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 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.

SdrC induces staphylococcal biofilm formation through a homophilic interaction.

The molecular pathogenesis of many Staphylococcus aureus infections involves growth of bacteria as biofilm. In addition to polysaccharide intercellular adhesin (PIA) and extracellular DNA, surface proteins appear to mediate the transition of bacteria from planktonic growth to sessile lifestyle as well as biofilm growth, and can enable these processes even in the absence of PIA expression. However, the molecular mechanisms by which surface proteins contribute to biofilm formation are incompletely understood. Here we demonstrate that self-association of the serine-aspartate repeat protein SdrC promotes both bacterial adherence to surfaces and biofilm formation. However, this homophilic interaction is not required for the attachment of bacteria to abiotic surfaces. We identified the subdomain that mediates SdrC dimerization and subsequent cell-cell interactions. In addition, we determined that two adjacently located amino acid sequences within this subdomain are required for the SdrC homophilic interaction. Comparative amino acid sequence analysis indicated that these binding sites are conserved. In summary, our study identifies SdrC as a novel molecular determinant in staphylococcal biofilm formation and describes the mechanism responsible for intercellular interactions. Furthermore, these findings contribute to a growing body of evidence suggesting that homophilic interactions between surface proteins present on neighbouring bacteria induce biofilm growth.

Postgenomic adventures with Rhodobacter sphaeroides.

This review describes some of the recent highlights taken from the studies of Rhodobacter sphaeroides 2.4.1. The review is not intended to be comprehensive, but to reflect the bias of the authors as to how the availability of a sequenced and annotated genome, a gene-chip, and proteomic profile as well as comparative genomic analyses can direct the progress of future research in this system.

Combining microarray and genomic data to predict DNA binding motifs.

The ability to detect regulatory elements within genome sequences is important in understanding how gene expression is controlled in biological systems. In this work, microarray data analysis is combined with genome sequence analysis to predict DNA sequences in the photosynthetic bacterium Rhodobacter sphaeroides that bind the regulators PrrA, PpsR and FnrL. These predictions were made by using hierarchical clustering to detect genes that share similar expression patterns. The DNA sequences upstream of these genes were then searched for possible transcription factor recognition motifs that may be involved in their co-regulation. The approach used promises to be widely applicable for the prediction of cis-acting DNA binding elements. Using this method the authors were independently able to detect and extend the previously described consensus sequences that have been suggested to bind FnrL and PpsR. In addition, sequences that may be recognized by the global regulator PrrA were predicted. The results support the earlier suggestions that the DNA binding sequence of PrrA may have a variable-sized gap between its conserved block elements. Using the predicted DNA binding sequences, a whole-genome-scale analysis was performed to determine the relative importance of the interplay between the three regulators PpsR, FnrL and PrrA. Results of this analysis showed that, compared to the regulation by PpsR and FnrL, a much larger number of genes are candidates to be regulated by PrrA. The study demonstrates by example that integration of multiple data types can be a powerful approach for inferring transcriptional regulatory patterns in microbial systems, and it allowed the detection of photosynthesis-related regulatory patterns in R. sphaeroides.

DNA sequence duplication in Rhodobacter sphaeroides 2.4.1: evidence of an ancient partnership between chromosomes I and II.

The complex genome of Rhodobacter sphaeroides 2.4.1, composed of chromosomes I (CI) and II (CII), has been sequenced and assembled. We present data demonstrating that the R. sphaeroides genome possesses an extensive amount of exact DNA sequence duplication, 111 kb or approximately 2.7% of the total chromosomal DNA. The chromosomal DNA sequence duplications were aligned to each other by using MUMmer. Frequency and size distribution analyses of the exact DNA duplications revealed that the interchromosomal duplications occurred prior to the intrachromosomal duplications. Most of the DNA sequence duplications in the R. sphaeroides genome occurred early in species history, whereas more recent sequence duplications are rarely found. To uncover the history of gene duplications in the R. sphaeroides genome, 44 gene duplications were sampled and then analyzed for DNA sequence similarity against orthologous DNA sequences. Phylogenetic analysis revealed that approximately 80% of the total gene duplications examined displayed type A phylogenetic relationships; i.e., one copy of each member of a duplicate pair was more similar to its orthologue, found in a species closely related to R. sphaeroides, than to its duplicate, counterpart allele. The data reported here demonstrate that a massive level of gene duplications occurred prior to the origin of the R. sphaeroides 2.4.1 lineage. These findings lead to the conclusion that there is an ancient partnership between CI and CII of R. sphaeroides 2.4.1.

Whole-genome shotgun optical mapping of Rhodobacter sphaeroides strain 2.4.1 and its use for whole-genome shotgun sequence assembly.

Rhodobacter sphaeroides 2.4.1 is a facultative photoheterotrophic bacterium with tremendous metabolic diversity, which has significantly contributed to our understanding of the molecular genetics of photosynthesis, photoheterotrophy, nitrogen fixation, hydrogen metabolism, carbon dioxide fixation, taxis, and tetrapyrrole biosynthesis. To further understand this remarkable bacterium, and to accelerate an ongoing sequencing project, two whole-genome restriction maps (EcoRI and HindIII) of R. sphaeroides strain 2.4.1 were constructed using shotgun optical mapping. The approach directly mapped genomic DNA by the random mapping of single molecules. The two maps were used to facilitate sequence assembly by providing an optical scaffold for high-resolution alignment and verification of sequence contigs. Our results show that such maps facilitated the closure of sequence gaps by the early detection of nascent sequence contigs during the course of the whole-genome shotgun sequencing process.