Mycoplasma promotes malignant transformation in vivo, and its DnaK, a bacterial chaperone protein, has broad oncogenic properties

Abstract

We isolated a strain of human mycoplasma that promotes lymphomagenesis in SCID mice, pointing to a p53-dependent mechanism similar to lymphomagenesis in uninfected p53^-/- SCID mice. Additionally, mycoplasma infection in vitro reduces p53 activity. Immunoprecipitation of p53 in mycoplasma-infected cells identified several mycoplasma proteins, including DnaK, a member of the Hsp70 chaperon family. We focused on DnaK because of its ability to interact with proteins. We demonstrate that mycoplasma DnaK interacts with and reduces the activities of human proteins involved in critical cellular pathways, including DNA-PK and PARP1, which are required for efficient DNA repair, and binds to USP10 (a key p53 regulator), impairing p53-dependent anticancer functions. This also reduced the efficacy of anticancer drugs that depend on p53 to exert their effect. mycoplasma was detected early in the infected mice, but only low copy numbers of mycoplasma DnaK DNA sequences were found in some primary and secondary tumors, pointing toward a hit-and-run/hide mechanism of transformation. Uninfected bystander cells took up exogenous DnaK, suggesting a possible paracrine function in promoting malignant transformation, over and above cells infected with the mycoplasma. Phylogenetic amino acid analysis shows that other bacteria associated with human cancers have similar DnaKs, consistent with a common mechanism of cellular transformation mediated through disruption of DNA-repair mechanisms, as well as p53 dysregulation, that also results in cancer-drug resistance. This suggests that the oncogenic properties of certain bacteria are DnaK-mediated.

Keywords: DNA repair; DnaK; cancer; mycoplasma; p53.

Fig. 1.

Mycoplasma infection induces tumorigenesis in SCID mice. (A) Mycoplasma infection in SCID mice. An inverted Kaplan–Meyer formula was used to generate a plot of the time to tumor development. CB17.SCID (n = 18) and NOD/SCID (n = 12) mice were infected with a strain of M. fermentans isolated at the IHV. The experiments were carried out for about 19–20 wk after infection, until the animals reached an age of about 27 wk. Of the 30 infected animals, 12 (eight CB17.SCID and four NOD/SCID) mice developed tumors by 27 wk of age, starting at about 8 wk after infection. The CB17.SCID animals belonged to a colony maintained in our animal facility under pathogen-free conditions. NOD/SCID and NSG mice were obtained from the Jackson Laboratory. Young animals (about 6 wk old) were infected by i.p. injection with mycoplasma (10⁷ pfu). Tumor development was observed in animals infected with mycoplasma grown in either aerobic or anaerobic conditions. As early as 7 wk post infection the spleen and lymph nodes were enlarged in animals infected with mycoplasma. In some animals tumor cells colonized the vestigial thymic area, and necropsy showed an enlarged tumor mass. About 30% of the animals died of wasting within 30 wk of infection. Age-matched uninfected CB17.SCID (n = 9) and NOD/SCID (n = 9) animals were kept in adjacent cages as controls. Control, uninfected CB17.SCID mice had a lifespan of about 40–50 wk, and NOD/SCID mice had a lifespan of 38–45 wk. Only one CB17.SCID mouse developed a spontaneous tumor at about 26 wk of age. Spontaneous T cell lymphoma was observed in more than 40% of both the CB17.SCID animals and the NOD/SCID animals after 33 wk of age. As a further control, we used NSG mice, which are resistant to lymphoma development even after sublethal irradiation treatment. None of the infected NSG animals (n = 8) developed tumors during the time of the experiment. In some experiments (n = 10 mice) we also used the prototype M. fermentans PG18 grown under standard conditions. Seven animals died of wasting within 30 wk after infection, and none of the remaining animals developed lymphoma. Eight animals were injected with nonviable mycoplasma, and none developed lymphoma up to 28 wk of age (see also Materials and Methods). (B) Splenomegaly and enlarged lymph nodes in mycoplasma-infected mice that developed tumors. Spleens from mycoplasma-infected animals and uninfected animals were collected and compared to determine size increase. Uninfected spleens showed very little variation in size and were considered as references in comparing the size of spleen from infected animals. (C) Analysis of spleens from a total of seven infected animals and five uninfected animals. Error bars indicate SD. *P < 0.01; Student’s t test. (D) Tumor infiltration of mycoplasma-infected mice. (1) Image of an H&E-stained section of a peripheral lymph node showing increased cellularity of tumor infiltration. (Magnification; 10×.) Increased vascularity is indicated by numerous slits. (2) Image of an H&E-stained section of tumor infiltration of a peripheral lymph node. (Magnification; 20×.) Note the prominent follicular hyperplasia with a poorly defined medullar zone. (3) Image of an H&E-stained section of the spleen with prominent red pulp showing increased cellularity of tumor infiltration. (Magnification; 10×.) Increased vascularity is indicated by numerous slits. (4) Image of an H&E-stained of a spleen with a tumor infiltration. (Magnification; 10×.)

Fig. 2.

DnaK negatively affects p53 activities, and mycoplasma infection reduces the effect of anticancer drugs. (A) DnaK reduces p53-associated activity in HCT116 cells. Levels of p53, p21, Bax, and PUMA proteins were analyzed in control and vector- and DnaK-transfected cells at different time points (2, 8, and 16 h). DnaK expression was verified using the anti-V5 antibody. β-act, β-actin; D, DMSO; DnaK, DnaK transfected; M, medium; N, Nutlin; NT, not transfected; VT, vector transfected. Band intensity was measured by densitometric analysis. Numbers above bands indicate the fold increase above the level of Nutlin-treated DnaK-transfected cells, normalized for the levels of β-actin. (B) DnaK increases cell-cycle progression. HCT116 cells were transfected with a DnaK-expressing vector and subsequently analyzed for cell-cycle progression. Data were collected 16–24 h after transfection. Results represent the mean and SDs of five different experiments. *P < 0.02; **P < 0.05; Fisher’s exact t test. (C) Mycoplasma infection reduces the effect of the chemotherapeutic drugs 5-FU and Nutlin. HCT116 cells were infected with mycoplasma. Results are expressed as percent cell viability over control (uninfected cells in medium alone were considered as 100%). Mean difference is shown. *P < 0.001 calculated using Poisson regression.

Fig. 3.

DnaK Immunoprecipitates USP10 and reduces the stability of p53 upon DNA damage. (A) Immunoprecipitation analysis shows binding of DnaK to USP10. HCT116 cells were transfected with DnaK-V5, and immunoprecipitation was performed using anti-V5 antibody and IgR [antibody isotype control (rabbit)]. After washing, the immunoprecipitated products were loaded on an acrylamide gel as described in Materials and Methods. αUSP10, anti-USP10 antibody. (B) DnaK induces p53 ubiquitination. HCT116 cells were cotransfected with DnaK-V5 together with HA-Ubiquitin (HA-Ub) and Flag-p53 expression vectors. Empty V5-vector was used as a negative control. Cells were treated with the proteasome inhibitor MG132 for 5 h before harvest. Flag-p53 and IgG isotype control immunoprecipitates (IP) or whole-cell lysates (Input) were immunoblotted with anti-Flag and anti-HA. Input lysates were also immunoblotted with anti-V5 and anti–β-actin antibodies. The immunoblot is representative of two independent experiments. (C) DnaK regulates p53 stability. CT116 cells transfected with DnaK-V5 or the control vector were treated with cycloheximide (CHX) (0.1 mg/mL) and were harvested at time points 0, 1, 2, and 4 h. Cell lysates were then blotted with anti-V5 (Top panel), anti-p53 (Middle panel), and anti–β-actin (Bottom panel) antibodies.

Fig. 4.

Interaction of DnaK with proteins implicated in the DNA-repair pathway and with DNAJA1. HCT116 cells were transfected with DnaK-V5, and immunoprecipitation was performed using anti-V5 antibody and antibody isotype control (Rabbit) (IgR). After washing, the immunoprecipitated products were loaded on an acrylamide gel as described in Materials and Methods. (A) Immunoprecipitation analysis shows binding of DnaK to PARP1. αPARP1, anti-PARP1 antibody. (B) Measurement of the catalytic activity of PARP1 shows reduced histone PARylation in the presence of DnaK. Purified PARP1 and DnaK were incubated together, and PARP1 activity was subsequently analyzed according to the protocol described in Materials and Methods. (C) Immunoprecipitation analysis shows binding of DnaK to DNA-PK_CS. αDNA-PK_CS, anti–DNA-PK_CS antibody. (D) Immunoprecipitation analysis shows binding of DnaK to DNAJA1. αDNAJA1, anti-DNAJA1 antibody; IP, immunoprecipitation; V5, tag for DnaK.

Fig. 5.

Intracellular uptake of exogenous DnaK-V5 by mycoplasma-free HCT116 cells. Confocal images of exogenous DnaK-V5 protein of M. fermentans in HCT116 cells treated or not treated with DnaK-V5 protein. The figures show the collected Z-stacks of the corresponding gallery of images, each presenting a 0.5-µm-thick slide. A mouse monoclonal antibody, anti-V5, was used for primary labeling, and a FITC fluoresce-labeled antibody was used for secondary labeling. (A) Nuclear localization. (B) Perinuclear localization. (C) Primary and secondary antibodies alone without DnaK-V5 protein. (D) Negative control: no antibodies and no protein. DAPI staining was used for nuclei detection. Insets in the lower right corners of A and B show a corresponding constructed 3D presentation of the protein uptake. (Scale bars: 5 μm in A and B; 20 μm in C and D.)

Fig. 6.

Phylogenetic analysis of bacterial DnaKs. Published bacterial amino acid DnaK sequences were used to construct this tree using MEGA 7.02.20 software (SI Appendix, ref. 3). In addition to DnaKs from several strains of E. coli, other DnaKs from intracellular pathogens currently associated with some human cancers are indicated. bss, base substitutions per site.