Modulation of the extracellular matrix by Streptococcus gallolyticus subsp. gallolyticus and importance in cell proliferation.

Streptococcus gallolyticus subspecies gallolyticus (Sgg) has a strong clinical association with colorectal cancer (CRC) and actively promotes the development of colon tumors. Previous work showed that this organism stimulates CRC cells proliferation and tumor growth. However, the molecular mechanisms underlying these activities are not well understood. Here, we found that Sgg upregulates the expression of several type of collagens in HT29 and HCT116 cells, with type VI collagen (ColVI) being the highest upregulated type. Knockdown of ColVI abolished the ability of Sgg to induce cell proliferation and reduced the adherence of Sgg to CRC cells. The extracellular matrix (ECM) is an important regulator of cell proliferation. Therefore, we further examined the role of decellularized matrix (dc-matrix), which is free of live bacteria or cells, in Sgg-induced cell proliferation. Dc-matrix prepared from Sgg-treated cells showed a significantly higher pro-proliferative activity than that from untreated cells or cells treated with control bacteria. On the other hand, dc-matrix from Sgg-treated ColVI knockdown cells showed no difference in the capacity to support cell proliferation compared to that from untreated ColVI knockdown cells, suggesting that the ECM by itself is a mediator of Sgg-induced cell proliferation. Furthermore, Sgg treatment of CRC cells but not ColVI knockdown CRC cells resulted in significantly larger tumors in vivo, suggesting that ColVI is important for Sgg to promote tumor growth in vivo. These results highlight a dynamic bidirectional interplay between Sgg and the ECM, where Sgg upregulates collagen expression. The Sgg-modified ECM in turn affects the ability of Sgg to adhere to host cells and more importantly, acts as a mediator for Sgg-induced CRC cell proliferation. Taken together, our results reveal a novel mechanism in which Sgg stimulates CRC proliferation through modulation of the ECM.

A type VII secretion system of Streptococcus gallolyticus subsp. gallolyticus contributes to gut colonization and the development of colon tumors.

Streptococcus gallolyticus subspecies gallolyticus (Sgg) has a strong clinical association with colorectal cancer (CRC) and actively promotes the development of colon tumors. However, the molecular determinants involved in Sgg pathogenicity in the gut are unknown. Bacterial type VII secretion systems (T7SS) mediate pathogen interactions with their host and are important for virulence in pathogenic mycobacteria and Staphylococcus aureus. Through genome analysis, we identified a locus in Sgg strain TX20005 that encodes a putative type VII secretion system (designated as SggT7SST05). We showed that core genes within the SggT7SST05 locus are expressed in vitro and in the colon of mice. Western blot analysis showed that SggEsxA, a protein predicted to be a T7SS secretion substrate, is detected in the bacterial culture supernatant, indicating that this SggT7SST05 is functional. Deletion of SggT7SST05 (TX20005Δesx) resulted in impaired bacterial adherence to HT29 cells and abolished the ability of Sgg to stimulate HT29 cell proliferation. Analysis of bacterial culture supernatants suggest that SggT7SST05-secreted factors are responsible for the pro-proliferative activity of Sgg, whereas Sgg adherence to host cells requires both SggT7SST05-secreted and bacterial surface-associated factors. In a murine gut colonization model, TX20005Δesx showed significantly reduced colonization compared to the parent strain. Furthermore, in a mouse model of CRC, mice exposed to TX20005 had a significantly higher tumor burden compared to saline-treated mice, whereas those exposed to TX20005Δesx did not. Examination of the Sgg load in the colon in the CRC model suggests that SggT7SST05-mediated activities are directly involved in the promotion of colon tumors. Taken together, these results reveal SggT7SST05 as a previously unrecognized pathogenicity determinant for Sgg colonization of the colon and promotion of colon tumors.

Streptococcus gallolyticus abrogates anti-carcinogenic properties of tannic acid on low-passage colorectal carcinomas.

The tannase-producing Gram-positive bacterial species Streptococcus gallolyticus subsp. gallolyticus (Sgg) is an opportunistic pathogen of the human gut and strongly associated with colorectal cancer (CRC). A unique feature of Sgg is its ability to degrade tannic acids (TA). TA constitute an important part of the human diet with known anti-tumorigenic properties. Here, we examined whether Sgg is able to protect tumor cells from the toxic effect of TA and thus drive tumorigenesis indirectly. Human CRC cell lines (n = 8) were treated with increasing concentrations of TA. We confirmed the cytotoxic activity of TA in a dose-dependent manner. In virtually all cell lines, viability decreased significantly (>60% inhibition). Moreover, pyrogallol, the degradation product of TA, had no effect on the tested cell lines. This suggests a specific effect of TA. Cytotoxicity was due to necrosis and induction of senescence in residual cells. Finally, when TA was degraded by Sgg, the cytotoxic effect could be abolished. Tumor cells even responded with boosted cell proliferation, highlighting the impact of Sgg on CRC progression. We here provide another piece of evidence for the active interplay between Sgg and cancer preventive components. These data will help to move forward in designing concepts for therapeutic and eventually also prophylactic approaches to combat gastrointestinal malignancies.

Increased Intracellular Cyclic di-AMP Levels Sensitize Streptococcus gallolyticus subsp. gallolyticus to Osmotic Stress and Reduce Biofilm Formation and Adherence on Intestinal Cells.

Cyclic di-AMP is a recently identified second messenger exploited by a number of Gram-positive bacteria to regulate important biological processes. Here, we studied the phenotypic alterations induced by the increased intracellular c-di-AMP levels in Streptococcus gallolyticus, an opportunistic pathogen responsible for septicemia and endocarditis in the elderly. We report that an S. gallolyticus c-di-AMP phosphodiesterase gdpP knockout mutant, which displays a 1.5-fold higher intracellular c-di-AMP levels than the parental strain UCN34, is more sensitive to osmotic stress and is morphologically smaller than the parental strain. Unexpectedly, we found that a higher level of c-di-AMP reduced biofilm formation of S. gallolyticus on abiotic surfaces and reduced adherence and cell aggregation on human intestinal cells. A genome-wide transcriptomic analysis indicated that c-di-AMP regulates many biological processes in S. gallolyticus, including the expression of various ABC transporters and disease-associated genes encoding bacteriocin and Pil3 pilus. Complementation of the gdpP in-frame deletion mutant with a plasmid carrying gdpP in trans from its native promoter restored bacterial morphology, tolerance to osmotic stress, biofilm formation, adherence to intestinal cells, bacteriocin production, and Pil3 pilus expression. Our results indicate that c-di-AMP is a pleiotropic signaling molecule in S. gallolyticus that may be important for S. gallolyticus pathogenesis.IMPORTANCEStreptococcus gallolyticus is an opportunistic pathogen responsible for septicemia and endocarditis in the elderly and is also strongly associated with colorectal cancer. S. gallolyticus can form biofilms, express specific pili to colonize the host tissues, and produce a specific bacteriocin allowing killing of commensal bacteria in the murine colon. Nevertheless, how the expression of these colonization factors is regulated remains largely unknown. Here, we show that c-di-AMP plays pleiotropic roles in S. gallolyticus, controlling the tolerance to osmotic stress, cell size, biofilm formation on abiotic surfaces, adherence and cell aggregation on human intestinal cells, expression of Pil3 pilus, and production of bacteriocin. This study indicates that c-di-AMP may constitute a key regulatory molecule for S. gallolyticus host colonization and pathogenesis.

Variations among Streptococcus gallolyticus subsp. gallolyticus strains in connection with colorectal cancer.

Streptococcus gallolyticus subsp. gallolyticus (Sg) has long been reported to display a strong association with colorectal cancer (CRC). It was recently demonstrated to actively promote the development of CRC, underscoring the importance of Sg in both clinical correlation and functional relevance in CRC. Here we investigated several clinical isolates of Sg in their interactions with human colon cancer cells and in mouse models. Some Sg strains were able to stimulate host cell proliferation (proliferation-promoting Sg, PP-Sg) whereas others were not (non-proliferation-promoting Sg, NP-Sg). PP-Sg strains adhered to colon cancer cells much better than NP-Sg strains, suggesting that close contact between Sg and host cells is important. In mice, PP-Sg is significantly better at colonizing the colon tissues of A/J mice compared to NP-Sg, however this difference was not observed in C57BL/6 mice, suggesting that Sg colonization of mouse colon tissues involves specific interactions between bacterial and host factors on the colonic epithelium. Finally, in an azoxymethane-induced mouse model of CRC, PP-Sg promoted tumor development whereas NP-Sg did not. These findings provide clues to the mechanism underlying the Sg-CRC association and have important implications to clinical studies that aim to correlate Sg with clinical and pathological features of CRC.

Strain-dependent interactions of Streptococcus gallolyticus subsp. gallolyticus with human blood cells.

BACKGROUND: Streptococcus gallolyticus subsp. gallolyticus (S. gallolyticus) is the causative pathogen in up to 20% of streptococcal-induced infective endocarditis (IE) cases. However, the underlying mechanisms of pathogenesis in S. gallolyticus have not yet been solved. Pathogens causing IE need to employ virulent strategies to initiate and establish infections, such as escape the bloodstream, invade the host-cell, and persist intracellularly. In this study, we examined the induction of inflammation by different S. gallolyticus strains in relation to their survival in whole blood and cell culture models as well as their ability to induce platelet aggregation. Phagocytosis of these bacteria by macrophages, followed by intracellular survival, was also quantified. METHODS: In whole blood and THP-1 cell culture assays bacterial growth kinetics was determined by plating, followed by colony counting. Induction of interleukin (IL)-6 expression in whole blood of three healthy volunteers, caused by different strains, was quantified by ELISA. Gene expression of cytokines (IL1B, IL6 and IL8) was quantified by real-time PCR after stimulating THP-1 monocytes with bacteria. Induction of platelet aggregation was analyzed by light transmission aggregometry using the BORN method. A macrophage model was used to analyze phagocytosis of strains and their survival in macrophages within 48 h. RESULTS: Strains promoted IL-6 secretion in a time-dependent fashion. For example, DSM16831 induced IL-6 secretion in whole blood earlier than other isolates, and was eliminated in the whole blood of one volunteer, whereas UCN34 could grow. Platelet aggregation depended on the different isolates used and on the individual platelet donor. Two strains (AC1181 and 010672/01) induced cytokine gene expression in THP-1 monocytes only marginally, compared to other strains. The phagocytosis rate of S. gallolyticus isolates differed significantly, and the isolates UCN34 and BAA-2069 could persist for a considerable time in the phagocytes. CONCLUSION: The strain-dependent differences of S. gallolyticus isolates, observed during interaction with human blood cells, support the hypotheses that divergences in individual virulence factors determine a distinct pathogenicity of the isolates. These data constitute an additional step towards the elucidation of mechanisms in the complex, multifactorial pathogenesis of this IE pathogen.

Streptococcus gallolyticus subsp. gallolyticus promotes colorectal tumor development.

Streptococcus gallolyticus subsp. gallolyticus (Sg) has long been known to have a strong association with colorectal cancer (CRC). This knowledge has important clinical implications, and yet little is known about the role of Sg in the development of CRC. Here we demonstrate that Sg promotes human colon cancer cell proliferation in a manner that depends on cell context, bacterial growth phase and direct contact between bacteria and colon cancer cells. In addition, we observed increased level of ß-catenin, c-Myc and PCNA in colon cancer cells following incubation with Sg. Knockdown or inhibition of ß-catenin abolished the effect of Sg. Furthermore, mice administered with Sg had significantly more tumors, higher tumor burden and dysplasia grade, and increased cell proliferation and ß-catenin staining in colonic crypts compared to mice receiving control bacteria. Finally, we showed that Sg is present in the majority of CRC patients and is preferentially associated with tumor compared to normal tissues obtained from CRC patients. These results taken together establish for the first time a tumor-promoting role of Sg that involves specific bacterial and host factors and have important clinical implications.