Significance: Infection withLeishmania(Viannia) parasites can have different manifestations, ranging from localized cutaneous to disseminated and mucocutaneous leishmaniasis, that are prone to relapse after the healing. We previously described the association of the endosymbiontLeishmaniaRNA virus 1 (LRV1) with increased disease severity. Here, we showed that coinfection with the lymphocytic choriomeningitis virus (LCMV) or Toscana virus exacerbated the outcome ofLeishmania guyanensis-induced murine leishmaniasis, favoring parasite persistence and dissemination resulting in metastasis. Both endogenous and exogenous coinfections were dependent upon type I interferon responses. Strikingly, LCMV coinfection after the healing of leishmaniasis induced disease reactivation, overriding the protective adaptive immune response. Thus, viral infections may be a significant risk factor contributing to the pathological spectrum of human leishmaniasis.

In eukaryotes, sphingolipids (SLs) are important membrane components and powerful signaling molecules. In Leishmania, the major group of SLs is inositol phosphorylceramide (IPC), which is common in yeast and Trypanosomatids but absent in mammals. In contrast, sphingomyelin is not synthesized by Leishmania but is abundant in mammals. In the promastigote stage in vitro, Leishmania use SL metabolism as a major pathway to produce ethanolamine (EtN), a metabolite essential for survival and differentiation from non-virulent procyclics to highly virulent metacyclics. To further probe SL metabolism, we identified a gene encoding a putative neutral sphingomyelinase (SMase) and/or IPC hydrolase (IPCase), designated ISCL (Inositol phosphoSphingolipid phospholipase C-Like). Despite the lack of sphingomyelin synthesis, L. major promastigotes exhibited a potent SMase activity which was abolished upon deletion of ISCL, and increased following over-expression by episomal complementation. ISCL-dependent activity with sphingomyelin was about 20 fold greater than that seen with IPC. Null mutants of ISCL (iscl⁻) showed modest accumulation of IPC, but grew and differentiated normally in vitro. Interestingly, iscl⁻ mutants did not induce lesion pathology in the susceptible BALB/c mice, yet persisted indefinitely at low levels at the site of infection. Notably, the acute virulence of iscl⁻ was completely restored by the expression of ISCL or heterologous mammalian or fungal SMases, but not by fungal proteins exhibiting only IPCase activity. Together, these findings strongly suggest that degradation of host-derived sphingomyelin plays a pivotal role in the proliferation of Leishmania in mammalian hosts and the manifestation of acute disease pathology. Leishmania are obligate intracellular parasites responsible for a spectrum of diseases in humans ranging from self-healing skin lesions to deadly visceral infections. To survive, they must downregulate the microbicidal activity and scavenge nutrients from the host. Although Leishmania parasites do not synthesize sphingomyelin, which is an abundant lipid in mammals, they do possess the ability to hydrolyze sphingomyelin. This neutral sphingomyelinase (SMase) activity is dependent on an ISCL gene, which is also required for the degradation of inositol phosphorylceramide, the dominant sphingolipid in Leishmania. Deletion of ISCL in L. major led to a complete loss of SMase activity but no obvious defects in growth or differentiation in vitro. Remarkably, null mutants of ISCL failed to induce any detectable pathology in mammals yet were able to persist at low levels indefinitely. Such defect was completely reversed when a functional neutral SMase was introduced into the mutant. In total, our results suggest that degradation of host-derived sphingomyelin by Leishmania is essential for acute virulence. Further studies will elucidate the pivotal role of sphingolipid homeostasis in the molecular interaction between Leishmania parasites and their mammalian hosts.

Enterotoxigenic Escherichia coli (ETEC), defined by their elaboration of heat-labile (LT) and/or heat-stable (ST) enterotoxins, are a common cause of diarrheal illness in developing countries. Efficient delivery of these toxins requires ETEC to engage target host enterocytes. This engagement is accomplished using a variety of pathovar-specific and conserved E. coli adhesin molecules as well as plasmid encoded colonization factors. Some of these adhesins undergo significant transcriptional modulation as ETEC encounter intestinal epithelia, perhaps suggesting that they cooperatively facilitate interaction with the host. Among genes significantly upregulated on cell contact are those encoding type 1 pili. We therefore investigated the role played by these pili in facilitating ETEC adhesion, and toxin delivery to model intestinal epithelia. We demonstrate that type 1 pili, encoded in the E. coli core genome, play an essential role in ETEC virulence, acting in concert with plasmid-encoded pathovar specific colonization factor (CF) fimbriae to promote optimal bacterial adhesion to cultured intestinal epithelium (CIE) and to epithelial monolayers differentiated from human small intestinal stem cells. Type 1 pili are tipped with the FimH adhesin which recognizes mannose with stereochemical specificity. Thus, enhanced production of highly mannosylated proteins on intestinal epithelia promoted FimH-mediated ETEC adhesion, while conversely, interruption of FimH lectin-epithelial interactions with soluble mannose, anti-FimH antibodies or mutagenesis of fimH effectively blocked ETEC adhesion. Moreover, fimH mutants were significantly impaired in delivery of both heat-stable and heat-labile toxins to the target epithelial cells in vitro, and these mutants were substantially less virulent in rabbit ileal loop assays, a classical model of ETEC pathogenesis. Collectively, our data suggest that these highly conserved pili play an essential role in virulence of these diverse pathogens. Enterotoxigenic Escherichia coli (ETEC) infections contribute substantially to death and morbidity due to diarrheal illness and are associated with serious sequelae including malnutrition, stunted growth, and intellectual impairment among young children in developing countries. Effective engagement of intestinal epithelial cells is essential for ETEC pathogenicity. Consequently, pathovar specific plasmid-encoded adhesin structures known as colonization factors (CFs) have been a principal target for vaccines. However, tremendous inter-strain variation in the carriage of gene clusters encoding different CFs and significant antigenic diversity of the CF adhesins has posed a challenge to vaccine development. In contrast, type 1 pili are encoded by the fim operon located in the chromosome of most ETEC strains and are highly conserved. While type 1 pili are known to play a critical role in virulence of extraintestinal pathogenic E. coli, the present studies represent the first detailed examination of the contribution of these pili in ETEC pathogenesis. Here we demonstrate that ETEC type 1 pili are essential for optimal interactions with intestinal epithelia and that they play a critical role in virulence. These data may inform additional approaches toward development of broadly protective vaccines for these pathogens of global importance.

Enterotoxigenic Escherichia coli (ETEC) infections are highly prevalent in developing countries, where clinical presentations range from asymptomatic colonization to severe cholera-like illness. The molecular basis for these varied presentations, which may involve strain-specific virulence features as well as host factors, has not been elucidated. We demonstrate that, when challenged with ETEC strain H10407, originally isolated from a case of cholera-like illness, blood group A human volunteers developed severe diarrhea more frequently than individuals from other blood groups. Interestingly, a diverse population of ETEC strains, including H10407, secrete the EtpA adhesin molecule. As many bacterial adhesins also agglutinate red blood cells, we combined the use of glycan arrays, biolayer inferometry, and noncanonical amino acid labeling with hemagglutination studies to demonstrate that EtpA is a dominant ETEC blood group A–specific lectin/hemagglutinin. Importantly, we have also shown that EtpA interacts specifically with glycans expressed on intestinal epithelial cells from blood group A individuals and that EtpA-mediated bacterial-host interactions accelerate bacterial adhesion and effective delivery of both the heat-labile and heat-stable toxins of ETEC. Collectively, these data provide additional insight into the complex molecular basis of severe ETEC diarrheal illness that may inform rational design of vaccines to protect those at highest risk.

Significance: Leishmaniaparasites can be infected withLeishmaniavirus(LRV1), a double-stranded RNA virus whose presence inLeishmania guyanensisparasites exacerbates disease severity in both mouse models and humans. Studies of the role of the virus on parasite biology and virulence are hampered by the dearth of isogenic lines bearing and lacking LRV, particularly in the clinically important speciesLeishmania braziliensis. Here, we describe a method to systematically generate LRV1-freeLeishmaniaparasites using the parasite RNA interference (RNAi) pathway. The ability of transgene-driven RNAi to overcome the ability of LRV1 to withstand the endogenous RNAi attack suggests a third paradigm of virus–RNAi interaction where RNAi and virus replication exist in balance to maintain persistent infection.

Significance: The endogenous double-stranded RNA virusLeishmaniavirus(LRV1) has been implicated as a pathogenicity factor for leishmaniasis in rodent models and human disease, and associated with drug-treatment failures. As a first step toward the identification of therapeutic LRV1 inhibitors, we identified two adenosine analogs that selectively inhibited LRV1 replication. These analogs were used as tools to confirm that viral inheritance is by random segregation, as well as to generate LRV1-cured lines ofLeishmania guyanensis,which correspondingly lost the increased pathogenicity conferred by LRV1. These compounds hold promise as leads to ameliorate the severity of LRV1-bearingLeishmaniainfections, and raise the possibility of targeting other protozoal infections whose pathogenicity may be exacerbated by similar endogenous viruses.