The role of type 1 fimbriae in Salmonella infections
Team: Maciej Ugorski, Krzysztof Grzymajło, Aleksandra Orłowska, Agata Mikołajczyk
Infection by Salmonella is initiated by the attachment and colonization of gut mucosa, which seems to be an essential step in the pathogenesis of salmonellosis. There is convincing evidence suggesting that type 1 fimbriae play an important role in bacterial survival and persistence in the host, although their exact role in adhesion, invasiveness, pathogenesis and host-specificity is still not known and controversial. Type 1 fimbriae are proteinaceous, filamentous structures present on the surface of many species of the Enterobacteriaceae, including genus Salmonella. They are composed primarily of protein subunits called FimA. However, their binding properties depend on FimH adhesin, responsible for bacteria binding to oligomannosidic structures carried by many eukaryotic membrane-bound and secreted glycoproteins [mannose-sensitive (MS) FimH adhesins]. FimH proteins of Salmonella are closely related structurally, but it has become increasingly clear that there is significant heterogeneity among type 1 fimbriae from different serovars and even from different strains of the same serovar (allelic variants). It has been suggested that minor differences in the structure of FimH are associated with differences in adhesion specificities and may determine the tropism of various Salmonella serovars to different species and tissues.
We have cloned fimH genes of S. Enteritidis, S. Typhimurium, S. Abortus-ovis, S. Choleraesuis, S. Dublin, S. Gallinarum biovar Gallinarum and S. Gallinarum biovar Pullorum. Analysis of these genes has revealed a high degree of conservation (about 97 – 99.9%) in sequenced fimH genes, with several nucleotide substitutions causing minor differences in amino acid sequences of FimH proteins. Using recombinant FimH proteins, it was shown that FimH adhesins from Salmonella enterica serovars with limited (Choleraesuis, Dublin) or restricted (Abortus-ovis) host range as well as from host unrestricted serovar Enteritidis were able to bind mannose-rich glycoproteins (RNase B, HRP and Man-BSA) with comparable affinity measured by surface plasmon resonance (Kisiela et al., 2006; Grzymajło et al., 2013). However, even though all of them represent low-adhesive variants of FimH protein, significant differences in the binding profiles of the FimH proteins of host restricted and host unrestricted serovars to enterocyte glycoproteins of sheep, pig and cattle, was observed (Grzymajło et al., 2013). When FimH adhesin from S. Enteritidis was subjected to Western blot analysis, it bound to surface membrane protein of about 130 kDa, and FimH adhesins from S. Abortus-ovis, S. Choleraesuis and S. Dublin bound to surface membrane protein of about 55 kDa. These results suggest that FimH adhesins of type 1 fimbriae are one of the factors responsible for different host-specificities of these Salmonella serovars. This hypothesis is also supported by studies on FimH adhesins from host-specific Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum, which infect only poultry. In contrast to other analyzed Salmonella serovars, they did not bind to high-mannose oligosaccharides [mannose-resistant (MR) FimH adhesins. The loss of sugar-binding properties by biovar Gallinarum and biovar Pullorum FimH adhesins is the result of a single T78I mutation, as was demonstrated by site-directed mutagenesis of FimH proteins (Kisiela et al., 2005).
It was suggested that the expression of MS type 1 fimbriae was associated with Salmonella infections which are limited to the alimentary tract, causing gastroenteritis rather than an invasive typhoid-like disease associated with expression of MR type 1 fimbriae (Kisiela et al., 2012). Therefore, to investigate the role of the MR variant of the FimH adhesin in the pathogenesis of S. Gallinarum, the isogenic mutant elaborating type 1 fimbriae with the MS variant of the FimH adhesin from S. Enteritidis and the mutant strain with no FimH expression were constructed, and their invasiveness in 1-day-old chicks was studied. Our results demonstrated that the S. Gallinarum expressing S. Enteritidis FimH adhesin decreased systemic dissemination of S. Gallinarum and colonization of internal organs in chicks indicating the importance of the endogenous MR variant of the FimH protein in the virulence of S. Gallinarum. Elaborating from these studies, we proposed that MS variants of FimH are advantageous in gastrointestinal infections in contrast to MR FimH variants which decrease intestinal colonization and promote Salmonella systemic spreading. This hypothesis was supported by our studies on mice infected with wild-type S. Enteritidis and its fimH gene knockout strain. We observed that the loss of MS FimH adhesin correlates with the highly increased colonization of mice by these bacteria. We also showed that expression and secretion of IL-1b, IL-6, IL-10 and IL-12b was significantly higher in mice intestinal cells infected with wild-type S. Enteritidis compared to cells infected with the mutant strain. Therefore, we propose that type 1 fimbriae may play an important role in the pathogenicity of S. Enteritidis and may contribute to an intestinal inflammatory response.
Research for new bacterial virulence determinants using Omics technologies
Team: Rafał Kolenda, Maciej Ugorski
Project in collaboration with: Peter Schierack (BTU Cottbus-Senftenberg, Germany), Gordon Dougan (Sanger Institute, UK), Josefine Bartholdson Scott (Sanger Institute, UK), Dereck Pickard (Sanger Institute, UK), Michał Burdukiewicz (Warsaw Technical University), Aamir Ali (National Institute for Biotechnology and Genetic Engineering, Pakistan), Sebastian Gunther (University of Greifswald, Germany), Torsten Semmler (Robert Koch Institute, Germany)
Advances in Omics technologies have provided us with new tools to investigate pathogenic bacteria. Adhesion and/or invasion to various epithelial cells are among the first steps during the pathogenesis of bacterial infections. Bacteria have evolved various strategies to adhere and invade host cells. To study the cell-bacteria interactions, in vitro adhesion/invasion models are often used. Currently used methods are not feasible for large scale studies of bacterial cell line infectivity. Therefore, the object was to develop an automated microscopy-based system for bacterial quantification and to screen Salmonella clinical isolates for cell line infecting phenotypes. With the use of VideoScan Technology, developed in Prof. Schierack’s group (BTU Cottbus-Senftenberg), Salmonella serovars with broad (S. Typhimurium, S. Enteritidis) and narrow (S. Gallinarum, S. Choleraesuis, S. Dublin) host ranges were screened for cell line infecting phenotypes. Bacteria were used in infection assays on three different cell lines including IPEC-J2 (porcine), Caco-2 (human) and CHIC-8E11 (avian). Adhering/infecting bacteria were counted after 1h and 4h of infection with the automated VideoScan instrument. Next, genomes of selected Salmonella isolates with the highest and lowest cell line infecting phenotypes were sequenced with next generation sequencing (NGS) at Sanger Institute in cooperation with Prof. Dougan. Currently, comparative analysis of these Salmonella genomes is being carried out. This will allow us to identify genes associated with adhesion and invasion for Salmonella clinical isolates.
The Salmonella isolates used for cell line infection assays were screened for type 1 fimbriae (T1F) expression using anti-FimH static adhesion assay (part of VideoScan). Expression of T1F was observed for more than 80% of isolates tested. Moreover, it was found that T1F are expressed in more isolates from serovars with broad host range, than in isolates from serovars with narrow host range. Next, genomes of selected Salmonella isolates with the highest and lowest T1F expression were sequenced with next generation sequencing (NGS) in cooperation with Prof. Sebastian Gunther and Dr. Torsten Semmler. Moreover, four Salmonella isolates were submitted for RNA sequencing. Subsequent analyses of Salmonella genomes and expression profiles will provide useful information about the role of genetic variability in clinical isolates of Salmonella.
The genes identified in both projects will be verified using appropriate bacterial mutants and in vitro cell line models, and in vivo animal models. The functional analysis of these genes will provide new information on the pathogenesis of Salmonella infections and will help to develop new strategies for the prevention and treatment of diseases caused by Salmonella.
The role of ceramide galactosyltransferase (UGT8) and galactosylceramide in breast cancer progression
Team: Maciej Ugorski, Jarosław Suchański, Maciej Zacharski
Numerous studies on cellular glycoconjugates have revealed that neoplastic transformation and tumor progression are almost invariably associated with changes in the expression profiles of surface glycoproteins and glycolipids. However, there is very little information available on galactosylceramide (GalCer) expression in human tumors. GalCer is synthesized by ceramide galactosyltransferase (UGT8). Transcriptomic profiling of breast cancer tissues showed that UGT8 is one of six genes whose elevated expression correlated with a significantly increased risk of lung metastases in breast cancer patients (Landemaine et al., 2008). Based on this finding, our studies with the use of immunohistochemistry and real-time PCR on the presence of UGT8 in breast cancer tissue specimens revealed a significant increase in UGT8 expression in (1) metastatic tumors v. primary tumors, (2) tumors of malignancy grades G3 v. G2 and G3 v. G1, and (3) node-positive v. node-negative tumors (Dziegiel et al., 2010). Therefore, our data suggests that UGT8 can be a significant index of tumor aggressiveness and a potential marker for prognostic evaluation of lung metastases in breast cancer (Dziegiel et al., 2010). We have also shown, using an athymic nu/nu mice model, that the suppression of UGT8 expression in breast cancer cells has a profound effect on their tumorigenic and metastatic properties (Owczarek et al., 2013). It was found that breast cancer cells with high expression of UGT8 and GalCer form tumors much more efficiently than breast cancer cells with suppressed synthesis of GalCer after their orthotropic transplantation. Further studies with immunohistochemical staining of tumor specimens revealed that high expression of UGT8 accompanied by accumulation of GalCer in breast cancer cells is associated with a much higher proliferative index and a lower number of apoptotic cells than cells with suppressed synthesis of GalCer. Also, breast cancer cells expressing higher levels of UGT8 and synthesizing larger amounts of GalCer had a higher ability to form metastatic colonies after intracardiac inoculation into nu/nu mice. Based on these results we proposed that the presence of GalCer protects breast cancer cells from cellular stresses induced by the tumor microenvironment, and probably its cytoprotective effect is associated with increased resistance to stress-induced apoptosis. Confirming this hypothesis, it was found that expression of UGT8 and accumulation of GalCer in human breast cancer cells increased their resistance to apoptosis induced by hypoxia and free radicals as well as doxorubicin and paclitaxel. The question remains as to the exact mechanism by which GalCer mediates cytoprotective effects during stress- and drug-induced apoptosis. To answer this question we analysed the intracellular concentration of ceramide, known as a pro-apoptotic molecule, by HILIC-ESI-MS/MS. However, no differences in ceramide concentrations were found between cells synthesizing high and low amounts of GalCer. Therefore, expression of genes involved in apoptosis-related pathways was analyzed using RT2 Profiler™ PCR Array Human Apoptosis assay and Real-Time PCR using self designed primers. The level of expression of selected genes was also analysed on the level of protein using Western blotting. Gene expression profiling revealed statistically significant down-regulation of TNFRSF1B, TNFRSF9 and up-regulation of BCL2 in breast cancer cell lines with high expression of UGT8 and GalCer. These data suggest that GalCer can affect the expression of apoptosis-related genes on the level of transcription.
This work is performed in collaboration with the L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences and the Medical University of Wrocław.
Molecular bases of heart failure
Team: Liliana Kiczak, Maciej Zacharski
In the framework of project “Wrovasc - Integrated Cardiovascular Centre” a porcine model of tachycardia-induced cardiomyopathy (TIC) was developed. This type of an animal model of heart failure (HF) seems to be a rational alternative for comprehensive studies of the whole spectrum of the natural history of HF, including the initial stages, which are largely under investigated (Pasławska 2011).
We investigated selected molecular elements of the parasympathetic system in the medulla oblongata (MO) in male pigs during subsequent stages of chronic TIC. Our results showed changes in the activity and levels of various elements in the parasympathetic system of MO, providing a functional link between the MO and the heart during the progression of HF. Importantly, the very early changes seen within the parasympathetic system in the MO preceded an increase in sympathetic drive. This assessment was based on high circulating catecholamines (Tomaszek 2013). Our results suggest a significant contribution of adrenals to the circulating pool of catecholamines in subjects with systolic HF (Tomaszek 2015).
Our TIC model was used to determine myocardial inflammatory factors, such as inducible NO synthase (iNOS) and oxidative stress indices during HF development. In contrast to rodents, inducible NO synthase was shown to be constitutively expressed within porcine LV. Its level decreased during the progression of systolic non-ischaemic heart failure in the pig TIC model. We found that the oxidative and inflammatory status of the failing LV myocardium did not change when compared to control animals. Thus, it can be assumed that an up-regulation of pro-inflammatory factors is not involved in HF in large mammals, and that the impact of oxidative stress may be restricted to the mitochondria in these organisms.
Another research area is myocardial remodeling in HF progression. The extracellular matrix (ECM) is supposed to play a fundamental role in this process, as well as matrix metalloproteinases (MMPs). We have demonstrated the presence of high molecular weight (HMW) complexes containing MMP9, MMP2, TIMP1 () and NGAL () in myocardium from both diseased and healthy animals. In skeletal muscles we found no HMW complexes. These HMW complexes are proteolytically inactive, but can spontaneously release active MMP9 and MMP2. This observation indicates that the HMW complexes may serve as the tissue reservoir of active MMPs, which seems to be unique for myocardial tissue (Kiczak 2013, Kiczak 2014). Investigation of sex differences in heart remodeling showed that significant LV dilatation occurred only in males, along with a strong induction of the pro-hypertrophic program. Total gelatinolytic activity in LV was higher in males than in females (irrespective of HF), and the HF progression was associated with reduced total gelatinolytic activity in the LV only in males. In females the LV dilated more slowly, without pronounced signs of eccentric hypertrophy, preserving initial diastolic function Our findings, showing sex differences in some aspects of the ECM turnover leading to a diverse myocardial response to RV pacing, highlight the need for sex-specific medicine. Furthermore, care should be taken in analyzing the results from animal HF models as some effects might be sex-specific.
Microbial multidrug resistance
Anna Kolaczkowska
Resistance to currently available drugs in microorganisms (bacteria, parasites, viruses and fungi) can be a serious threat to global public health. Infections caused by drug resistant microorganisms are difficult to treat and cause longer duration of illness. Opportunistic pathogens from the genus Candida, e.g. C. albicans and C. glabrata, are the most frequently isolated etiological factors of fungal infections. C. glabrata is mainly isolated from patients with a compromised immune system and may cause superficial but also life threatening systemic infections. In addition, C. glabrata is intrinsically less susceptible to azole drugs. It also rapidly develops resistance to antifungal agents during therapy treatment. An increased expression of genes coding membrane ATP- binding transporters (ABC transporters) extruding drugs out of the cell is the main mechanism responsible for intrinsic as well as acquired azole resistance. Apart from ABC transporter genes, other loci are involved in the development of drug resistance. To this group belong genes of the LTE (Lipid Translocating Exporters) family, loci involved in cell wall remodeling and those influencing mitochondrial status. One of the main goals is to investigate their role in drug resistance development and to understand the regulatory signals and cascades connected with this process.
