Capsular polysaccharide of Mycoplasma mycoides subsp. capri contributes to phenotypic diversity promoting distinctive immune responses

Abstract

Mollicutes are minute cell wall less bacteria encompassing important pathogens. We show that pathogenic Mycoplasma mycoides subsp. capri (Mmc) can switch expression of capsular polysaccharide (CPS), which creates phenotypic diversity and has dramatic repercussions on immune responses. To characterize the immune responses, we employed the highly virulent Mmc strain GM12 as well as an engineered CPS-deficient mutant in a set of assays employing primary blood cells from its native caprine host and cattle. Primary blood cells stimulated with GM12 showed only very moderate effects on cell viability as well as activation marker expression supporting an immunological furtiveness-like lifestyle. Interestingly, GM12 showed the capacity to survive and resist inside monocyte-derived macrophages (MDMs), which fosters dissemination and persistence in the host. Stimulation with the CPS-deficient mutant which exposes surface proteins including lipoproteins, increased cell death, strongly suppressed expression of major histocompatibility complex on antigen-presenting cells and induced secretion of several pro-inflammatory cytokines/chemokines which is a clinical hallmark in infected animals. Moreover, the CPS-deficient mutant elicited inflammatory cell death in MDMs. In conclusion, we showed that Mmc can switch the expression of CPS, which leads to different immunological trajectories paving the way for clinical disease, dissemination and persistence in the host.

Fig 1. Effect of different growth conditions on Mycoplasma colony size and presence of polysaccharide.

After four days of incubation the colonies were transferred onto a nitrocellulose membrane and subsequently stained using Periodic Acid-Schiff (PAS). Colony sectors stained for polysaccharide are indicated by black arrows. Scale bare is 1 mm. (A) Mycoplasmas were incubated at different temperatures on high nutrient agar. (B) Mycoplasmas were incubated at different temperatures on low nutrient agar.

Fig 2. Cell morphology and activation/maturation of caprine monocytes-derived macrophages (MDMs) after stimulation with live mycoplasmas.

(A) The MDMs (2 × 10⁵/mL in 12-well plate) were stimulated with mycoplasmas for 24 hours and their cell morphology was investigated by microscopy. Experiments were done in three biological replicates and representative data are displayed. Magnification of the pictures was 20x. (B) MDMs surface marker expression in response to stimulation with live mycoplasmas at 24 hours (upper panel) and 48 hours (lower panel), analyzed by flow cytometry. (C) Histogram of relative cell count versus activation markers (MHC-II, MHC-I and CD25) for MDMs stimulated with GM12 and GM12::YCpMmyc1.1-Δglf compared to no stimulation. (D) Cytokines and chemokines secreted at 6, 24, and 48 hours after exposure to mycoplasmas, measured by multiplex immunoassay.

Fig 3. Viability of caprine monocyte-derived macrophages (MDMs) and Mycoplasma mycoides subsp. capri GM12 in co-culture.

(A) Percentage of viable MDMs 48 hours post-infection with GM12 at an MOI of 100 was analyzed using the LIVE/DEAD Fixable Yellow Dead Cell Stain Kit. (B) Viability of mycoplasmas following co-culture with MDMs was determined by measurement of colony-forming units (CFUs) at 48 hours post-infection. (C) Flow cytometry analysis of phagocytosis after 48 hours of coincubation with mCherry-labeled GM12::YCp1.1. (D) Fluorescence microscopy images of infected MDMs 48 hours post-infection. Scale bare is 125 μm. (E) Viability of mycoplasmas following co-culture with MDMs determined by comparing color-changing units (CCUs) with or without treatment employing 500 μg/mL gentamicin (control = untreated). (F) The CFUs of intracellular mycoplasmas released from MDMs (H₂O treatment) 48 hours post-infection.

Fig 4. The absence of capsular polysaccharide impairs antigen presentation.

(A) Viability of mycoplasmas following co-culture with caprine peripheral blood mononuclear cells (PBMCs) by comparing colony-forming units (CFUs) at T = 0 and T = 16 hours. A star indicates statistically significance by paired t-test. (B–E) Modulation of activation markers on (B) caprine monocytes (MHC-I, MHC-II, CD25), (C) bovine monocytes, (D) dendritic cells (MHC-II, CD25, CCR7), (E) natural killer (NK) cells and γδ T cells (CD25). Cells were exposed to mycoplasmas at a MOI = 100 for 16 hours. (F) GM12::YCpMmyc1.1-Δglf induced MHC-II downregulation is due to a retention in the intracellular compartment on cattle PBMCs. The fold changes analysis was determined by FCM with FlowJo. Cells from the individual animals are represented by different color codes. Data are presented as mean ± standard deviation (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

Fig 5. Cytokines and chemokines secreted by peripheral blood mononuclear cells (PBMCs) stimulated with mycoplasmas.

Supernatants were harvested 16 hours post infection and measured by a commercial multiplex immunoassay. (A) Cytokines/chemokines secreted by caprine PBMCs, (B) cytokines/chemokines secreted by bovine PBMCs. Stars indicate significance levels (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

Fig 6. Intracellular cytokines produced by bovine plasmacytoid dendritic cells (pDCs) and natural killer (NK) cells in response to GM12::YCpMmyc1.1-Δglf.

Intracellular cytokine staining of TNF-α and IFN-γ. Following stimulation with mycoplasmas for 16 hours, intracellular TNF-α and IFN-γ were determined by flow cytometry. Stars indicate significance levels (*p < 0.05 and **p < 0.01).

Fig 7. Number of differentially expressed genes (DEGs) in caprine peripheral blood mononuclear cells (PBMCs) stimulated with mycoplasmas for 6 hours (left panel) and 16 hours (right panel).

(A) Number of DEGs in infected PBMCs over no stimulation at 6 hours and 16 hours. (B) Venn diagram of DEGs among four comparisons (GM12 vs. no stimulation, GM12::YCpMmyc1.1 vs. no stimulation, GM12::YCpMmyc1.1-Δ68 vs. no stimulation and GM12::YCpMmyc1.1-Δglf vs. no stimulation). The number in each large circle represents the total number of DEGs between combinations, the overlap part of the circles represents common DEGs. DEGs were annotated on the basis of gene ontology (GO) using DAVID (Database for Annotation, Visualization and Integrated Discovery).

Fig 8. Number of differentially expressed genes (DEGs) in caprine peripheral blood mononuclear cells (PBMCs) stimulated with mycoplasmas for 6 hours (left panel) and 16 hours (right panel).

(A) Volcano plot and (B) KEGG pathway of 246 DEGs at 6 hours and 748 DEGs at 16 hours compared between GM12::YCpMmyc1.1-Δglf and GM12. The line represents the significant genes at adjusted p value of < 0.05 and absolute log₂ fold change > 2.