Inflammation, DNA damage and Staphylococcus aureus infection Nadejda Berkova UMR1253 STLO (Science Technologie du Lait et de l’ Oeuf) INRA Agrocampus Ouest, Rennes) .01
INFLAMMATION • Microbial infection Cause of • Noxious substances • Tissue stress inflammation • Tissue injury • Defense from microbial in fection Physiological • Expulsion of noxious substances purpose • Tissue repaire • Adaptation to stress • Tissue dammage • Autoimmune diseases Outcome • Disease of homeostasis • Fibrosis, metaplasie, cancer .02
HOMEOSTATIS AND INFLAMMATION • Maintains the stability of biological system Homeostasis • Result of perturbations that Inflammation exceed the homeostatic capacity of the system Inflammation Inflammation Homeostasis Pathological Pathological state state Normal state Medzhitov R. IBiology .03
Metchnikoff's theory of inflammation Prolonged inflammation causes harm in bystander normal tissues and promotes inflammatory diseases Physiological inflammation is a protective response mediating the 1845-1916 elimination of injurious agents Homeostasis Alfred Tauber .04
Chronic inflammation Acute inflammation Adaptive Innate Concentration of immunity immunity cytokines Inflammation Inflammation Moderate Severe tissue tissue damage damage Resolution Chronic tissue damage Endless End/Healing Pro-inflammatory cytokines Anti-inflammatory cytokines .05
INFECTION MEDIATED-INFLAMMATION Pathogens induce various levels of inflammation in the host Inflammation caused by infection leads to increased production of mutagenic compounds as reactive oxygen species (ROS)/reactive nitrogen species (RNS) and subsequent oxidative DNA damage These in turn cause further inflammation and exacerbation of DNA damage .06
CHRONIC INFLAMMATION AS A DRIVING FORCE IN THE GENESIS OF DNA DAMAGE Chronic inflammation is an important modulator of mutation susceptibility Nakad and Schumacher Frontiers in Genetics 2016 .07
THE IMMUNE SYSTEM RESPONDS TO DNA DAMAGE Chronic Premature aging Cancer inflammation Damaged endogenous DNA triggers inflammatory gene expression . DNA damage response (DDR) orchestrates DNA damage checkpoint activation DNA SENSING/ACTIVATION OF IMMUNE SIGNALING Protein complex of replication protein A (RPA) Activation of TLR9 signalling DNA damage-binding protein 2, DNA glycosylases Mre11-Rad50-Nbs1 (MRN) complex In duction of IFN-stimulated genes ERK1/2 MAPK signaling MutS proteins .08
Staphylococcus aureus Gram-positive bacterium that is carried by up to 50% of healthy people .09
Staphylococcus aureus is responsible for a wide range of infections in human and animals Human: Dairy cattle: Pneumonia, osteomyelitis, meningitis, endocarditis, Chronic mastitis sepsis, chronic infections as bone and joint infections .010
S. aureus, FACULTATIVE INTRACELLULAR PATHOGEN Foster T. et al, Nat Rev Microbio, 2016 .011
Many pathogens such as Helicobacter sp ., Chlamydia sp ., Salmonella sp ., or Escherichia coli induce DNA damage in the host, demonstrating that it may lead to genomic alterations and cancer-associated transformation Does Staphylococcus aureus induce DNA damage in host cells? .012
S. aureus INFECTION Control cells S. aureus -infected cells 45 min 6 days 12 days TEM .013
S. aureus -induced DNA damage SA SA Oxidative stress ROS Cell cycle arrest ɣH2AX DDR activation DSBs 53BP1 ATM Cell death .014
S. aureus induces DNA damage HeLa ɣH2AX ɣH2AX ɣH2AX Control Control Control Control 6 h 6 h 20 h S. aureus MOI 1:25 S. aureus MOI 1:50 Etoposide 6 h Etoposide Etoposide Etoposide 20 h Cell count S. aureus S. aureus S. aureus S. aureus ɣH2AX ɣH2AX is a marker for DNA damage in the absence of apoptosis .015
S. aureus triggers DNA repair HeLA S. aureus MW2 S. aureus MW2 Control 6 h S. aureus MW2 6 h Control 24 h S. aureus MW2 24 h + KU 55922 6 h + KU 55922 24 h 53 BP1 MG-63 S. aureus MW2 S. aureus MW2 Control 6 h S. aureus MW2 6 h Control 24 h S. aureus MW2 24 h + KU 55922 6 h + KU 55922 24 h 53 BP1 53BP1 is the early repair protein To define whether the formation of 53BP1 foci was associated with a canonical DDR comprising the triggering of the ATM kinase-signaling pathway, HeLa and MG-63 cells were treated with the ATM inhibitor KU-55933. .016
S. aureus induced ROS-mediated DNA damage Relative phosphorylation (%) 6 h 250% * * 200% 150% 100% 50% 20 h Cells+ S. aureus Cells+NAC+ S. aureus Relative phosphorylation (%) * 250% * 200% 150% 100% 50% Cells+ S. aureus Cells+NAC+ S. aureus ROS can trigger DNA damage in the host cells N-acetyl-L-cysteine (NAC) is an inhibitor of ROS .017
S. aureus prompts a ROS production, which induces 8-oxoG DNA lesion HeLA Control 6 h Etoposide 6 h S. aureus MW2 6 h 8-oxoG Control 24 h Etoposide 24 h S. aureus MW2 24 h Mutagenic lesion 8-oxoG is most often involved in oxidative DNA damage .018
Are S. aureus virulence factors PSMs and LPL involved in DNA damage? Secreted virulence factors Phenol-solubles modulines S.aureus (PSM) PSM α , PSM β , δ -toxine Lipoproteins A pathogenicity island that contains a cluster of lipoprotein-encoding genes, lpl Membrane bound virulence factors .019
Phenol-soluble modulins peptides (PSMs) define the virulence potential of S. aureus . PSM α 1-PSM α 4 δ -toxin 20-25 amino acids PSM β 44 amino acids PSM α 1-PSM α 4 are encoded in the psm α operon PSM β 1 and PSM β 2 are encoded in the psm β operon δ -toxin is encoded within the coding sequence for RNAIII, the RNA effector molecule of the accessory gene regulator (AGR) quorum-sensing system Peschel and Michael Otto, 2013 .020
OVERVIEW OF PHENOL-SOLUBLE MODULIN ACTIVITIES Cell cycle arrest Almeida S. (post-doc) Filho R et al. (PhD) Inflammasomes induction Leite E et al. (PhD) Peschel and Michael Otto, 2013 .021
Pivotal role of S. aureus PSMα1– 4 toxins in the induction of host DNA damage a b c d Cell count ɣH2AX Control Control Control Control LAC (WT) pTX D 16 LAC (WT) pTX D 16 LAC (WT) pTX D 16 LAC (WT) pTX D 16 LAC D psm ab hld pTX D 16 LAC D psm ab hld pTX D 16 LAC D psm ab hld pTX D 16 LAC D psm ab hld pTX D 16 Etoposide LAC D psm ab hld pTX D α1 -4 LAC D psm ab hld pTX D β1 -2 LACD psm ab hld pTX D hld S. aureus LAC (WT) pTXΔ16 contains plasmid pTXΔ16 (blue line ) PSM-deficient deletion mutant LAC Δ psm αβ hld pTXΔ16 (yellow line ) Complemented mutant LAC Δ psm αβ hld pTX Δα1 -4 (blue-green line, b) LAC Δ psm αβ hld pTX Δβ ( blue-green line, c) and LACΔ psm αβ hld pTXΔhld (blue -green line d) Constructed in the Michael Otto laboratory, NIH, USA .022
Bacterial Lipoproteins After crossing the cytoplasmic membrane Lgt (prolipoprotein diacyl glyceryl transferase) transfers a diacylglyceride to the polypeptide chain and Lsp (lipoprotein signal peptidase) cleaves the signal peptide .023
Lpls dampen DNA damage of infected cells a b Cell count ɣH2AX Control USA300 wt Control USA300 wt USA300Δ lpl (pTX30- lpl ) USA300 Δ lpl Etoposide Mutant USA300Δ lpl in which the entire lpl cluster was deleted and the complemented mutant USA300Δ lp l (pTX30- lpl ) were constructed in the Fritz Goetz laboratory, University of Tubingen, Germany .024
S. aureus clinical isolats Three couples of S. aureus isolates were selected from patients P1, P2, P3 who were diagnosed with initial acute (i) and recurrent (r) staphylococcal BJI Patient P1 Patient P2 Patient P3 45i 47i 51i 46r 48r 52r Genomic comparison did not reveal mutations in the major regulatory systems ( agr , sar , sigma genes) or in virulence genes between initial and recurrent strains .025
S. aureus recurrent isolates induce stronger DNA damage than initial acute isolates 6 h Patient 1 Patient 2 Patient 3 Relative phosphorylation (%) 45i vs 46r 47i vs 48r 51i vs 52r * 150% 150% * 150% * * 125% 125% 125% 100% 100% 100% 75% 75% 75% 50% 50% 50% 20 h 150% * * * 150% Relative phosphorylation (%) * 150% * * 125% 125% 125% 100% 100% 100% 75% 75% 75% 50% 50% 50% High content screening analysis .026
S. aureus recurrent isolates express a lower amount of Lpls than initial acute isolates Patient 1 Patient 2 Patient 3 45i 46r 47i 48r 51i 52r a MW kDa 70 55 40 35 25 P1: 45i vs 46r P2: 47i vs 48r P3: 51i vs 52r 15 b Lpl 40 35 .027
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DNA DAMAGE DURING INFECTION DNA repair Cell cycle arrest Apoptosis Level of DNA damage .029
Does Staphylococcus aureus induce cell cycle arrest in host cells? .030
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