Peter E. Kima

Leishmania promastigotes activate PI3K/Akt signalling to confer host cell resistance to apoptosis.

Previous reports have shown that cells infected with promastigotes of some Leishmania species are resistant to the induction of apoptosis. This would suggest that either parasites elaborate factors that block signalling from apoptosis inducers or that parasites engage endogenous host signalling pathways that block apoptosis. To investigate the latter scenario, we determined whether Leishmania infection results in the activation of signalling pathways that have been shown to mediate resistance to apoptosis in other infection models. First, we showed that infection with the promastigote form of Leishmania major, Leishmania pifanoi and Leishmania amazonensis activates signalling through p38 mitogen‐activated protein kinase (MAPK), NFκB and PI3K/Akt. Then we found that inhibition of signalling through the PI3K/Akt pathway with LY294002 and Akt IV inhibitor reversed resistance of infected bone marrow‐derived macrophages and RAW 264.7 macrophages to potent inducers of apoptosis. Moreover, reduction of Akt levels with small interfering RNAs to Akt resulted in the inability of infected macrophages to resist apoptosis. Further evidence of the role of PI3K/Akt signalling in the promotion of cell survival by infected cells was obtained with the finding that Bad, which is a substrate of Akt, becomes phosphorylated during the course of infection. In contrast to the observations with PI3K/Akt signalling, inhibition of p38 MAPK signalling with SB202190 or NFκB signalling with wedelolactone had limited effect on parasite‐induced resistance to apoptosis. We conclude that Leishmania promastigotes engage PI3K/Akt signalling, which confers to the infected cell, the capacity to resist death from activators of apoptosis.

Perforin and gamma interferon are critical CD8+ T-cell-mediated responses in vaccine-induced immunity against Leishmania amazonensis infection.

ABSTRACT Previous studies have demonstrated that protection against New World leishmaniasis caused by Leishmania amazonensis can be elicited by immunization with the developmentally regulated Leishmania amastigote antigen, P-8. In this study, several independent experimental approaches were employed to investigate the protective immunological mechanisms involved. T-cell subset depletion experiments clearly indicate that elicitation of CD8+ (as well as CD4+) effector responses is required for protection. Further, mice lacking β2-microglobulin (and hence deficient in major histocompatibility complex class I antigen presentation) were not able to control a challenge infection after vaccination, indicating an essential protective role for CD8+ T effector responses. Analysis of the events ongoing at the cutaneous site of infection indicated a changing cellular dynamic involved in protection. Early postinfection in protectively vaccinated mice, a predominance of CD8+ T cells, secreting gamma interferon (IFN-γ) and expressing perforin, was observed at the site of infection; subsequently, activated CD4+ T cells producing IFN-γ were primarily found. As protection correlated with the ratio of total IFN-γ-producing cells (CD4+ and CD8+ T cells) to macrophages found at the site of infection, a role for IFN-γ was evident; in addition, vaccination of IFN-γ-deficient mice failed to provide protection. To further assess the effector mechanisms that mediate protection, mice deficient in perforin synthesis were examined. Perforin-deficient mice vaccinated with the P-8 antigen were unable to control infection. Thus, the elicitation of CD8+ T cell effector mechanisms (perforin, IFN-γ) are clearly required in the protective immune response against L. amazonensis infection in vaccinated mice.

Leishmania pifanoi amastigotes avoid macrophage production of superoxide by inducing heme degradation.

ABSTRACT Whereas infections of macrophages by promastigote forms of Leishmania mexicana pifanoi induce the production of superoxide, infections by amastigotes barely induce superoxide production. Several approaches were employed to gain insight into the mechanism by which amastigotes avoid eliciting superoxide production. First, in experiments with nitroblue tetrazolium, we found that 25% of parasitophorous vacuoles (PVs) that harbor promastigotes are positive for the NADPH oxidase complex, in contrast to only 2% of PVs that harbor amastigotes. Second, confocal microscope analyses of infected cells labeled with antibodies to gp91phox revealed that this enzyme subunit is found in PVs that harbor amastigotes. Third, in immunoblots of subcellular fractions enriched with PVs from amastigote-infected cells and probed with antibodies to gp91phox, only the 65-kDa premature form of gp91phox was found. In contrast, subcellular fractions from macrophages that ingested zymosan particles contained both the 91- and 65-kDa forms of gp91phox. This suggested that only the immature form of gp91phox is recruited to PVs that harbor amastigotes. Given that gp91phox maturation is dependent on the availability of heme, we found that infections by Leishmania parasites induce an increase in heme oxygenase 1 (HO-1), the rate-limiting enzyme in heme degradation. Infections by amastigotes performed in the presence of metalloporphyrins, which are inhibitors of HO-1, resulted in superoxide production by infected macrophages. Taken together, we propose that Leishmania amastigotes avoid superoxide production by inducing an increase in heme degradation, which results in blockage of the maturation of gp91phox, which prevents assembly of the NADPH oxidase enzyme complex.

Activation of PI3K/Akt signaling has a dominant negative effect on IL-12 production by macrophages infected with Leishmania amazonensis promastigotes.

Infection of macrophages with Leishmania parasites does not result in the production of IL-12. In addition, infection with Leishmania suppresses IL-12 production elicited by otherwise potent activators of IL-12. We provide evidence that engagement of phosphatidyl inositol-3 kinase (PI3K) signaling during Leishmania amazonensis infection leads to the prevention of IL-12 p70 production at the level of transcription of its p40 subunit in bone marrow derived macrophages (BMDMPhi). Inhibition of PI3K signaling with specific inhibitors of PI3K or the downstream kinase Akt, reverses the IL-12 blockade. Although the MAP kinase ERK (p44 and p42) was transiently activated by infection with L. amazonensis, inhibition of MEK, the kinase upstream of ERK, with PD98059, did not reverse the blockade of IL-12. Furthermore, inhibition of the other MAP kinases JNK and p38 as well as treatment of cells with pertussis toxin that blocks G protein mediated signaling, did not reverse the prevention of IL-12 production by Leishmania infection. Interestingly, activation of PI3K/Akt signaling had differential effects on ERK and p38 activation. Taken together we propose that infection of BMDMPhi with Leishmania promastigotes activates both positive and negative signaling pathways that control IL-12 production. PI3K signaling activated by the infection is the negative signaling pathway that prevents IL-12 production.

Leishmania pifanoi Pathogenesis: Selective Lack of a Local Cutaneous Response in the Absence of Circulating Antibody

ABSTRACT Recently, a role for B cells in the pathogenesis associated with infection by Leishmania (Leishmania mexicana complex and L. donovani) has been established. In the case of L. mexicana complex parasites (L. mexicana, L. pifanoi, and L. amazonensis), a critical role for immunoglobulin G-mediated mechanisms for the amastigote stage in the host is evident; however, the immunological mechanisms involved remain to be established. In vitro analysis of the kinetics of parasite uptake by macrophages failed to indicate a major effect of antibody opsonization. Given the importance of CD4+ T cells in the development of disease caused by these parasites, the possibility that the lack of pathogenesis was due to the lack of development of an immune response at the local site (draining lymph node and/or cutaneous site) was explored. Interestingly, the level of CD4+-T-cell activation (proliferation and cytokine) in draining lymph nodes from mice lacking circulating antibody (resistant) was found to be comparable to that in nodes from wild-type mice (susceptible) at 2, 5, and 10 weeks postinfection. However, antibody-deficient animals had markedly reduced numbers of monocytes and lymphocytes recruited or retained at the site of cutaneous infection in comparison to wild-type mice, indicating a selective impairment in the local cutaneous immune response. In vitro antigen presentation studies employing tissue-derived (opsonized) amastigotes demonstrated that L. pifanoi-infected FcR−/− macrophages, in contrast to comparably infected wild-type cells, failed to activate Leishmania antigen-specific T lymphocytes. These data, taken together, suggest that one possible mechanism for the role of antibody in pathogenesis may be to mediate parasite uptake and regulate the immune response at the local cutaneous site of infection.

Leishmania parasitophorous vacuoles interact continuously with the host cell's endoplasmic reticulum; parasitophorous vacuoles are hybrid compartments

Macrophages that express representative endoplasmic reticulum (ER) molecules tagged with green fluorescence protein were generated to assess the recruitment of ER molecules to Leishmania parasitophorous vacuoles (PVs). More than 90% of PVs harbouring Leishmania pifanoi or Leishmania donovani parasites recruited calnexin, to their PV membrane. An equivalent proportion of PVs also recruited the membrane‐associated soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs), Sec22b. Both ER molecules appeared to be recruited very early in the formation of nascent PVs. Electron microscopy analysis of infected Sec22b/YFP expressing cells confirmed that Sec22b was recruited to Leishmania PVs. In contrast to PVs, it was found that no more than 20% of phagosomes that harboured Zymosan particles recruited calnexin or Sec22b to their limiting phagosomal membrane. The retrograde pathway that ricin employs to access the cell cytosol was exploited to gain further insight into ER–PV interactions. Ricin was delivered to PVs in infected cells incubated with ricin. Incubation of cells with brefeldin A blocked the transfer of ricin to PVs. This implied that molecules that traffic to the ER are transferred to PVs. Moreover the results show that PVs are hybrid compartments that are composed of both host ER and endocytic pathway components.

Interferon gamma in leishmaniasis.

Leishmaniasis is a complex disease that is caused by parasites of the Leishmania genus. Leishmania are further classified into several complexes, each of which can engage in distinct interactions with mammalian hosts resulting in differing disease presentations. It is therefore not unexpected that host immune responses to Leishmania are variable. The induction of interferon gamma (IFN-γ) and response to it in these infections has received considerable attention. In this review, we summarize our current understanding of some of the host responses during Leishmania infections that are regulated by IFN-γ. In addition, studies that explore the nature of parasite-derived molecular mediators that might affect the host response to IFN-γ are also discussed.

Biochemical and Biological Characterization of the Protective Leishmania pifanoi Amastigote Antigen P-8

ABSTRACT The Leishmania pifanoi amastigote antigen P-8 has been previously shown to induce protective immunity in a murine model of cutaneous leishmaniasis (L. Soong, S. M. Duboise, P. Kima, and D. McMahon-Pratt, Infect. Immun. 63:3559–3566, 1995). As this antigen is of interest for further vaccine studies, the biochemical characterization of P-8 was undertaken. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western-blot analysis, and gel filtration chromatography revealed that P-8 antigen consisted of two proteoglycolipid complexes. The P-8 epitope is associated with theL. pifanoi amastigote-specific glycolipid components found in the two complexes. The P-8 complex 1 (P-8c1) consists of a 56-kDa serine metalloproteinase, apolipoprotein E (derived from fetal bovine serum), and amastigote-specific glycolipids. The P-8 complex 2 (P-8c2) consists of a 31-kDa cysteine proteinase associated with amastigote glycolipids. Biochemical analyses suggest that the P-8 antigenic glycolipids may be distinct from previously describedLeishmania glycolipids (glycosylinositolphospholipids and sphingoglycolipids). Protective immunity studies revealed that P-8c1 (serine metalloproteinase-glycolipid complex) confers comparable protection against infection as immunopurified P-8. The isolated P-8c2 (cysteine proteinase-glycolipid complex) does not provide significant protection, nor does stimulation with P-8c2 result in significant T-cell activation in P-8- or P-8c2-vaccinated mice. Consequently, the P-8c1 complex appears to be the immunodominant component of P-8.

Targeting Host Syntaxin-5 Preferentially Blocks Leishmania Parasitophorous Vacuole Development in Infected Cells and Limits Experimental Leishmania Infections

Our previous observations established a role for syntaxin-5 in the development of Leishmania parasitophorous vacuoles (LPVs). In this study, we took advantage of the recent identification of Retro-2, a small organic molecule that can cause the redistribution of syntaxin-5; we show herein that Retro-2 blocks LPV development within 2 hours of adding it to cells infected with Leishmania amazonensis. In infected cells incubated for 48 hours with Retro-2, LPV development was significantly limited; furthermore, infected cells harbored four to five times fewer parasites than infected cells incubated in vehicle alone. In vivo studies revealed that Retro-2 curbed experimental L. amazonensis infections in a dose-dependent manner. Retro-2 did not have any appreciable effect on the host cell physiological characteristics; furthermore, it had no apparent toxicity in experimental animals. An unexpected, but welcome, finding was that Retro-2 inhibited the replication of Leishmania parasites in axenic cultures. This study is significant because it identifies an endoplasmic reticulum/Golgi SNARE as a potential target for the control of Leishmania infections; moreover, it suggests that small organic molecules can be identified that can selectively disrupt the vesicle fusion machinery that promotes the development of pathogen-containing compartments without exerting toxic effects on the host.

Identification of Leishmania Proteins Preferentially Released in Infected Cells Using Change Mediated Antigen Technology (CMAT)

Although Leishmania parasites have been shown to modulate their host cells responses to multiple stimuli, there is limited evidence that parasite molecules are released into infected cells. In this study, we present an implementation of the change mediated antigen technology (CMAT) to identify parasite molecules that are preferentially expressed in infected cells. Sera from mice immunized with cell lysates prepared from L. donovani or L. pifanoi-infected macrophages were adsorbed with lysates of axenically grown amastigotes of L. donovani or L. pifanoi, respectively, as well as uninfected macrophages. The sera were then used to screen inducible parasite expression libraries constructed with genomic DNA. Eleven clones from the L. pifanoi and the L. donovani screen were selected to evaluate the characteristics of the molecules identified by this approach. The CMAT screen identified genes whose homologs encode molecules with unknown function as well as genes that had previously been shown to be preferentially expressed in the amastigote form of the parasite. In addition a variant of Tryparedoxin peroxidase that is preferentially expressed within infected cells was identified. Antisera that were then raised to recombinant products of the clones were used to validate that the endogenous molecules are preferentially expressed in infected cells. Evaluation of the distribution of the endogenous molecules in infected cells showed that some of these molecules are secreted into parasitophorous vacuoles (PVs) and that they then traffic out of PVs in vesicles with distinct morphologies. This study is a proof of concept study that the CMAT approach can be applied to identify putative Leishmania parasite effectors molecules that are preferentially expressed in infected cells. In addition we provide evidence that Leishmania molecules traffic out of the PV into the host cell cytosol and nucleus.