin

Two interesting candidate genes that may be more directly involved in persister formation:

YgfA, which can inhibit nucleotide synthesis,

YigB, which may block metabolism by depleting the pool of flavinmononucleotide

A progressive increase in the exposure to fluoroquinolones kills regular cells but has little effect on the survival of persisters.

This means that the dormant persisters rather than regular cells with induced repair will ultimately survive the DNA-damaging antibiotic.

ANTIBIOTICS CAN INDUCE FORMATIONOFMULTIDRUG-TOLERANT PERSISTERS

REPAIR MECHANISM

COMBINING A REGULAR ANTIBIOTIC WITH A COMPOUND DISABLING A PERSISTER MAINTENANCE FUNCTION COULD PROVIDE A STERILIZING THERAPEUTIC.

DEVELOP PRODRUGS, COMPOUNDS THAT ENTER INTO THE CELL AND ARE CONVERTED TO A GENERALLY REACTIVE COMPOUND BY A BACTERIA-SPECIFIC ENZYME.

THE ACTIVE SPECIES WILL THEN ATTACK UNRELATED TARGETS,INCLUDING DNA AND THE MEMBRANE, AND KILL DORMANT CELLS.

An anti-persister - produced by combining a antibiotic,such as a fluoroquinolone, and an inhibitor of an essential persister protein.

Mutation in the essential E. coli gene encoding PlsB, which increases the Km of the PlsB protein, results in a 2–3-log drop in the frequency of persister formation.

Proteins such as PlsB that are essential for maintaining the persister are attractive targets for anti-persister drug development

We should look for antibiotic that kills sensitive as well as

persister cells

CONCLUSION