A specific class of infectious agents isolated from bovine serum and dairy products and peritumoral colon cancer tissue

Villiers, Ethel‐Michele de; Gunst, Karin; Chakraborty, Deblina; Ernst, Claudia; Bund, Timo; Hausen, Harald zur

doi:10.1080/22221751.2019.1651620

Cited by 26 publications

(94 citation statements)

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“…In silico analyses of the DNA sequence of the original H1MSB.1 isolate itself differs in nt2, nt73, and nt328 from all H1MSB.1 variants from colon tissue described here. Additional single nucleotide changes in the multiple isolates obtained, led to modifications in the genome organization, which in turn opens the possibility of altered gene functions and posttranscriptional modifications (12). Additional LMD-isolates obtained in this study substantiated previous results.…”

Section: Resultssupporting

confidence: 86%

“…Early results were rather disappointing and somewhat discouraging: Initial data obtained from DNA derived from colon carcinoma cell lines failed to provide evidence for BMMF sequences. Recently, testing of surgically removed primary colon cancer biopsies now supported the initial anticipation: DNAs analyzed from colon tissue of colon cancer patients, and in particular from peritumor "normal" tissue, showed the presence of circular DNAs which, after cloning and sequencing, showed similarity with isolates of the BMMF1 group (12). The sequences are almost identical to one of our previous isolates (H1MSB.1) from a lesion of a multiple sclerosis patient (10).…”

Section: Discussionsupporting

confidence: 54%

“…Isolation of BMMF DNA from peritumor colon cancer tissue. In parallel to the detection of Rep antigens in colon tissues by IHC, LMD permitted DNA retrieval from tissue areas with apparent BMMF enrichment (12). DNA was isolated, followed by rolling circle amplification, two rounds of PCR with BMMF1-specific backto-back primers, cloning of products, and final sequencing.…”

Section: Resultsmentioning

confidence: 99%

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Analysis of chronic inflammatory lesions of the colon for BMMF Rep antigen expression and CD68 macrophage interactions

Bund

Nikitina

Chakraborty

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Consumption of Eurasian bovine meat and milk has been associated with cancer development, in particular with colorectal cancer (CRC). In addition, zoonotic infectious agents from bovine products were proposed to cause colon cancer (zur Hausen et al., 2009). Bovine meat and milk factors (BMMF) are small episomal DNA molecules frequently isolated from bovine sera and milk products, and recently, also from colon cancer (de Villiers et al., 2019). BMMF are bioactive in human cells and were proposed to induce chronic inflammation in precancerous tissue leading to increased radical formation: for example, reactive oxygen and reactive nitrogen species and elevated levels of DNA mutations in replicating cells, such as cancer progenitor cells (zur Hausen et al., 2018). Mouse monoclonal antibodies against the replication (Rep) protein of H1MSB.1 (BMMF1) were used to analyze BMMF presence in different cohorts of CRC peritumor and tumor tissues and cancer-free individuals by immunohistochemistry and Western blot. BMMF DNA was isolated by laser microdissection from immunohistochemistry-positive tissue regions. We found BMMF Rep protein present specifically in close vicinity of CD68+ macrophages in the interstitial lamina propria adjacent to CRC tissues, suggesting the presence of local chronic inflammation. BMMF1 (modified H1MSB.1) DNA was isolated from the same tissue regions. Rep and CD68+ detection increased significantly in peritumor cancer tissues when compared to tissues of cancer-free individuals. This strengthens previous postulations that BMMF function as indirect carcinogens by inducing chronic inflammation and DNA damage in replicating cells, which represent progress to progenitor cells for adenoma (polyps) formation and cancer.

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Section: Resultssupporting

confidence: 86%

Section: Discussionsupporting

confidence: 54%

Section: Resultsmentioning

confidence: 99%

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Analysis of chronic inflammatory lesions of the colon for BMMF Rep antigen expression and CD68 macrophage interactions

Bund

Nikitina

Chakraborty

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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“…3 and 5), cytotoxicity is necessarily an intrinsic property of the aggregates themselves, because, a priori, no function is associated with this bacterial prion-like protein in the heterologous mammalian host. Very recently, RepA-like proteins with a conserved WH1 domain (60) were described as nearly the only gene products encoded by small mobile genetic elements that were isolated in dairies and in meat (bovine meat and milk factors [BMMFs]), with a probable origin in cattle microbiota, but also in samples from patients with cancer and multiple sclerosis (61). These findings open the intriguing possibility that the cytotoxicity described here for RepA-WH1 might have broader relevance in human disease.…”

Section: Figmentioning

confidence: 99%

Intercellular Transmission of a Synthetic Bacterial Cytotoxic Prion-Like Protein in Mammalian Cells

Revilla-García

Fernández

Álamo

et al. 2020

mBio

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RepA is a bacterial protein that builds intracellular amyloid oligomers acting as inhibitory complexes of plasmid DNA replication. When carrying a mutation enhancing its amyloidogenesis (A31V), the N-terminal domain (WH1) generates cytosolic amyloid particles that are inheritable within a bacterial lineage. Such amyloids trigger in bacteria a lethal cascade reminiscent of mitochondrial impairment in human cells affected by neurodegeneration. To fulfill all the criteria to qualify as a prion-like protein, horizontal (intercellular) transmissibility remains to be demonstrated for RepA-WH1. Since this is experimentally intractable in bacteria, here we transiently expressed in a murine neuroblastoma cell line the soluble, barely cytotoxic RepA-WH1 wild type [RepA-WH1(WT)] and assayed its response to exposure to in vitro-assembled RepA-WH1(A31V) amyloid fibers. In parallel, murine cells releasing RepA-WH1(A31V) aggregates were cocultured with human neuroblastoma cells expressing RepA-WH1(WT). Both the assembled fibers and donor-derived RepA-WH1(A31V) aggregates induced, in the cytosol of recipient cells, the formation of cytotoxic amyloid particles. Mass spectrometry analyses of the proteomes of both types of injured cells pointed to alterations in mitochondria, protein quality triage, signaling, and intracellular traffic. Thus, a synthetic prion-like protein can be propagated to, and become cytotoxic to, cells of organisms placed at such distant branches of the tree of life as bacteria and mammalia, suggesting that mechanisms of protein aggregate spreading and toxicity follow default pathways. IMPORTANCE Proteotoxic amyloid seeds can be transmitted between mammalian cells, arguing that the intercellular exchange of prion-like protein aggregates can be a common phenomenon. RepA-WH1 is derived from a bacterial intracellular functional amyloid protein, engineered to become cytotoxic in Escherichia coli. Here, we have studied if such bacterial aggregates can also be transmitted to, and become cytotoxic to, mammalian cells. We demonstrate that RepA-WH1 is capable of entering naive cells, thereby inducing the cytotoxic aggregation of a soluble RepA-WH1 variant expressed in the cytosol, following the same trend that had been described in bacteria. These findings highlight the universality of one of the central principles underlying prion biology: No matter the biological origin of a given prion-like protein, it can be transmitted to a phylogenetically unrelated recipient cell, provided that the latter expresses a soluble protein onto which the incoming protein can readily template its amyloid conformation.

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“…In the case of the phenotype caused in the mammalian cells by the expression of WH1(A31V) amyloids (Figures 1 and 2), or by their uptake and subsequent templating on WH1(WT) in the recipient cells (Figures 3–5), cytotoxicity is necessarily an intrinsic property of the aggregates themselves, because a priori no function is associated with this bacterial prion-like protein in the heterologous mammalian host. Very recently, RepA-like proteins with a conserved WH1 domain (Kilic et al, 2019) have been described as nearly the only gene products encoded by small mobile genetic elements that were isolated in dairies and meat (BMMFs), with a probable origin in cattle microbiota, but also in samples from patients with cancer and multiple sclerosis (de Villiers et al, 2019). These findings open the intriguing possibility that the cytotoxicity described here for RepA-WH1 might have broader relevance in human disease.…”

Section: Discussionmentioning

confidence: 99%

Intercellular transmission of a bacterial cytotoxic prion-like protein in mammalian cells

Revilla-García

Fernández

Álamo

et al. 2019

Preprint

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1RepA is a bacterial protein that builds intracellular amyloid oligomers acting as inhibitory complexes of 2 plasmid DNA replication. When carrying a mutation enhancing its amyloidogenesis (A31V), the N-2007; Torreira et al., 2015). When expressed in E. coli, the hyper-amyloidogenic domain variant RepA-1 WH1(A31V) forms particles of various sizes, distributed across the bacterial cytosol (Fernández-2 Tresguerres et al., 2010). These particles are transmissible vertically (during cell division, i.e. from 3 mother to daughter cells) as two distinct aggregate strains with remarkable appearances and phenotypes: 4 elongated and mildly cytotoxic, and compact and acutely cytotoxic, respectively (Gasset-Rosa et al., 5 2014). Co-expression of soluble and aggregation-prone RepA-WH1 variants in E. coli demonstrated that 6 the A31V variant can template its conformation on the parental WT protein (Molina-García and 7 Giraldo, 2014). Systems analyses (Molina-García et al., 2017), together with in vitro reconstruction in 8 cytomimetic lipid vesicles (Fernández et al., 2016b; Fernández and Giraldo, 2018), suggest that RepA-9 WH1(A31V) oligomers target the internal bacterial membrane, hampering proton motive force and thus 10 ATP synthesis and transport through membranes, and enhance oxidative stress. In parallel, protein 11 factors mounting the defence against stress and envelope damage co-aggregate with RepA-WH1(A31V) 12 amyloids (Molina-García et al., 2017). Altogether, bacteria viability is severely compromised by RepA-13 WH1 amyloidosis, in a way resembling some of the central mitochondrial routes found in human 14 amyloidoses (Haelterman et al., 2014; Norambuena et al., 2018; Mathys et al., 2019; Wang et al., 2019).15 However, E. coli is not suitable for addressing the issues of cell-to-cell transmissibility of protein 16 aggregates and the subsequent intracellular amyloid cross-aggregation, since this Gram-negative 17 bacterium does not uptake large protein particles due to the insurmountable obstacle of its three-layered 18 cell envelope. 19To explore the capability of the prion-like protein RepA-WH1 to propagate in a heterologous host, 20 here we exposed murine neuroblastoma cells, transiently expressing mCherry-tagged soluble RepA-21 WH1(WT), to in vitro assembled RepA-WH1(A31V) amyloid fibres. In addition, we studied the 22 intercellular induction of protein aggregation by co-culturing murine cells releasing RepA-WH1(A31V) 23 aggregates with human neuroblastoma cells stably expressing soluble RepA-WH1(WT). Confocal 24 microscopy and biochemical studies showed that the mammalian cells can take up RepA-WH1(A31V) 25 amyloid fibres, that cross-seed the cytosolic aggregation of the endogenous RepA-WH1(WT) in the 26 recipient cells. Moreover, co-culture of cells releasing the RepA-WH1(A31V) variant also induced the 27 aggregation of RepA-WH1(WT) in bystander cells, suggesting intercellular transmission of RepA-28 5 WH1(A31V) seeds. In both setups of experimental transmission, the induced RepA-WH1(WT) 1 aggregates were cytoto...

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A specific class of infectious agents isolated from bovine serum and dairy products and peritumoral colon cancer tissue

Cited by 26 publications

References 58 publications

Analysis of chronic inflammatory lesions of the colon for BMMF Rep antigen expression and CD68 macrophage interactions

Analysis of chronic inflammatory lesions of the colon for BMMF Rep antigen expression and CD68 macrophage interactions

Intercellular Transmission of a Synthetic Bacterial Cytotoxic Prion-Like Protein in Mammalian Cells

Intercellular transmission of a bacterial cytotoxic prion-like protein in mammalian cells

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