Identification of Genes Contributing to the Virulence of Francisella tularensis SCHU S4 in a Mouse Intradermal Infection Model

Background: Francisella tularensis is a highly virulent human pathogen. The most virulent strains belong to subspecies tularensis and these strains cause a sometimes fatal disease. Despite an intense recent research effort, there is very limited information available that explains the unique features of subspecies tularensis strains that distinguish them from other F. tularensis strains and that explain their high virulence. Here we report the use of targeted mutagenesis to investigate the roles of various genes or pathways for the virulence of strain SCHU S4, the type strain of subspecies tularensis. Methodology/Principal Findings: The virulence of SCHU S4 mutants was assessed by following the outcome of infection after intradermal administration of graded doses of bacteria. By this route, the LD\u2085\u2080 of the SCHU S4 strain is one CFU. The virulence of 20 in-frame deletion mutants and 37 transposon mutants was assessed. A majority of the mutants did not show increased prolonged time to death, among them notably \u394pyrB and \u394recA. Of the remaining, mutations in six unique targets, tolC, rep, FTT0609, FTT1149c, ahpC, and hfq resulted in significantly prolonged time to death and mutations in nine targets, rplA, wbtI, iglB, iglD, purL, purF, ggt, kdtA, and glpX, led to marked attenuation with an LD\u2085\u2080 of >10\ub3 CFU. In fact, the latter seven mutants showed very marked attenuation with an LD\u2085\u2080 of 6510\u2077 CFU. Conclusions/Significance: The results demonstrate that the characterization of targeted mutants yielded important information about essential virulence determinants that will help to identify the so far little understood extreme virulence of F. tularensis subspecies tularensis.Peer reviewed: YesNRC publication: Ye

Resistance of Francisella tularensis Strains against Reactive Nitrogen and Oxygen Species with Special Reference to the Role of KatG

Francisella tularensis is a facultative intracellular bacterial pathogen capable of proliferating within host macrophages. The mechanisms that explain the differences in virulence between various strains of the species are not well characterized. In the present study, we show that both attenuated (strain LVS) and virulent (strains FSC200 and SCHU S4) strains of the pathogen replicate at similar rates in resting murine peritoneal exudate cells (PEC). However, when PEC were activated by exposure to gamma interferon (IFN-γ), they killed LVS more rapidly than virulent strains of the pathogen. Addition of N(G)-monomethyl-l-arginine, an inhibitor of inducible nitric oxide synthase, to IFN-γ-treated PEC, completely inhibited killing of the virulent strains, whereas it only partially blocked the killing of LVS. Similarly, in a cell-free system, SCHU S4 and FSC200 were more resistant to killing by H(2)O(2) and ONOO(−) than F. tularensis LVS. Catalase encoded by katG is a bacterial factor that can detoxify bactericidal compounds such as H(2)O(2) and ONOO(−). To investigate its contribution to the virulence of F. tularensis, katG deletion-containing mutants of SCHU S4 and LVS were generated. Both mutants demonstrated enhanced susceptibility to H(2)O(2) in vitro but replicated as effectively as the parental strains in unstimulated PEC. In mice, LVS-ΔkatG was significantly attenuated compared to LVS whereas SCHU S4-ΔkatG, despite slower replication, killed mice as quickly as SCHU S4. This implies that clinical strains of the pathogen have katG-independent mechanisms to combat the antimicrobial effects exerted by H(2)O(2) and ONOO(−), the loss of which could have contributed to the attenuation of LVS

Tumour colonisation of Parvimonas micra is associated with decreased survival in colorectal cancer patients

Increasing evidence suggests that the gut microbiota may impact colorectal cancer (CRC) development and progression. In this study, the tumour colonisation of two CRC-associated bacteria, Parvimonas micra and Fusobacterium nucleatum, was studied in relation to patient survival in a cohort of 257 CRC patients. Colonisation of P. micra and F. nucleatum was analysed in fresh frozen tumour tissue (n = 112) and in faeces (n = 250) by qPCR. When analysing tumour tissues, both P. micra and F. nucleatum were found to be associated with decreased five-year cancer-specific survival, an association that remained significant in multivariable analysis for P. micra. Furthermore, we found significant associations of high levels of P. micra and F. nucleatum with tumour molecular characteristics, i.e., tumours mutated in BRAFV600E, and tumours of the MSI subtype. The analysis of faecal samples showed weaker associations with prognosis and tumour molecular characteristics. In conclusion, our findings support a novel association of tumour colonisation of P. micra with decreased patient survival. A better understanding of the role of the gut microbiota in CRC might contribute to the advancement of prognostic tools and new targets for therapy

Metabolic factors and the risk of colorectal cancer by KRAS and BRAF mutation status

Factors related to energy metabolism and the metabolic syndrome, such as higher body mass index (BMI), blood glucose, or blood lipids, and blood pressure, are associated with an increased risk of colorectal cancer (CRC). However, CRC is a heterogeneous disease, developing through distinct pathways with differences in molecular characteristics and prognosis, and possibly also in risk factors. For subtypes defined by KRAS and BRAF mutation status, BMI is the only metabolic factor previously studied, with inconsistent findings. We investigated whether associations between BMI, blood glucose, blood lipids, and blood pressure and CRC risk differed by tumor KRAS and BRAF mutation status in 117,687 participants from two population-based cohorts within the Northern Sweden Health and Disease Study (NSHDS). Hazard ratios (HRs) for overall CRC and CRC subtypes by metabolic factors were estimated with Cox proportional hazards regression, using multiple imputation to handle missing exposure and tumor data. During a median follow-up of 15.6 years, we acquired 1,250 prospective CRC cases, of which 766 cases had complete baseline and molecular tumor data. Consistent with previous evidence, higher BMI, total cholesterol, triglyceride levels, and blood pressure were associated with an increased risk of overall CRC (HRs per 1 standard deviation increase: 1.07 to 1.12). These associations were similar regardless of CRC subtype by KRAS and BRAF mutation status (all pheterogeneity &gt; 0.05). The same was true for subtypes based on microsatellite instability status. Poor metabolic health may therefore be a universal mechanism for colorectal cancer, acting across multiple developmental pathways.Originally included in thesis in manuscript form with title [Metabolic factors and colorectal cancer risk by KRAS and BRAF mutation status]</p

A modified protein marker panel to identify four consensus molecular subtypes in colorectal cancer using immunohistochemistry

Colorectal cancer (CRC) is a heterogeneous disease with different genetic and molecular backgrounds, leading to a diverse patient prognosis and treatment response. Four consensus molecular subtypes (CMS 1–4) have recently been proposed based on transcriptome profiling. A clinically practical immunohistochemistry (IHC) based CMS classifier consisting of the four markers FRMD6, ZEB1, HTR2B, and CDX2 was then demonstrated. However, the IHC-CMS classifier did not distinguish between CMS2 and CMS3 tumours. In this study, we have applied the proposed transcriptome based and IHC-based CMS classifiers in a CRC cohort of 65 patients and found a concordance of 77.5 %. Further, we modified the IHC-CMS classifier by analysing the differentially expressed genes between CMS2 and CMS3 tumours using RNA-sequencing data from the TCGA dataset. The result showed that WNT signalling was among the most upregulated pathways in CMS2 tumours, and the expression level of CTNNB1 (encoding β-catenin), a WNT pathway hallmark, was significantly upregulated (P = 1.15 × 10−6). We therefore introduced nuclear β-catenin staining to the IHC-CMS classifier. Using the modified classifier in our cohort, we found a 71.4 % concordance between the IHC and RNA-sequencing based CMS classifiers. Moreover, β-catenin staining could classify 16 out of the 19 CMS2/3 tumours into CMS2 or CMS3, thereby showing an 84.2 % concordance with the RNA-sequencing-based classifier. In conclusion, we evaluated CMS classifiers based on transcriptome and IHC analysis. We present a modified IHC panel that categorizes CRC tumours into the four CMS groups. To our knowledge, this is the first study using IHC to identify all four CMS groups

Porphyromonas gingivalis in colorectal cancer and its association to patient prognosis

Microbiota dysbiosis may affect both the development and progression of colorectal cancer (CRC). Large metagenomic studies have highlighted specific oral bacteria linked to CRC including Porphyromonas gingivalis. Few studies have however analysed the implications of this bacterium in CRC progression and survival. In this study, we investigated the intestinal presence of P. gingivalis by qPCR in both faecal and mucosal samples from two different patient cohorts, including patients with precancerous dysplasia or CRC, as well as controls. P. gingivalis was detected in 2.6-5.3% of CRC patients and significantly different levels of P. gingivalis were found in faeces of CRC patients compared to controls (P = 0.028). Furthermore, an association was found between the presence of P. gingivalis in faeces and tumour tissue (P &lt; 0.001). Our findings further suggested a potential link between mucosal P. gingivalis and tumours of MSI subtype (P = 0.040). Last but not least, patients with faecal P. gingivalis were found to have a significantly decreased cancer-specific survival (P = 0.040). In conclusion, P. gingivalis could be linked to patients with CRC and to a worse patient prognosis. Further studies are needed to elucidate the role of P. gingivalis in CRC pathogenesis

Francisella noatunensis subspecies noatunensis clpB deletion mutant impairs development of francisellosis in a zebrafish model

Keywords: Atlantic cod, Francisella noatunensis, Francisellosis, ClpB, Immunization, Zebrafis

A longitudinal study of prediagnostic metabolic biomarkers and the risk of molecular subtypes of colorectal cancer

Body fatness increases the risk of colorectal cancer (CRC). Insulin resistance and altered adipokines are potential mechanisms, but previous biomarker studies have been inconsistent. Intertumoral heterogeneity might provide an explanation. We investigated insulin, C-peptide, adiponectin, and leptin in relation to CRC molecular subtypes using a nested case-control design (1010 cases, 1010 matched controls, median 12.3 years from baseline to CRC diagnosis) from the population-based Northern Sweden Health and Disease Study. Repeated samples were available from 518 participants. Risks of CRC and subtypes, defined by tumor BRAF and KRAS mutations and microsatellite instability (MSI) status, were estimated using conditional logistic regression and linear mixed models. Higher C-peptide and lower adiponectin were associated with increased CRC risk (odds ratios per standard deviation increase (95% CI): 1.11 (1.01, 1.23) and 0.91 (0.83, 1.00), respectively), though weakened when adjusted for body mass index. Insulin and leptin were not associated with CRC risk. Within-individual time trajectories were similar in cases and controls, and no subtype-specific relationships were identified (all Pheterogeneity &gt; 0.1). Adiponectin was weakly inversely associated with the risk of KRAS-mutated (P = 0.08) but not BRAF-mutated or KRAS/BRAF-wildtype CRC, consistent with the one previous study. These findings contribute to an increased understanding of the complex role of body size in CRC

Ex Vivo Organoid Cultures Reveal the Importance of the Tumor Microenvironment for Maintenance of Colorectal Cancer Stem Cells

Colorectal cancer (CRC) is a heterogeneous disease, with varying clinical presentations and patient prognosis. Different molecular subgroups of CRC should be treated differently and therefore, must be better characterized. Organoid culture has recently been suggested as a good model to reflect the heterogeneous nature of CRC. However, organoid cultures cannot be established from all CRC tumors. The study examines which CRC tumors are more likely to generate organoids and thus benefit from ex vivo organoid drug testing. Long-term organoid cultures from 22 out of 40 CRC tumor specimens were established. It was found that organoid cultures were more difficult to establish from tumors characterized as microsatellite instable (MSI), BRAF-mutated, poorly differentiated and/or of a mucinous type. This suggests that patients with such tumors are less likely to benefit from ex vivo organoid drug testing, but it may also suggest biological difference in tumor growth. RNA sequencing analysis of tumor sections revealed that the in vivo maintenance of these non-organoid-forming tumors depends on factors related to inflammation and pathogen exposure. Furthermore, using TCGA data we could show a trend towards a worse prognosis for patients with organoid-forming tumors, suggesting also clinical differences. Results suggest that organoids are more difficult to establish from tumors characterized as MSI, BRAF-mutated, poorly differentiated and/or of a mucinous type. We further suggest that the maintenance of cell growth of these tumors in vivo may be promoted by immune-related factors and other stromal components within the tumor microenvironment