Characterization of an Immunoglobulin Binding Protein (IbpM) From Mycoplasma pneumoniae

Abstract

Bacteria evolved many ways to invade, colonize and survive in the host tissue. Such complex infection strategies of other bacteria are not present in the cell-wall less Mycoplasmas. Due to their strongly reduced genomes, these bacteria have only a minimal metabolism. Mycoplasma pneumoniae is a pathogenic bacterium using its virulence repertoire very efficiently, infecting the human lung. M. pneumoniae can cause a variety of conditions including fever, inflammation, atypical pneumoniae, and even death. Due to its strongly reduced metabolism, M. pneumoniae is dependent on nutrients from the host and aims to persist as long as possible, resulting in chronic diseases. Mycoplasmas evolved strategies to subvert the host immune system which involve proteins fending off immunoglobulins (Igs). In this study, we investigated the role of MPN400 as the putative factor responsible for Ig-binding and host immune evasion. MPN400 is a cell-surface localized protein which binds strongly to human IgG, IgA, and IgM. We therefore named the protein MPN400 immunoglobulin binding protein of Mycoplasma (IbpM). A strain devoid of IbpM is slightly compromised in cytotoxicity. Taken together, our study indicates that M. pneumoniae uses a refined mechanism for immune evasion.

Keywords: Mycoplasma pneumoniae; host–pathogen interaction; immune evasion; mollicutes; protein–protein interaction.

FIGURE 1

Structural analysis of IbpM (MPN400). (A) Structural alignment of the predicted tertiary structure of IbpM from M. pneumoniae (green) to the resolved crystal structure of the antibody-binding region of Protein M (PDB: 4NZR, Grover et al., 2014) from M. genitalium (orange). The figure was created by PyMOL. (B) The ProtterBlot shows the domain architecture of IbpM (using UniProt accession number P75383) anchored in the plasma membrane. Amino acids in red indicate the signal peptide and yellow the predicted transmembrane domain. Colored residues in green indicate a leucine rich region-like domain (LRR domain) and gray the C-terminal disordered domain. Thr insertion point for the mini-transposon is indicated in orange (aa 391–392) and the truncation point for the recombinant mutant in blue (aa 446).

FIGURE 2

Localization of IbpM (MPN400) on the surface of M. pneumoniae cells. (A) Results of colony blot of freshly grown M. pneumoniae colonies. Blotted colonies were treated with guinea pig α-MPN400, α-P14 (positive control), and α-NADH oxidase (Nox, negative control), respectively. (B) Reactivity of SDS-PAGE-separated and blotted whole M. pneumoniae proteins after mild treatment with increasing concentrations of trypsin. Lane 1: 0 μg ml^{– 1}, lane 2: 10 μg ml^{– 1}, lane 3: 40 μg ml^{– 1} and lane 4: 100 μg ml^{– 1} trypsin. Western blots were incubated with guinea pig α-MPN400, α-Nox and α-P14, respectively. The bands corresponding to the non-degraded proteins of interest are highlighted by red arrows. Guinea pig antibodies were detected using rabbit α-guinea pig HRP conjugated antibody.

FIGURE 3

Isolation of a mpn400 transposon insertion mutant. (A) Schematic representation of the genomic region of mpn400 in M. pneumoniae and the transposon insertion site in the mpn400:Tn-4001 mutant GPM113. The location of the probes is indicated by dashed lines. (B) Southern blot analysis to confirm single transposon integration using chromosomal DNA of the wild type (wt) and strain GPM113 were digested using SacI. Detection was carried out with a probe specific for the aac-ahpD resistance cassette and a probe hybridizing to the gene mpn400 (right) which is upshifted after transposon integration. The relevant bands are highlighted by red arrows. λ-marker (HindIII/EcoRI) served as a size standard.

FIGURE 4

Crystal violet stain of HeLa cells after mycoplasmal infection. Cytotoxicity of M. pneumoniae strains toward HeLa cell cultures. Confluent HeLa cell culture without infection or with M129 wild type cells and with GPM113 (mpn400:Tn) mutant cells. (A) After 20 h, 48 h, and 96 h post infection HeLa cells were stained with crystal violet and photographed. (B) OD₅₉₅ measurements were plotted and statistical significance of the cytotoxicities in different strains at the two time points was calculated. For the original data, see Supplementary Data Sheet S1.

FIGURE 5

Detection of proteins bound by immobilized MPN400. The binding was performed with recombinant MPN400 (from E. coli BL21:pGP3215) immobilized to StrepTactin matrix, which was incubated with different concentrations of human serum (HuSe). Columns were washed extensively, and bound proteins eluted four times with D-desthiobiotin. We used the fractions from elution 3 to analyze the protein content by 12% SDS-PAGE stained with silver. Lane 1, pre-stained protein ladder plus (Thermo Fisher); lanes 2–5, rMPN400 incubated with 5, 3, 2, and 1 μg protein from HuSe, respectively; lane 6, empty vector crude extract from E. coli incubated with 5 μg HuSe.

FIGURE 6

Binding of different Ig’s toward IbpM in an ELISA experiment. (A) Purified full-length IbpM, C-terminal truncated IbpM, and human serum albumin (HSA) were coated in different concentrations onto 96-well plates and incubated with IgG, IgA, or IgM. Binding of Ig’s was quantified by the detection with rabbit α-human Ig-AP antibody responsible for ABTS color formation and detection at 420 nm. Squares, circles, and triangles, each indicate a biological replicate mean, each biological replicate itself consisting of three technical replicates. The solid bar represents the average of all measurements. Full-length IbpM in green, truncated IbpM in pink, and HSA in orange. Values from IgG measurement in the graphic are representative as well for IgA and IgM (for graphs of IgA and IgM binding please refer Supplementary Figure S1). (B) Binding profiles of full length IbpM to IgA, IgG, and IgM.

FIGURE 7

Interaction of fibronectin and plasminogen with IbpM. Purified IbpM (blue and brown) or whole cells of M. pneumoniae (gray and green) were coated onto 96-well plates and incubated with different concentrations of fibronectin (left) or plasminogen (right). Interaction of IbpM with the human proteins was quantified with peroxidase-conjugated antibodies detecting the corresponding human proteins. Absorbance was detected at 405 nm. BSA (black bars) served as negative control.