Ingeborg Becker

Salmonella porins induce a sustained, lifelong specific bactericidal antibody memory response

We examined the ability of porins from Salmonella enterica serovar typhi to induce a long‐term antibody response in BALB/c mice. These porins triggered a strong lifelong production of immunoglobulin G (IgG) antibody in the absence of exogenous adjuvant. Analysis of the IgG subclasses produced during this antibody response revealed the presence of the subclasses IgG2b, IgG1, IgG2a and weak IgG3. Despite the high homology of porins, the long‐lasting anti‐S. typhi porin sera did not cross‐react with S. typhimurium. Notably, the antiporin sera showed a sustained lifelong bactericidal‐binding activity to the wild‐type S. typhi strain, whereas porin‐specific antibody titres measured by enzyme‐linked immunosorbent assay (ELISA) decreased with time. Because our porin preparations contained the outer membrane proteins C and F (OmpC and OmpF), we evaluated the individual contribution of each porin to the long‐lasting antibody response. OmpC and OmpF induced long‐lasting antibody titres, measured by ELISA, which were sustained for 300 days. In contrast, although OmpC induced sustained high bactericidal antibody titres for 300 days, postimmunization, the bactericidal antibody titre induced by OmpF was not detected at day 180. These results indicate that OmpC is the main protein responsible for the antibody‐mediated memory bactericidal response induced by porins. Taken together, our results show that porins are strong immunogens that confer lifelong specific bactericidal antibody responses in the absence of added adjuvant.

The innate immune response to Entamoeba histolytica lipopeptidophosphoglycan is mediated by toll-like receptors 2 and 4

Entamoeba histolytica is a human pathogen that may invade the intestinal mucosa, causing amoebic colitis or hepatic abscesses when the trophozoites travel through the portal circulation to the liver. Lipopeptidophosphoglycan (LPPG) is a molecular pattern of E. histolytica recognized by the human immune system. Here we report that LPPG is exposed on the cell surface of E. histolytica trophozoites, and is recognized by the host through toll‐like receptor (TLR) 2 and TLR4. Correspondingly, human embryonic kidney (HEK)‐293 cells were rendered LPPG responsive through overexpression of TLR2 or TLR4/MD2. Moreover, co‐expression of CD14 enhanced LPPG signal transmission through TLR2 and TLR4. The interaction of LPPG with TLR2 and TLR4 resulted in activation of NF‐κB and release of interleukin (IL)‐10, IL‐12p40, tumour necrosis factor (TNF)‐α, and IL‐8 from human monocytes. Consistent with these findings, responsiveness of mouse macrophages lacking TLR2 expression (TLR2–/–) or functional TLR4 (TLR4d/d) to E. histolytica LPPG challenge was impaired while double deficient macrophages were unresponsive. In contrast to wild‐type control and TLR2–/–animals succumbing to lethal shock syndrome, TLR4d/dmice were resistant to systemic LPPG challenge‐induced pathology.

Using evolutionary changes to achieve species-specific inhibition of enzyme action--studies with triosephosphate isomerase.

BACKGROUND Many studies that attempt to design species-specific drugs focus on differences in the three-dimensional structures of homologous enzymes. The structures of homologous enzymes are generally well conserved especially at the active site, but the amino-acid sequences are often very different. We reasoned that if a non-conserved amino acid is fundamental to the function or stability of an enzyme from one particular species, one should be able to inhibit only the enzyme from that species by using an inhibitor targeted to that residue. We set out to test this hypothesis in a model system. RESULTS We first identified a non-conserved amino acid (Cys14) whose integrity is important for catalysis in triosephosphate isomerase (TIM) from Trypanosoma brucei. The equivalent residues in rabbit and yeast TIM are Met and Leu, respectively. A Cys14Leu mutant of trypanosomal TIM had a tendency to aggregate, reduced stability and altered kinetics. To model the effects of a molecule targeted to Cys14, we used methyl methanethiosulfonate (MMTS) to derivatize Cys14 to a methyl sulfide. This treatment dramatically inhibited TIMs with a Cys residue at a position equivalent to Cys14, but not rabbit TIM (20% inhibition) or yeast TIM (negligible inhibition), which lack this residue. CONCLUSIONS Cys14 of trypanosomal TIM is a non-conserved amino acid whose alteration leads to loss of enzyme structure and function. TIMs that have a cysteine residue at position 14 could be selectively inhibited by MMTS. This approach may offer an alternative route to species-specific enzyme inhibition.

CD8 cytotoxic T cells in cutaneous leishmaniasis.

CD8 T cells are essential in the defence against viruses, yet little is known of their participation in the host defence against parasites, such as Leishmania, which can cause a variety of clinical diseases, such as localized cutaneous, diffuse cutaneous, mucocutaneous and visceral leishmaniasis. Murine models of leishmaniasis suggest that CD8 T cells participate through IFN‐γ production, yet their cytotoxic capacity also plays an important role, as has been found in patients infected with various Leishmania strains, where CD8 T cell cytotoxicity and apoptosis of autologous Leishmania‐infected macrophages correlate with cure. Yet the mechanisms underlying the CD8 T activation in patients with leishmaniasis remain an enigma. It is possible that dendritic cells activate CD8 T cells through mechanisms that include antigen cross‐presentation. Here we summarize the recent findings of CD8 T cells in cutaneous leishmaniasis and discuss their significance in the control of the disease. Further knowledge in this field will undoubtedly improve the design of therapeutic and vaccine strategies.

CD8 Cells of Patients with Diffuse Cutaneous Leishmaniasis Display Functional Exhaustion: The Latter Is Reversed, In Vitro, by TLR2 Agonists

Leishmania mexicana (Lm) causes localized (LCL) and diffuse (DCL) cutaneous leishmaniasis. DCL patients have a poor cellular immune response leading to chronicity. It has been proposed that CD8 T lymphocytes (CD8) play a crucial role in infection clearance, although the role of CD8 cytotoxicity in disease control has not been elucidated. Lesions of DCL patients have been shown to harbor low numbers of CD8, as compared to patients with LCL, and leishmanicidal treatment restores CD8 numbers. The marked response of CD8 towards Leishmania parasites led us to analyze possible functional differences between CD8 from patients with LCL and DCL. We compared IFNγ production, antigen-specific proliferation, and cytotoxicity of CD8 purified from PBMC against autologous macrophages (MO) infected with Leishmania mexicana (MOi). Additionally, we analyzed tissue biopsies from both groups of patients for evidence of cytotoxicity associated with apoptotic cells in the lesions. We found that CD8 cell of DCL patients exhibited low cytotoxicity, low antigen-specific proliferation and low IFNγ production when stimulated with MOi, as compared to LCL patients. Additionally, DCL patients had significantly less TUNEL+ cells in their lesions. These characteristics are similar to cellular “exhaustion” described in chronic infections. We intended to restore the functional capacity of CD8 cells of DCL patients by preincubating them with TLR2 agonists: Lm lipophosphoglycan (LPG) or Pam3Cys. Cytotoxicity against MOi, antigen-specific proliferation and IFNγ production were restored with both stimuli, whereas PD-1 (a molecule associated with cellular exhaustion) expression, was reduced. Our work suggests that CD8 response is associated with control of Lm infection in LCL patients and that chronic infection in DCL patients leads to a state of CD8 functional exhaustion, which could facilitate disease spread. This is the first report that shows the presence of functionally exhausted CD8 T lymphocytes in DCL patients and, additionally, that pre-stimulation with TLR2 ligands can restore the effector mechanisms of CD8 T lymphocytes from DCL patients against Leishmania mexicana-infected macrophages.

Polymorphism analysis of the internal transcribed spacer and small subunit of ribosomal RNA genes of Leishmania mexicana.

Abstract.Leishmania mexicana causes a wide spectrum of clinical diseases. In spite of the variety of clinical forms, no data exist regarding genetic polymorphism of L. mexicana. We analyzed the polymorphism of the internal transcribed spacer (ITS) and the small subunit rRNA genes of 3 reference strains and 24 Mexican isolates of L. mexicana, by means of polymerase chain reaction and subsequent digestion by restriction enzymes.All strains of L. mexicana had invariant patterns for both the ITS and the small subunit of rRNA genes. Leishmania amazonensis and Leishmania venezuelensis displayed polymorphism only in the ITS. The high degree of identity of this region was confirmed by sequencing DNA from three L. mexicana isolates. There was almost complete identity of the sequence for the ITS region of L. venezuelensis and that of strains of Leishmania major, suggesting that these species may be more closely related than previously thought.

Perturbation of the Dimer Interface of Triosephosphate Isomerase and its Effect on Trypanosoma cruzi

Background Chagas disease affects around 18 million people in the American continent. Unfortunately, there is no satisfactory treatment for the disease. The drugs currently used are not specific and exert serious toxic effects. Thus, there is an urgent need for drugs that are effective. Looking for molecules to eliminate the parasite, we have targeted a central enzyme of the glycolytic pathway: triosephosphate isomerase (TIM). The homodimeric enzyme is catalytically active only as a dimer. Because there are significant differences in the interface of the enzymes from the parasite and humans, we searched for small molecules that specifically disrupt contact between the two subunits of the enzyme from Trypanosoma cruzi but not those of TIM from Homo sapiens (HTIM), and tested if they kill the parasite. Methodology/Principal Findings Dithiodianiline (DTDA) at nanomolar concentrations completely inactivates recombinant TIM of T. cruzi (TcTIM). It also inactivated HTIM, but at concentrations around 400 times higher. DTDA was also tested on four TcTIM mutants with each of its four cysteines replaced with either valine or alanine. The sensitivity of the mutants to DTDA was markedly similar to that of the wild type. The crystal structure of the TcTIM soaked in DTDA at 2.15 Å resolution, and the data on the mutants showed that inactivation resulted from alterations of the dimer interface. DTDA also prevented the growth of Escherichia coli cells transformed with TcTIM, had no effect on normal E. coli, and also killed T. cruzi epimastigotes in culture. Conclusions/Significance By targeting on the dimer interface of oligomeric enzymes from parasites, it is possible to discover small molecules that selectively thwart the life of the parasite. Also, the conformational changes that DTDA induces in the dimer interface of the trypanosomal enzyme are unique and identify a region of the interface that could be targeted for drug discovery.

Catalytic classes of proteinases of Entamoeba histolytica.

Endopeptidase inhibitors were used to determine the catalytic classes of proteinases present in extracts of Entamoeba histolytica (strain HM 1:IMSS) axenically grown in vitro. Cysteine proteinases account for most of the proteolytic activity; one or more proteinases with different catalytic mechanisms are also present but could not be unambiguously assigned to a particular catalytic class. Proteinases in amebic lysates were resolved by polyacrylamide gel electrophoresis with sodium dodecyl sulfate. The detergent was exchanged with Triton X-100 and the proteolytic activity in the gels was demonstrated by overlaying it on another gel containing the substrate. Four lysis zones were observed corresponding to molecular weights of 66,000, 56,000, 40,000 and 27,000. The first cannot be classified yet, but the last three showed properties consistent with those of cysteine proteinases. Finally, a novel technique is described which uses purified human alpha-2-macroglobulin to trap, purify and characterize proteases from amebic lysates. The results obtained with this technique confirm those of the overlay technique, since both methods reveal four distinct proteinases in the two different amebic preparations examined.

Translating innate response into long-lasting antibody response by the intrinsic antigen-adjuvant properties of papaya mosaic virus

Identifying the properties of a molecule involved in the efficient activation of the innate and adaptive immune responses that lead to long‐lasting immunity is crucial for vaccine and adjuvant development. Here we show that the papaya mosaic virus (PapMV) is recognized by the immune system as a pathogen‐associated molecular pattern (PAMP) and as an antigen in mice (Pamptigen). A single immunization of PapMV without added adjuvant efficiently induced both cellular and specific long‐lasting antibody responses. PapMV also efficiently activated innate immune responses, as shown by the induction of lipid raft aggregation, secretion of pro‐inflammatory cytokines, up‐regulation of co‐stimulatory molecules on dendritic cells and macrophages, and long‐lasting adjuvant effects upon the specific antibody responses to model antigens. PapMV mixed with Salmonella enterica serovar Typhi (S. typhi) outer membrane protein C increased its protective capacity against challenge with S. typhi, revealing the intrinsic adjuvant properties of PapMV in the induction of immunity. Antigen‐presenting cells loaded with PapMV efficiently induced antibody responses in vivo, which may link the innate and adaptive responses observed. PapMV recognition as a Pamptigen might be translated into long‐lasting antibody responses and protection observed. These properties could be used in the development of new vaccine platforms.

Disease Severity in Patients Infected with Leishmania mexicana Relates to IL-1β

Leishmania mexicana can cause both localized (LCL) and diffuse (DCL) cutaneous leishmaniasis, yet little is known about factors regulating disease severity in these patients. We analyzed if the disease was associated with single nucleotide polymorphisms (SNPs) in IL-1β (−511), CXCL8 (−251) and/or the inhibitor IL-1RA (+2018) in 58 Mexican mestizo patients with LCL, 6 with DCL and 123 control cases. Additionally, we analyzed the in vitro production of IL-1β by monocytes, the expression of this cytokine in sera of these patients, as well as the tissue distribution of IL-1β and the number of parasites in lesions of LCL and DCL patients. Our results show a significant difference in the distribution of IL-1β (−511 C/T) genotypes between patients and controls (heterozygous OR), with respect to the reference group CC, which was estimated with a value of 3.23, 95% CI = (1.2, 8.7) and p-value = 0.0167), indicating that IL-1β (−511 C/T) represents a variable influencing the risk to develop the disease in patients infected with Leishmania mexicana. Additionally, an increased in vitro production of IL-1β by monocytes and an increased serum expression of the cytokine correlated with the severity of the disease, since it was significantly higher in DCL patients heavily infected with Leishmania mexicana. The distribution of IL-1β in lesions also varied according to the number of parasites harbored in the tissues: in heavily infected LCL patients and in all DCL patients, the cytokine was scattered diffusely throughout the lesion. In contrast, in LCL patients with lower numbers of parasites in the lesions, IL-1β was confined to the cells. These data suggest that IL-1β possibly is a key player determining the severity of the disease in DCL patients. The analysis of polymorphisms in CXCL8 and IL-1RA showed no differences between patients with different disease severities or between patients and controls.