With more than 12 million people affected worldwide, 2 million new cases occurring per year, and the rapid emergence of drug resistance and treatment failure, leishmaniasis is an infectious disease for which research on drug and vaccine development, host-pathogen, and vector-parasite interactions are current international priorities. Upon Leishmania-macrophage interaction, activation of the protein tyrosine phosphatase (PTP) SHP-1 rapidly leads to the down-regulation of Janus kinase and mitogen-activated protein kinase signaling, resulting in the attenuation of host innate inflammatory responses and of various microbicidal macrophage functions. We report that, in addition to SHP-1, the PTPs PTP1B and TCPTP are activated and posttranslationally modified in infected macrophages, and we identify an essential role for PTP1B in the in vivo progression of Leishmania infection. The mechanism underlying PTP modulation involves the proteolytic activity of the Leishmania surface protease GP63. Access of GP63 to macrophage PTP1B, TCPTP, and SHP-1 is mediated in part by a lipid raft–dependent mechanism, resulting in PTP cleavage and stimulation of phosphatase activity. Collectively, our data present a mechanism of cleavage-dependent activation of macrophage PTPs by an obligate intracellular pathogen and show that internalization of GP63, a key Leishmania virulence factor, into host macrophages is a strategy the parasite uses to interact and survive within its host.

Leishmania parasites have evolved sophisticated mechanisms to subvert macrophage immune responses by altering the host cell signal transduction machinery, including inhibition of JAK/STAT signalling and other transcription factors such as AP-1, CREB and NF-κB. AP-1 regulates pro-inflammatory cytokines, chemokines and nitric oxide production. Herein we show that upon Leishmania infection, AP-1 activity within host cells is abolished and correlates with lower expression of 5 of the 7 AP-1 subunits. Of interest, c-Jun, the central component of AP-1, is cleaved by Leishmania. Furthermore, the cleavage of c-Jun is dependent on the expression and activity of the major Leishmania surface protease GP63. Immunoprecipitation of c-Jun from nuclear extracts showed that GP63 interacts, and cleaves c-Jun at the perinuclear area shortly after infection. Phagocytosis inhibition by cytochalasin D did not block c-Jun down-regulation, suggesting that internalization of the parasite might not be necessary to deliver GP63 molecules inside the host cell. This observation was corroborated by the maintenance of c-Jun cleavage upon incubation with L. mexicana culture supernatant, suggesting that secreted, soluble GP63 could use a phagocytosis-independent mechanism to enter the host cell. In support of this, disruption of macrophage lipid raft microdomains by Methyl β-Cyclodextrin (MβCD) partially inhibits the degradation of full length c-Jun. Together our results indicate a novel role of the surface protease GP63 in the Leishmania-mediated subversion of host AP-1 activity. Leishmaniasis is a tropical disease affecting more than 12 million people around the world. The disease is caused by the Leishmania parasites that are transmitted to the mammalian host by a sandfly vector when it takes a blood meal. The parasites are able to survive and multiply inside of cells that comprise the primary defence of the host, the macrophages. We have extensively studied the mechanism whereby Leishmania escapes from macrophage microbicidal functions. Herein we report that the parasite can inactivate these cells by decreasing the activity of transcription factors such as Activated Protein-1(AP-1) that are involved in transcription of genes coding for antimicrobial functions of macrophages. In this study, we showed that Leishmania parasites use their most abundant surface protein GP63 to inactivate the AP-1 transcription factor. Furthermore, we found that GP63 enter into the macrophages independently of parasite internalization using lipid rich microdomains localized in the cellular membrane. In addition, GP63 was observed to reach the nuclear compartment where it cleaves AP-1 subunit proteins. Collectively, our findings reveal a novel mechanism used by Leishmania to facilitate its survival and propagation within its mammalian host cells. Better knowledge concerning the mechanisms whereby this pathogen can escape the innate immune response may help to develop new anti-Leishmania therapy.

Leishmania spp. are obligate intracellular parasites that inhabit the phagolysosomes of macrophages. Manipulation of host cell signaling pathways and gene expression by Leishmania is critical for Leishmania’s survival and resultant pathology. Here, we show that infection of macrophages with Leishmania promastigotes in vitro causes specific cleavage of the NF‐κB p65^RelA subunit. Cleavage occurs in the cytoplasm and is dependent on the Leishmania protease gp63. The resulting fragment, p35^RelA, migrates to the nucleus, where it binds DNA as a heterodimer with NF‐κB p50. Importantly, induction of chemokine gene expression (MIP‐2/CXCL2, MCP‐1/CCL2, MIP‐1α/CCL3, MIP‐1β/CCL4) by Leishmania is NF‐κB dependent, which implies that p35^RelA/p50 dimers are able to activate transcription, despite the absence of a recognized transcriptional transactivation domain. NF‐κB cleavage was observed following infection with a range of pathogenic species, including L. donovani, L. major, L. mexicana, and L. (Viannia) braziliensis, but not the non‐pathogenic L. tarentolae or treatment with IFN‐γ. These results indicate a novel mechanism by which a pathogen can subvert a macrophage's regulatory pathways to alter NF‐κB activity.

Host-pathogen interactions are of great importance in understanding the pathogenesis of infectious microorganisms. We developed in vitro models to study the host-pathogen interactions of porcine respiratory tract pathogens using two immortalized epithelial cell lines, namely, the newborn pig trachea (NPTr) and St. Jude porcine lung (SJPL) cell lines. We first studied the interactions ofActinobacillus pleuropneumoniae, an important swine pathogen, using these models. Under conditions where cytotoxicity was absent or low, we showed thatA. pleuropneumoniaeadheres to both cell lines, stimulating the induction of NF-κB. The NPTr cells consequently secrete interleukin 8, while the SJPL cells do not, since they are deprived of the NF-κB p65 subunit. Cell death ultimately occurs by necrosis, not apoptosis. The transcriptomic profile ofA. pleuropneumoniaewas determined after contact with the porcine lung epithelial cells by using DNA microarrays. Genes such astadBandrcpA, members of a putative adhesin locus, and a gene whose product has high homology to the Hsf autotransporter adhesin ofHaemophilus influenzaewere upregulated, as were the genespgaBC, involved in biofilm biosynthesis, while capsular polysaccharide-associated genes were downregulated. The in vitro models also proved to be efficient with other swine pathogens, such asActinobacillus suis,Haemophilus parasuis, andPasteurella multocida. Our results demonstrate that interactions ofA. pleuropneumoniaewith host epithelial cells seem to involve complex cross talk which results in regulation of various bacterial genes, including some coding for putative adhesins. Furthermore, our data demonstrate the potential of these in vitro models in studying the host-pathogen interactions of other porcine respiratory tract pathogens.

Leishmaniaparasites are able to secure their survival and propagation within their host by altering signalling pathways involved in the ability of macrophages to kill pathogens or to engage adaptive immune system. An important step in this immune evasion process is the activation of host protein tyrosine phosphatase SHP-1 byLeishmania. SHP-1 has been shown to directly inactivate JAK2 and Erk1/2 and to play a role in the negative regulation of several transcription factors involved in macrophage activation. These signalling alterations contribute to the inactivation of critical macrophage functions (e.g., Nitric oxide, IL-12, and TNF-α). Additionally, to interfere with IFN-γreceptor signalling,Leishmaniaalso alters several LPS-mediated responses. Recent findings from our laboratory revealed a pivotal role for SHP-1 in the inhibition of TLR-induced macrophage activation through binding to and inactivating IL-1-receptor-associated kinase 1 (IRAK-1). Furthermore, we identified the binding site as an evolutionarily conserved ITIM-like motif, which we named kinase tyrosine-based inhibitory motif (KTIM). Collectively, a better understanding of the evasion mechanisms utilized byLeishmaniaparasite could help to develop more efficient antileishmanial therapies in the near future.