Novel Concepts of Mycobacterial Evasion Mechanisms

Mycobacterium tuberculosis -bakteerin aiheuttama tuberkuloosi on tällä hetkellä maailman tappavin infektiotauti. Nykyinen tuberkuloosin antibioottihoito on useamman lääkkeen yhdistelmä ja se kestää useita kuukausia, mutta ei siitäkään huolimatta takaa onnistunutta mykobakteerin häätämistä. Tehottomien antibioottien käyttö sekä pitkät hoitoajat keskeytyksineen edesauttavat antibioottiresistenttien kantojen syntyä. Lisäksi turvallisen ja tehokkaan rokotteen puute tekee tuberkuloosin ehkäisemisestä hankalaa. Yksi syy näihin haasteisiin tuberkuloosin ehkäisemisessä ja hoidoissa on mykobakteerin kyky kehittää krooninen infektio ja suojella itseään ympäristöhaitoilta kuten aktiiviselta immuunivasteelta sekä antimikrobiaalisilta lääkkeiltä. Tämän tutkimuksen tavoitteena on tutkia mykobakteerin mekanismeja, joiden avulla se pystyy estämään antibioottien ja immuunivasteiden tehokkaan toiminnan, sekä etsiä uusia strategioita näihin mekanismeihin puuttumiseen. Mycobacterium marinum -bakteeria ja seeprakalaa käytettiin tutkimaan isännän ja patogeenin välistä vuorovaikutusta. Mykobakteeri kykenee aktiivisesti vaimentamaan immuunivasteita ja myöhästyttämään hankittujen immuunivasteiden alkamista. Tutkimuksessa testattiin immunomodulaation tehoa näiden mykobakteerin ominaisuuksien estämisessä. Ilman hoitoja noin 10 % aikuisista seeprakaloista pystyy luonnollisesti pitämään mykobakteerimäärän alle qPCR:n detektiorajan. Tätä osuutta voitiin kuitenkin nostaa 25 %:iin aktivoimalla immuunivaste lämpötapetulla Listeria monocytogenes -bakteerilla ennen mykobakteeri-infektiota. Aikaansaatu suojaava immuunivaste vähensi merkitsevästi mykobakteerimääriä mitattuna neljä viikkoa infektion jälkeen ja jopa steriloi mykobakteerin osasta populaatiota sekä villityypin kalassa että rag1-/- -mutanttikaloissa, mikä viittaa synnynnäisten immuunivasteiden tärkeään rooliin mykobakteerin steriloivassa vasteessa. Suojaavassa vasteessa mpeg1, tnf ja nos2b -geenien ilmentyminen oli merkitsevästi lisääntynyt, kun taas sod2 oli vähentynyt verrattuna kontrolliryhmään. Nämä tulokset viittaavat mykobakteerin häätämiseen infektion varhaisessa vaiheessa lisääntyneen tulehdusvasteen ja tehokkaamman mykobakteerin tuhoamisen avulla. Huomattavaa on, että immunolodulaatio lämpötapettua L. monocytogenes -bakteeria hyödyntäen ei saanut aikaan suojaavaa vastetta, jos infektio oli jo olemassa. Vastaavaa immuuniterapiaa voitaisiin kuitenkin käyttää ennaltaehkäisevänä menetelmänä korkean tuberkuloosi- ilmaantuvuuden alueilla. Toinen tutkittu mykobakteerin suojautumiskeino on biofilmin muodostaminen. Immuunivasteiden heikentämisen ja infektion aikaansaamisen jälkeen mykobakteeri muodostaa bakteeriyhteisöjä granuloomissa ja tuottaa solunulkoista matriksia, joka antaa bakteeripopulaatiolle tukea ja suojaa. Nopeakasvuista bioluminoivaa M. marinum -bakteeria käytettiin biofilmin kypsymisen ja koostumuksen tutkimuksessa in vitro. Tulokset osoittivat, että biofilmin kypsyminen ei muuttanut rifampisiinin MIC-arvoa (minimum inhibitory concentration), mutta nosti MBC-arvoa (minimum bactericidal concentration) 63-kertaisesti verrattuna planktoniseen yksisoluisena kasvavaan mykobakteeriin kahden päivän biofilmin kasvattamisen jälkeen. Toistuvilla antibioottialtistuksilla pystyttiin varmistamaan, että biofilmin lisääntynyt antibioottitoleranssi ei johtunut geneettisestä resistenssistä. Elävien mykobakteerien lukumäärää biofilmissä pystyttiin vähentämään hajottamalla solunulkoisen matriksin tärkeimpiä polymeerejä yhdessä rifampisiini-hoidon kanssa, mikä osoitti biofilmin matriksin tärkeyden antibioottitoleranssissa. Nopea bioluminesenssimittaukseen perustuva tappokäyräanalyysi validoitiin mittaamaan erityisesti persistoivien mykobakteerien alapopulaatiota. Analyysissa seurataan bakteeripopulaation tappokinetiikkaa korkealla antibioottikonsentraatiolla, ja voidaan mitata persisteribakteerien alapopulaatiota, joka kykenee sietämään antibioottia keskimääräistä pidemmän ajan. Tappokinetiikka oli merkitsevästi hitaampi yhden viikon ikäisellä M. marinum biofilmillä verrattuna planktoniseen yksisoluiseen kasvatukseen. Lisäksi rifampisiinin konsentraatio saturoitui 400 μg/ml:ssa, jonka jälkeen antibioottikonsentraation nostaminen ei nopeuttanut bakteerien tappoa. Tätä menetelmää on mahdollista käyttää seulomaan hoitoja, jotka vaikuttavat erityisesti persistoiviin soluihin. Lopuksi tutkittiin mykobakteerin biofilmin solunulkoisen matriksin proteiineihin, GroEL1 ja GroEL2, kohdentuvia sybodeja in vitro M. marinum ja M. tuberculosis biofilmeissä sekä ex vivo seeprakalan granuloomissa. Sybodit ovat synteettisiä vasta- aineen sitoutumisosaa matkivia molekyylejä, jonka sitoutumista voidaan helposti muokata ja seuloa halutun kohteen mukaan olemassa olevista sybody-kirjastoista. Seulonnasta saatujen potentiaalisten sybodien sitoutumista mykobakteerin biofilmiin arvioitiin konfokaalimikroskopian avulla. Fluoresenssileimatut anti-GroEL-sybodit tunnistivat GroEL-proteiineja biofilmin päältä sekä in vitro että ex vivo tehden sybodista houkuttelevan vaihtoehdon erilaisten molekyylien kohdentamisessa mykobakteerin biofilmeihin. Nykyisiä antibioottihoitoja voidaan mahdollisesti tehostaa ja hoitojen pituutta lyhentää ottamalla huomioon granuloomien sisällä biofilmeissä olevat mykobakteeripersisterit. Ymmärtämällä paremmin mykobakteerin menetelmiä, joilla se suojelee itseään ympäristön haittavaikutuksilta, tulevaisuuden hoitoja voidaan kohdentaa tehokkaammin Mycobacterium tuberculosis -bakteeria vastaan.Tuberculosis is caused by bacterium Mycobacterium tuberculosis, and it is currently the deadliest infectious disease worldwide. The standard antimicrobial regimen is a cocktail of antimicrobials taken for several months, which still does not guarantee a successful eradication of the bacteria. Treatment dropouts and the use of antimicrobials that are inefficient in eradicating the infection have led to emerged drug-resistance. Also, the lack of safe and effective vaccine makes the prevention of tuberculosis difficult. One reason for the challenges in both prevention and treatment of tuberculosis is the ability of mycobacterium to cause chronic infection and protect itself from environmental hazards such as active immune responses and antimicrobials. The aim of this study was to examine mycobacterial evasion mechanisms against antimicrobials and immune responses and propose new intervention strategies. Mycobacterium marinum and zebrafish were used to study the interaction of the host and the pathogen. Mycobacterium can actively suppress immune responses and delay the onset of adaptive responses, and to prevent this immune evasion, an immunomodulation was tested. In the adult zebrafish population, around 10% of the population is able to naturally retain the M. marinum load below the detection limit of qPCR. This proportion could be increased up to 25% by priming with heat- killed Listeria monocytogenes prior the mycobacterial infection. The induced protective immune response significantly reduced mycobacterial loads after four weeks of infection and induced clearance in both wildtype and rag1-/- mutant fish showing the important role of innate immune responses in the sterilizing response. The protective immune response was characterized with an increased expression of mpeg1, tnf and nos2b and decreased expression of sod2. These results indicate early clearance mediated via pro-inflammatory responses and enhanced killing of mycobacteria. Importantly, the immunomodulation was ineffective if the infection was already established. However, a similar approach could be used as a prophylactic treatment in high burden areas. Another mycobacterial evasion strategy is the formation of biofilms. After suppressing immune responses and establishing the infection, mycobacteria form bacterial communities in granulomas and produce extracellular matrix that gives structure and protection for the bacterial population. The fast-growing M. marinum with a bioluminescent cassette was used to study the biofilm maturation and composition in vitro. The results showed that the biofilm maturation did not alter the minimum inhibitory concentration (MIC) but increased minimum bactericidal concentration (MBC) 63 times compared to planktonic cells within two days of biofilm culturing. It was further confirmed with repeated antimicrobial exposures that the increased tolerance of biofilm cultures was not due to genetic resistance. Degrading any of the major extracellular matrix components in combination with rifampicin reduced the number of live bacteria in a biofilm, demonstrating the important role of the biofilm matrix in the antimicrobial tolerance. Time-kill curve analysis with a quick bioluminescence read-out was established to specifically measure the subpopulation of persister mycobacteria. In the analysis, the killing kinetics of a bacterial population was followed after the exposure to high concentration of a bactericidal antimicrobial, here rifampicin, to measure the persister subpopulation that can tolerate the drug for a prolonged time. For maturing M. marinum biofilm, the killing kinetics were significantly different after one week of culturing compared to planktonic cells, and the rifampicin concentration was saturated at 400 μg/ml after which increasing the antimicrobial concentration did not alter the killing kinetics. This method has a potential in screening for treatments that specifically target mycobacterial persisters. Finally, sybodies against biofilm extracellular matrix proteins GroEL1 and GroEL2 were used to specifically target M. marinum and M. tuberculosis biofilms in vitro and in ex vivo zebrafish granulomas. Sybodies are synthetic molecules mimicking the binding domain of antibodies. The binding properties of the sybodies can be easily modified and screened against the wanted target in sybody libraries. The potential sybodies acquired from the screens were assessed by confocal imaging. Fluorescence labelled anti-GroEL sybodies were able to bound GroEL on both in vitro and ex vivo biofilms making sybodies an attractive molecular carrier to target mycobacterial biofilms. A treatment that specifically targets mycobacterial persisters in biofilms inside granulomas could enhance the efficacy of antimicrobial therapy and shorten the current treatment time. By understanding better, the mycobacterial evasion mechanisms, future treatments can more effectively be targeted against Mtb

In vitro and ex vivo proteomics of Mycobacterium marinum biofilms and the development of biofilm-binding synthetic nanobodies

The antibiotic-tolerant biofilms present in tuberculous granulomas add an additional layer of complexity when treating mycobacterial infections, including tuberculosis (TB). For a more efficient treatment of TB, the biofilm forms of mycobacteria warrant specific attention. Here, we used Mycobacterium marinum (Mmr) as a biofilm-forming model to identify the abundant proteins covering the biofilm surface. We used biotinylation/streptavidin-based proteomics on the proteins exposed at the Mmr biofilm matrices in vitro to identify 448 proteins and ex vivo proteomics to detect 91 Mmr proteins from the mycobacterial granulomas isolated from adult zebrafish. In vitro and ex vivo proteomics data are available via ProteomeXchange with identifiers PXD033425 and PXD039416, respectively. Data comparisons pinpointed the molecular chaperone GroEL2 as the most abundant Mmr protein within the in vitro and ex vivo proteomes, while its paralog, GroEL1, with a known role in biofilm formation, was detected with slightly lower intensity values. To validate the surface exposure of these targets, we created in-house synthetic nanobodies (sybodies) against the two chaperones and identified sybodies that bind the mycobacterial biofilms in vitro and those present in ex vivo granulomas. Taken together, the present study reports a proof-of-concept showing that surface proteomics in vitro and ex vivo proteomics combined is a valuable strategy to identify surface-exposed proteins on the mycobacterial biofilm. Biofilm surface–binding nanobodies could be eventually used as homing agents to deliver biofilm-targeting treatments to the sites of persistent biofilm infection.Peer reviewe

Identification of protective postexposure mycobacterial vaccine antigens using an immunosuppression-based reactivation model in the zebrafish

Abstract Roughly one third of the human population carries a latent Mycobacterium tuberculosis infection, with a 5–10% lifetime risk of reactivation to active tuberculosis and further spreading the disease. The mechanisms leading to the reactivation of a latent Mycobacterium tuberculosis infection are insufficiently understood. Here, we used a natural fish pathogen, Mycobacterium marinum, to model the reactivation of a mycobacterial infection in the adult zebrafish (Danio rerio). A low-dose intraperitoneal injection (∼40 colony-forming units) led to a latent infection, with mycobacteria found in well-organized granulomas surrounded by a thick layer of fibrous tissue. A latent infection could be reactivated by oral dexamethasone treatment, which led to disruption of the granuloma structures and dissemination of bacteria. This was associated with the depletion of lymphocytes, especially CD4⁺ T cells. Using this model, we verified that ethambutol is effective against an active disease but not a latent infection. In addition, we screened 15 mycobacterial antigens as postexposure DNA vaccines, of which RpfB and MMAR_4207 reduced bacterial burdens upon reactivation, as did the Ag85-ESAT-6 combination. In conclusion, the adult zebrafish-M. marinum infection model provides a feasible tool for examining the mechanisms of reactivation in mycobacterial infections, and for screening vaccine and drug candidates

Surface-Shaving Proteomics of Mycobacterium marinum Identifies Biofilm Subtype-Specific Changes Affecting Virulence,Tolerance, and Persistence

The complex cell wall and biofilm matrix (ECM) act as key barriers to antibiotics in mycobacteria. Here, the ECM and envelope proteins of Mycobacterium marinum ATCC 927, a nontuberculous mycobacterial model, were monitored over 3 months by label-free proteomics and compared with cell surface proteins on planktonic cells to uncover pathways leading to virulence, tolerance, and persistence. We show that ATCC 927 forms pellicle-type and submerged-type biofilms (PBFs and SBFs, respectively) after 2 weeks and 2 days of growth, respectively, and that the increased CelA1 synthesis in this strain prevents biofilm formation and leads to reduced rifampicin tolerance. The proteomic data suggest that specific changes in mycolic acid synthesis (cord factor), Esx1 secretion, and cell wall adhesins explain the appearance of PBFs as ribbon-like cords and SBFs as lichen-like structures. A subpopulation of cells resisting 64x MIC rifampicin (persisters) was detected in both biofilm subtypes and already in 1-week-old SBFs. The key forces boosting their development could include subtype-dependent changes in asymmetric cell division, cell wall biogenesis, tricarboxylic acid/glyoxylate cycle activities, and energy/redox/iron metabolisms. The effect of various ambient oxygen tensions on each cell type and nonclassical protein secretion are likely factors explaining the majority of the subtype-specific changes. The proteomic findings also imply that Esx1-type protein secretion is more efficient in planktonic (PL) and PBF cells, while SBF may prefer both the Esx5 and nonclassical pathways to control virulence and prolonged viability/persistence. In conclusion, this study reports the first proteomic insight into aging mycobacterial biofilm ECMs and indicates biofilm subtype-dependent mechanisms conferring increased adaptive potential and virulence of nontuberculous mycobacteria. IMPORTANCE Mycobacteria are naturally resilient, and mycobacterial infections are notoriously difficult to treat with antibiotics, with biofilm formation being the main factor complicating the successful treatment of tuberculosis (TB). The present study shows that nontuberculous Mycobacterium marinum ATCC 927 forms submergedand pellicle-type biofilms with lichen- and ribbon-like structures, respectively, as well as persister cells under the same conditions. We show that both biofilm subtypes differ in terms of virulence-, tolerance-, and persistence-conferring activities, highlighting the fact that both subtypes should be targeted to maximize the power of antimycobacterial treatment therapies.Peer reviewe

Low Th2/Th1 and high Treg are associated with activity of disease at late-stage mycobacterial infection.

<p>(A) The proportion of dormant bacteria in each non-stimulated subgroup was assessed by measuring the expression of a mycobacterial dormancy-associated gene <i>GltA1</i>. (B, D&E) The T cell inductions of <i>Reactivated</i> group (fish showing symptoms between 8 and 20 weeks after an initial controlled phase) and <i>High</i> group at 5 mpi. (C) ROC analysis of the <i>gata3/tbx21</i> ratio as a biomarker to distinguish individuals with a high bacterial burden from the individuals with a lower bacterial load (AUC = area under the curve). (F) Induction of <i>foxp3</i> normalized to <i>cd3</i> induction in the subgroups at 5 mpi and fish showing symptoms between 2 and 5 mpi.</p

Controlled mycobacterial infection is characterized by Th2-type response from 4 weeks post infection.

<p>(A–C) <i>Tbx21</i> (<i>t-bet</i>) and <i>gata3</i> induction was measured in the different subpopulations at 2, 4 wpi and 5 mpi (months post infection). The <i>gata3/tbx21</i> ratio was calculated to determine the dominant Th type. (D–F) The induction of selected type cytokines for Th1 (<i>IFNγ1-2</i>) and Th2 response (<i>IL4b</i>) was measured in the different subpopulations. The <i>il4/IFNγ</i> ratio of induction was calculated. (G–I) <i>Rag1</i> (−/−) mutant zebrafish (n = 20) were infected with 35±18 cfu of <i>M. marinum</i> and analyzed at 4 wpi. (G) Grouping of mutant fish was carried out according to bacterial load similarly to wt fish (See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004190#ppat.1004190.s001" target="_blank">Figure S1E</a>). The association of <i>gata3/tbx21</i> with the bacterial load was assessed. (H) The induction of <i>il4</i> at 4 wpi was compared between wt and <i>rag1</i> (−/−) fish. (I) The induction of <i>ifnγ</i> at 4 wpi was compared between wt and <i>rag1</i> (−/−) fish. (J) Semi-quantitative western blots were carried out at 4 wpi from a population of 20 fish. Gata3 antibody was used as the Th2 marker, and CXCR3 as the Th1 marker. The bacterial loads were measured from the corresponding DNA samples to allow grouping to subpopulations. (K) As a control experiment for assessing the effect of initially high bacterial load on <i>gata3/tbx21</i> ratio, WT zebrafish were infected with a high dose (2691±520 cfu) and the <i>gata3/tbx21</i> ratio of this group (n = 25) was compared to those of the group (n = 30) infected with a low dose (21±7 cfu) at 4 wpi. (L) To assess the natural polarization pattern of T cells with regard to <i>gata3/tbx21</i>, WT zebrafish (n = 14) were stimulated by an intraperitoneal injection of heat-killed <i>M. marinum</i>. <i>Gata3/tbx21</i> ratio was determined 10 days post injection.</p

T cell numbers are higher in individuals with low bacterial loads.

<p>(A) In an adoptive transfer experiment, we transferred spleen and kidney cells to low-dose infected <i>rag1</i> (−/−) mutant fish 12 dpi. The donors were WT immunocompetent fish treated with heat-killed <i>M. marinum</i> (Hk.<i>M.m.</i>) or PBS and <i>rag1</i> (−/−) fish treated with Hk.<i>M.m.</i> 10 d prior to the adoptive transfer. The bacterial loads of the recipient fish were measured 4 wpi by q-PCR, n = 8–10/group. (B–D) At all time points of this study, zebrafish infected with a low dose of <i>M. marinum</i> (21±7 cfu) were divided in subpopulations according to the bacterial load into upper quarter (<i>High</i>) (n = 7), lower quarter (<i>Low</i>) (n = 7) and a <i>Medium</i> group (n = 15). The changes in the total T cell numbers were assessed in the different subpopulations of low-dose infected WT fish by measuring <i>cd3</i> transcription by q-RT-PCR during a primary (2 and 4 weeks) or a late stage (5 months) mycobacterial infection (E) As a control experiment for assessing the effect of initially high bacterial load on <i>cd3</i>, WT zebrafish were infected with a high dose (2691±520 cfu) and the <i>cd3</i> levels of this group (n = 25) were compared to those of the group (n = 30) infected with a low dose (21±7 cfu) at 4 wpi.</p

Controlled mycobacterial infection is distinguished from progressive infection by higher induction of Th2 markers.

<p>The groups with different bacterial loads were analyzed for typical Th2 (A–F) and Th1 (G–J) markers by q-RT-PCR at 4 wpi. (K) The ratio of the inductions of <i>il13</i> to <i>ifnγ</i> was also calculated in the different subgroups at 4 wpi.</p

In vitro and ex vivo proteomics of Mycobacterium marinum biofilms and the development of biofilm-binding synthetic nanobodies

The antibiotic-tolerant biofilms present in tuberculous granulomas add an additional layer of complexity when treating mycobacterial infections, including tuberculosis (TB). For a more efficient treatment of TB, the biofilm forms of mycobacteria warrant specific attention. Here, we used Mycobacterium marinum (Mmr) as a biofilm-forming model to identify the abundant proteins covering the biofilm surface. We used biotinylation/streptavidin-based proteomics on the proteins exposed at the Mmr biofilm matrices in vitro to identify 448 proteins and ex vivo proteomics to detect 91 Mmr proteins from the mycobacterial granulomas isolated from adult zebrafish. In vitro and ex vivo proteomics data are available via ProteomeXchange with identifier PXD033425 and PXD039416, respectively. Data comparisons pinpointed the molecular chaperone GroEL2 as the most abundant Mmr protein within the in vitro and ex vivo proteomes, while its paralog, GroEL1, with a known role in biofilm formation, was detected with slightly lower intensity values. To validate the surface exposure of these targets, we created in-house synthetic nanobodies (sybodies) against the two chaperones and identified sybodies that bind the mycobacterial biofilms in vitro and those present in ex vivo granulomas. Taken together, the present study reports a proof-of-concept showing that surface proteomics in vitro and ex vivo proteomics combined are a valuable strategy to identify surface-exposed proteins on the mycobacterial biofilm. Biofilm-surface-binding nanobodies could be eventually used as homing agents to deliver biofilm-targeting treatments to the sites of persistent biofilm infection.With the currently available antibiotics, the treatment of tuberculosis takes months. The slow response to treatment is caused by antibiotic tolerance, which is especially common among bacteria that forms biofilms. Such biofilms are composed of bacterial cells surrounded by the extracellular matrix. Both the matrix and the dormant lifestyle of the bacterial cells are thought to hinder the efficacy of antibiotics. To be able to develop faster-acting treatments against tuberculosis, the biofilm forms of mycobacteria deserve specific attention. In this work, we characterise the protein composition of Mycobacterium marinum biofilms in bacterial cultures and in mycobacteria extracted from infected adult zebrafish. We identify abundant surface-exposed targets and develop the first synthetic nanobodies that bind to mycobacterial biofilms. As nanobodies can be linked to other therapeutic compounds, in the future, they can provide means to target therapies to biofilms.Peer reviewe