Exploitation of the host cell ubiquitin machinery by microbial effector proteins.

Pathogenic bacteria are in a constant battle for survival with their host. In order to gain a competitive edge, they employ a variety of sophisticated strategies that allow them to modify conserved host cell processes in ways that favor bacterial survival and growth. Ubiquitylation, the covalent attachment of the small modifier ubiquitin to target proteins, is such a pathway. Ubiquitylation profoundly alters the fate of a myriad of cellular proteins by inducing changes in their stability or function, subcellular localization or interaction with other proteins. Given the importance of ubiquitylation in cell development, protein homeostasis and innate immunity, it is not surprising that this post-translational modification is exploited by a variety of effector proteins from microbial pathogens. Here, we highlight recent advances in our understanding of the many ways microbes take advantage of host ubiquitylation, along with some surprising deviations from the canonical theme. The lessons learned from the in-depth analyses of these host-pathogen interactions provide a fresh perspective on an ancient post-translational modification that we thought was well understood.This article is part of a Minifocus on Ubiquitin Regulation and Function. For further reading, please see related articles: 'Mechanisms of regulation and diversification of deubiquitylating enzyme function' by Pawel Leznicki and Yogesh Kulathu (J. Cell Sci.130, 1997-2006). 'Cell scientist to watch - Mads Gyrd-Hansen' (J. Cell Sci.130, 1981-1983).

Evaluation of a cross contamination model describing transfer of Salmonella spp. and Listeria monocytogenes during grinding of pork and beef.

In a previous study, a model was developed to describe the transfer and survival of Salmonella during grinding of pork (Møller, C.O.A., Nauta, M.J., Christensen, B.B., Dalgaard, P., Hansen, T.B., 2012. Modelling transfer of Salmonella typhimurium DT104 during simulation of grinding of pork. Journal of Applied Microbiology 112 (1), 90-98). The robustness of this model is now evaluated by studying its performance for predicting the transfer and survival of Salmonella spp. and Listeria monocytogenes during grinding of different types of meat (pork and beef), using two different grinders, different sizes and different numbers of pieces of meats to be ground. A total of 19 grinding trials were collected. Acceptable Simulation Zone (ASZ), visual inspection of the data, Quantitative Microbiological Risk Assessment (QMRA), as well as the Total Transfer Potential (TTP) were used as approaches to evaluate model performance and to access the quality of the cross contamination model predictions. Using the ASZ approach and considering that 70% of the observed counts have to be inside a defined acceptable zone of ±0.5 log10CFU per portion, it was found that the cross contamination parameters suggested by Møller et al. (2012) were not able to describe all 19 trials. However, for each of the collected grinding trials, the transfer event was well described when fitted to the model structure proposed by Møller et al. (2012). Parameter estimates obtained by fitting observed trials performed at different conditions, such as size and number of pieces of meat to be ground, may not be applied to describe cross contamination of unlike processing. Nevertheless, the risk estimates, as well as the TTP, revealed that the risk of disease may be reduced when the grinding of meat is performed in a grinder made of stainless steel (for all surfaces in contact with the meat), using a well-sharpened knife and holding at room temperatures lower than 4°C.

Validation of Toxtree and SciQSAR in silico predictive software using a publicly available benchmark mutagenicity database and their applicability for the qualification of impurities in pharmaceuticals.

The draft ICH M7 guidance (US FDA, 2013) recommends that the computational assessment of bacterial mutagenicity for the qualification of impurities in pharmaceuticals be performed using an expert rule-based method and a second statistically-based (Q)SAR method. The public nonproprietary 6489 compound Hansen benchmark mutagenicity data set was used as an external validation data set for Toxtree, a free expert rule-based SAR software. This is the largest known external validation of Toxtree. The Toxtree external validation specificity, sensitivity, concordance and false negative rate for this mutagenicity data set was 66%, 80%, 74% and 20%, respectively. This mutagenicity data set was also used to create a statistically-based SciQSAR-Hansen mutagenicity model. In a 10% leave-group-out internal cross validation study the specificity, sensitivity, concordance and false negative rate for the SciQSAR mutagenicity model was 71%, 83%, 77% and 17%, respectively. Combining Toxtree and SciQSAR predictions and scoring a positive finding in either software as a positive mutagenicity finding reduced the false negative rate to 7% and increased sensitivity to 93% at the expense of specificity which decreased to 53%. The results of this study support the applicability of Toxtree, and the SciQSAR-Hansen mutagenicity model for the qualification of impurities in pharmaceuticals.

Diversity of culturable microorganisms and occurrence of Listeria monocytogenes and Salmonella spp. in Tuber aestivum and Tuber melanosporum ascocarps.

The aim of this study was to investigate the total mesophilic microorganisms, Pseudomonas genus, Enterobacteriaceae family, mold and yeast counts and the presence of Listeria monocytogenes and Salmonella spp on Tuber aestivum and Tuber melanosporum ascocarps. The results confirmed that the major percentage of the microorganisms, approximately 9.0 log ufc/g, were present in the peridium, the glebas of healthy truffles being practically free of microorganisms. The predominant microbial group was the Pseudomonas averaging 8.3 and 8.4 log cfu/g on T. aestivum and T. melanosporum whole ascocarps, respectively. The Enterobacteriaceae also achieved high populations, especially in T. aestivum truffles, with 6.3 log cfu/g. Molds and yeasts never exceeded 5.0 log cfu/g. The characterization of the isolates revealed that the fluorescens pseudomonads were the most prevalent. Raoultella terrigena and Enterobacter intermedius were the dominant Enterobacteriaceae. The identification of the yeast isolates revealed five species: Debaryomyces hansenii, Issatchenkia scutulata, Rhodotorula aurantiaca, Saccharomyces dairensis and Trichosporon beigelii subspecies A and B. The mold genera detected in both species of truffles were Aspergillus, Cladosporium, Penicillium and Fusarium, Trichoderma being present only in T. aestivum. L. monocytogenes was found in 10% of the samples of T. aestivum analysed but Salmonella spp. was not detected. Knowledge of the microbial population in terms of possible food borne and pathogen microorganisms is very useful for establishing successful disinfection and storage methods to prolong the shelf-life of ascocarps of T. aestivum and T. melanosporum.

Proteolytic yeasts isolated from raw, ripe tomatoes and metabiotic association of Geotrichum candidum with Salmonella.

Metabiotic associations between food-borne fungi and bacteria capable of causing human diseases are a public health concern. A survey of decayed and damaged, uncooked, ripe tomatoes was done to determine the presence and prevalence of yeasts capable of increasing the pH pulp tissue, thus creating a more favorable environment for survival and growth of enteric pathogens. Sixty-two of the 371 (16.7%) fungi isolated from 215 decayed or damaged tomatoes; 12 of the 62 (19.4%) yeasts showed proteolytic activity on gelatin agar (GA) and/or standard methods caseinate (SMC) agar. The pH of tomato pericarp (pulp) tissue from which 9 of the 12 yeasts were isolated ranged from 4.3 to 7.5 (mean=5.3) compared to 4.2-5.1 (mean=4.8) for sound pulp tissue in the same tomatoes. The 12 proteolytic yeasts consisted of four strains of Cryptococcus albidus, two strains each of Debaryomyces hansenii and Trichosporon pullulans, and one strain each of Cryptococcus humicolus, Cryptococcus laurentii, Geotrichum candidum, and Sporidiobolus pararoseus. Survival and growth characteristics of a five-serotype mixture of Salmonella co-inoculated with G. candidum into sound (not chill injured) and chill-injured tomatoes were studied. Storage of sound tomatoes at 15 degrees C for 10 days resulted in an increase in population of 7.6 log(10) cfu of Salmonella/g of a 2-g sample of co-infected pulp tissue. Increases were less in tissue inoculated with Salmonella only, Salmonella on day 0 followed by G. candidum on day 3, or G. candidum on day 0 followed by Salmonella on day 3. Trends were similar in sound inoculated tomatoes stored at 25 degrees C. Growth of Salmonella was enhanced in chill-injured tomatoes compared to sound tomatoes; a population of 10 log(10) cfu/g of chill-injured pulp tissue was reached within 10 days at 25 degrees C. Results clearly show that growth of a proteolytic, alkalinizing yeast such as G. candidum in raw tomatoes enhances conditions for growth of Salmonella. The removal of tomatoes infected with proteolytic yeasts and other fungi from lots intended for minimally processed tomato products is an essential step in reducing the risk of human diseases caused by pathogenic bacteria favored by increased pH of decayed pulp tissue.

Chronic bacterial infections: living with unwanted guests.

Some bacterial pathogens can establish life-long chronic infections in their hosts. Persistence is normally established after an acute infection period involving activation of both the innate and acquired immune systems. Bacteria have evolved specific pathogenic mechanisms and harbor sets of genes that contribute to the establishment of a persistent lifestyle that leads to chronic infection. Persistent bacterial infection may involve occupation of a particular tissue type or organ or modification of the intracellular environment within eukaryotic cells. Bacteria appear to adapt their immediate environment to favor survival and may hijack essential immunoregulatory mechanisms designed to minimize immune pathology or the inappropriate activation of immune effectors.

Synovial fluid antigen-presenting cell function in rheumatoid arthritis.

We have previously demonstrated enhanced synovial fluid (SF) antigen-presenting cell (APC) function in inflammatory arthritis patients selected on the basis of marked SF mononuclear cell (MNC) responsiveness to reactive arthritis-associated bacteria (Clin Exp Immunol 1990; 79:189-94). In this study we have assessed whether similarly enhanced synovial APC function is present in other inflammatory arthritis patients by using two assay systems to study 18 rheumatoid arthritis patients whose MNC responsiveness had not been determined in advance. We demonstrate that rheumatoid SF APC are much more potent than peripheral blood (PB) APC in stimulating the responses of autologous PB T cells to a range of recall antigens. In addition, SF APC are shown to be efficient stimulators of the antigen-specific responses of MHC-compatible, cloned T cells. Enhanced synovial APC function is thus likely to be a general feature of inflammatory arthritis and may play an important role in its pathogenesis.

Epitope specificity and MHC restriction of rheumatoid arthritis synovial T cell clones which recognize a mycobacterial 65 kDa heat shock protein.

CD4+ T cell clones specific for the mycobacterial hsp 65 were obtained from synovial fluid of a DR4 homozygous rheumatoid arthritis (RA) patient. A stimulatory epitope was defined using both deletion mutants of the mycobacterial hsp 65 and synthetic peptides and proved to be in a highly conserved region of the molecule. Despite this, however, there was no recognition by these clones of either the recombinant human homologue of mycobacterial hsp 65, P60, nor of a synthetic peptide containing an amino acid sequence from P60 corresponding to the epitope defined in the mycobacterial hsp 65. When the pattern of HLA restriction shown by the hsp-65-specific T cell clones was investigated, all clones tested proved to be restricted by HLA-DP rather than the more usual HLA-DR. Inhibition experiments suggested that this restriction also applied to the polyclonal synovial T cell response to hsp 65, but not to other antigens. Exclusive restriction of T cell recognition of an antigen by HLA-DP has not been reported previously, and strongly suggests that in this case the T cell repertoire for recognizing hsp 65 in the context of DR4 is deficient. Such an association between DR4 and the inability to respond to an immunodominant bacterial antigen may have implications for the pathogenesis of RA.

In vitro responses to a 65-kilodalton mycobacterial protein by synovial T cells from inflammatory arthritis patients.

Bacterial Ag, especially those of mycobacteria, have been implicated in the pathogenesis of experimental inflammatory arthritis in rodents, while in man, reactive arthritis has a clear temporal relationship to infection with particular bacteria. To investigate the role of immune responses to bacterial Ag in inflammatory arthritis, we have examined the proliferative responses of paired synovial fluid and PBMC when stimulated with 1) suspensions of irradiated or heat-killed bacteria associated with reactive arthritis (ReA), 2) purified protein derivative, 3) a recombinant 65-kDa heat shock protein of Mycobacterium leprae. The 65-kDa Ag was stimulatory to synovial fluid mononuclear cells, but not PBMC, from patients with different arthropathies, including most of those with ReA, but also some with rheumatoid arthritis. Furthermore, the magnitude of these responses correlated more closely with responses to ReA-associated bacteria (such as Salmonella), than with responses to the mycobacterial Ag represented in purified protein derivative. These results suggest that the 65-kDa molecule, which is common to a wide range of bacteria, may be an important immunogen for the T cell-mediated immune responses within the joint in different clinically defined inflammatory arthropathies.

The O antigenic relationships amongst Dekkera, Hanseniaspora, Escherichia coli O:75, Salmonella cholerae-suis and Salmonella aberdeen.

The immune sera for Dekkera bruxellensis, D. intermedia and Hanseniaspora osmophila agglutinated Salmonella cholerae-suis (O:62,7) and vice versa. The immune serum for H. osmophila also agglutinated E. coli (O:75), S. aberdeen (O:11) and vice versa. Cross agglutination and absorption tests demonstrated common antigenic factor(s) between above mentioned yeasts and Salmonella O:62, O:7 antigens. H. osmophila also showed a common antigenic factor(s) with E. coli (O:75) and S. aberdeen (O:11).

Impairment of Jones-Mote hypersensitivity and specific antibody response against depolymerized flagellin in lepromatous leprosy.

Cutaneous hypersensitivity and antibody-producing capacity were assessed in patients with lepromatous leprosy with defective immunity, by immunizing them with monomeric flagellin from Salmonella adelaide. Results were compared with those of controls, matched for age and sex, derived from similar socioeconomic stratum, but without any defect of the immunological system. In contrast to the normal individuals, who showed Jones-Mote type of hypersensitivity, no lepromatous patient could mount any 'delayed-in-time' cutaneous hypersensivivity reaction against an intradermal challenge of monomeric flagellin. However, when immunized through the subcutaneous route, both groups could produce adequate amounts of specific serum antibody. In addition to this unique split tolerance found in all lepromatous patients, some patients showed low levels of 'natural' IgM antibody, reduced formation of specific antibody when immunized through the subcutaneous route, and incomplete maturation of IgG class of anti-flagellin antibody. When immunized by the intradermal route, however, production of both anti-flagellin antibody and maturation of IgG antibody was significantly inhibited in normal adults but not in lepromatous patients. Thus, contrary to the earlier concept of hyperactivity of the humoral immune apparatus in lepromatous leprosy, the present study detected B-cell hypofunction in some patients.

[The antigenic relationship between Salmonella 0:7 antigen and Debaromyces hansenii and its synonym strains]. / Debaryomyces hansenii ve bazi sinonimlerinin Salmonella 0:7 antijeni ile iliskisi.

In this publication antigenic relationship between Salmonella 0:7 antigen and Debaryomyces marama, Debaryomyces hansenii and some synonym strains of Debaryomyces hansenii are shown together.

The antigenic relationship between Brettanomyces-Debaryomyces strains and the Salmonella cholerae-suis O antigen.

The immune sera for Brettanomyces lambicus, B. claussenii, Debaryomyces hansenii and D. marama agglutinated Salmonella cholerae-suis (0:6(2), 7). The immune serum for S. cholerae-suis agglutinated B. lambicus, B. clausenni, D. hansenii and D. marama. Absorption and agglutination cross-tested demonstrated common antigen factor(s) in the tested yeasts and Salmonella 0:7 antigen.