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1.
SAR QSAR Environ Res ; 28(7): 609-620, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28789565

ABSTRACT

Fluorimetric analysis is still a growing line of research in the determination of a wide range of organic compounds, including pharmaceuticals and pesticides, which makes necessary the development of new strategies aimed at improving the performance of fluorescence determinations as well as the sensitivity and, especially, the selectivity of the newly developed analytical methods. In this paper are presented applications of a useful and growing tool suitable for fostering and improving research in the analytical field. Experimental screening, molecular connectivity and discriminant analysis are applied to organic compounds to predict their fluorescent behaviour after their photodegradation by UV irradiation in a continuous flow manifold (multicommutation flow assembly). The screening was based on online fluorimetric measurement and comprised pre-selected compounds with different molecular structures (pharmaceuticals and some pesticides with known 'native' fluorescent behaviour) to study their changes in fluorescent behaviour after UV irradiation. Theoretical predictions agree with the results from the experimental screening and could be used to develop selective analytical methods, as well as helping to reduce the need for expensive, time-consuming and trial-and-error screening procedures.


Subject(s)
Fluorescence , Pesticides/chemistry , Pharmaceutical Preparations/chemistry , Photolysis , Quantitative Structure-Activity Relationship , Ultraviolet Rays
2.
Genes Immun ; 17(1): 19-29, 2016.
Article in English | MEDLINE | ID: mdl-26562079

ABSTRACT

The outcome of infection with Salmonella Typhimurium in mouse models of human typhoid fever is dependent upon a coordinated complex immune response. A panel of recombinant congenic strains (RCS) derived from reciprocal backcross of A/J and C57BL/6J mice was screened for their susceptibility to Salmonella infection and two susceptibility loci, Ity4 (Immunity to Typhimurium locus 4) and Ity5, were identified. We validated Ity5 in a genetic environment free of the impact of Ity4 using a cross between A/J and 129S6. Using a time-series analysis of genome-wide transcription during infection, comparing A/J with AcB60 mice having a C57BL/6J-derived Ity5 interval, we have identified the differential expression of the positional candidate gene Cd40, Cd40-associated signaling pathways, and the differential expression of numerous genes expressed in neutrophils. CD40 is known to coordinate T cell-dependent B-cell responses and myeloid cell activation. In fact, CD40 signaling is altered in A/J mice as seen by impaired IgM upregulation during infection, decreased Ig class switching, neutropenia, reduced granulocyte recruitment in response to infection and inflammation, and decreased ERK1/2 activity. These results suggest that altered CD40 signaling and granulocyte recruitment in response to infection are responsible for the Ity5-associated Salmonella susceptibility of A/J mice.


Subject(s)
CD40 Antigens/immunology , Cation Transport Proteins/genetics , Disease Models, Animal , Mice , Salmonella Infections, Animal/immunology , Animals , Cation Transport Proteins/immunology , Crosses, Genetic , Gene Expression Profiling , Genetic Predisposition to Disease , Immunoglobulins/immunology , MAP Kinase Signaling System , Mice/classification , Mice/genetics , Mice/immunology , Mice, Inbred C57BL , Neutrophil Activation
3.
Genes Immun ; 15(5): 320-32, 2014.
Article in English | MEDLINE | ID: mdl-24848930

ABSTRACT

Genetic and immunological analysis of host-pathogen interactions can reveal fundamental mechanisms of susceptibility and resistance to infection. Modeling human infectious diseases among inbred mouse strains is a proven approach but is limited by naturally occurring genetic diversity. Using N-ethyl-N-nitrosourea mutagenesis, we created a recessive loss-of-function point mutation in Unc93b1 (unc-93 homolog B1 (C. elegans)), a chaperone for endosomal Toll-like receptors (TLR)3, TLR7 and TLR9, which we termed Letr for 'loss of endosomal TLR response'. We used Unc93b1(Letr/Letr) mice to study the role of Unc93b1 in the immune response to influenza A/PR/8/34 (H1N1), an important global respiratory pathogen. During the early phase of infection, Unc93b1(Letr/Letr) mice had fewer activated exudate macrophages and decreased expression of CXCL10, interferon (IFN)-γ and type I IFN. Mutation of Unc93b1 also led to reduced expression of the CD69 activation marker and a concomitant increase in the CD62L naive marker on CD4(+) and CD8(+) T cells in infected lungs. Finally, loss of endosomal TLR signaling resulted in delayed viral clearance that coincided with increased tissue pathology during infection. Taken together, these findings establish a role for Unc93b1 and endosomal TLRs in the activation of both myeloid and lymphoid cells during the innate immune response to influenza.


Subject(s)
Lymphocyte Activation , Macrophage Activation , Membrane Transport Proteins/genetics , Mutation , Orthomyxoviridae Infections/immunology , Alternative Splicing , Animals , CD8-Positive T-Lymphocytes/metabolism , Chemokine CXCL10/genetics , Chemokine CXCL10/metabolism , Endosomes/metabolism , Ethylnitrosourea , Immunity, Innate , Influenza A Virus, H1N1 Subtype/pathogenicity , Interferon Type I/genetics , Interferon Type I/metabolism , Interferon-gamma/genetics , Interferon-gamma/metabolism , L-Selectin/genetics , L-Selectin/metabolism , Lung/metabolism , Lung/pathology , Membrane Transport Proteins/metabolism , Mice , Mice, Inbred C57BL , Orthomyxoviridae Infections/genetics , Toll-Like Receptors/genetics , Toll-Like Receptors/metabolism
4.
Genes Immun ; 15(5): 282-92, 2014.
Article in English | MEDLINE | ID: mdl-24807690

ABSTRACT

Host genetics has a key role in susceptibility to Salmonella Typhimurium infection. We previously used N-ethyl-N-nitrosourea (ENU) mutagenesis to identify a loss-of-function mutation within the gene ubiquitin-specific peptidase 18 (Usp18(Ity9)), which confers increased susceptibility to Salmonella Typhimurium. USP18 functions to regulate type I interferon (IFN) signaling and as a protease to remove ISG15 from substrate proteins. Usp18(Ity9) mice are susceptible to infection with Salmonella Typhimurium and have increased expression and function of ISG15, but Usp18(Ity9) mice lacking Isg15 do not show improved survival with Salmonella challenge. Type I IFN signaling is increased in Usp18(Ity9) mice and inhibition of type I IFN signaling is associated with improved survival in mutant mice. Hyperactivation of type I IFN signaling leads to increased IL-10, deregulated expression of autophagy markers and elevated interleukin (IL)-1ß and IL-17. Furthermore, Usp18(Ity9) mice are more susceptible to infection with Mycobacterium tuberculosis, have increased bacterial load in the lung and spleen, elevated inflammatory cytokines and more severe lung pathology. These findings demonstrate that regulation of type I IFN signaling is the predominant mechanism affecting the susceptibility of Usp18(Ity9) mice to Salmonella infection and that hyperactivation of signaling leads to increased IL-10, deregulation of autophagic markers and increased proinflammatory cytokine production.


Subject(s)
Cytokines/metabolism , Interferon Type I/metabolism , Mutation , Salmonella Infections/genetics , Signal Transduction , Ubiquitin Thiolesterase/metabolism , Animals , Autophagy , Cytokines/genetics , Interleukin-17/genetics , Interleukin-17/metabolism , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Lung/metabolism , Lung/microbiology , Lung/pathology , Mice , Mice, Inbred C57BL , Mycobacterium Infections/genetics , Mycobacterium Infections/metabolism , Salmonella Infections/metabolism , Spleen/metabolism , Spleen/microbiology , Ubiquitin Thiolesterase/genetics , Ubiquitins/genetics , Ubiquitins/metabolism
5.
Genes Immun ; 13(2): 175-83, 2012 Feb.
Article in English | MEDLINE | ID: mdl-21956657

ABSTRACT

Typhoid fever, which is caused by Salmonella typhi and paratyphi, is a severe systemic disease that remains a major public health issue in several areas of the world. We can model the human disease using mice infected with a related bacterium, Salmonella typhimurium. This model recapitulates several clinical aspects of the human disease and allows for the study of the host response to Salmonella typhimurium infection in vivo. Previous work in our laboratory has identified three Immunity to typhimurium loci (Ity, Ity2 and Ity3) in the wild-derived MOLF/Ei mice, influencing survival after infection with Salmonella typhimurium. The MOLF/Ei alleles at Ity and Ity2 are protective, while the MOLF/Ei allele at Ity3 confers susceptibility. In this paper, we have generated a novel cross combination between the highly susceptible strain, MOLF/Ei, and the resistant strain, 129S6, to better define the genetic architecture of susceptibility to infection in MOLF/Ei. Using this cross, we have replicated the locus on chr 11 (Ity2) and identified a novel locus on chr 13 (Ity13). Using microarrays and transcriptional profiling, we examined the response of uninfected and infected Ity2 congenic mice. These analyses demonstrate a role for both type-1-interferon (IFN) and TRP53 signaling in the pathogenesis of Salmonella infection.


Subject(s)
Cation Transport Proteins/immunology , Salmonella Infections/genetics , Salmonella typhimurium , Signal Transduction , Alleles , Animals , Cation Transport Proteins/metabolism , Female , Genetic Predisposition to Disease , Interferon Type I/immunology , Male , Mice , Oligonucleotide Array Sequence Analysis , Salmonella Infections/immunology , TRPC Cation Channels/immunology , TRPC Cation Channels/metabolism , Tumor Suppressor Protein p53/immunology , Tumor Suppressor Protein p53/metabolism
6.
Genes Immun ; 12(7): 531-43, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21614019

ABSTRACT

In humans, Salmonella infection causes two major clinical diseases, typhoid fever and a self-limiting gastro-enteritidis. Salmonella transmission occurs by the fecal-oral route and the interactions between the bacteria and the digestive tract epithelium are central to the outcome of the infection. Using a mouse model of typhoid fever, we previously identified a mutation in USP18 affecting type I interferon (IFN) signaling resulting in increased susceptibility to systemic Salmonella infection. In this study, we demonstrate the effects of this mutation during the early response to Salmonella using a model of typhlitis. Mutant Usp18 mice showed a minimal inflammatory response early after Salmonella Typhimurium infection that was associated with low pathologic scores and low IFN-γ production. This resulted in an increased interaction of Salmonella with the cecal epithelium and earlier systemic dissemination of the bacteria. The global transcriptional signature in the cecum of mouse during Salmonella infection showed normal expression of tissue specific genes and upregulation of type I IFN pathway in mutant mice. In control mice, there was a significant over-representation of genes involved in cellular recruitment and antibacterial activity paralleling the histopathological features. These results show the impact of USP18 in the development of Salmonella-induced typhlitis.


Subject(s)
Endopeptidases/metabolism , Interferons/metabolism , Salmonella Infections/metabolism , Signal Transduction , Typhlitis/metabolism , Animals , Cecum/metabolism , Cecum/pathology , Disease Models, Animal , Endopeptidases/genetics , Gene Expression Profiling , Gene Expression Regulation , Kaplan-Meier Estimate , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Transgenic , Mutation , Salmonella Infections/genetics , Salmonella Infections/mortality , Salmonella Infections/pathology , Salmonella typhimurium , Typhlitis/genetics , Typhlitis/mortality , Typhlitis/pathology , Ubiquitin Thiolesterase
7.
Genes Immun ; 12(4): 280-90, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21326319

ABSTRACT

Infection of inbred mouse strains with Citrobacter rodentium represents an ideal model to reveal the genetic factors controlling host resistance to noninvasive enteric bacterial pathogens. We have chosen a positional cloning approach to identify putative gene(s) that control the known difference in survival between resistant C57BL/6J and susceptible C3H/HeJ and C3H/HeOuJ mice. Our work has identified one major locus within proximal chromosome 15 that is responsible for the marked susceptibility of both C3H strains, and we formally exclude Tlr4 from control of survival to this pathogen. We have named this new host resistance locus Cri1 (Citrobacter rodentium infection 1). The Cri1 genetic interval currently spans ∼16 Mb and it confers survival to the infection in a recessive manner. Transfer of the Cri1 locus from the surviving B6 mice into a congenic mouse with a C3Ou genetic background confirms its overall chromosomal localization and its highly significant effect on host survival. The C3Ou.B6-Cri1 mice thus produced have also enabled us to dissociate the control of mouse survival from the control of bacterial load early in the infection as well as from control of colonic hyperplasia.


Subject(s)
Citrobacter rodentium/immunology , Enterobacteriaceae Infections/genetics , Animals , Enterobacteriaceae Infections/immunology , Enterobacteriaceae Infections/pathology , Genetic Loci , Genetic Markers , Mice , Phenotype , Toll-Like Receptor 4/immunology
8.
Annu Rev Immunol ; 26: 81-132, 2008.
Article in English | MEDLINE | ID: mdl-17953509

ABSTRACT

Forward genetics is an experimental approach in which gene mapping and positional cloning are used to elucidate the molecular mechanisms underlying phenotypic differences between two individuals for a given trait. This strategy has been highly successful for the study of inbred mouse strains that show differences in innate susceptibility to bacterial, parasitic, fungal, and viral infections. Over the past 20 years, these studies have led to the identification of a number of cell populations and critical biochemical pathways and proteins that are essential for the early detection of and response to invading pathogens. Strikingly, the macrophage is the point of convergence for many of these genetic studies. This has led to the identification of diverse pathways involved in extracellular and intracellular pathogen recognition, modification of the properties and content of phagosomes, transcriptional response, and signal transduction for activation of adaptive immune mechanisms. In models of viral infections, elegant genetic studies highlighted the pivotal role of natural killer cells in the detection and destruction of infected cells.


Subject(s)
Immunity/immunology , Infections/immunology , Animals , Communicable Diseases/genetics , Communicable Diseases/immunology , Humans , Immunity/genetics , Immunity, Innate/genetics , Immunity, Innate/immunology , Infections/genetics , Lymphocytes/immunology , Lymphocytes/metabolism , Macrophages/immunology , Macrophages/metabolism , Mice , Models, Immunological
9.
Talanta ; 73(4): 718-25, 2007 Oct 15.
Article in English | MEDLINE | ID: mdl-19073093

ABSTRACT

This manuscript deals with the application of molecular connectivity calculations to predict the photo-induced chemiluminescent behaviour of the family of herbicides grouped as amides. Several compounds of this group were theoretically studied by means of a general discriminant equation formerly obtained, being 18 of them (plus eight from the chloroacetanilide sub-group) predicted with a high probability as photo-induced chemiluminescent. Empirical confirmation of the chemiluminometric behaviour was performed with some few commercially available amide herbicides. On the basis of these results, a new multicommutation-photo-chemiluminescent method is proposed for the determination of the diphenamid. The method is based on the photodegradation of the pesticide and then the resulting photo-fragment solutions are oxidized by K(3)[Fe(CN)(6)] in sodium hydroxide medium, at room temperature and 80 degrees C, for the photodegradation and chemiluminometric oxidation, respectively. The studied calibration up to 5.0mgl(-1), revealed a linear dynamic graph up to 20mgl(-1). The reproducibility between days resulted in a R.S.D. (in slope %) of 2.8 (n=5) and the repeatability with a %R.S.D. (n=20) of 4.3. LOD (s/n=3) of 1mugl(-1) and sample throughput of 20h(-1). The influence of foreign compounds is also tested and the optimized flow assembly is applied to different kind of samples.

10.
Genes Immun ; 7(8): 655-66, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17024130

ABSTRACT

The host response to Salmonella infection is controlled by its genetic makeup. Using the mouse model of typhoid fever, several genes were found to influence the outcome of Salmonella infection, including Nramp1 (Slc11a1). In order to improve our knowledge of genetic determinants of the mouse response to acute Salmonella Typhimurium infection, we performed a systematic screening of a set of A/J and C57BL/6J recombinant congenic strains (RCS) for their resistance to infection. While we knew that the parental strains differ in their susceptibility to Salmonella because C57BL/6J mice carry a non-functional allele at Nramp1, we hypothesized that other genes would influence the response to Salmonella and segregate in the RCS. We identified several RCS that showed a non-expected phenotype given their known Nramp1 genotype proving that the response to Salmonella in A/J and C57BL/6J mice is complex. Based on these findings, we selected two RCS for generation of fully informative F2 crosses, (AcB61 x 129S6) and (AcB64 x DBA/2J). Genetic analyses performed on these crosses identified five novel Salmonella susceptibility QTL mapping to chromosomes 3 (Ity4), 2 (Ity5), 14 (Ity6), 7 (Ity7) and 15 (Ity8). These results illustrate the genetic complexity associated with the mouse response to Salmonella Typhimurium.


Subject(s)
Cation Transport Proteins/genetics , Immunity, Innate/genetics , Quantitative Trait Loci , Salmonella Infections/genetics , Salmonella Infections/immunology , Salmonella typhimurium , Animals , Crosses, Genetic , Liver/microbiology , Lod Score , Mice , Mice, Congenic , Spleen/microbiology , Survival Analysis , Time Factors
11.
Genes Immun ; 7(5): 372-83, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16738669

ABSTRACT

The mouse response to Salmonella Typhimurium infection is partly controlled through detection of the bacterium lipopolysaccharide by the host pattern recognition receptor, Toll-like receptor 4 (Tlr4). Mice deficient in Tlr4 signaling are extremely susceptible to Salmonella infection with a 1,000-fold reduction in LD(50). In a previous study, we showed, using transgenic mice carrying one, three, six and >30 copies of Tlr4, that the level of expression of this gene influences the outcome of Salmonella infection, with a plateau effect starting at three copies. In the present study, we further investigate the impact of Tlr4 during Salmonella infection in mice expressing Tlr4 at slightly sub-normal, normal and slightly supra-normal levels by comparing host responses in mice carrying one, two and three copies of Tlr4 on the same genetic background. We describe in detail the in vivo host response to pathogenic Salmonella and show for the first time, in this narrow range of Tlr4 expression, an incremental protective effect against Salmonella due to improved control of bacterial growth in target organs and increased expression of important immune response genes in the spleen.


Subject(s)
Gene Expression Regulation/immunology , Salmonella Infections, Animal/genetics , Salmonella Infections, Animal/immunology , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism , Alleles , Animals , Gene Dosage , Mice , Mice, Transgenic , RNA, Messenger/analysis , Salmonella Infections, Animal/microbiology , Salmonella Infections, Animal/mortality , Transgenes
12.
Genes Immun ; 6(6): 500-8, 2005 Sep.
Article in English | MEDLINE | ID: mdl-15973461

ABSTRACT

The host response to infection in humans is multifactorial and involves the complex interaction between two genomes (the host and the pathogen) and the environment. Using an experimental mouse model of chronic infection, we have previously identified the individual effect of three significant and one suggestive quantitative trait loci (QTLs) (Ses1, Ses2, Ses3 and Ses1.1) on Salmonella Enteritidis persistence in target organs of 129S6/SvEvTac mice. Congenic strain construction was performed by transferring each of these QTLs from C57BL/6J onto the 129S6/SvEvTac background, and phenotypic analysis confirmed that Ses1 and Ses1.1 contribute to bacterial clearance. Additional QTLs regulating Salmonella carriage in 129S6/SvEvTac mice were identified using a two-locus epistasis QTL linkage mapping approach conducted separately in females and males. The epistatic model for females included the individual effect of Ses3 and two significant interactions (Ses1-D7Mit267 and Ses1-DXMit48) accounting for 47% of the total phenotypic variance. The model for males included the individual effect of Ses1.1, three interactions (Ses1-D9Mit218, D2Mit197-D4Mit2 and D3Mit256-D13Mit36) and explained 47% of the phenotypic variance. Our results suggest that the oligogenic nature of Salmonella persistence and epistasis are important constituents of the genetic architecture of the host response to chronic Salmonella infection.


Subject(s)
Epistasis, Genetic , Quantitative Trait Loci , Salmonella Infections/genetics , Salmonella enteritidis , Animals , Chromosome Mapping , Female , Male , Mice , Mice, Inbred BALB C , Salmonella Infections/immunology , Sex Factors
13.
Poult Sci ; 82(5): 721-6, 2003 May.
Article in English | MEDLINE | ID: mdl-12762392

ABSTRACT

Selection for increased resistance to Salmonella carrier-state (defined as the persistency of the bacteria 4 wk after inoculation) could reduce the risk for the consumer of food toxi-infections. The effects of two genomic regions on chromosomes 7 and 17 harboring two genes, NRAMP1 (SLC11A1) and TLR4, known to be involved in the level of chicken infection 3 d after inoculation by Salmonella were thus tested on a total of 331 hens orally inoculated at the peak of lay with 10(9) bacteria. The animals and their parents were genotyped for a total of 10 microsatellite markers mapped on chromosomes 7 and 17. Using maximum likelihood analysis and interval mapping, it was found that the SLC11A1 region was significantly involved in the control of the probability of spleen contamination 4 wk after inoculation. Single nucleotide polymorphisms (SNP) within the SLC11A1 and TLR4 gene were tested on those animals as well as on a second batch of 279 hens whose resistance was assessed in the same conditions. As the former was significantly associated with the risk of spleen contamination and the number of contaminated organs, SLC11A1 appears to be involved in the control of resistance to Salmonella carrier state. The involvement of the TLR4 gene was also highly suspected as a significant association between SNP within the gene, and the number of contaminated organs was detected.


Subject(s)
Carrier State/veterinary , Chickens/genetics , Genetic Predisposition to Disease , Poultry Diseases/genetics , Salmonella Infections, Animal/genetics , Animals , Carrier State/microbiology , Chickens/microbiology , Chromosome Mapping/veterinary , Chromosomes/genetics , Colony Count, Microbial/veterinary , Female , Likelihood Functions , Microsatellite Repeats , Polymorphism, Genetic , Poultry Diseases/microbiology , Salmonella , Salmonella Infections, Animal/microbiology , Spleen/microbiology
14.
Genes Immun ; 3(7): 381-93, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12424619

ABSTRACT

Salmonella spp are Gram-negative bacteria capable of infecting a wide range of host species, including humans, domesticated and wild mammals, reptiles, birds and insects. The outcome of an encounter between Salmonella and its host is dependent upon multiple factors including the host genetic background. To facilitate the study of the genetic factors involved in resistance to this pathogen, mouse models of Salmonella infection have been developed and studied for years, allowing identification of several genes and pathways that may influence the disease outcome. In this review, we will cover some of the genes involved in mouse resistance to Salmonella that were identified through the study of congenic mouse strains, cloning of spontaneous mouse mutations, use of site-directed mutagenesis or quantitative trait loci analysis. In parallel, the relevant information pertaining to genes involved in resistance to Salmonella in humans will be discussed.


Subject(s)
Drosophila Proteins , Gene Expression Regulation/immunology , Immunity, Innate/genetics , Salmonella Infections, Animal/genetics , Salmonella Infections, Animal/immunology , Agammaglobulinaemia Tyrosine Kinase , Animals , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Cloning, Molecular , Cytokines/genetics , Cytokines/metabolism , Immunity, Innate/physiology , Lipopolysaccharide Receptors/genetics , Lipopolysaccharide Receptors/immunology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , Mice, Congenic/immunology , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type II , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Quantitative Trait Loci/immunology , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Toll-Like Receptors
15.
Infect Immun ; 70(11): 6032-42, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12379679

ABSTRACT

The roles of the genes feoB (ABC ferrous iron transporter), mntH (proton-dependent manganese transporter), and sitABCD (putative ABC iron and/or manganese transporter) in Salmonella pathogenicity were investigated by using mutant strains deficient in one, two, or three transporters. Our results indicated that sitABCD encodes an important transporter of Mn(II) and Fe(II) which is required for full virulence in susceptible animals (Nramp1(-/-)) and for replication inside Nramp1(-/-) macrophages in vitro. The mntH sitABCD double mutant (mutant MS) showed minimal Mn(II) uptake and increased sensitivity to H(2)O(2) and to the divalent metal chelator 2,2'-dipyridyl (DP) and was defective for replication in macrophages. In vivo MS appeared to be as virulent as the sitABCD mutant in Nramp1(-/-) animals. The ferrous iron transporter Feo was required for full virulence in 129/Sv Nramp1(-/-) mice, and infection with multiple mutants lacking FeoB was not fatal. The sitABCD feoB mutant (mutant SF) and the mntH sitABCD feoB mutant (mutant MSF) showed minimal Fe(II) uptake and were slightly impaired for replication in susceptible macrophages. MSF showed reduced growth in minimal medium deficient in divalent cations. The role of the mntH gene, which is homologous to mammalian Nramp genes, was also investigated after overexpression in the double mutant MS. MntH preferred Mn(II) over Fe(II) and could suppress MS sensitivity to H(2)O(2) and to DP, and it also improved the intracellular survival in Nramp1(-/-) macrophages. This study indicates that acquisition of Mn(II), in addition to Fe(II), is required for intracellular survival and replication of Salmonella enterica serovar Typhimurium in macrophages in vitro and for virulence in vivo.


Subject(s)
Bacterial Proteins , Carrier Proteins/genetics , Cation Transport Proteins , Genes, Bacterial/physiology , Iron/metabolism , Manganese/metabolism , Salmonella typhimurium/genetics , Animals , Biological Transport , Culture Media , Hydrogen Peroxide/pharmacology , Macrophages/microbiology , Mice , Operon , Salmonella typhimurium/growth & development , Salmonella typhimurium/pathogenicity , Virulence
16.
Genes Immun ; 3(4): 196-204, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12058254

ABSTRACT

The Gram-negative bacteria, Salmonella, cause a broad spectrum of clinical diseases in both animals and humans ranging from asymptomatic carriage to life-threatening sepsis. We have developed a model to study the contribution of genetic factors to the susceptibility of 129sv and C57BL/6J inbred mice to Salmonella enteritidis during the late phase of infection. C57BL/6J mice were able to eliminate completely sublethal inoculums of S. enteritidis from their reticuloendothelial system, whereas 129sv mice could not even after 60 days post inoculation. A genome scan performed on 302 (C57BL/6J x 129sv) F2 progeny identified three dominant loci (designated Ses1 to Ses3) that are associated with disease susceptibility in 129sv mice. Two highly significant linkages were identified on chromosomes 1 (Ses1) and 7 (Ses2) with respective LOD scores of 9.9 (P = 1.4 x 10(-11)) at D1Mcg5 and 4.0 (P = 1.9 x 10(-5)) at D7Mit62. One highly suggestive QTL was located on chromosomes15 (Ses3) with a LOD score 3.4 (P = 1.2 x 10(-4)). The estimated effects of Ses1, Ses2 and Ses3 on the bacterial clearance were greater in females. Using a model of three loci, with interaction between Ses1 and Ses2 and sex as a covariate, the three QTLs explained 32% of the phenotypic variance. The candidacy of Nramp1 as the gene for Ses1 was evaluated using mice carrying a null allele at Nramp1 (129sv-Nramp1(tm1Mcg)). These mice have a significantly lower spleen bacterial load compared to the wild-type 129sv mice, strongly suggesting the involvement of Nramp1 in controlling S. enteritidis clearance during the late phase of infection.


Subject(s)
Cation Transport Proteins/genetics , Salmonella Infections, Animal/genetics , Salmonella Infections/genetics , Salmonella enteritidis , Animals , Cation Transport Proteins/physiology , Chromosome Mapping , Chronic Disease , Crosses, Genetic , Disease Models, Animal , Female , Lod Score , Mice , Mice, Inbred C57BL , Microsatellite Repeats , Quantitative Trait Loci
17.
Genomics ; 74(1): 21-35, 2001 May 15.
Article in English | MEDLINE | ID: mdl-11374899

ABSTRACT

Complex genetic traits can be dissected in mice, using well-defined sets of recombinant inbred strains, congenic strains, and recombinant congenic strains (RCS). We report the creation of a series of 37 independent RCS derived from the commonly used inbred strains of laboratory mouse A/J (A) and C57BL/6J (B6). These RCS were derived by systematic inbreeding of independent pairs of animals from a (F1 x A) x A and a (F1 x B) x B double backcross (N3), to create AcB and BcA strains, respectively. Fifteen AcB strains and 22 BcA strains at between 18 and 30 generations of inbreeding have been generated, are healthy, and show stable breeding performance. These strains have been genotyped for a total of 625 informative microsatellite DNA markers covering the entire genome, with an average spacing of 2.6 cM. Haplotype analyses indicate that on average, AcB and BcA strains contain 13.25% of the donor genome, a value close to the 12.5% expected from the breeding scheme used in their creation. In the AcB set, approximately 79% of the B6 genome has been transferred in independent strains, while in the BcA set approximately 84% of the A genome is represented on the B6 background. This represents an excellent coverage of congenic segments from both parental genomes in the two sets of strains, which can now be used to map simple and complex traits in a genome-wide fashion. As an example of the power of AcB/BcA strains as a mapping tool, the 37 strains were typed for susceptibility to infection with Legionella pneumophila, a monogenic trait controlled by the Lgn1 locus on Chromosome 13. Analysis of the strain distribution pattern of L. pneumophila susceptibility allowed direct mapping of Lgn1 to a 3-cM interval. The AcB/BcA set should prove a useful tool with which to investigate the complex genetic basis of known interstrain differences between A and B6 for many important diseases.


Subject(s)
Mice, Congenic/genetics , Quantitative Trait, Heritable , Animals , Chromosomes/genetics , Crosses, Genetic , DNA/genetics , Female , Gene Frequency , Genome , Genotype , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Microsatellite Repeats , Recombination, Genetic
18.
Genes Immun ; 2(1): 56-9, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11294571

ABSTRACT

Toll-like receptors (Tlrs) are transmembrane proteins that have recently been shown to play a critical role in the innate immune recognition of microbial constituents. Among this family, Tlr4 is a crucial signal transducer for lipopolysaccharide (LPS), the major component of the Gram-negative bacteria outer cell membrane. In this paper, we report that C57BL/6.KB2-mnd mice, a model of neuronal ceroid lipofuscinosis, do not respond to LPS. This defect is associated with a spontaneous mutation in Tlr4 consisting of a large insertion within exon 2 predicting a frameshift mutation and a truncated protein.


Subject(s)
Drosophila Proteins , Lipopolysaccharides/pharmacology , Membrane Glycoproteins/genetics , Mutation , Receptors, Cell Surface/genetics , Animals , Base Sequence , DNA Primers , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Neuronal Ceroid-Lipofuscinoses/genetics , Phenotype , Toll-Like Receptor 4 , Toll-Like Receptors
19.
Infect Immun ; 68(8): 4485-91, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10899846

ABSTRACT

In Escherichia coli, the waaP (rfaP) gene product was recently shown to be responsible for phosphorylation of the first heptose residue of the lipopolysaccharide (LPS) inner core region. WaaP was also shown to be necessary for the formation of a stable outer membrane. These earlier studies were performed with an avirulent rough strain of E. coli (to facilitate the structural chemistry required to properly define waaP function); therefore, we undertook the creation of a waaP mutant of Salmonella enterica serovar Typhimurium to assess the contribution of WaaP and LPS core phosphorylation to the biology of an intracellular pathogen. The S. enterica waaP mutant described here is the first to be both genetically and structurally characterized, and its creation refutes an earlier claim that waaP mutations in S. enterica must be leaky to maintain viability. The mutant was shown to exhibit characteristics of the deep-rough phenotype, despite its ability to produce a full-length core capped with O antigen. Further, phosphoryl modifications in the LPS core region were shown to be required for resistance to polycationic antimicrobials. The waaP mutant was significantly more sensitive to polymyxin in both wild-type and polymyxin-resistant backgrounds, despite the decreased negative charge of the mutant LPSs. In addition, the waaP mutation was shown to cause a complete loss of virulence in mouse infection models. Taken together, these data indicate that WaaP is a potential target for the development of novel therapeutic agents.


Subject(s)
Glucosyltransferases/genetics , Phosphotransferases (Alcohol Group Acceptor) , Polymyxins/pharmacology , Salmonella typhimurium/pathogenicity , Animals , Carbohydrate Sequence , Drug Resistance, Microbial , Escherichia coli Proteins , Lipopolysaccharides/chemistry , Mice , Mice, Inbred C57BL , Microbial Sensitivity Tests , Molecular Sequence Data , Mutation , Oligosaccharides/chemistry , Salmonella typhimurium/drug effects , Salmonella typhimurium/genetics , Sequence Analysis , Virulence/genetics
20.
Genomics ; 64(3): 230-40, 2000 Mar 15.
Article in English | MEDLINE | ID: mdl-10756091

ABSTRACT

Toll-like receptors (TLRs) are a group of evolutionarily conserved pattern recognition receptors involved in the activation of the immune system in response to various pathogens. In this paper, we describe the cloning and characterization of the mouse homologue of human TLR5. Mouse Tlr5 encodes a 859-amino-acid protein that contains an N-terminal signal sequence, a leucine-rich repeat extracellular domain, a short transmembrane domain typical of type I transmembrane proteins, and a Toll/interleukin-1R signaling domain characteristic of all TLR proteins. The mouse Tlr5 protein shows 81% homology to human TLR5 and approximately 40% similarity to other TLR family members. Northern blot analysis reveals that Tlr5 is expressed predominantly in liver and lung with low-level expression in most other tissues examined. We have mapped Tlr5 to distal chromosome 1 using the (C57BL/6J x Mus spretus) x C57BL/6J Jackson BSB panel as well as a (C57BL/6J x MOLF/Ei)F(2) panel with the following position: D1Mit112-8.0 cM-Tlr5-9.6 cM-D1Mit17. The presence of a quantitative trait locus for susceptibility to Salmonella typhimurium on distal chromosome 1 prompted the examination of Tlr5 in susceptible MOLF/Ei mice. Polymorphic sequence variants in Tlr5 allowed us to identify a unique 4-allele haplotype in MOLF/Ei. Furthermore, using both Northern blot analysis and reverse transcription-polymerase chain reaction, we have shown a reduced expression of Tlr5 during infection of MOLF/Ei mice with Salmonella. The assignment of Tlr5 to a chromosomal region known to harbor a Salmonella-susceptibility locus together with decreased expression of Tlr5 mRNA in liver of susceptible MOLF/Ei mice suggests the possibility that, as with other members of this family, Tlr5 may play a role in host response to bacterial gram-negative infections.


Subject(s)
Drosophila Proteins , Membrane Glycoproteins/genetics , Receptors, Cell Surface/genetics , Salmonella Infections, Animal/genetics , Salmonella typhimurium , Amino Acid Sequence , Animals , Blotting, Northern , Chromosome Mapping , Cloning, Molecular , Genetic Predisposition to Disease , Haplotypes , Humans , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Molecular Sequence Data , RNA, Messenger/analysis , Receptors, Cell Surface/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Toll-Like Receptor 5 , Toll-Like Receptors
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