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

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 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

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 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

Adequate Th2-Type Response Associates with Restricted Bacterial Growth in Latent Mycobacterial Infection of Zebrafish

Tuberculosis is still a major health problem worldwide. Currently it is not known what kind of immune responses lead to successful control and clearance of Mycobacterium tuberculosis. This gap in knowledge is reflected by the inability to develop sufficient diagnostic and therapeutic tools to fight tuberculosis. We have used the Mycobacterium marinum infection model in the adult zebrafish and taken advantage of heterogeneity of zebrafish population to dissect the characteristics of adaptive immune responses, some of which are associated with well-controlled latency or bacterial clearance while others with progressive infection. Differences in T cell responses between subpopulations were measured at the transcriptional level. It was discovered that a high total T cell level was usually associated with lower bacterial loads alongside with a T helper 2 (Th2)-type gene expression signature. At late time points, spontaneous reactivation with apparent symptoms was characterized by a low Th2/Th1 marker ratio and a substantial induction of foxp3 reflecting the level of regulatory T cells. Characteristic gata3/tbx21 has potential as a biomarker for the status of mycobacterial disease.Public Library of Science open acces