Characterization of cytokine binding and its implications in the physiology of Aggregatibacter actinomycetemcomitans

Bacterial pathogens cause severe infections in humans. The emergence of multiresistant bacteria requires the development of new antimicrobial strategies. Bacteria utilize various virulence mechanisms to promote their survival in the host organism. Among these virulence mechanisms, we may find novel targets for antimicrobial treatment. This thesis concerns a virulence mechanism of an oral bacterium that may disturb the human immune response and potentially increase bacterial virulence. The model organism for this study is Aggregatibacter actinomycetemcomitans, a gram-negative opportunistic pathogen that forms biofilms on the surface of teeth and causes an inflammatory oral disease called periodontitis. Bacteria living in biofilms are more resistant to antimicrobial compounds and the mechanisms associated with their virulence are poorly known. Previously, it was discovered that A. actinomycetemcomitans binds and uptakes human inflammatory cytokines, which may modulate the local inflammatory milieu and weaken the host defense. This thesis consists of four parts, each published as a separate article in scientific journals. In the beginning of this thesis I discovered a novel cytokine-binding outer membrane protein, BilRI, in A. actinomycetemcomitans. BilRI was located on the outer membrane of A. actinomycetemcomitans and interacted with the human cytokine IL-1β. The second article showed that BilRI binds multiple cytokines and has an intrinsically disordered structure. In the third article, I showed that a major bacterial cell wall component, lipopolysaccharide, interacted with certain cytokines. This interaction was shown in many A. actinomycetemcomitans serotypes using intact bacterial cells, outer membrane vesicles and isolated lipopolysaccharides. Further, some information was obtained about the location of the interaction site. In the fourth article, I found that a channel protein of A. actinomycetemcomitans, HofQ, binds human cytokines, such as IL-1β and IL-8. This protein has been previously associated with DNA uptake, providing a possible link between the naturally occurring uptake of extracellular DNA and the cytokine uptake mechanism in bacteria. This thesis showed the cytokine binding of A. actinomycetemcomitans outer membrane molecules and how they affected the physiology of the pathogen. Further research is needed regarding the roles of these molecules in the virulence of A. actinomycetemcomitans.Patogeeniset bakteerit aiheuttavat ihmisille vakavia infektioita. Nykyisille antibiooteille multiresistenttien bakteerien yleistyminen vaatii uusien antimikrobistrategioiden kehittämistä. Bakteerit käyttävät useita virulenssimekanismeja, jotka auttavat niitä selviytymään isäntäorganismissa. Näiden virulenssimekanismien joukosta saattaisi löytyä uusia antimikrobihoidon kohteita. Väitöskirjassa tutkittiin suubakteerin virulenssimekanismia, joka saattaa häiritä ihmisen immuunipuolustusta ja lisätä bakteerin virulenssia. Työssä käytettiin malliorganismina Aggregatibacter actinomycetemcomitans – bakteeria, joka on gram-negatiivinen opportunistinen patogeeni. Se muodostaa biofilmejä hampaiden pinnalle ja aiheuttaa suun tulehdussairautta parodontiittia. Biofilmeissä elävät bakteerit ovat vastustuskykyisempiä antimikrobisille aineille ja niiden virulenssiin liittyviä mekanismeja tunnetaan huonosti. Aiemmin on havaittu, että A. actinomycetemcomitans sitoo ja siirtää sisäänsä ihmisen tulehduksenvälittäjäaineita, sytokiineja. Tämä saattaa muuttaa paikallista tulehdusympäristöä ja heikentää isännän puolustusta. Väitöskirjatutkimukseni koostuu neljästä osatyöstä, jotka on julkaistu erillisinä artikkeleina tieteellisissä lehdissä. Väitöskirjatutkimukseni alussa löysin A. actinomycetemcomitansista uuden sytokiineja sitovan ulkokalvon proteiinin, BilRI:n. BilRI tuottui bakteerin ulkokalvolle ja sitoutui IL-1β:aan. Toisessa osatyössä BilRI:n havaittiin sitovan useita sytokiineja ja olevan rakenteeltaan luonnostaan järjestäytymätön. Kolmannessa osatyössä osoitin, että bakteerin soluseinän komponetti lipopolysakkaridi vuorovaikuttaa joidenkin sytokiinien kanssa. Vuorovaikutus osoitettiin useiden eri A. actinomycetemcomitans serotyyppien kanssa kokonaisilla bakteerisoluilla, ulkokalvon vesikkeleillä sekä eristetyillä lipopolysakkarideillä. Lisäksi vuorovaikutuskohdan sijainnista saatiin tietoa. Neljännessä osatyössä havaitsin, että HofQ-kanavaproteiini sitoo myös ihmisen sytokiineja, kuten IL-1β:aa ja IL-8:aa. Aiemmin tämän kanavaproteiinin on havaittu osallistuvan DNA:n sisäänottoon, joten työ osoittaa mahdollisen yhteyden luonnollisen solunulkoisen DNA:n sisäänoton ja sytokiinien sisäänoton välillä. Väitöskirjassa osoitettiin A. actinomycetemcomitansin ulkokalvon molekyylien sitoutuminen sytokiineihin sekä niiden vaikutuksia bakteerin fysiologiaan. Näiden molekyylien vaikutuksista A. actinomycetemcomitansin virulenssiin tarvitaan vielä lisää tutkimusta

Aggregatibacter actinomycetemcomitans LPS binds human interleukin-8

Various gram-negative species sequester host cytokines using outer membrane proteins or surface modulation by sulfated polysaccharides. An outer membrane lipoprotein (BilRI) of the periodontal pathogen Aggregatibacter actinomycetemcomitans binds several cytokines, including interleukin (IL)-8. Because IL-8 is positively charged at physiological pH, we aimed to determine whether IL-8 interacts with negatively charged lipopolysaccharide (LPS). Binding was investigated using electrophoretic mobility shift assays and microwell-based time-resolved fluorometric immunoassay. LPS from each tested strain of A. actinomycetemcomitans (N = 13), Pseudomonas aeruginosa (N = 1) and Escherichia coli (N = 1) bound IL-8. The Kd value of the A. actinomycetemcomitans LPS-IL-8 interaction varied between 1.2–17 μM irrespective of the serotype and the amount of phosphorus in LPS and was significantly lower than that of the BilRI-IL-8 interaction. Moreover, IL-8 interacted with whole A. actinomycetemcomitans cells and outer membrane vesicles. Hence, LPS might be involved in binding of IL-8 to the outer membrane of A. actinomycetemcomitans. This raises an interesting question regarding whether other gram-negative periodontal pathogens use LPS for IL-8 sequestering in vivo.</p

Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake

Naturally competent bacteria acquire DNA from their surroundings to survive in nutrient-poor environments and incorporate DNA into their genomes as new genes for improved survival. The secretin HofQ from the oral pathogen Aggregatibacter actinomycetemcomitans has been associated with DNA uptake. Cytokine sequestering is a potential virulence mechanism in various bacteria and may modulate both host defense and bacterial physiology. The objective of this study was to elucidate a possible connection between natural competence and cytokine uptake in A. actinomycetemcomitans. The extramembranous domain of HofQ (emHofQ) was shown to interact with various cytokines, of which IL-8 exhibited the strongest interaction. The dissociation constant between emHofQ and IL-8 was 43nM in static settings and 2.4M in dynamic settings. The moderate binding affinity is consistent with the hypothesis that emHofQ recognizes cytokines before transporting them into the cells. The interaction site was identified via crosslinking and mutational analysis. By structural comparison, relateda type I KH domain with a similar interaction site was detected in the Neisseria meningitidis secretin PilQ, which has been shown to participate in IL-8 uptake. Deletion of hofQ from the A. actinomycetemcomitans genome decreased the overall biofilm formation of this organism, abolished the response to cytokines, i.e., decreased eDNA levels in the presence of cytokines, and increased the susceptibility of the biofilm to tested -lactams. Moreover, we showed that recombinant IL-8 interacted with DNA. These results can be used in further studies on the specific role of cytokine uptake in bacterial virulence without interfering with natural-competence-related DNA uptake.Peer reviewe

A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8

Intrinsically disordered proteins (IDPs) do not have a well-defined and stable three-dimensional fold. Some IDPs can function as either transient or permanent binders of other proteins and may interact with an array of ligands by adopting different conformations. A novel outer membrane lipoprotein, bacterial interleukin receptor I (BilRI) of the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans binds a key gatekeeper proinflammatory cytokine interleukin (IL)-1β. Because the amino acid sequence of the novel lipoprotein resembles that of fibrinogen binder A of Haemophilus ducreyi, BilRI could have the potential to bind other proteins, such as host matrix proteins. However, from the tested host matrix proteins, BilRI interacted with neither collagen nor fibrinogen. Instead, the recombinant non-lipidated BilRI, which was intrinsically disordered, bound various pro/anti-inflammatory cytokines, such as IL-8, tumour necrosis factor (TNF)-α, interferon (IFN)-γ and IL-10. Moreover, BilRI played a role in the in vitro sensing of IL-1β and IL-8 because low concentrations of cytokines did not decrease the amount of extracellular DNA in the matrix of bilRI- mutant biofilm as they did in the matrix of wild-type biofilm when the biofilms were exposed to recombinant cytokines for 22 hours. BilRI played a role in the internalization of IL-1β in the gingival model system but did not affect either IL-8 or IL-6 uptake. However, bilRI deletion did not entirely prevent IL-1β internalization, and the binding of cytokines to BilRI was relatively weak. Thus, BilRI might sequester cytokines on the surface of A. actinomycetemcomitans to facilitate the internalization process in low local cytokine concentrations.</p

Identification of a Novel Bacterial Outer Membrane Interleukin-1B-Binding Protein from Aggregatibacter actinomycetemcomitans

Peer reviewe

Outer membrane channel protein of an oral pathogen binds human cytokine IL-8

Opportunistic pathogen Aggregatibacter actinomycetemcomitans resides in the multispecies biofilm in dento-gingival junction. A. actinomycetemcomitans binds and uptakes human proinflammatory cytokines, which may increase the bacterial virulence. Outer membrane  secretin channel (here OMS), a DNA binder, is involved in uptake of DNA by A. actinomycetemcomitans. However, OMS homologue in Neisseria meningitidis binds cytokines. ELISA was used to characterize the binding of cytokines to extramembranous domain of OMS (emOMS). Binding of IL-8 to emOMS was studied with multiple methods: EuLISA, Thermofluor and Biacore. NMR and cross-linking were used to study the interaction sites. As OMS was previously described as a DNA binder, the interaction between IL-8 and DNA was studied with EMSA. emOMS bound multiple cytokines, IL-8 being the strongest binder with Kd values from nM to μM. Binding of IL-8 stabilized the structure of emOMS. NMR revealed binding to five residues in IL-8 near Lys15 that was close emOMS in the cross-linking experiment. IL-8 interacted with DNA in EMSA. OMS might form the channel that transfers IL-8 inside the bacterial cells which could be coupled to the DNA uptake. This bacterial mechanism seems both to affect the virulence of the bacterium and have potential to interfere with the host defense by binding cytokines

Aggregatibacter actinomycetemcomitans LPS binds human interleukin-8

Various gram-negative species sequester host cytokines using outer membrane proteins or surface modulation by sulfated polysaccharides. An outer membrane lipoprotein (BilRI) of the periodontal pathogen Aggregatibacter actinomycetemcomitans binds several cytokines, including interleukin (IL)-8. Because IL-8 is positively charged at physiological pH, we aimed to determine whether IL-8 interacts with negatively charged lipopolysaccharide (LPS). Binding was investigated using electrophoretic mobility shift assays and microwell-based time-resolved fluorometric immunoassay. LPS from each tested strain of A. actinomycetemcomitans (N = 13), Pseudomonas aeruginosa (N = 1) and Escherichia coli (N = 1) bound IL-8. The K-d value of the A. actinomycetemcomitans LPS-IL-8 interaction varied between 1.2-17 mu M irrespective of the serotype and the amount of phosphorus in LPS and was significantly lower than that of the BilRI-IL-8 interaction. Moreover, IL-8 interacted with whole A. actinomycetemcomitans cells and outer membrane vesicles. Hence, LPS might be involved in binding of IL-8 to the outer membrane of A. actinomycetemcomitans. This raises an interesting question regarding whether other gram-negative periodontal pathogens use LPS for IL-8 sequestering in vivo

A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8

Abstract Intrinsically disordered proteins (IDPs) do not have a well-defined and stable 3-dimensional fold. Some IDPs can function as either transient or permanent binders of other proteins and may interact with an array of ligands by adopting different conformations. A novel outer membrane lipoprotein, bacterial interleukin receptor I (BilRI) of the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans binds a key gatekeeper proinflammatory cytokine interleukin (IL)-1β. Because the amino acid sequence of the novel lipoprotein resembles that of fibrinogen binder A of Haemophilus ducreyi, BilRI could have the potential to bind other proteins, such as host matrix proteins. However, from the tested host matrix proteins, BilRI interacted with neither collagen nor fibrinogen. Instead, the recombinant non-lipidated BilRI, which was intrinsically disordered, bound various pro/anti-inflammatory cytokines, such as IL-8, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-10. Moreover, BilRI played a role in the in vitro sensing of IL-1β and IL-8 because low concentrations of cytokines did not decrease the amount of extracellular DNA in the matrix of bilRI− mutant biofilm as they did in the matrix of wild-type biofilm when the biofilms were exposed to recombinant cytokines for 22 hours. BilRI played a role in the internalization of IL-1β in the gingival model system but did not affect either IL-8 or IL-6 uptake. However, bilRI deletion did not entirely prevent IL-1β internalization, and the binding of cytokines to BilRI was relatively weak. Thus, BilRI might sequester cytokines on the surface of A. actinomycetemcomitans to facilitate the internalization process in low local cytokine concentrations