Cholesterol is required for Leishmania donovani infection: implications in leishmaniasis

Leishmania donovani is an obligate intracellular parasite that infects macrophages of the vertebrate host, resulting in visceral leishmaniasis in humans, which is usually fatal if untreated. The molecular mechanisms involved in host-parasite interaction leading to attachment on the cell surface and subsequent internalization of the parasite are poorly characterized. Cholesterol is a major constituent of eukaryotic membranes and plays a crucial role in cellular membrane organization, dynamics, function, and sorting. It is often found distributed non-randomly in domains in membranes. Recent observations suggest that cholesterol exerts many of its actions by maintaining a specialized type of membrane domain, termed "lipid rafts", in a functional state. Lipid rafts are enriched in cholesterol and sphingolipids, and have been thought to act as platforms through which signal transduction events are coordinated and pathogens gain entry to infect host cells. We report here that cholesterol depletion from macrophage plasma membranes using methyl-beta-cyclodextrin (MbetaCD) results in a significant reduction in the extent of leishmanial infection. Furthermore, the reduction in the ability of the parasite to infect host macrophages can be reversed upon replenishment of cell membrane cholesterol. Interestingly, these effects were not observed when parasites were serum-opsonized, indicating a specific requirement of cholesterol to mediate entry via the non-opsonic pathway. Importantly, we show that entry of Escherichia coli remains unaffected by cholesterol depletion. Our results therefore point to the specific requirement of plasma membrane cholesterol in efficient attachment and internalization of the parasite to macrophage cells leading to a productive infection. More importantly, these results are significant in developing novel therapeutic strategies to tackle leishmaniasis.     

Oral putrescine restores virulence of ornithine decarboxylase-deficient Leishmania donovani in mice

Administration of putrescine as a 1% solution in the drinking water ameliorated the profound loss of virulence exhibited by ornithine decarboxylase (ODC) deficient Leishmania donovani in mice. Furthermore, supplying α-difluoromethylornithine, an ODC inhibitor, at 2% in the drinking water reduced but did not eliminate infection with wild type L. donovani in the mouse model. Taken collectively, these findings: (1) demonstrate that oral putrescine can access the phagolysosome of macrophages in which the parasite resides in mice; (2) establish that the loss of virulence due to the Δodc lesion is a consequence of the inability of the mutant parasite to synthesize sufficient polyamines de novo; (3) imply that the L. donovani amastigote cannot access host polyamines in sufficient amounts for survival and growth; (4) and validate ODC as a drug target, although oral administration of DFMO is an unlikely therapeutic paradigm for visceral leishmaniasis.     

Expression of multiple CPB genes encoding cysteine proteases is required for Leishmania mexicana virulence in vivo

Leishmania mexicana mutants deficient in the multicopy CPB gene array have reduced virulence, demonstrated by poor lesion growth in BALB/c mice and induction of a protective Th1 response. Reinsertion of the amastigote-specific CPB2.8 or metacyclic stage-specific CPB2 gene into a CPB-deficient mutant L. mexicana failed to restore either a Th2 response or sustained virulence. However, reexpression of multiple CPB genes from a cosmid significantly restored virulence. This was characterized by increased lesion and parasite growth and the acquisition of a Th2 response, as determined by measuring interleukin-4 production and immunoglobulin G1 (IgG1) and IgE levels. These studies confirm that L. mexicana cysteine proteases are important virulence factors and provide an explanation for the presence in L. mexicana of a multicopy tandem array of CPB genes.     

Observations on human bone-marrow for Leishmania donovani

During the recent epidemic in North Bihar, a total of 65 cases which were clinically designated as Kala-azar had been subjected to bone-marrow aspiration from the iliac crest, out of which the L.D. body was demonstrated in 55 cases (84.61 per cent). Culture analysis of bone-marrow on modified Tobie's medium, revealed that ten cases (15.39 per cent) which were negative for L.D. body did not show any leptomonads, whereas out of 55 L.D. body positive cases, the promastigote forms developed in 22 cases (40.00 per cent). The probable reasons for the negative findings and also the age and sex distribution in Kala-azar are discussed in brief.     

The yjeQ gene is required for virulence of Staphylococcus aureus

Gene products required for in vivo growth and survival of Staphylococcus aureus and other pathogens represent new targets for antimicrobial chemotherapy. In this study we created a Staphylococcus aureus yjeQ deletion strain and tested its virulence using a mouse kidney abscess infection model. The yjeQ deletion strain was compromised for growth in vitro and severely attenuated for virulence. We concluded that yjeQ is an attractive and novel new drug target.     

Chromosome size polymorphisms of Leishmania donovani

A minimum of 22 chromosomes were found in all Leishmania donovani stocks examined by orthogonal field alternation gel electrophoresis (OFAGE). Chromosome sizes ranged from approximately 270 to 4000 kb. Certain chromosomes were polymorphic in size between stocks and chromosomes present in some stocks had no apparent equivalent in others. Specific polymorphisms were useful in distinguishing the subspecies L. d. donovani, L. d. infantum and L. d. chagasi and African L. donovani stocks but there were karotypic differences within these taxa. Radiolabelled DNA derived from whole chromosomes was hybridised to OFAGE Southern blots. Chromosome 1 of L. d. donovani was homologous to two larger chromosomes in all stocks. Chromosome 2 of certain L. d. chagasi and L. d. infantum stocks was homologous to both chromosomes 2 and 3 of L. d. donovani: this suggested that translocation between chromosomes may have contributed to the size polymorphisms. The smallest chromosome seen (270 kb) was unique to the African stock HU3. It was not homologous to small chromosomes in L. d. donovani, L. d. infantum or L. d. chagasi. The small chromosome did hybridise to two small chromosomes in another African stock, Khartoum, and to a large chromosome present in all stocks. The beta-tubulin gene was mapped to chromosomes 21/22, 13 and 7 with strongest hybridisation to 21/22. alpha-Tubulin was mapped to chromosomes 9. The alpha- and beta-tubulin arrangement was highly conserved.     

Methylglyoxal-catabolizing enzymes of Leishmania donovani promastigotes

Methylglyoxal is a toxic metabolite with growth inhibitory properties against Leishmania donovani promastigotes. We have shown in the present study that both log and stationary phase promastigotes of L. donovani can catabolize methylglyoxal to D-lactate as the major end product. The specific activity of methylglyoxal reductase was found to be the highest of all the catabolic enzymes. In contrast, the anabolic pathway for methylglyoxal could not be detected. Moreover, when control promastigotes or promastigotes in which the glycolytic pathway was inhibited were incubated with glucose, glycerol or dihydroxyacetone phosphate as energy source, neither methylglyoxal nor D-lactate could be detected.     

Alpha-difluoromethylornithine resistance in Leishmania donovani is associated with increased ornithine decarboxylase activity

The promastigote form of Leishmania donovani is sensitive to growth inhibition by DL-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), the first enzyme of the polyamine biosynthetic pathway, with an EC50 value of approximately 30 microM. Exposure of a wild type (DI700) cell population to gradually increasing concentrations of DFMO resulted in the selection of a strain of Leishmania, DFMO-10, which was capable of proliferating in 10 mM DFMO. DFMO-10 cells possessed an EC50 value for DFMO greater than 4 mM, and were cross-resistant to alpha-methylornithine, alpha-monofluoromethyl-3,4-dehydroornithine methyl ester, and delta-methyl-acetylenic putrescine, three other inhibitors of ODC activity. DI700 and DFMO-10 cells accumulated and/or transported [3H]DFMO and a spectrum of basic, neutral, and acidic amino acids at comparative rates. However, the DFMO-resistant Leishmania, if suspended in culture medium in the absence of DFMO for several days, expressed up to 15-fold greater levels of ODC activity than did wild-type cells. The overexpressed ODC in mutant cells appeared kinetically normal, since the ODC activities from DI700 and DFMO-10 cells possessed similar apparent Km values for ornithine and were equally sensitive to inactivation by DFMO. Incubation of extracts of DFMO-10 cells, but not of wild-type parental cells, with [3H]DFMO for 1 h resulted in the labeling of a polypeptide, presumably ODC, which migrated with a molecular weight of 76,000 +/- 4000 on SDS-gel electrophoretograms. As a consequence of the elevated ODC activities, the levels of putrescine in mutant cells released from DFMO exposure were also elevated by about 15-fold over those of wild-type cells, although spermidine levels in DI700 and DFMO-10 cells were similar. In the absence of prolonged selective pressure, the resistance to DFMO, the ODC activity, and the putrescine levels of DFMO-10 cells all returned to those of wild type cells, indicating that the mutant phenotype of DFMO-selected L. donovani was unstable.     

A vir-repressed gene of Bordetella pertussis is required for virulence.

Coordinate regulation of gene expression in Bordetella pertussis is controlled by the products of the vir locus, BvgA and BvgS. In the presence of modulating signals such as MgSO4 and nicotinic acid, expression of vir-activated genes (vag) is reduced, while expression of vir-repressed genes (vrg) is maximal. We have cloned one of these vir-repressed genes, vrg-6, in Escherichia coli. DNA sequencing has shown that vrg-6 is contained on a single EcoRI restriction endonuclease fragment and is predicted to code for a protein of 105 amino acids with a molecular weight of 11,441. The predicted protein product appears to have two domains, one consisting of seven hydrophobic proline-rich pentameric repeats and the other consisting of five alkaline trimeric repeats. Southern blot analysis has revealed vrg-6-homologous sequences in the chromosomes of Bordetella bronchiseptica and Bordetella parapertussis, but, unlike Bordetella pertussis, these species do not express vrg-6-homologous RNA when grown under modulating conditions. In order to assess the role of vrg gene products in B. pertussis pathogenesis, two 18323 derivatives which harbor TnphoA insertions in vrg genes were analyzed in a mouse model of respiratory infection. Strain SK6, which carries a vrg-6::TnphoA mutation, failed to induce lymphocytosis and was significantly less able to colonize lungs and trachea than its parent strain 18323 or than SK18, which harbors a TnphoA fusion in the vrg-18 locus. This is the first evidence that a vir-repressed gene may play an important role in the virulence of B. pertussis and the pathogenesis of whooping cough.     

TonB is required for intracellular growth and virulence of Shigella dysenteriae

To assess the importance of TonB-dependent iron transport systems to growth of Shigella in vivo, a tonB mutant of Shigella dysenteriae was isolated and tested in cultured cells. The tonB mutant invaded epithelial cells, but did not form plaques in confluent monolayers of Henle cells, indicating an inability of this mutant to spread from cell to cell. The rate of intracellular multiplication of the tonB mutant was reduced significantly compared to that of the wild type. The loss of virulence in the tonB mutant was not due to loss of either Shu or Ent, the TonB-dependent systems which allow for transport of heme and ferrienterobactin, respectively. A shuA mutant lacking the outer membrane receptor for heme, an entB mutant defective in enterobactin synthesis, and a shuA entB double mutant each were able to invade cultured cells, multiply intracellularly, and form wild-type plaques. The ability of S. dysenteriae to access iron during intracellular growth was assessed by flow cytometric analysis of an iron- and Fur-regulated shuA-gfp reporter construct. Low levels of green fluorescent protein expression in the intracellular environment were observed in all strains, indicating that iron is available to intracellular bacteria, even in the absence of TonB-dependent iron transport. The failure of the tonB mutant to grow well in an iron-replete intracellular environment suggests that TonB plays a role in addition to heme- and siderophore-mediated iron acquisition in vivo, and this function is required for the intracellular growth and intercellular spread of S. dysenteriae.     

Oxidant-mediated damage of Leishmania donovani promastigotes.

Dissemination of Leishmania within the host is related to parasites undergoing unchecked proliferation. We therefore studied the effects of oxidant generating systems on promastigote multiplication by (i) direct determinations of organism proliferation and (ii) the incorporation of [3H]uracil into promastigote nucleoprotein. These two parameters correlated closely as measures of organism replication as demonstrated by parallel suppression of them by the protein synthesis inhibitors puromycin and cycloheximide and the nucleic acid synthesis inhibitors actinomycin D and mitomycin C. Promastigotes showed dose-related susceptibility to reagent and generated hydrogen peroxide (H2O2) as reflected in quantitatively similar decreases in multiplication and [3H]uracil incorporation. These effects were specific for H2O2 as catalase abrogated the dimunition in multiplication. The generation of superoxide anion by acetaldehyde-xanthine oxidase (10 mU/ml) did not alter promastigote replication or nucleoprotein synthesis. These results indicate that Leishmania donovani promastigotes are damaged by H2O2 and that the incorporation of [3H]uracil into promastigote nucleoprotein may be useful for studying the interaction of this parasite with host effector cells.     

A Francisella tularensis locus required for spermine responsiveness is necessary for virulence

Tularemia is a debilitating febrile illness caused by the category A biodefense agent Francisella tularensis. This pathogen infects over 250 different hosts, has a low infectious dose, and causes high morbidity and mortality. Our understanding of the mechanisms by which F. tularensis senses and adapts to host environments is incomplete. Polyamines, including spermine, regulate the interactions of F. tularensis with host cells. However, it is not known whether responsiveness to polyamines is necessary for the virulence of the organism. Through transposon mutagenesis of F. tularensis subsp. holarctica live vaccine strain (LVS), we identified FTL_0883 as a gene important for spermine responsiveness. In-frame deletion mutants of FTL_0883 and FTT_0615c, the homologue of FTL_0883 in F. tularensis subsp. tularensis Schu S4 (Schu S4), elicited higher levels of cytokines from human and murine macrophages compared to wild-type strains. Although deletion of FTL_0883 attenuated LVS replication within macrophages in vitro, the Schu S4 mutant with a deletion in FTT_0615c replicated similarly to wild-type Schu S4. Nevertheless, both the LVS and the Schu S4 mutants were significantly attenuated in vivo. Growth and dissemination of the Schu S4 mutant was severely reduced in the murine model of pneumonic tularemia. This attenuation depended on host responses to elevated levels of proinflammatory cytokines. These data associate responsiveness to polyamines with tularemia pathogenesis and define FTL_0883/FTT_0615c as an F. tularensis gene important for virulence and evasion of the host immune response.     

Leishmania infection and virulence

Leishmaniasis is among the most important infectious diseases of the developing world. With the advent of effective culture conditions for the various stages of the life cycle, together with state of the art biochemical, genomics, and reverse genetic approaches, it has been possible to identify virulence factors required for the infection process. Several of these virulence factors are discussed within including lipophosphoglygan, A2, cysteine proteinases, and gp63.     

Iron-vibriobactin transport system is not required for virulence of Vibrio cholerae.

The possible requirement of a functional siderophore (vibriobactin)-mediated iron transport system in the pathogenicity of Vibrio cholerae was determined. Two mutants of V. cholerae defective in the iron-vibriobactin transport system were examined for their ability to multiply and elicit diarrhea in infant mice. One mutant, 40130, was unable to synthesize vibriobactin. The second mutant, 1510, was able to synthesize, but not transport, the siderophore. Both mutants retained the ability to multiply and produce disease in the infant mouse, and virulence was indistinguishable from the parent strains. This indicates that a functional iron-vibriobactin transport system is not essential for cholera pathogenesis. These mutants, like the wild-type strains, were found to have a ferric citrate iron uptake system and could utilize citrate or asparagine for growth in low-iron medium. Compounds of this type may increase the availability of iron to V. cholerae in the host.     

Tubulin is hyperphosphorylated on serine and tyrosine residues in arsenite-resistant Leishmania donovani promastigotes

An arsenite-resistant strain of Leishmania donovani was generated in vitro by the sequential exposure of a wild type strain to increasing concentrations of sodium m-arsenite. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of whole cell lysates of the two strains revealed that a protein band at the 55 kDa position showed slower migration in the resistant samples. This band was identified as tubulin by immunoblotting, with both α- and β-tubulin showing retarded migration in the resistant strain. Investigations into the reason for the observed slower migration revealed that phosphorylation of tubulin on both serine and tyrosine residues was enhanced in the resistant strain when compared to the wild type strain. Received: 5 January 2000 / Accepted: 15 March 2000     

Biochemical alterations in paromomycin-treated Leishmania donovani promastigotes

Paromomycin is used for the treatment of leishmaniasis in humans, but little is known about its mechanism of action. Investigating the effect of this antibiotic on promastigotes of Leishmania donovani, we showed that inhibition of the multiplication of these parasites could be related to its effect on RNA synthesis and to modifications of membranous polar lipids and membrane fluidity, leading to altered membrane permeability. Received: 13 June 1996 / Accepted: 11 September 1996