The Use of Immunodeficient Male (CBA/N x BALB/c) F1 Mice to Produce Monoclonal Antibodies Directed to Proteins of <i>Leptospira interrogans</i> Rather than to Immunodominant Lipopolysaccharides

Segers, R. P. A. M.; Nijs, A. de; Kooten, Peter J. S. van; Gaastra, Wim; Zeijst, Bernard A.M. van der

doi:10.1089/hyb.1990.9.275

Cited by 11 publications

(11 citation statements)

References 22 publications

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“…L. interrogans LPS is also an immunodominant molecule (26,27) and is unique in that it is recognized by Toll-like receptor 2 (TLR2) and not TLR4 in human cells (LPS from the majority of other Gram-negative bacteria is recognized by TLR4) (28). Pathogenic Leptospira strains demonstrate more abundant and longer LPS than the saprophyte Leptospira biflexa (29), and mutations affecting the native biosynthesis of LPS affect both virulence in hamsters (30) and colonization of target organs in the mouse model (31).…”

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confidence: 99%

Leptospira interrogans lpxD Homologue Is Required for Thermal Acclimatization and Virulence

et al. 2015

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Leptospirosis is an emerging disease with an annual occurrence of over 1 million human cases worldwide. Pathogenic Leptospira bacteria are maintained in zoonotic cycles involving a diverse array of mammals, with the capacity to survive outside the host in aquatic environments. Survival in the diverse environments encountered by Leptospira likely requires various adaptive mechanisms. Little is known about Leptospira outer membrane modification systems, which may contribute to the capacity of these bacteria to successfully inhabit and colonize diverse environments and animal hosts. Leptospira bacteria carry two genes annotated as UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase genes (la0512 and la4326 [lpxD1 and lpxD2]) that in other bacteria are involved in the early steps of biosynthesis of lipid A, the membrane lipid anchor of lipopolysaccharide. Inactivation of only one of these genes, la0512/lpxD1, imparted sensitivity to the host physiological temperature (37°C) and rendered the bacteria avirulent in an animal infection model. Polymyxin B sensitivity assays revealed compromised outer membrane integrity in the lpxD1 mutant at host physiological temperature, but structural analysis of lipid A in the mutant revealed only minor changes in the lipid A moiety compared to that found in the wild-type strain. In accordance with this, an in trans complementation restored the phenotypes to a level comparable to that of the wild-type strain. These results suggest that the gene annotated as lpxD1 in Leptospira interrogans plays an important role in temperature adaptation and virulence in the animal infection model. Members of the genus Leptospira are spirochete bacteria encompassing saprophytic and pathogenic species and are considered to be the most widespread zoonotic bacteria worldwide (1). Leptospira is the etiological agent of the disease leptospirosis, which in severe manifestations leads to hemorrhage in the lungs, meningitis, and liver and/or kidney failure (1). Leptospirosis is an emerging disease, and the worldwide annual occurrence is estimated to be over 1 million human cases, with a 5 to 20% mortality rate (2, 3). Leptospira cannot breach the host epidermal lining, and transmission requires direct contact of the bacteria with cuts or abrasions in the skin (4, 5). Rats and other rodent species serve as reservoir hosts for Leptospira, which colonizes the urinary systems of these animals (6). Leptospira is shed back into the environment through the urine of reservoir hosts and can persist in freshwater and soil until direct contact with an animal recommences an infection cycle (7,8).A prominent Leptospira feature is the ability to proliferate under significantly different environmental conditions. Other Gram-negative bacterial pathogens with this ability include species of the genera Escherichia (9), Salmonella (10), Yersinia (11), Vibrio (12), and Pseudomonas (13). The capacity of bacteria to adapt to disparate environments is likely imparted by numerous evolved strategies that probably include modi...

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Leptospira interrogans lpxD Homologue Is Required for Thermal Acclimatization and Virulence

et al. 2015

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“…SM. Bacterial SMases are thought to be a potent virulent factor and play a key role in the lysis of blood cells through hydrolysis of SM located on the outer leaflet of the plasma membranes (52). Very recently, Clostridium perfringens SMase was found to induce hemolysis of sheep erythrocytes by activating the endogenous SM-metabolizing pathway in host cells, by which SM on the cell surface was converted to Cer, Sph and finally S1P (53).…”

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Involvement of Neutral Ceramidase in Ceramide Metabolism at the Plasma Membrane and in Extracellular Milieu

Tani

Igarashi

Ito

2005

Journal of Biological Chemistry

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Neutral ceramidase is a type II integral membrane protein, which is occasionally secreted into the extracellular milieu after the processing of its N-terminal anchor (Tani, M., Iida, H., and Ito, M. (2003) J. Biol. Chem. 278, 10523-10530). We found that when overexpressed in CHOP cells, neutral ceramidase hydrolyzed cell surface ceramide, which increased in amount after the treatment of cells with bacterial sphingomyelinase, leading to an increase in the cellular level of sphingosine and sphingosine 1-phosphate. On the other hand, knockdown of the endogenous enzyme by siRNA decreased the cellular level of both sphingolipid metabolites. The treatment of cells with bovine serum albumin significantly reduced the cellular level of sphingosine, but not sphingosine 1-phosphate, generated by overexpression of the enzyme. The cellular level of sphingosine 1-phosphate increased with overexpression of the cytosolic sphingosine kinase. These results suggest that sphingosine 1-phosphate is mainly produced inside of the cell after the incorporation of sphingosine generated on the plasma membranes. The enzyme also seems to participate in the hydrolysis of serum-derived ceramide in the vascular system. Significant amounts of sphingosine as well as sphingosine 1-phosphate were generated in the cellfree conditioned medium of ceramidase transfectants, compared with mock transfectants. No increase in these metabolites was observed if serum or bacterial sphingomyelinase was omitted from the conditioned medium, suggesting that the major source of ceramide is the serum-derived sphingomyelin. A sphingosine 1-phosphate receptor, S1P 1 , was internalized much faster by the treatment of S1P 1 -overexpressing cells with conditioned medium of ceramidase transfectants than that of mock transfectants. Collectively, these results clearly indicate that the enzyme is involved in the metabolism of ceramide at the plasma membrane and in the extracellular milieu, which could regulate sphingosine 1-phosphate-mediated signaling through the generation of sphingosine.

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“…Western blots were performed using 10 Ag of total protein, as described by Segers et al (1990). The monoclonal antibody 881116 (a gift of A.-M.Svennerholm) was used to detect the CFA/I subunit protein.…”

Section: Western Blotsmentioning

confidence: 99%

The positive regulator CfaD overcomes the repression mediated by histone-like protein H-NS (H1) in the CFA/I fimbrial operon of Escherichia coli.

et al. 1992

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CFA/I fimbriae of enterotoxigenic Escherichia coli are expressed at 37 degrees C and not at 20 degrees C. Expression of CFA/I fimbriae requires two DNA regions (regions 1 and 2) which are separated by 40 kb on the wild type plasmid. Region 2 encodes a protein (CfaD) which activates the promoter in region 1. We investigated whether the histone‐like protein H‐NS (H1) of E.coli is involved in the temperature regulated expression of CFA/I fimbriae. As demonstrated recently with other temperature regulated genes, a mutation in the gene coding for this nucleoid‐associated H‐NS (H1) protein resulted in derepression of CFA/I expression. CFA/I fimbriae were now expressed both at 20 degrees C and 37 degrees C. More strinkingly, the positive regulator CfaD was not needed for CFA/I expression in an H‐NS‐ strain. This indicates that CfaD diminishes an inhibitory effect of the H‐NS nucleoid‐associated protein. We also showed that in the H‐NS‐ strain the CfaD protein still has a positive effect on the transcription of CFA/I.

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The Use of Immunodeficient Male (CBA/N x BALB/c) F1 Mice to Produce Monoclonal Antibodies Directed to Proteins of Leptospira interrogans Rather than to Immunodominant Lipopolysaccharides

Cited by 11 publications

References 22 publications

Leptospira interrogans lpxD Homologue Is Required for Thermal Acclimatization and Virulence

Leptospira interrogans lpxD Homologue Is Required for Thermal Acclimatization and Virulence

Involvement of Neutral Ceramidase in Ceramide Metabolism at the Plasma Membrane and in Extracellular Milieu

The positive regulator CfaD overcomes the repression mediated by histone-like protein H-NS (H1) in the CFA/I fimbrial operon of Escherichia coli.

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